The pearl jubilee of microcin J25: thirty years of research on an exceptional lasso peptide

Contents

• 1 Introduction

• 2 The founding story of MccJ25
  • 2.1 The birth of the microcins
  • 2.2 The discovery of MccJ25

• 3 The properties of the threaded lasso fold of MccJ25
  • 3.1 MccJ25, from the early structure to the lasso fold
  • 3.2 The high stability of the lasso fold of MccJ25
  • 3.3 Naturally occurring diversity in the lasso peptide framework

• 4 The biosynthesis of MccJ25
  • 4.1 The MccJ25 biosynthetic gene cluster
  • 4.2  In vitro reconstitution of MccJ25 and first biosynthesis rules of lasso peptides

• 5 The biological activity of MccJ25
  • 5.1 Bacterial resistance to MccJ25
  • 5.2 Uptake of MccJ25
  • 5.3 The targets of MccJ25

• 6 The further study of MccJ25 activity using in vitro and in vivo models and conceiving applications
  • 6.1 Activity of MccJ25 in in vitro and in vivo models
  • 6.2 MccJ25 effects on the intestinal barrier function and the gut microbiota
  • 6.3 Occurrence of MccJ25 producing strains in the genus Escherichia in diverse microbiota
  • 6.4 Design of applications of MccJ25: consortia, formulations, synergies

• 7 Bioengineering utilizing the MccJ25 lasso peptide scaffold
  • 7.1 Library screens of MccJ25
  • 7.2 MccJ25-derived αvβ3 integrin antagonists
  • 7.3 MccJ25-derived ClpP binders
  • 7.4 Topological engineering with MccJ25

• 8 Conclusion and outlook

• 9 Author contributions

• 10 Conflicts of interest

• Acknowledgements

• Notes and references

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Abstract

Covering: 1992 up to 2023

Since their discovery, lasso peptides went from peculiarities to be recognized as a major family of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that were shown to be spread throughout the bacterial kingdom. Microcin J25 was first described in 1992, making it one of the earliest known lasso peptides. No other lasso peptide has since then been studied to such an extent as microcin J25, yet, previous review articles merely skimmed over all the research done on this exceptional lasso peptide. Therefore, to commemorate the 30th anniversary of its first report, we give a comprehensive overview of all literature related to microcin J25. This review article spans the early work towards the discovery of microcin J25, its biosynthetic gene cluster, and the elucidation of its three-dimensional, threaded lasso structure. Furthermore, the current knowledge about the biosynthesis of microcin J25 and lasso peptides in general is summarized and a detailed overview is given on the biological activities associated with microcin J25, including means of self-immunity, uptake into target bacteria, inhibition of the Gram-negative RNA polymerase, and the effects of microcin J25 on mitochondria. The in vitro and in vivo models used to study the potential utility of microcin J25 in a (veterinary) medicine context are discussed and the efforts that went into employing the microcin J25 scaffold in bioengineering contexts are summed up.

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Julian D. Hegemann

During his Diploma and PhD theses, Julian D. Hegemann investigated the biosynthesis and characteristic features of lasso peptides in the research group of Professor Mohamed Marahiel at the Philipps-Universität Marburg. For his postdoctoral research on the biochemistry of lanthipeptides, he joined the laboratory of Professor Wilfred van der Donk at the University of Illinois at Urbana-Champaign, where he worked from 2016 to 2019. After a second Postdoc with Professor Roderich Süssmuth at the Technische Universität Berlin, he started his independent research career as a junior research group leader at the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) in Saarbrücken in 2021.

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1 Introduction

The year 2022 marked the 30th anniversary of the first publication describing microcin J25 (MccJ25),¹ which opened a long, rich, and still ongoing story, which we want to commemorate through this review. Whereas the early history of MccJ25 in context of the research on low molecular weight bacteriocins from Gram-negative bacteria (called microcins) took place in Spain and Argentina, the continuing story played out in a much larger international setting in the field of ribosomally synthesized and post-translationally modified peptides (RiPPs). As a matter of fact, MccJ25 can be placed into both of these different groupings of natural products. Due to its origin and activity, MccJ25 is classified as a microcin, but because of its three-dimensional structure and biosynthesis, it also is considered a lasso peptide. Thus, both natural product groups are tightly connected to each other, but are most often studied independently.

MccJ25 is considered a microcin since it is a low molecular weight bacteriocin produced by Escherichia coli. As defined in the early 1900's, bacteriocins are proteinaceous antibacterial compounds of ribosomal origin. The term microcins was coined in 1976 to distinguish them from the colicins, the higher molecular weight antibacterial proteins also produced by E. coli.² Later on, microcins were found to be ubiquitously distributed in various other Gram-negative bacterial genera, essentially Enterobacteriaceae, including Shigella, Salmonella, Klebsiella, Enterobacter, and Citrobacter species.³ Like most known bacteriocins, microcins are either unmodified or post-translationally modified peptides that often exhibit a narrow spectrum of activity directed against bacteria closely related to the producers.^4,5 Moreover, microcins have been shown to play important roles in bacterial communities, both as actors in bacterial competition as well as in the communication in various microbiota and holobionts.^4,6–8 These features have contributed to the high interest these natural products currently raise in medical and veterinary contexts, in particular in the fight against the rise of multidrug-resistant pathogens, which is now considered as one of the biggest threats for human health.^3,9–11

Additionally, MccJ25 is considered a RiPP as its biosynthesis starts with the ribosomal assembly of a peptidic precursor that is then further modified enzymatically to acquire its biologically active structure. It was only in the early 2000's that MccJ25 was shown to be a lasso peptide^12–15 and that the basic rules of its biosynthesis in E. coli were identified,¹⁶ thus paving the way for further studies. Since then, MccJ25 became the archetype for the RiPP subfamily of lasso peptides that sparked (and still sparks) a huge interest, due to both their fascinating structural characteristics resulting from unique biosynthetic pathways and enzymatic machineries, as well as their important biological properties including potent antibacterial activities and complex mechanisms of action.

Therefore, to talk about MccJ25 means to talk about lasso peptides, and whereas this review is focused on MccJ25, occasional comparisons to other lasso peptides need to be drawn. But what makes a lasso peptide? The defining feature of this RiPP subfamily lies in their threaded structures that are reminiscent of lariat knots and therefore also the reason for their naming.^17–19 In general, lasso peptides feature a macrolactam ring formed between the N-terminal α-amine and the carboxylic acid side chain of an aspartate or glutamate residue. For all so far known lasso peptides, the Asp/Glu residues were observed to be residing at positions seven, eight, or nine of the compound. This makes sense considering that in lasso peptides, these rings are threaded by a linear, C-terminal tail region, yielding their typical, interlocked [1]rotaxane structures (in general, rotaxanes are molecules featuring threaded macrocycles. The number [n] denotes how many molecules a rotaxane consists of. Lasso peptides are therefore [1]rotaxanes, as the macrocycle and the threading portion of the C-terminal peptide chain are covalently linked to each other). These structures are in turn stabilized by the positioning of bulky amino acid side chains, so-called plug residues, above and below where the tail threads the ring; their steric bulk preventing the tail from slipping out. Hence, six residue rings are likely too small to allow threading, while in larger rings, the threading might be too hard to maintain by mere steric means.

Four different classes of lasso peptides have been described thus far that can be differentiated by the presence and localization of disulfide bonds as an additional feature for their stabilization (Fig. 1A).^17–23 In this respect, MccJ25 is a typical class II lasso peptide that exhibits the common interlocked [1]rotaxane structure shared by all lasso peptides, while not containing any disulfide crosslinks. It consists of 21 amino acids that are organized in a macrolactam ring formed between the N-terminus and the side chain carboxylate of a Glu residue at position 8. The resulting peptidic tail is locked inside the ring by the bulky side chains of two aromatic residues located above (Tyr19) and below (Phe20) the ring and by the C-terminal residue that complements the plug to prevent the tail slipping out of ring. The resulting lasso structure is thus composed of an eight-residue ring topped by a large loop featuring eleven amino acids (Fig. 1B and C) and followed by two additional residues.^12–14

Fig. 1 (A) Overview of the four lasso peptide classes and their specific modes of stabilization. Class I (one ring-to-tail plus one ring-to-loop disulfide bridge), class II (two bulky amino acid side chains forming plugs below and above the ring; no disulfide bridges), class III (one ring-to-tail disulfide bridge), and class IV (one tail-to-tail disulfide bridge forming a plug below the ring). The ring residues are shown in teal, the ring forming Asp/Glu residues in orange, and the tail residues in black. Cys residues forming disulfide bridges are highlighted in red. (B) Schematic representation and (C) three-dimensional structure (PDB 1Q71) of MccJ25.

In this review, a complete overview of the research related to MccJ25 from 1992–2023 will be given, which will include some more general studies about lasso peptide biosynthesis and activity but will also cover the more unique aspects relating to MccJ25 that are commonly skipped over in lasso peptide reviews. The article will therefore start on the discovery of MccJ25 and the work leading to the realization that MccJ25 exhibits a threaded lasso fold and what this structural feature implies. It will also include a general overview of the current knowledge pertaining to MccJ25 biosynthesis, its biosynthetic gene cluster, and how the producing strain protects itself against this antimicrobial lasso peptide. Research about the mode(s)-of-action of MccJ25 will be summarized, as will the research focused on utilizing the MccJ25 scaffold in a synthetic biology context.

Taken together, we hope that we can entice our readers to delve with us into the complexities and intricacies of the research on this fascinating lasso peptide. We aim to demonstrate how this research is still important even after 30 years since the initial discovery of this natural product and why we think it will remain important to address future challenges ahead of us in this field.

2 The founding story of MccJ25

2.1 The birth of the microcins

The discovery of MccJ25 cannot be dissociated from the story of the microcins, the antimicrobial peptides that were specifically characterized in Enterobacteria. Microcins were “born” in the Children University Hospital La Paz (The Peace) in Madrid, Spain, when during the work on his PhD thesis in 1973 (50 years ago), Fernando Baquero (one of the authors of this review) started to develop a keen interest in understanding the rapid bacterial replacements of Enterobacteriaceae in the gut microbiota of newborns (less than one month of age).

The hypothesis that replacements could be due to antimicrobial substances produced by different organisms was explored considering two major premises: (1) that these substances should be stable to the gut proteases, unlike most known bacteriocins, and that this stability could be facilitated by a small molecular weight; and (2) that these substances should be produced and should be able to act in a minimal culture medium, since the free nutrients in the gut would also be very limited in the highly colonized environment.

Consequently, screening for such substances was performed under conditions able to address these requirements:²⁴ the antibiosis assays were carried out on agar plates containing M63 minimal medium (0.2% glucose, pH 7), which were inoculated with E. coli marker strains (K12, B, W, and 405 (McLeod strain)) using the soft agar double-layer technique. Upon hardening of the medium, a film (6 × 5 cm) of cellophane was placed on the agar surface of each plate. Smears of potential antibiotic producing enterobacterial strains isolated from different newborns were seeded in different spots on the cellophane surface that only allowed the passing of molecules with a molecular weight up to ∼10 kDa. After being incubated for 24 hours at 37 °C, the plates were checked for antibiosis (Fig. 2). The bioactive substances, which were named “microcins” to differentiate them from colicins on the basis of their lower molecular weight, were then purified and preliminarily classified by cross-activity on a collection of Enterobacteriaceae.

Fig. 2 The plates used in the first screening for microcins. Agar plates with M63 minimal medium were overlayed with M63 soft agar inoculated with E. coli marker strains and, simultaneously, heavy spots of Enterobacteriaceae isolates were seeded over a cellophane membrane excluding large molecules like colicins. On the left, a plate is shown with the cellophane membrane carrying the colonies of the spotted isolates. Inhibition halos are seen around some of the colonies. On the right, the cellophane membrane was removed and is shown at the lower left corner next to the Petri dish.

An important part of this process was performed by a top biochemist, Prof. Carlos Asensio, and his group, working at the Autonomous University in Madrid. Prof. Asensio was highly interested in the molecular effectors of microbial ecology and was heavily influenced by discussions with Cornelius van Niel when attending the famous Courses in General Microbiology at Stanford University's Marine Laboratory, Bahía de Monterrey, California (1930–1962). This renowned meeting was known to also inspire other prominent microbiologists of that time like Roger Stanier, Max Delbrück, Seymour Lederberg, Robert Hungate, Arthur Kornberg, Bruce Ames, and Elie Wollman. The first public presentation on microcins was given in 1975 at the International Symposium on Enzymatic Mechanisms in Biosynthesis and Cell Function in Madrid, which had been co-organized by the illustrious biochemists Arthur Kornberg and Bernard Horecker. This symposium was also honoring one of the key breakers of the genetic code, the 1959 Nobel Prize awarded Severo Ochoa, who himself, in a separate meeting, delivered a lecture on microcins, under the title “Bacterial Wars”. The first publication on microcins (“Low molecular weight antibiotics from Enterobacteria”) was then released in 1976.²⁵

Many of the early works on the genetics and the modes of action of, as well as on the resistances to microcins were carried out in the Baquero's Department of Microbiology at the Ramón y Cajal Hospital in Madrid under the leadership of Felipe Moreno. Moreno and co-workers continued the work started by Baquero and Bouanchaud at the Pasteur Institute about the roles of plasmids in microcin production.²⁶ The distinction of microcins from colicins, conventionally known antibacterial proteins produced by E. coli, required personal contacts between Fernando Baquero and the retired Belgian microbiologist Pierre Fredericq (who as early as in 1949 coined the name “bacteriocin” to name the first bacterial antagonistic substance previously described by Gratia in 1925). Another international workshop organized by Fernando Baquero and Felipe Moreno in Granada, Spain, in 1983, which included top international experts in the field, including Felipe Moreno, Roberto Kolter, Jordan Konisky, Claude Lazdunsky, Maxime Schwartz, Volkmar Braun, and Anthony Pugsley, (Fig. 3) was also most crucial.

Fig. 3 Picture of the participants of the microcin kick-off meeting in La Alhambra, Granada (Spain), in 1983, including members of the Fernando Baquero and Felipe Moreno groups and invited world top scientists in the fields of bacteriocins and microbial interactions.

2.2 The discovery of MccJ25

Since the present review commemorates the 30th anniversary of the first publication describing MccJ25, it is perhaps the right time to present a brief description of the story behind the discovery of this remarkable molecule, including many of the tales untold that led to the seminal publication from 1992.¹ For this purpose, let's travel back to a day at the end of 1984, when Raúl Salomón (one of the authors of this review) was still a biochemist working at the Universidad Nacional de Tucumán in Argentina, and was browsing a bacteriology journal coming across an article that caught his attention. The paper provided an overview of microcins,²⁷ a then new family of low molecular weight antibiotic peptides from Enterobacteria, discovered only a few years earlier by Fernando Baquero and Carlos Asensio in Madrid. Further reading of the microcin literature brought Salomón to learn that Felipe Moreno, at that time Head of the Unidad de Genetica Molecular, part of the Baquero's Department of Microbiology at the Hospital Ramón y Cajal in Madrid, was one of the researchers who continued to actively work on microcins. Inspired by this article Raúl Salomón wrote a letter (no email then!) to Felipe Moreno, asking him to join his group and, indeed, in April 1985, Raúl Salomón was welcomed in the Moreno lab as a doctoral student. Unfortunately, Salomón had to return to Argentina for personal reasons in late 1987. Although this brought his thesis project to an abrupt end, he did not return empty-handed, but with a strong background and technical skills in microbiology and recombinant DNA technology.

Back in his hometown of San Miguel de Tucumán, Salomón, still wanting to obtain a PhD degree, thought that looking for a new microcin could be a good doctoral research project. He therefore arranged a meeting with the Head of the Biological Chemistry Department at the University of Tucumán, Dr Ricardo Farías, to speak about his idea and ask him to be his PhD supervisor. However, while Farías was a renowned researcher in the field of membranes and lipids, he had no expertise in molecular microbiology or antibiotic peptides. At first, he doubted that Salomón's proposal would lead to worthwhile results and it is of note that at that time no one else in Argentina seemed to be working on microcins. Yet, despite his initial skepticism, Farías still agreed to become Salomón's supervisor and let him pursue his idea in his lab, as he knew that Salomón was well trained and was already capable of working independently. Fortunately, it turned out that the doubts of Farías were unfounded. While Salomón was the first person to work on microcins in Argentina, Magela Laviña in Uruguay was studying microcin H47, and Rosalba Lagos in Chile worked with microcin E492. Including these compounds, only about six microcins had been described, more or less in-depth, when Salomón started his project and he was convinced that there would be more waiting to be discovered!⁴

Thus, Salomón's hunt for a new microcin started in the middle of 1989. However, the initial progress was very slow, since the lab available was poorly equipped for microbiological work. To obtain starting material for the screening work, Salomón asked a friend to collect a small stool sample from her baby daughter's diaper. Using the screening approach of Asensio and Baquero, which only detected compounds of low molecular weight, he tested 200 Gram-negative isolates. Only one strain producing a microcin-like activity was detected and it was named AY25 (the letters are the initials of the baby's name, while the number indicates that it was the 25th colony picked from the master plate). Biochemical reactions and serological tests typified strain AY25 as Escherichia coli, but this was not confirmed by further analyses. It is of note that the incidence of microcinogenic strains in this screen (0.5%) was much lower than that reported by Baquero and Asensio in Spain (about 15%).^25,27 However, this discrepancy could be explained due to the fact that Salomón only tested colonies from the fecal sample of a single baby, not colonies from fecal samples of many different babies, as Baquero and Asensio did.

The initial purification procedure for MccJ25, based on the adsorption of the active compound found in the concentrated supernatants to activated charcoal, followed by elution with acetone and reversed phase HPLC, was cumbersome and inefficient. Yet, it still yielded a few precious milligrams of pure material for the first analyses. Since the old rotary evaporator of the lab did not work properly, Salomón used a flat oven pan heated by a Bunsen burner to concentrate the supernatants. Luckily, the antimicrobial substance could withstand boiling temperatures (and even autoclaving) without losing any activity. Later, a protocol using direct adsorption of the culture medium on a preparative reversed-phase C8 cartridge, followed by elution with methanol and C18 reversed-phase HPLC, provided a faster, simpler, and more efficient way to obtain relatively high amounts of the antimicrobial molecule. Compositional analysis of MccJ25 revealed that it was a hydrophobic peptide comprising about 20–21 amino acid residues. The peptide could not be directly sequenced by Edman degradation, suggesting that the N-terminus was blocked. The first indication therefore was that it was indeed a microcin.¹

Curing experiments indicated that the peptide was encoded on a plasmid. Most known microcins were plasmid-borne, but in contrast to them, this plasmid was not conjugative. After several attempts, Salomón could transform it by the calcium chloride method into the laboratory strain E. coli K-12 MC4100. He named the wild-type (WT) plasmid pTUC100 (TUC for Tucumán). The transformant was susceptible to all other known microcins. Since immunity is specific for the cognate microcin, this test provided clear evidence that strain AY25 was producing a novel microcin.

In the first publication, the newly identified compound was called microcin 25.¹ Later, a new immunity group (J) was defined, and it was renamed into microcin J25 (MccJ25).²⁸ Furthermore, genetic analysis of mutants resistant to MccJ25 allowed Salomón to establish that the components of the translocation machinery of the antibiotic were the multifunctional outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA.^29,30 With the help of doctoral student José Solbiati, the genes of the MccJ25 system were cloned and a genetic complementation analysis defined three genes necessary for the biosynthesis of MccJ25.²⁸ These genes were designated mcjA, mcjB, and mcjC (for microcin J25). A fourth gene just downstream from mcjC, named mcjD, conferred immunity to the antibiotic. The elucidation of the nucleotide sequence of the four MccJ25 genes was initiated in Salomón's lab and then completed in 1999 by Solbiati in Felipe Moreno's lab.³¹

Studies with lacZ-fusions, carried out by Maria Jose Chiuchiolo, a PhD student, showed that expression of the MccJ25 precursor gene was induced as cells entered stationary phase, confirming what had been seen years before by measuring the antimicrobial activity in culture supernatants.³² In the meantime, Salomón had began a collaboration with Sylvie Rebuffat (also an author of this review), at the Laboratory of Chemistry and Biochemistry of Natural Substances from the National Museum of Natural History in Paris, France, at the beginning of the 2000's, with the purpose of determining the structure of MccJ25.³³ Yet, this first proposed structure was still far from the true lasso peptide fold.^12–14,34

Mapping of a rare MccJ25 resistant mutant on the 90 minutes chromosomal region of E. coli led Salomón to suggest in his doctoral thesis that the MccJ25 target could be the bacterial RNA polymerase (RNAP). Later, work of Monica Delgado, a doctoral student, confirmed this hypothesis, showing that the mutation was located in the rpoC gene, which encodes the largest subunit of the enzyme. The publication of this finding³⁵ attracted the interest of Konstantin Severinov (another author of this review), at Rutgers University, New Jersey, and paved the way for a collaboration to define the nature of the MccJ25 binding site on the RNAP.

As most of this and subsequent work was performed with a transformed strain carrying recombinant plasmids enabling MccJ25 production, little attention was paid to the original wild strain isolated from the newborn. With the occasion of a recent review on microcins in 2019,⁴ Baquero et al. performed a search for the presence of MccJ25 in about 14 000 genomes (including plasmids) from Enterobacterales available in public databases. Surprisingly, MccJ25 was present in less than 1% of E. coli isolates in the collection, but was abundant (about one-third of isolates) in the genomes of Escherichia marmotae (recognized in 2015 as a novel species, formerly belonging to “cryptic Escherichia”). Even considering the possible bias related to the low number of E. marmotae strains sequenced so far, we can suspect that the MccJ25-producing original strain could belong to this minority species, which would be in line with the observation that it was the “only microcinogenic strain” isolated by Salomón among the many E. coli colonies of a single newborn.

3 The properties of the threaded lasso fold of MccJ25

3.1 MccJ25, from the early structure to the lasso fold

MccJ25 was one of the first lasso peptides for which the three-dimensional structure was determined. Credit to the first correctly solved threaded lasso peptide structure must however be given to the class I lasso peptide RP 71955; a member of the siamycin-type lasso peptides.^18,36

The efforts towards elucidating the MccJ25 structure were spearheaded by several groups of researchers, including Sylvie Rebuffat, David J. Craik, Seth A. Darst, Richard H. Ebright, and their respective co-workers. The involvement of the Craik group in the work started after reading an early paper by Blond et al. in the European Journal of Biochemistry in 2001,³³ where the three-dimensional structure of MccJ25 was first proposed. The primary structure of MccJ25 had earlier been deduced to be a 21-residue cyclic peptide (cyclo(GGAGHVPEYFVGIGTPISFYG)) on the basis of biochemical and NMR studies.³⁷ At the time, the structure was determined by NMR to be a head-to-tail cyclic peptide that folded up into a compact three-dimensional shape (Fig. 4A). As Blond et al.³⁷ compared the structure of MccJ25 to that of kalata B1 (Fig. 4B), a disulfide-rich 29 amino acid peptide structurally characterised by Craik and co-workers in 1995,³⁸ the interest of Craik in MccJ25 was awoken as well.

Fig. 4 Structural comparison of MccJ25 and kalata B1. (A) The originally reported structure of MccJ25 as a head-to-tail cyclized peptide (PDB code 1HG6), compared to (B) the cyclotide kalata B1 (PDB code 1KAL), and (C) the corrected MccJ25 structure with the threaded lasso fold (PDB code 1Q71).^13,33,40 On top, the three-dimensional structures are presented as sticks. In the middle, the primary structures are shown using the one letter code for amino acids. Below, the stick structures are superimposed with the electron density maps of the respective molecules.

Kalata B1 was later categorised as being part of a larger family of peptides called cyclotides,³⁹ which are characterised by a head-to-tail cyclic backbone and a knotted arrangement of three disulfide bonds, which together form a motif known as the cyclic cystine knot. This motif imparts cyclotides with exceptional stability.^40,41 It was therefore surprising to see a peptide like MccJ25, which had no disulfide bonds, adopting a similar three-dimensional fold and also having a high stability while lacking the underlying support of the three knotted disulfide bonds. This stirred up the question how this could occur and so Craik and co-workers examined the evidence for the reported structure in more detail.

The original three-dimensional structure of MccJ25 was proposed to comprise a compact fold and, accordingly, a large number of medium and long-range NOEs between residues would be expected in the structural core. However, few such NOEs were detected and used in the published structure calculations. Indeed, an analysis of the three-dimensional structure in more detail suggested that it did not fully agree with the experimental data. This was particularly evident from the numerous short inter-proton distances present in the proposed structure, i.e., closer than 3.5 Å, for which no corresponding NOEs were visible in the spectra or reported in the published restraints list, and whose absence could not be explained by signal overlaps alone. Additionally, there were no sequential NOEs linking the two Gly residues that were supposed to form the N- and C-terminal residues deduced from the gene sequence. However, such NOEs would be expected for a backbone cyclized peptide. Sequential Hαi-HNi+1 NOEs were observed throughout the entire sequence except for across the proposed peptide bond between Gly1 and Gly21, strongly suggesting that the termini are in fact not covalently linked.

To find an explanation for the observed NMR data, other than in terms of a cyclic backbone, the possibility of side chain-to-backbone linkages was explored. The NMR spectra revealed the presence of several NOEs between the amide proton of Gly1 and the side chain protons of Glu8 and it was hypothesized that the cyclization event might involve Gly1 and Glu8 rather than Gly1 and the C-terminus. This would result in the structure containing an eight-residue ring in its N-terminal region with a linear tail for the C-terminus. Craik and co-workers re-calculated the structure¹³ using the original NOE data from the Blond et al. paper³³ but with the alternative possibility that there was a ring in the structure formed by linkage between the N-terminus and the carboxyl side chain of Glu8. The published data fitted this new structure perfectly,¹³ and was fully consistent with an extensive set of MS fragmentation data. Sylvie Rebuffat kindly provided a sample of MccJ25 to the Craik lab, and they recorded a full set of new NMR data and calculated a high-resolution structure (PDB ID 1Q71) confirming that the lasso structure was indeed correct (Fig. 4C).

At the same time, another group¹⁴ was also questioning the validity of the originally reported MccJ25 structure. David Craik corresponded with Tom Muir from that group and found that they had done extensive mass spectral studies that had led them to a similar conclusion about the lasso structure. Their interest had originally been aroused with their observation that synthetic peptides based on the reported structure were biologically inactive. Using a combination of biochemical studies, mass spectrometry, and NMR, they also confirmed that the reported head-to-tail cyclic structure was incorrect,¹⁴ and they determined a structure similar to the one elucidated by Craik and co-workers,¹³ referring to it as a lassoed tail.¹⁴

Both groups of researchers agreed to coordinate the publication of their respective findings by sending papers to the Journal of the American Chemical Society. Unknown to them, a third team of researcher led by Prof. Richard Ebright¹² was also working on a revised structure of MccJ25 at the same time. That group used triple-resonance NMR experiments to directly confirm the presence of a backbone-side chain amide linkage between Gly1 and Glu8 and determined a three-dimensional structure they referred to as a lariat pseudoknot. In the end, three back-to-back papers were published in Volume 125 of the Journal of the American Chemical Society in 2003 describing the revised structure of MccJ25 as a threaded lasso peptide.^12–14

Part of the reason why the original structure was misinterpreted was that the authors had undertaken thermolysin-based cleavage⁴² and found that the peptide increased in mass by 18 Da, consistent with a head-to-tail cyclic structure. In light of the revised MccJ25 structure, it turned out that thermolysin cuts in the loop of the lasso peptide, thereby yielding two peptide segments strongly coupled to each other. The Craik lab was able to demonstrate this was the case in a later paper co-authored with the Rebuffat group, by showing that the molecule resulting from thermolysin cleavage of MccJ25 had a wheel and axle type arrangement; a [2]rotaxane.³⁴

An inspection of the structure of MccJ25 reveals the underlying basis for its remarkable stability and unusual fragmentation upon thermolysin cleavage. The threading of the peptide chain through the embedded ring formed between the linkage of Glu8 and the N-terminus is very tight and associated with a large number of intra chain contacts that stabilise it. In addition, two bulky residues sit on either side of the embedded ring explaining why cleavage in the loop region with thermolysin does not allow the newly formed chain segment to be released from the ring. It is also notable that the C-terminal glycine residue is stabilised by a charge–charge interaction with the side chain of His5.

Taken together, the work summarized in this segment helped not only to identify MccJ25 as a bona fide example of a threaded lasso peptide, but also paved the way for future lasso peptide structure elucidations by teaching important lessons from the initially misassigned head-to-tail cyclic structure and what mistakes need to be avoided to end up with a true, threaded lasso structure.

3.2 The high stability of the lasso fold of MccJ25

One of the most fascinating features of the structure of lasso peptides is how it is maintained, at least in class II lasso peptides, merely by the steric interactions between the N-terminal ring and the plug residues in the linear C-terminal region.^43–47 For representatives of the lasso peptide subfamily outside of class II (Fig. 1), disulfide bridges can provide further structural stabilization by yielding an additional covalent linkage between the ring and the tail (classes I and III)^20,48–50 or by increasing the steric bulk through the formation of a second macrocycle following the lower plug amino acid (class IV).^21–23

In context of lasso peptide stability, MccJ25 is yet again a remarkable representative as its structure even withstands autoclaving at 120 °C.^1,43 This observation originated the belief that thermal stability is an intrinsic feature inherent to all lasso peptides and it took more than 20 years until it was demonstrated how there are indeed also naturally-occurring lasso peptides that are heat sensitive.^43,51–53 For these compounds, the increase in the molecular movement at high temperatures also increases the statistical likelihood of the residues situated in both ring and tail of the lasso peptide to adopt conformations that enable the tail to slip out of the ring.^44–46 This conversion of a lasso into a branched-cyclic peptide has since been called lasso peptide unthreading (Fig. 5A).

Fig. 5 (A) The possible behaviors of heat sensitive (left side) and heat stable (right side) lasso peptides upon exposure to elevated temperatures.⁴³ While a heat stable lasso peptide remains unchanged, a heat sensitive lasso peptide will unthread into a branched-cyclic peptide. (B) Two exemplary scenarios for the generation of [2]rotaxanes from a [1]rotaxane lasso peptide by one (right side) or two (left side) peptide bond hydrolysis events. Note, that each hydrolysis event adds one water molecule to the mass of the resulting [2]rotaxane,^54–56 while corresponding bond breakages induced by fragmentation in the gas phase are mass neutral events.^49,57,58

After the initial report of a heat sensitive lasso peptide, various studies have delved deeper into the physicochemical properties that decide on the thermal stability of lasso peptides.⁴³

In general, a combination of the size and the nature of both the macrolactam ring and the plug residues are the key factors that determine the behaviour of a lasso peptide at elevated temperatures.^{43–47,51,59,60} With its intermediate sized ring formed between Gly1 and Glu8 as well as the considerably big and rigid side chains of the upper (Phe19) and lower (Tyr20) plug amino acids, MccJ25 is able to maintain its [1]rotaxane structure even at high temperatures,^1,43 thus being a paragon for heat stable lasso peptides.

As a matter of fact, the steric hindrance induced through the macrolactam ring and Phe19/Tyr20 is so strong in MccJ25 that the ring and tail remain attached to each other even after the breakage of a peptide bond in the lasso peptide loop region. This observation (Fig. 5B) has been made both in the gas phase^14,43,61,62 (see Section 3.2.1) and in solution^34,55,63,64 (see Section 3.2.2) and will be discussed in detail in the subsequent subsections of this review article that explain how MccJ25 became such an important model system for understanding what forces are crucial for the stabilization of lasso peptide structures.

3.2.1 Stability of MccJ25 in the gas phase and rules for the structural characterization of lasso peptides by mass spectrometry. The lasso structure of MccJ25 results in an unusual behaviour following its hydrolysis in solution (see Section 3.2.2) or its fragmentation in the gas phase in MS/MS experiments.⁶⁵ Both processes convert the [1]rotaxane peptide into a [2]rotaxane entity upon bond breakages in the loop region; i.e., a pair of non-covalently linked peptide fragments is generated and the fragments remain attached to each other through the steric entrapment of the C-terminal tail within the macrolactam ring (Fig. 5B).

The [2]rotaxane product ions of MccJ25 generated by MS/MS constitute a signature of the lasso structure. Such product ions can be generated in positive ion mode upon collision induced dissociation (CID), infrared multiple photon dissociation (IRMPD), electron capture dissociation (ECD), and electron transfer dissociation (ETD)^{13,14,58,61,66} as well as in negative ion mode through electron detachment dissociation (EDD) and activated-electron photodetachment dissociation (a-EPD).⁶⁷

The detection of [2]rotaxane product ions relies on the occurrence of at least two bond breakage events in the loop region, since one bond breakage would only yield a [2]rotaxane entity isobaric to and therefore undiscernible from the [1]rotaxane precursor ion. Thus, the formation of [2]rotaxane product ions is statistically favoured for lasso peptides with long loop regions such as MccJ25. The analysis of a collection of lasso peptides revealed that the detection of [2]rotaxane product ions resulting from CID can constitute a robust signature of a lasso structure for class II lasso peptides with loop regions of at least five amino acids.⁵⁸ For lasso peptides with shorter loops, the detection of such [2]rotaxane species in the gas phase is usually not possible.

A more subtle, yet more broadly applicable signature of lasso peptide structures, as it is independent of the size of the loop, is the extent of hydrogen migration events (formation of from ) observed between residues in the loop region, which can be accessed via ECD or ETD.^58,61,66 Time-resolved double-resonance experiments under ECD conditions can be used to measure the formation rate constants of ions resulting from this process.⁶⁶ Lasso peptides, for which the complementary ions remain associated longer, exhibit a high rate of hydrogen migration. This is in stark contrast to a mere branched-cyclic peptide, where the prolonged association of ions is not possible to this extent.

ECD of the [M + 3H]³⁺ ion of MccJ25 and its homologous branched-cyclic isomer also generates a series of atypical radical b product ions resulting from a dual c/z˙ and y/b dissociation in the ring and in the tail, respectively.⁶¹ These product ions are proposed to constitute a signature of branched-cyclic peptides.

An efficient MS method to separate lasso and branched-cyclic peptides with the same primary structures is provided by ion mobility (Fig. 6).^68–70 The differentiation of lasso and branched-cyclic isomers by ion mobility-mass spectrometry (IM-MS) is particularly efficient at high charge states, which can be obtained in the presence of supercharging agents such as sulfolane,⁶⁸ although state-of-the-art IM-MS instruments with high resolving power can already accomplish a good separation of these isomers at low charge states as well. As compared to their branched-cyclic isomers, lasso peptides display: (i) a lower abundance for highly charged ions, (ii) a more or less linear change in their collision cross sections (CCS) in relationship to their charge states, and (iii) narrower ion mobility peak widths.^68,70

Fig. 6 (A) Schematic depicting the separation of a lasso peptide from its branched-cyclic topoisomer by traversing an IMS drift tube while having the same charge states. (B) As the lasso peptide is more compact than its branched-cyclic homolog (especially at high charge states), it has a lower drift time/a smaller CCS.^{49,57,62,68,70–72} (C) The relationship between the drift time/CCS and the charge state is approximately linear for a lasso peptide. In contrast, electrostatic repulsion at higher charge states yields less compact species in the more flexible branched-cyclic peptide.

This IM-MS based prediction method was enhanced by nanoelectrospray ionization-trapped ion mobility spectrometry-mass spectrometry (nESI-TIMS-MS) and the use of alkali metalation reagents.⁶⁹ The use of high-resolving, state-of-the-art TIMS-MS instruments furthermore allowed the identification and characterization of conformational changes contributed to the cis/trans isomerization of the Pro7 and Pro16 residues of MccJ25,⁶² which was corroborated by the generation and analysis of a series of MccJ25 variants (MccJ25(P7A), MccJ25(P16A), and MccJ25(P7A/P16A)).

IRMPD spectroscopy was used to characterize hydrogen bonding in the gas phase conformations of MccJ25 and other lasso peptides.⁷³ With these experiments, it became possible to connect smaller/larger CCSs measured by IM-MS to the extent of existing hydrogen-bonding-networks.

Finally, the cleavage sites in the hydrolysed forms of MccJ25 and other lasso peptides generated in solution (see Section 3.2.2) can be identified via MS/MS.^61,63 Single hydrolysis events in the loop region of [1]rotaxane lasso peptide species generates isomeric [2]rotaxane entities with a molecular weight increased by 18 Da. The location of the +18 Da increment, provided by the CID spectra, permits the localization the cleavage site unambiguously.

The manifold of information that can be taken from MS combined with the short duration of MS measurements and the low amounts of sample required makes MS-based techniques ideal methods for the characterization of threaded lasso structures explaining the huge impact these techniques had on the research on MccJ25 and lasso peptides in general.

3.2.2 In vitro stability of the MccJ25 lasso scaffold. As mentioned before, MccJ25 is a remarkably stable lasso peptide, whose threaded structure withstands high temperatures and exhibits a good stability in a pH range from 2 to 12.¹ The MccJ25 scaffold also tolerates bond cleavages in the loop region^{1,14,34,43,61,63} and the high stability of thermolysin-cleaved MccJ25 enabled the solution of the three-dimensional structure of this [2]rotaxane derivative of MccJ25 by NMR spectroscopy.³⁴ Yet, the activity of thermolysin-cleaved MccJ25 is significantly decreased compared to MccJ25, albeit not completely abolished.^34,56,64

Besides thermolysin, some other proteases are also able to cleave inside the loop region of MccJ25, e.g., elastase and, to a lesser extent, chymotrypsin,⁶³ which are part of the pancreatin, one of the main proteolytic components in the gastro-intestinal (GI) tract (see also Section 6.1). In contrast, MccJ25 is completely resistant to treatment with either carboxypeptidase Y or pepsin.³⁷

Interestingly, most lasso peptides do not allow for proteolytic cleavage of residues situated in their loop.^{47,51,53,60,74} The reason this can be observed for MccJ25 is likely due to the fact that it features one of the longest, most protruding loop regions of the known lasso peptides. Most other representatives of this RiPP subfamily contain much smaller and hence more compact and rigid loops that therefore become sterically inaccessible to protease active sites.

Due to the N-terminal α-amine being involved in the isopeptide bond, MccJ25, like all other lasso peptides, is also stable against aminopeptidases and chemical Edman degradation.¹ The side chains and the C-terminus of MccJ25 can however be modified according to their chemical characteristics (Fig. 7). E.g., glycine amidation of the C-terminus has been reported and it was demonstrated that this modification abolishes the ability of MccJ25 to inhibit the RNAP, but not the cellular uptake.^75,76 Carbethoxylation of the His5 imidazole ring has also been carried out and decreased the biological activity as well.⁷⁶ However, the ability to inhibit the RNAP in vitro was not affected,⁷⁶ hence this modification has to instead interfere with cellular uptake, a claim that is corroborated by the important interaction between the His5 residue of MccJ25 with the binding pocket of the FhuA receptor as revealed through their co-crystal structure (see also Section 5.2).⁷⁷

Fig. 7 Chemical modifications of MccJ25 reported in the literature.^76,78 (A) Glycine amidation of the C-terminus. (B) Carbethoxylation of the His5 side chain. (C) Nitration of the Tyr9 and Tyr20 residues. EDC = 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.

Moreover, specific conversion of the Tyr9 and Tyr20 residues of MccJ25 into 3-nitrotyrosine was accomplished using special dinitroimidazole reagents that were activated by irradiation at a 390 nm wavelength.⁷⁸

A class of lasso peptide isopeptidases have been reported that selectively cleave the isopeptide bonds of their substrate lasso peptides.^44,79–82 However, it has been shown that these isopeptidases are only acting on the lasso peptides produced by biosynthetic gene clusters found in close proximity to the respective isopeptidase-encoding gene (Fig. 8).^44,79–82 The isopeptidases accomplish their high substrate specificity not by using specific interactions with the side chains of the substrate lasso peptides but by utilizing a shape complementarity between their binding pockets and their substrates.^79,80 Hence, not even a branched-cyclic peptide with an identical primary structure to the substrate lasso peptide is degraded. There is no corresponding isopeptidase gene found in proximity to the MccJ25 operon and due to its quite unique three-dimensional structure amongst lasso peptides, it is rather unlikely that any of the other lasso peptide isopeptidases would have evolved towards recognizing a lasso peptide with a close enough shape to tolerate MccJ25 as a substrate as well.

Fig. 8 Typical organization of a lasso peptide isopeptidase-containing gene cluster found adjacent to a corresponding lasso peptide biosynthetic gene cluster.^{44,51,53,59,79–83}

3.3 Naturally occurring diversity in the lasso peptide framework

MccJ25 was one of the first lasso peptides discovered through bioactivity-guided screenings. In contrast, the discovery of novel lasso peptides for the last 15 years has been mainly driven by genome mining approaches,^{18,21,83–87} extending greatly the number of representatives (more than 80 different lasso peptide isolated so far) as well as the diversity within this RiPP subfamily.

The size of known lasso peptides ranges from 13 to 33 amino acids and they show great variations in the size of the ring, loop, and tail regions (Fig. 9). The macrolactam ring is formed by an isopeptide bond formed between the N-terminal α-amine and the side chain of a Glu or Asp residue at positions seven, eight or nine. The shortest ring (Gly1-Asp7, 22 atoms) is observed for huascopeptin,⁸⁸ while the largest (29 atoms) is observed for various lasso peptides containing a ring involving a Glu9 side chain, such as the caulonodins.⁵⁹ More drastic variations are observed for the size of the loop (e.g., three residues in huascopeptin;⁸⁸ seven residues in pandanodin²² and caulonodin V;⁵⁹ eleven residues in MccJ25; 18 residues in ubonodin⁸⁹) and tail regions (e.g., two residues in MccJ25, caulonodin V,⁵⁹ and ubonodin;⁸⁹ three residues in huascopeptin,⁸⁸ 18 residues in pandonodin²²).

Fig. 9 Overview of the three-dimensional structures of a selection of exemplary lasso peptides. On the bottom of the figure, the primary structures of the respective lasso peptides are depicted and their loop regions are highlighted in gray. Directly above, the corresponding three-dimensional structures of the lasso peptides are shown as sticks. On the top, the stick structures, viewed either from the side or from below, are overlayed with the electron density maps of the macrolactams and the elements required for the steric stabilization of each lasso fold. The structures are from huascopeptin,⁸⁸ caulonodin V (PDB code 2MLJ),⁵⁹ microcin J25 (PDB code 1Q71),¹³ pandonodin (PDB code 6Q1X),²² and ubunodin (PDB code 6POR).

Known lasso peptides range from highly polar^83,84 to highly hydrophobic representatives, such as fuscanodin/fusilassin.^90,91 Moreover, certain lasso peptides carry additional post-translational modifications (Fig. 10),⁹² such as an aspartimide in cellulonodin-2 and lihuanodin,⁹³ hydroxylations in canucin A^94,95 and RES-701-4,⁹⁶ phosphorylation of the side chain of a C-terminal Ser in paeninodin,^97,98 a deiminiation turning an Arg into a citrulline residue in citrulassin A,⁹⁹ acetylation in albusnodin,¹⁰⁰ C-terminal methylation in lassomycin¹⁰¹ and other lassomycin-like lasso peptides,¹⁰² epimerization of the α-carbon of the C-terminal amino acid residues in MS-271 ¹⁰³ and specialicin,¹⁰⁴ and phosphorylation and glycosylation in pseudomycoidin.¹⁰⁵ Most of these modifications are located in the C-terminal regions of the respective lasso peptides, possibly in part reinforcing the steric restraints involved in the stabilization of their lasso folds. In general, the addition of post-translational modifications to lasso peptides constitutes an attractive bioengineering route to create further diversity and potential new activities.

Fig. 10 Overview of all the additional tailoring reactions that have been observed for lasso peptide scaffolds so far.^{93,94,97–101,103–105,108} The modifications introduced are highlighted in orange.

The diversity within this RiPP subfamily relies not only on the diversity of the mature lasso peptides, but also on the organization of their biosynthetic gene clusters (BGCs) and their underlying maturation mechanisms. Unconventional trends have been reported such as the absence of a leader peptide for triculamin,¹⁰⁶ the maturation of core peptide sequences catalyzed only by the macrolactam synthetase for pseudomycoidin,¹⁰⁵ or the presence of bifunctional proteins (B2/D) featuring N-terminal leader peptidase (B2) and C-terminal ABC transporter (D) domains for the cochonodins.¹⁰⁷

New lasso peptide BGCs were also used to provide further insight into the biosynthesis of lasso peptides. In particular the processing enzymes from the thermophilic bacteria Thermobifida fusca involved in the production of the lasso peptide fuscanodin/fusilassin provided robust in vitro activity.^90,91 Moreover, additional genes in lasso peptide BGCs, such as genes encoding two-component regulation systems,^48,50,107 additional multimeric ABC exporter protein complexes,¹⁰⁷ lasso peptide isopeptidases,^44,79–82 or toxin/antitoxin systems,^109,110 suggest a complex regulation and catabolism for certain lasso peptides and delineate trends to assess lasso peptide evolution.¹¹⁰

Finally, diversity is also observed with regards to the biological activities of lasso peptides. The main activity reported for MccJ25 is its antibacterial properties against certain strains closely related to the producing strain, mainly Salmonella, Shigella, and Escherichia species.^3,111 Analogs of MccJ25 have been isolated from Enterobacteria, such as klebsidin (produced by a Klebsiella pneumoniae sp.¹¹²) and microcin Y (MccY; produced by a Salmonella enterica sp.¹¹³).

In spite of the variations in their primary sequences and three-dimensional structures, various other antimicrobial lasso peptides from Proteobacteria (capistruin,^114,115 citrocin,¹⁰⁹ ubonodin,⁸⁹ acinetodin and klebsidin,¹¹² MccY¹¹⁶) share the same intracellular target as MccJ25, the RNAP, while having a different activity spectrum. This suggests that the spectrum of activity is mainly driven by the uptake process, as already reported for MccJ25.³ Certain uptake mechanisms have been solved, such as that of ubonodin,¹¹⁷ which involves PupB as outer membrane receptor and YddA as inner membrane receptor in sensitive Burkholderia species. For citrocin, resistant mutants carry mutations in the gene sbmA and the uptake seems not to rely on TonB-dependent transporters, in contrast to MccJ25.¹⁰⁹

Antibacterial activities against critical pathogens have been reported for ubonodin (against Burkholderia cepacia complex⁸⁹) and cloacaenodin (against Enterobacter cloacae complex¹¹⁸) as well as for lassomycin, lariatins, and triculamin (all active against mycobacteria).^101,106

Antibacterial lasso peptides can also have different targets than the RNAP, such as the ATP-dependent proteolytic ClpC1P1P2 complex in case of lassomycin,¹⁰¹ the prolyl endopeptidase in case of propeptin,¹¹⁹ and the cell wall biosynthesis in case of siamycin I.^120,121

Finally, other biological activities have been reported for lasso peptides, such as antifungal activity for humidimycin (caspofungin activity potentiator),¹²² anti-HIV activity for siamycin-type lasso peptides,^123,124 antitumoral activity for the felipeptins,²³ and anti-migratory activity against cancer cells for the three closely-related lasso peptides sungsanpin,¹²⁵ chaxapeptin,⁷⁴ and ulleungdin.¹²⁶ This highlights the potential of the lasso structure as a source for new drug leads and as bioengineering scaffolds.

4 The biosynthesis of MccJ25

4.1 The MccJ25 biosynthetic gene cluster

The gene cluster involved in the biosynthesis of MccJ25 was the first lasso peptide BGC described in the literature. It was discovered to be encoded on a 60 kb plasmid (pTUC100) found in the producing strain E. coli AY25.¹ A 6.2 kb fragment cut out from pTUC100 was subcloned into a pACYC184-derived backbone, yielding a smaller, 10.15 kb plasmid, named pTUC202.^28,31 It was demonstrated that this plasmid contains all the genetic information necessary for the production of MccJ25 in other E. coli strains.

Further analysis of this plasmid revealed that the MccJ25 BGC comprises four genes encoding a precursor peptide (McjA), two maturation enzymes (McjB and McjC), and an ABC transporter (McjD) that confers immunity to MccJ25. The genes mcjBCD form an operon, whereas mcjA is transcribed in the opposite direction upstream of mcjB and has its own, distinct promoter (Fig. 11).

Fig. 11 Schematic depiction of the BamHI/SalI fragment excised from pTUC100 that contains the complete MccJ25 BGC. This DNA fragment was cloned into a pACYC184 backbone to yield the widely used MccJ25-production plasmid pTUC202.²⁸ Below, the primary structure of the McjA precursor peptide is shown.

It was observed that MccJ25 production in E. coli is initiated upon transition to the early stationary phase.¹ Gene expression of the MccJ25 BGC was further investigated by translational fusion of the β-galactosidase gene lacZ to mcjA, mcjB, or mcjC in order to identify factors governing the growth phase-dependent regulation.³² These experiments revealed that the expression of mcjA is triggered by carbon and phosphate starvation, consistent with the nutrient depletion encountered in the stationary phase where MccJ25 production is observed. On the contrary, mcjB and mcjC expression already occur during the exponential growth, although expression levels further increase during later growth phases.

Using corresponding regulator deficient strains, it has been shown that mcjA, mcjB and mcjC genes are positively regulated by some phase-responsive global regulators, including (p)ppGpp, the leucine-responsive protein (Lrp), and the integrative host factor (IHF). Indeed, sequence analysis confirmed that both the mcjA and mcjBCD promoter regions harbor putative recognition sites of Lrp and IHF, respectively.³² These insights into the regulatory mechanisms are of great value when employing the MccJ25 BGC for bioengineering efforts.

The observation that MccJ25 production is tied to the entry into the stationary growth phase/nutrient depletion can potentially be explained when considering that MccJ25 exhibits antimicrobial activity against bacteria living in the same ecological niche as E. coli AY25. Since MccJ25 production would help to fend off competing microorganisms in the fight over limited resources, the production of this lasso peptide likely provides a competitive advantage to the producing organism.

4.2 In vitro reconstitution of MccJ25 and first biosynthesis rules of lasso peptides

4.2.1 The post-translational modification (PTM) enzymes required for acquiring the lasso peptide fold. MccJ25 has been used for a long time as a model system to study lasso peptide biosynthesis, providing the first paradigm into this complex process. Early transposon mutagenesis showed that mcjA, mcjB, and mcjC are required for the production of MccJ25.^28,31 The mcjA gene encodes a 58 amino acid precursor peptide, comprising an N-terminal leader and the C-terminal MccJ25 core peptide sequence. McjB is composed of two domains, among which the C-terminal one resembles a Cys protease and contains a highly conserved catalytic Asp-His-Cys triad, while the N-terminal domain belongs to the previously identified RiPP precursor recognition element (RRE) protein family.^{21,90,91,127–134} McjC shows homology to asparagine synthetases and contains a conserved ATP-binding site.

Intuitively, it was proposed that McjA would first undergo cleavage by McjB to remove the leader peptide and thereby release the N-terminal α-amine needed for macrolactam formation (Fig. 12). Next, McjC would first catalyze an ATP-dependent activation of the carboxylic acid side chain of Glu8 as an AMP ester and subsequently the cyclization with the free N-terminal amine of Gly1 (Fig. 12). It was further hypothesized that McjC would transfer the primary amine to the activated carboxylic acid side chain similar to how asparagine synthetases transfer an ammonium ion (obtained from the hydrolysis of Gln into Glu and ammonia) to an activated Asp side chain for the generation of Asn. At one point before the ring formation, a pre-folding of McjA or the linear core sequence must occur to enable that the ring can close around the linear C-terminal tail, thereby entrapping it in the macrolactam to yield the threaded lasso structure. However, there is so far no experimental insight into how this process is accomplished.

Fig. 12 Scheme depicting the general principles of lasso peptide biosynthesis identified so far.^17,18,86,141

Duquesne et al. performed the first in vitro reconstitution of the biosynthetic machinery of MccJ25 and showed that McjB and McjC are sufficient to convert McjA into a mature lasso peptide in the presence of ATP.¹⁶ Point mutations of two of the three catalytic triad residues in McjB (Cys150 and His182) as well as of the conserved residues involved in ATP binding in McjC (Ser199, Asp203, and Asp302) abolished MccJ25 production in vivo.^135,136 Using these catalytic deficient variants in vitro allowed unambiguous characterization of McjB as a Cys protease and of McjC as a macrolactam synthetase. Most importantly, it was shown that the activity of both McjB and McjC requires the physical presence of the other partner (even if it is only in form of a catalytically inactive variant), which suggests that they need to form a complex for proper functioning.¹³⁵ Another study supported this notion by demonstrating that a membrane protein extract from E. coli expressing the MccJ25 BGC can also transform recombinant McjA into the mature lasso peptide.¹³⁷ This indicates that McjB and McjC are likely associated together with the membrane protein McjD to facilitate rapid export once MccJ25 is synthesized. The recent discovery of naturally occurring fusions of leader peptidases with ABC transporter domains in the BGCs of the cochonodin-type lasso peptides also points towards the likely formation of membrane-associated lasso synthetase complexes.¹⁰⁷

The role of the leader peptide is a key question in RiPP biosynthesis. Most of the leader sequence of McjA, except the last six C-terminal residues, were found to be dispensable for MccJ25 production in vivo.^138,139 Consistently, it was observed in vitro that McjB and McjC can generally transform the McjA core peptide into mature MccJ25 with or without the leader peptide present in trans, albeit both reactions yield only about 1% of the amount of MccJ25 compared to the reaction when using full length McjA as a substrate.¹³⁵ These data suggest that the leader sequence might play a role in the formation or the activation of the McjB/McjC lasso synthetase complex.

With regard to the substrate specificity of McjB, a strict requirement for Thr at the penultimate position and Gly at position 1 was observed for in vitro cleavage,¹³⁵ consistent with the fact that a penultimate Thr residue of the leader is found in most lasso peptide precursors. Substitution of the Thr at the penultimate position of the leader is tolerated with only very few other amino acids for MccJ25 production in vivo.^138,140

McjC requires Glu8 for activity, as an assay using an E8D variant of McjA resulted in only the leader cleavage.¹³⁵ The reasons for the preference of McjC for the Gly at position 1 are still unknown, due to the lack of structural information about the protein active site. Yet, mutational analysis of lasso peptide precursors demonstrated how there is often a very pronounced preference for the naturally occurring position 1 residue (which can indeed be different to Gly) and substitution usually significantly decreases, if not completely abolishes, lasso peptide production.^{51,53,59,60,97,128,135,142,143}

Interestingly, it has been shown that McjC can be replaced by SviC to synthesize MccJ25 from McjA together with McjB.⁴⁸ SviC is originally encoded in the genome of Streptomyces sviceus DSM 924 and is involved in the biosynthesis of the class I lasso peptide sviceucin, where SviC normally catalyzes the cyclization between the N-terminal α-amine of Cys1 and the carboxylic acid side chain of Asp9. The surprising ability of SviC to take part in the biosynthesis of a very distantly related lasso peptide certainly warrants further investigation in the future.

Due to the difficulty to obtain high-quality proteins for biophysical and structural analysis, the molecular mechanisms of MccJ25 biosynthesis still remain elusive. However, recent studies using lasso peptide BGCs that encode discrete RRE proteins and cysteine proteases provide some insights that could be extrapolated to MccJ25 biosynthesis. Indeed, the MccJ25 BGC belongs to the lasso peptide subclade using two-domain B proteins for biosynthesis. In contrast, there are also many other lasso peptide BGCs, where the leader peptidase and the RRE are encoded as discrete proteins, named B2 and B1, respectively. The leader sequences of the precursors from BGCs with genes for discrete B1 and B2 proteins harbor a conserved recognition motif (YxxP; sometimes WxxP), while precursors from BGCs using two-domain B proteins lack this recognition motif. In vitro binding studies have demonstrated how important the presence of the YxxP motif is for the recognition of the leader peptide by the B1 protein and that exchange of either the Tyr or Pro residue with Ala drastically reduces the binding affinity of this interaction.^91,128 In contrast, the Thr-2 residue has no influence on B1 binding and hence the main function of this residue is to guide the B2 leader peptidase to the correct cleavage site in the precursor.^91,128

Crystal structures of the B1-leader peptide complex from Thermobifida fusca and Thermobaculum terrenum are available,^127,132 both showing how the B1 protein engages the leader peptide via hydrophobic interactions involving the YxxP motif. Moreover, a hydrophobic patch is jointly formed by residues from both the B1 protein and the leader peptide. Recently, homology modelling by AlphaFold and sequence covariance analysis provided evidence that the B2 leader peptidases interact with their cognate leader peptide-B1 protein complexes through the above-mentioned hydrophobic surface. Through this interaction the protease active site is formed jointly from residues of the B2 leader peptidase with residues from B1,^133,134 explaining previous observations that B2 proteins are unable to catalyze precursor cleavage in the absence of their partner B1 proteins.^128,129

To generalize this B1/B2 interaction to also include two-domain B proteins, Kretsch et al.¹³³ extended their analysis to BurB from the burhizin lasso peptide BGC. Corresponding residues involved in the hydrophobic interactions between B1 and B2 were identified in BurB and their roles were confirmed by mutagenesis and activity assays. Hence, the RREs in lasso peptide biosynthesis fulfill two functions: (i) they recognize the precursor substrates by highly selective binding to a recognition site in the leader region, and (ii) upon binding of the leader peptide, they facilitate the peptidase activity by contributing to the formation of a jointly active site with the cysteine protease (domain).

To date, very little is known about the molecular interactions with the C proteins. Worth noting is that a previous NMR study showed that both McjB and McjC bind to the N-terminus of the leader sequence and that McjC interacts predominantly with the V6-S18 region of the McjA core sequence.¹⁴⁴ Since this region largely defines the β-hairpin structure in MccJ25, it was proposed that McjC binding to the peptide may facilitate the pre-folding process. These data hint at the important roles of the C protein in substrate–protein interactions that remain to be discovered.

Despite of all the research on MccJ25 and lasso peptides in general, there are still many unknowns with regard to the underlying mechanisms of lasso peptide biosynthesis, some of which can probably only be answered by obtaining structural information about the processing enzymes, ideally with bound peptide and the needed co-substrates. Hence, this remains an important challenge to be addressed in the future.

4.2.2 MccJ25 export and self-immunity of the producing strain. Bacteria which biosynthesize antibacterial compounds have to overcome two critical issues. First, they need some means to provide self-immunity against their own toxic compounds. Second, they have to export their antimicrobial molecules to permit them to exert their expected roles in microbial communities as mediators for competition or communication. In many cases, these two issues are addressed by distinct means, i.e., the separate production of immunity proteins to ensure self-immunity and transport systems for export. However, in a few cases, a unique strategy can provide both functions, as is the case for MccJ25. The self-immunity to MccJ25 is accomplished via the dedicated ATP-binding cassette (ABC) transporter McjD, which is the third component encoded by the MccJ25 BGC (see also Sections 4.1 and 4.2.1). It ensures the export of MccJ25 into the periplasmic space and simultaneously provides self-immunity by keeping the concentration of MccJ25 below toxic levels inside the producing cells.¹⁴⁵ The final export step out of the periplasmic space is ensured by the outer-membrane protein TolC that forms a channel which facilitates the transport out of the cell.¹⁴⁶

The crystal structure of McjD has been determined in the presence and the absence of nucleotides. It displays a characteristic homodimeric ABC transporter architecture (type IV according to a recent classification¹⁴⁷) with a transmembrane domain (TMD) consisting of twelve transmembrane helices and two nucleotide-binding domains (NBDs)^145,148 (Fig. 13). McjD forms a homodimer that displays a specific outward-occluded conformation without intertwinning of the transmembrane domains, which has the particularity to be occluded on both sides of the membrane, thereby distinguishing it from previously characterized ABC transporter conformations. The crystal structure also revealed the presence of a large binding cavity of around 5900 ų with the length of approximately 40 Å that would be able to accommodate one MccJ25 molecule.¹⁴⁵ MccJ25 was modeled within the cavity and mutational analysis showed that Phe86, Asn134, and Asn302 of McjD are important for its recognition.¹⁴⁵

Fig. 13 Structure of the self-immunity ABC transporter McjD.^{145,148–152} (A) McjD displays a type IV ABC transporter architecture, consisting of a transmembrane domain and two nucleotide-binding domains. The transmembrane domain displays an occluded conformation that is driving substrate specificity as it requires both ATP and substrate for opening. McjD is shown in cartoon and one protomer is colored in rainbow (blue to red) and the other in light grey. The ATP analogue AMP-PNP is shown as black sticks and the membrane as a grey box. (B) The MccJ25 residues important for interaction with McjD, as identified by NMR-CEST, have been mapped onto the MccJ25 NMR structure and are shown in orange sticks, other residues are shown in green sticks.

Together with functional and biochemical studies, the structure allowed deciphering the mechanism of transport by McjD and its specificity.^145,149 A specific class of lipids was shown to be essential for the structure, stability, and activity of McjD.¹⁵⁰ The structure of phosphatidylglycerol associated with McjD was resolved at a 3.4 Å resolution. Non-denaturing and tandem mass spectrometry together with molecular dynamics simulations revealed that McjD associates with various lipids, that McjD can bind MccJ25 without interference from lipids, and that phosphatidylethanolamine and phosphatidylglycerol play synergistic roles facilitating the structure stabilization and function, respectively.¹⁵⁰

To identify the regions of the lasso peptide crucial for the interaction with McjD, the recognition mechanism of MccJ25 by McjD was investigated by NMR spectroscopy in bicelles.¹⁴⁹ Four residues at the very beginning of the lasso peptide loop (Glu8, Tyr9, Phe10, Val11) constitute the primary site of interaction with McjD.

Furthermore, a combination of functional assays, crystal structures in different conformations, pulsed electron double resonance (PELDOR) measurements, single molecule Förster resonance energy transfer (sm-FRET), and molecular dynamics simulations was used to probe the conformational changes and the dynamics of the transporter along the transport cycle.^{145,148,151,152} Based on the results of these experiments, a specific mechanism for substrate transport and selectivity was proposed that was called the ‘occluded-mechanism with transient opening’.

In this model, McjD is in an occluded conformation in the absence of MccJ25, where the transmembrane domain remains shielded to both the cytoplasmic and periplasmic sides of the membrane, regardless of the absence or the presence of nucleotides. In the presence of MccJ25, McjD acquires an inward-open conformation where its binding cavity becomes accessible to the peptide. Both MccJ25 and ATP binding induce a transient outward-open conformation for MccJ25 release to the periplasm.^148,152 Subsequently, the transmembrane domain returns to an occluded conformation and ATP hydrolysis resets the transporter to its inward conformation. Unlike other multidrug resistance ABC transporters that can adopt the outward-open conformation in the absence of a substrate, McjD requires both MccJ25 and ATP to open.

Finally, the specificity of the McjD ABC exporter was examined. It is particularly high compared to other ABC transporters that can be extremely promiscuous.^149,153 Indeed, many other drugs, antimicrobial peptides, or bacteriocins, including other lasso peptides, are not accepted as substrates by McjD.¹⁴⁹ Conversely, another ABC transporter, CapD, whose original function is the export of the RNAP-inhibiting lasso peptide capistruin, was also unable to transport MccJ25.¹⁴⁹ The high selectivity of McjD for MccJ25 is very likely due to the tight coupling of ATP and substrate binding for the export across the inner membrane.

Another ABC transporter, YojI, is also involved in MccJ25 export,¹⁵⁴ although it fulfills this function much less efficiently than McjD. YojI is chromosomally encoded in E. coli and is under the control of the leucine-response regulatory protein Lrp.¹⁵⁵ Similar to McjD, YojI is anchored at the inner membrane and works in concert with TolC at the outer membrane.^146,156

The actual physiological role of YojI is currently unknown and still needs to be deciphered. Although MccJ25 was identified as the first substrate known for YojI, expelling of this lasso peptide cannot be the normal function of this ABC transporter. Yet, conventional antibiotics and other chemical compounds translocated by the AcrAB pump or other major drug transporters are not exported by YojI,¹⁵⁷ suggesting a quite narrow substrate specificity.

More recently, as elegantly demonstrated in the meningitis pathogenesis context, YojI appears as a virulence factor.¹⁵⁸ Here, YojI was shown to act as a specific membrane protein for the interaction of E. coli with the blood–brain barrier. This interaction further results in the penetration of the bacteria into the brain and thereby triggers intracranial inflammation. The interferon α/β receptor IFNAR2, which, among many other significant functions, plays an important role in antiviral defense mechanisms, was identified as the host receptor for E. coli YojI and acts as a sensitive sensor of YojI.

Therefore, YojI enables on the one side the export of MccJ25 out of E. coli strains when McjD is failing (or absent), allowing competition and conferring a certain level of resistance against this antimicrobial lasso peptide, while on the other side, it contributes to pathogen–host-interactions. These observations suggest that YojI could combine differentially a protective role or act as a virulence factor for E. coli cells, depending on the context.

5 The biological activity of MccJ25

5.1 Bacterial resistance to MccJ25

Resistance to microcins should be clearly distinguished from self-immunity which permits producing strains being protected against their own weapons. Independent of self-immunity systems that are encoded in bacteriocin BGCs, bacteria have evolved mechanisms of acquired resistance against external antimicrobials. For a given bacteriocin/microcin, different mechanisms are at play. They often involve mutations or transporters that differ from those used for self-immunity. In general, they have significant biological costs for the bacterial cell. In some cases, resistance mechanisms may also complement self-immunity in producer strains.

Uptake decrease of the toxic entity and pumping it out of susceptible cells are efficient strategies to confer resistance to antimicrobial compounds. Indeed, changes in the uptake machinery are the first source of resistance to MccJ25. It was early demonstrated by the genetic analysis of spontaneous MccJ25-resistant mutants of E. coli that their mutations were located in the fhuA, exbB, exbD, and sbmA genes, revealing that the outer membrane receptor FhuA (which is energized by the Ton system that includes ExbB and ExbD) and the inner membrane transporter SbmA are primarily involved in the resistance to MccJ25.²⁹ Later on, membrane permeabilization by a synthetic cationic peptide, which made the MccJ25 entry independent of FhuA and SbmA, was shown to induce MccJ25 susceptibility in resistant fhuA and sbmA mutant strains,¹⁵⁹ thus further confirming the critical roles of these two proteins for the resistance against MccJ25.

Efflux pumps, which expel the toxic compounds out of bacteria and thereby preventing them from reaching their toxic intracellular concentration, can also constitute a first line of resistance against various antimicrobials.¹⁶⁰ They can be specific for a single substrate or more promiscuous, thus conferring resistance to multiple structurally different antimicrobials. For instance, the overexpression of efflux pumps constitutes one of the mechanisms of resistance to β-lactams and quinolones.^161,162

For MccJ25, the ABC exporter McjD, which is highly specific to this lasso peptide and expels it to the periplasm, where the TolC channel then ensures the last export step to the extracellular space, facilitates both MccJ25 export and self-immunity for the producing cells (see Section 3.4 above).^145,148,153 Furthermore, the ABC transporter YojI, which is located in the inner membrane and is coupled to the TolC protein in the outer membrane as well, also mediates resistance to MccJ25 by pumping it out of the cells.¹⁵⁴

The possibility that polysaccharides, and especially colanic acid, which is involved in the E. coli capsule that forms a permeability barrier to external compounds, could contribute to resistance was discarded¹⁵⁴ after the preeminence of McjD and YojI efflux pumps in the resistance to MccJ25 was demonstrated.

The observation that E. coli cells become more resistant to MccJ25 when entering the stationary phase led to finding a direct correlation between the resistance to MccJ25 and the accumulation of the bacterial alarmones ppGpps (global abbreviation to define guanosine tetraphosphate and guanosine pentaphosphate), which occurs in the stationary phase and is usually known to affect the growth rate and traits important for virulence. The ppGpps were shown to upregulate yojI expression, thus increasing the production of YojI and subsequently favouring the expulsion of MccJ25 out of the cells, thereby keeping its intracellular concentration below a toxic level.¹⁶³ In addition, it was noticed that high ppGpps levels could also interfere with the binding of MccJ25 to the RNAP.¹⁶³

A last mechanism of resistance involves mutations in the intracellular target of MccJ25, the RNAP. A T931I mutation in the conserved segment of the rpoC gene that codes for the largest RNAP subunit β′ confers resistance to MccJ25.³⁵ This is in agreement with the MccJ25 mechanism of action that involves the occlusion of the RNAP secondary channel leading to interference with trafficking NTPs to accessing the catalytic active site.¹⁶⁴ The crystal structure of the MccJ25-RNAP complex further confirmed the binding of MccJ25 within the RNAP secondary channel.¹¹⁴ Additional rpoC mutations affecting amino acids exposed into the RNAP secondary channel in the conserved segments G, G′, and F also led to MccJ25 resistance in vivo and in vitro.

Therefore, both the reduced uptake of the toxic entity via FhuA and SbmA and the increased efflux by YojI are the prevailing mechanisms that can confer resistance to MccJ25 and the expulsion via McjD adds to these mechanisms in MccJ25 producing cells. Mutations in the rpoC gene further complement the resistance landscape.

However, resistance routes appear to be modulated in the gastrointestinal tract due to the numerous and complex interaction networks established between the members of the microbiota and with the host.^4,7 The process (termed colonization resistance) by which commensal bacteria compete with invading nonindigenous microorganisms (including pathogens) for the niche and nutrients via employing specific antimicrobial molecules and by stimulating the host immune defense^165,166 is a driving force for controlling the emergence of resistant strains against both conventional antibiotics and bacteriocins/microcins in an ecological context.

The ‘rock-paper-scissors’ model elaborated for colicins in E. coli describes the fine-tuned equilibrium between bacteriocin producing and non-producing cells, including strains that do or do not possess resistance systems against such compounds.¹⁶⁷ The winner which dominates the niche is the one that minimizes the costs by only having the resistance attributes, but the persistence and coexistence of the different strains also depend on the structuration of the environment, either well-mixed and homogeneous, or more structured, such as in biofilms.^167–169 Otherwise, the rise of resistance to MccJ25 has been essentially observed in vitro at very high concentrations of this compound.

5.2 Uptake of MccJ25

Unlike other antimicrobial peptides that exert their modes of action by damaging the integrity of the phospholipid membrane bilayers, MccJ25 can only reach its intra-cytoplasmic target, the RNAP, by crossing the typical protective outer and inner membranes. Since the early pioneering studies on MccJ25, it is known that the activity of MccJ25 requires the outer membrane siderophore receptor FhuA, which is energized through the TonB-ExbB-ExbD multi-subunit protein machinery (also referred to as the Ton system),^29,30 as well as the inner membrane protein SbmA.²⁹ It was also demonstrated that the production of MccJ25 is increased under iron-limiting conditions and hence that the MccJ25 biosynthesis is regulated in an iron-dependent manner.¹⁷⁰ These results were confirmed, evidenced, and further explained in subsequent years.

The tight correlation between the extracellular iron concentration and MccJ25 uptake is explained by the essential role played by iron as an indispensable nutrient in bacterial life. Since in aerobic environments iron is present in its insoluble Fe(III) form, which cannot be efficiently internalized, bacteria evolved to produce both high affinity iron-chelating agents called siderophores¹⁷¹ and outer membrane receptors with high affinity and high selectivity for the uptake of Fe(III)-siderophore complexes. Thereby, many bacteria became able to scavenge iron from the environment and internalize it.¹⁷²

These receptors are generally coupled to the Ton system, which is anchored at the inner membrane and transfers energy from the proton motive force to TonB-dependent outer membrane receptors. ExbB and ExbD harness the proton motive force and channel it to the TonB subunit, which has a C-terminal domain spanning the periplasm. This C-terminal domain of TonB is in turn interacting with a short sequence of seven amino acids that is conserved in outer membrane transporters (the TonB box), thus physically connecting outer membrane receptors to the inner membrane and enabling active transport.^173,174

FhuA is one of these TonB-dependent receptors, which mediates the uptake of the hydroxamate siderophore ferrichrome. It is a monomeric β-barrel comprising 22 antiparallel β-strands that spans the outer membrane, wrapping around to form a large extracellular ligand binding pocket open to the external medium. This binding pocket is gated by a cork-like domain folded inside the β-barrel from the periplasmic side that occludes the cavity in the ground state.^77,175

Interestingly, siderophore receptors are often hijacked by diverse molecules and thus unwittingly facilitate their uptake into cells. FhuA is for example also the receptor for the antibiotic albomycin, colicin M, and some bacteriophages (T5, T1, and Φ80). Furthermore, iron transport systems are highly regulated to prevent intracellular iron accumulation, which is also toxic to the cells as it causes oxidative damage. In iron-depleted environments, TonB-dependent siderophore receptors and siderophore biosynthesis genes are upregulated to improve iron acquisition, while in iron-rich environments, these genes are downregulated. This fine-tuned regulation mechanism is mediated by the Fur (ferric uptake regulator) transcriptional repressor, which ensures iron homeostasis¹⁷⁶ and explains how the cellular uptake of MccJ25, and subsequently its activity, is increased in iron-depleted environments and decreased in iron-rich environments.

MccJ25 was confirmed to hijack the TonB-dependent ferrichrome siderophore uptake system by binding to FhuA with a 2 : 1 stoichiometry and a K_d of 1.2 μM despite the lack of structural similarity between MccJ25 and the siderophore. MccJ25 is furthermore able to compete with phage T5 infection since the bacteriophage also uses the FhuA receptor for docking onto the bacterial cell. FhuA external loops oriented toward the extracellular space were identified as being involved in the primary docking between FhuA and MccJ25.¹⁷⁷ In turn, the intact lasso structure of MccJ25 was shown to be essential for recognition by FhuA,^{64,77,177,178} while the macrolactam ring and the C-terminal tail were involved in the interaction with the RNAP.^35,179

Finally, the FhuA-MccJ25 co-crystal structure provided the molecular basis of how MccJ25 interacts with FhuA and is subsequently internalized⁷⁷ (Fig. 14). Upon binding, MccJ25 completely occludes the FhuA channel and occupies a location very similar to the natural ligand, ferrichrome. For the interaction with FhuA, MccJ25 undergoes a small conformational change in its β-hairpin loop region, thereby losing the short β-strand found in the solution structure and adopting a more flexible conformation. Interestingly, whereas ferrichrome forms an extensive array of hydrogen bonds with the FhuA cork domain, only two residues of MccJ25 form hydrogen bonds with this domain in addition to numerous hydrophobic contacts along the length of the peptide.

Fig. 14 MccJ25 hijacks outer and inner membrane proteins for internalization.^{29,30,77,177,180} (A) Crystal structure of the siderophore receptor FhuA in complex with MccJ25. MccJ25 mimics the binding of the ferrichrome siderophore and its interactions with the plug domain via specific hydrogen bonds to initiate a transport event. FhuA is shown in grey cartoons and MccJ25 in orange sticks. The membrane is depicted as a grey box. (B) Cryo-EM structure of SbmA. The structure is a homodimer with a transmembrane domain flanked by two TM0 domains (colored dark to light blue). SbmA adopts an outward-open conformation with its cavity open to the periplasm for substrate binding and transport. SbmA is shown in cartoon with one protomer colored in rainbow (blue to red). The membrane is depicted as a grey box.

Predominantly His5, but to some extent also Ala3 are involved in two of the three key hydrogen bonds. The His5 imidazole ring and backbone carbonyl group make hydrogen bonds with the Phe115 backbone carbonyl group and the Tyr116 side chain of FhuA.⁷⁷ Moreover, the side chain of the His5 residue is buried inside the FhuA cavity and is critical for FhuA recognition, as evidenced by the reduced binding affinity of a H5A variant of MccJ25.⁷⁷ The hydrogen bonds formed between His5 and FhuA are furthermore essential to trigger the displacement of the cork domain, which elicits a transport event via the Ton system that facilitates MccJ25 internalization. Conversely, the loss of the hydrogen bond in the H5A variant of MccJ25 prevented internalization. An additional but less stable hydrogen bond is established between the Ala3 carbonyl and the Gln100 residue of FhuA. This model of the MccJ25/FhuA association, and especially the key roles of His5 and the location of important binding sites in the ring and lower tail region, were confirmed by computer simulations including computational mutational analyses.¹⁸⁰ Moreover, MccJ25 uptake is dependent on the FhuA-TonB system, as reflected by the resistance of tonB deletion strains, thus providing further evidence that MccJ25 can induce the displacement of the cork domain for translocation through the Ton pathway.⁷⁷

The specific interactions between MccJ25 and FhuA explain the narrow spectrum of antibacterial activity of MccJ25, which is directed essentially against E. coli, Shigella, and Salmonella species. Indeed, only certain FhuA homologues are capable of MccJ25 uptake in Salmonella enterica subsp. enterica serovars, with isolates ranging from highly sensitive to highly resistant.^181,182 Thus, resistant strains of S. enterica Typhimurium become susceptible to MccJ25 when expressing E. coli FhuA.¹⁸¹

Alignments of the FhuA sequences from high-susceptibility and low-susceptibility variants of S. enterica Newport showed that variations in the amino acid sequence, and especially deletions in two segments (regions 354–370 and 451–455) that correspond to the extracellular loops in FhuA,⁷⁷ are strongly associated with susceptibility to MccJ25. These segments must therefore be crucial for the MccJ25/FhuA interaction. This observation clearly establishes that the structure of FhuA determines the level of susceptibility to MccJ25 and this was shown as being independent of the S. enterica serovar and the resistance phenotype.¹⁸²

After uptake into the periplasmic space, MccJ25 still has to pass through the inner membrane to reach the RNAP in the cytoplasm. The shuttling through the inner membrane is mediated by the SbmA transport system^29,178,183 and the His5 residue in MccJ25 is likely also playing an important role in this step.^178,183 The exact native function and substrate(s) of SbmA have not yet been identified, although it is known that it acts as a virulence factor in pathogenic E. coli¹⁸⁴ and that SbmA-null mutants are resistant to many antimicrobial peptides, including MccJ25.²⁹

Sensitization of MccJ25 resistant strains by a synthetic cationic membrane-permeabilizing peptide ([KFFF]3K), allowed MccJ25 entry independent of FhuA and SbmA under assay conditions.¹⁵⁹ This caused the loss of the initial narrow spectrum antibacterial activity of MccJ25 and extended it to also include several other Gram-negative genera, such as Enterobacter, Citrobacter, Klebsiella, Pseudomonas, Moraxella, Acinetobacter, and S. enterica Thyphimurium.¹⁵⁹

Microcin Y (MccY) is a naturally occurring homolog of MccJ25 (MccJ25: GGAGHVPEYFVGIGTPISFYG; MccY: GGRGHIAEYFSGPITQVSFYG; eight positions carry different amino acids) that is produced by a BGC found in S. enterica Birkenhead. MccY also uses FhuA and SbmA for uptake and, similar to what was observed with MccJ25, differences in the FhuA sequence appear to be responsible for the target specificity of MccY in Salmonella species.¹¹³ The spectra of antibacterial activity of MccJ25 and MccY are different to some extent. In addition to targeting Salmonella, MccY also inhibits a panel of other Gram-negative bacteria, and notably even Gram-positive bacteria, like Bacillus subtilis, Clostridium perfringens, and Staphylococcus aureus, suggesting that for MccY other receptors could also be involved in the bacterial uptake.

5.3 The targets of MccJ25

5.3.1 Microcin J25 inhibits the Gram-negative RNA polymerase. In 2001, a spontaneous E. coli mutant resistant to MccJ25 was isolated and it was revealed that it harbored a single point mutation in the rpoC gene leading to a T931I substitution in the RNAP β′ subunit.³⁵ Shortly afterwards, in vitro experiments demonstrated that the synthesis of short abortive transcripts by the RNAP purified from WT E. coli was inhibited by micromolar concentrations of MccJ25.¹⁶⁴ Conversely, the RNAP variant harboring the T931I substitution in the β′ subunit was not inhibited by MccJ25. These initial observations established that the RNAP is indeed a direct intracellular target of MccJ25. Additional experiments furthermore demonstrated that MccJ25 inhibited the abortive transcription whether added before or after the promoter DNA, suggesting that it is not the promoter recognition by the RNAP that is affected by MccJ25.

To identify the MccJ25 binding site on the enzyme, additional mutations in the RNAP coding genes conferring resistance were specifically looked for. Dozens of mutants with varying degree of resistance were obtained.^185,186 All residues affected by resistance mutations (including the prototypical β′ Thr931) were located at the circumference of the secondary channel – an evolutionary conserved structural element present in all multisubunit DNA-dependent RNAPs. This channel opens up on the “downstream” (with respect to the direction of the transcription) face of the RNAP and leads towards the enzyme's active site where a tightly bound catalytic Mg²⁺ ion is located. Modeling of the MccJ25 structure into the binding site predicted by the resistance mutations suggested that MccJ25 should completely block the secondary channel,¹⁸⁵ acting like a “cork in a bottle”.

The RNAP secondary channel funnels NTP substrates towards the growing 3′ end of the nascent RNA located at the active center. During transcript elongation, the RNAP sometimes slides back along the DNA template, forming inactive (dead-end) backtracked elongation complexes, where the 3′ end of the transcript is disengaged from the catalytic center. In the backtracked conformation, the 3′-end proximal part of the nascent transcript is positioned in the secondary channel. Transcript cleavage factors (GreA and GreB in bacteria) rescue backtracked complexes by stimulating the endonucleolytic activity of the RNAP catalytic center, which generates a new 3′ end aligned with the enzyme's catalytic site. To accomplish this feat, Gre factors insert their elongated coiled-coil domain into the secondary channel to reach the catalytic center, where they then stimulate the cleavage activity.

The location of the predicted MccJ25 binding site suggested very specific effects of MccJ25 binding on RNAP elongation, backtracking, and factor-dependent transcript cleavage activities. The expected effects were indeed observed in discriminating in vitro transcription assays.

Real-time single-molecule transcript elongation experiments revealed that the addition of MccJ25 induced extended periods of apparently complete cessation of transcript elongation. In between these periods of inactivity, transcription elongation was carried out at the same rate as in the absence of MccJ25. The result is consistent with the proposed “cork in a bottle” model: bound MccJ25 prevents NTP access to the catalytic center; when MccJ25 dissociates from the elongation complex, the access of NTPs to the active site is restored and the transcription elongation proceeds normally until the same or another MccJ25 molecule binds to the RNAP.

As expected, MccJ25 also prevented RNAP backtracking, presumably because there is no space in the secondary channel to accommodate the 3′ end proximal portion of the transcript when MccJ25 is bound. Finally, MccJ25 inhibited transcript cleavage by Gre factors likely for the same reason, i.e., occlusion of the secondary channel.

It should be noted that at the time the corresponding studies were carried out, most of the proposed functions of the secondary channel were purely hypothetical and that the use of MccJ25 thus provided important support for identifying its role in transcription, which was subsequently validated by numerous three-dimensional structures.

Residues of MccJ25 important for transcription inhibition (presumably, through RNAP binding) were also studied. The fact that the antibacterial activity and the transcription inhibition could be dissected became apparent when a thermolysin-treated MccJ25 with excised residues 13–17 (located in the loop region) was shown to retain the ability to inhibit the RNAP in vitro, while completely losing the ability to inhibit cell growth.¹⁷⁹ Systematic structure–activity analysis of MccJ25 subsequently further confirmed that practically the entire loop region is dispensable for transcription inhibition.¹⁸⁷

After the described series of genetic studies, mutagenesis and enzyme assays had pinpointed the secondary channel of the Gram-negative RNAP as the binding site for MccJ25, a co-crystal structure of MccJ25 bound to the E. coli RNAP was elucidated.¹¹⁴ This structure required a new crystal form of the RNAP in the P4₁2₁2 space group, in contrast to previous crystals in the P2₁2₁2₁ space group that did not support the binding of MccJ25. Another key technical breakthrough that facilitated the determination of the co-crystal structure was the generation of bromine-labeled MccJ25 variants using non-canonical amino acid incorporation into the McjA precursor peptide as further discussed in Section 7.1.

The co-crystal structure provided an atomic view of MccJ25-RNAP interaction and is generally consistent with data obtained from mutational and functional analyses. The structure of MccJ25 and the E. coli RNAP revealed that MccJ25 bound deep within the secondary channel with the MccJ25 loop region facing away from the RNAP active site (Fig. 15A).¹⁸⁵ This orientation is the opposite of the one proposed by the initial modeling¹⁸⁵ and the closure of the secondary site is incomplete. Though the remaining opening is too small to allow NTP access, it is possible that, because of the thermal motion of the enzyme, the MccJ25 cork may be leaky allowing occasional access of NTPs to the catalytic center. If such events were to occur, the synthesis of a phosphodiester bond should be generally possible since bound MccJ25 does not have direct contacts with the catalytic site. This observation may also explain why at high concentrations of NTP substrates residual synthesis of abortive transcripts can be detected even in the presence of MccJ25.^114,185 However, because MccJ25 binding, as judged from single-molecule experiments, is a slow process, it cannot be excluded that residual synthesis occurs in between MccJ25 dissociation/association from the transcription complex.¹⁸⁶

Fig. 15 Transcription by the E. coli RNAP is inhibited as binding of the lasso peptides, (A) MccJ25 or (B) capistruin, is blocking the NTP uptake through the secondary channel.^35,114,115 For both lasso peptides, the three-dimensional structures overlayed with electron density maps and a schematic representation of their primary structures are shown on the left. In the middle, an overview of the E. coli RNAP structure with bound DNA and either MccJ25 (PDB code 6N60) or capistruin (PDB code 6N61) is shown. The binding site for the lasso peptides is highlighted with a red circle. On the right, a close-up of the lasso peptides bound in the secondary channel is shown.

The binding of MccJ25 sterically occludes the secondary channel, constricting this conduit for NTPs from a diameter of ∼10 Å to less than 5 Å. The ring and tail portions of MccJ25 make the most extensive contacts with the secondary channel, with the His5 of MccJ25 being positioned within a 6.5 Å distance of the Mg²⁺ ion in the RNAP active site. Key interactions include hydrogen bonding between the RNAP and the hydroxyl groups of the Tyr9 and Tyr20 side chains of MccJ25 as well as a salt bridge between the RNAP and the C-terminus of MccJ25. Consistently, substitution of these Tyr residues (with exception of a Y20F exchange) as well as glycine amidation of the C-terminus abrogates the ability of MccJ25 to inhibit the transcription.¹⁸⁷

Beyond this steric blockade of the secondary channel, MccJ25 also disrupts the catalytic activity of the RNAP by interfering with the folding of the trigger loop. The trigger loop is a structurally conserved element of the β′ subunit that undergoes folding/unfolding cycles upon the binding of a correct NTP in the active site.¹⁸⁸ In the RNAP catalytic cycle, the trigger loop starts out in an unfolded state, allowing access of NTP substrates to the active site. Once the correct NTP substrate is present in the active site, the trigger loop folds, thus accelerating the incorporation of the NTP into the growing RNA chain.¹⁸⁹ The binding of MccJ25 within the secondary channel prevents the folding of the trigger loop and therefore even the efficiency of the incorporation of those NTPs that managed to reach the RNAP active site in the presence of bound MccJ25 will be strongly decreased, compared to the free enzyme.

Over the years, several lasso peptides that target the bacterial RNAP were identified. These include capistruin,¹¹⁵ acinetodin,¹¹² klebsidin,¹¹² citrocin,¹⁰⁹ ubunodin,⁸⁹ and cloacaenodin.¹¹⁸ They all inhibit the WT E. coli RNAP in vitro and have no effect on the MccJ25-resistant T931I enzyme. Thus, the binding sites for all of these lasso peptides must (at least somewhat) overlap with that of MccJ25.

Citrocin and ubonodin both contain a Tyr at a position corresponding to the key RNAP-interacting MccJ25 residue Tyr9, while klebsidin has a Phe residue at the respective position. Klebsidin and ubunodin inhibit the transcription as efficiently as MccJ25, while citrocin was shown to be a more potent RNAP inhibitor.^109,112 It is therefore likely that the mode of RNAP-binding and transcription inhibition of these three lasso peptides are the same as those of MccJ25 despite the differences in their primary structures and three-dimensional lasso folds. Capistruin and acinetodin were shown to be considerably less efficient as inhibitors in vitro.^89,112,115 The first exocyclic residue in these peptides are Thr9 (in acinetodin) and Ala10 (in capistruin).

Capistruin and cloacaenodin are the only RNAP inhibiting lasso peptides known so far that contain nine-residue macrocycles, whereas all the other RNAP-inhibiting lasso peptides feature an eight-residue macrolactam. The structure of capistruin in complex with the E. coli RNA polymerase holoenzyme was also determined (Fig. 15B)¹¹⁴ and revealed significant differences compared to the MccJ25/RNAP complex. Most importantly, the capistruin binding site in the secondary channel is located farther away from the catalytic center and occludes the channel only partially, such that NTP access remains possible. Hence, capistruin probably exerts its activity mainly via the steric blockade of trigger loop folding.

5.3.2 MccJ25-triggered overproduction of superoxide. By the early 2000s, the Gram-negative RNAP has been firmly established as the intracellular target of MccJ25.^{35,164,185,186} Yet, around the same time, other potential mechanisms of action of MccJ25 were identified.

First, Rintoul et al.¹⁹⁰ showed that MccJ25 interacts with artificial small unilamellar phospholipid vesicles whose composition mimics that of the E. coli membrane, inducing microviscosity changes, disorders of the bilayer structure, and increased permeability. There was however no evidence showing that these in vitro effects were relevant to the in vivo antimicrobial action of MccJ25,¹⁹⁰ as the observed liposome effects were observed at concentrations (15 μM) much higher than those inhibiting some strains of Salmonella and E. coli in vivo (MICs in the nanomolar range).¹

The same authors later found that in Salmonella enterica serovar Newport, which is intrinsically hypersusceptible to MccJ25, the lasso peptide affected the inner membrane permeability, dissipating the membrane potential, and interfered with electron transport, which caused a decrease in oxygen consumption.¹⁹¹ Remarkably, none of these actions could be observed in the closely related MccJ25-susceptible E. coli K-12 strain when treated with concentrations sufficient to prevent bacterial growth.

Another intriguing observation was made three years later with a MccJ25 derivative, called MccJ25-Ga (Ga stands for glycine amidated), in which the C-terminal carboxylate was amidated with a glycine methyl ester group.⁷⁶ This modification specifically blocks the ability of MccJ25 to inhibit the RNAP and hence MccJ25-Ga has no antibiotic activity on E. coli strains. Yet, MccJ25-Ga was still able to inhibit the cell respiration in S. Newport,¹⁹² implying that distinct regions of MccJ25 are involved in RNAP inhibition and cell respiration interference. Thus, MccJ25 appeared to have two modes of action depending on the targeted bacteria: (1) inhibition of the RNAP in E. coli, and (2) inhibition of both the RNAP and the cell respiration in S. Newport. This was surprising given the phylogenetic proximity of the two species.

It took a few years until it was discovered that the lack of effect of MccJ25 on E. coli respiration was a matter of concentration. Bellomio et al.¹⁹³ demonstrated that artificially increasing the intracellular concentration of MccJ25 in E. coli by overexpressing the outer membrane siderophore receptor FhuA from the multicopy plasmid pGC01, led to the same membrane effects and respiration inhibition previously observed in S. Newport. Curiously, this was observed only in the E. coli K-12 substrain AB1133, which is moderately more susceptible to MccJ25 than other K-12 derivatives. Moreover, a 10 μM MccJ25 concentration, which is 500-fold higher than the 0.02 μM MIC for E. coli AB1133 (pGC01), was required to achieve a 30% respiration inhibition in this strain. It was nonetheless proposed that in E. coli MccJ25 hits both targets, the RNAP and the electron transport chain, and that the high susceptibility of E. coli AB1133 (pGC01) and S. Newport to MccJ25 could result from the additive effect of these two modes of action.

It is therefore important to point out that Socías et al.¹⁵⁵ realized that this E. coli AB1133 susceptibility phenotype originates in part from the leucine supplementation of the culture medium necessary to overcome the auxotrophy of this strain (E. coli has a chromosomally encoded MccJ25 efflux pump, YojI, which is positively regulated by the Leucine-responsive regulatory protein (Lrp). High concentrations of exogenous leucine antagonize the effect of the Lrp, thus lowering the YojI expression levels, which in turn increases the susceptibility to MccJ25).

To offer additional evidence of the dual-targeting notion, Bellomio et al.¹⁹³ transduced an MccJ25-resistance conferring RNAP mutation (rpoC931; carrying the aforementioned T931I substitution in the β′ RNAP subunit)³⁵ into E. coli AB1133 using the phage P1vir. The transductant was then transformed with the plasmid pGC01 to overexpress FhuA, thereby increasing the uptake of MccJ25. The resulting strain, E. coli AB1133 rpoC931 (pGC01), was still as hypersensitive to MccJ25 as the control strain without the rpoC mutation (i.e., E. coli AB1133 (pGC01)).

This result appears somewhat surprising, as the presence of two effective (lethal or inhibitory) targets in a bacterial cell, each able to interact independently with MccJ25, implies that the overall effect of MccJ25 would be the sum of its actions on each target. If now one target becomes resistant by mutation, one would expect this to produce at least a partial resistance and that the remaining susceptibility would only be the result of the action of MccJ25 on the second target. Yet in the cited experiment,¹⁹³ there was no significant difference observed for the MccJ25 sensitivity of E. coli AB1133 rpoC931 (pGC01) and E. coli AB1133 (pGC01).

It is of note that the T931I substitution in the β′ RNAP subunit endows bacteria with a very high resistance to MccJ25. For example, when the mutant strain E. coli AB1133 rpoC931 is transformed with the plasmid pTUC348, a pACYC184 derivative carrying the MccJ25 BGC lacking the self-immunity and export determinant mcjD, the transformants grow normally,¹⁹² whereas this plasmid is lethal for cells carrying a WT RNAP. A reanalysis of the transductant constructed in Bellomio et al. would be advisable, to verify that the rpoC931 mutation was correctly transduced. This could possibly help to explain these unexpected results.

Subsequent studies demonstrated that under the conditions of high intracellular MccJ25 concentration achieved in E. coli AB1133 (pGC01), there was a surge in superoxide radicals (O₂⁻).¹⁹³ The authors proposed that this oxidizing species is involved in the damage to an unidentified electron transport chain component (the putative second target), causing the impairment in respiratory activity. It is not clear, however, how the produced superoxide radicals could damage the respiratory chain. Furthermore, these effects were again only observed in E. coli AB1133, but not in other E. coli K-12 genetic backgrounds.

Since reactive oxygen species (ROS) are generated in direct proportion to the oxygen concentration, the intracellular production of ROS must be minimal or nonexistent under anaerobic conditions. The possible involvement of superoxide formation in the antibacterial activity of MccJ25 therefore naturally led to the idea that under oxygen-limited conditions, MccJ25 should have no effect on E. coli AB1133 rpoC931 (pGC01).

This hypothesis was seemingly confirmed in experiments by Bellomio et al.¹⁹³ However, their assays were not appropriate to prove the role of superoxide in cell death, since the entry of MccJ25 via the TonB-dependent outer membrane receptor FhuA requires the proton motive force, which is generated by electron flow through the respiratory chain in a similar way to what happens with aminoglycosides.¹⁹⁴ In fact, it was shown that the protonophore 2,4-dinitrophenol decreases MccJ25 uptake by 80%.¹⁹⁵ Thus, resistance to MccJ25 in anaerobiosis would most likely result from failure of antibiotic uptake. Although an oxygen-poor environment decreased the toxicity of MccJ25 on strain E. coli AB1133 (pGC01), the antibiotic activity was mostly restored when nitrate was used as an alternative electron acceptor, in place of oxygen (Raúl Salomón, unpublished). Thus, oxidative injury seems to be not essential for MccJ25 toxicity.

In subsequent studies, Chalon et al.¹⁹⁶ showed that an MccJ25 variant with a Y9F substitution was still able to inhibit the RNAP but lost the ability to stimulate superoxide production and inhibit oxygen consumption. It was concluded that Tyr9 was somehow necessary for the inhibitory action of MccJ25 on the respiratory chain. Later, the authors showed that Tyr9 behaves in vitro as a redox-active group that could form a tyrosyl radical, suggesting that this residue is directly involved in the process of superoxide generation.¹⁹⁷

Nevertheless, all of these studies addressing a dual mechanism of action of MccJ25 fell short of explaining the precise molecular basis of superoxide generation or the mechanism of inhibition of bacterial respiration by MccJ25. Actually, there was no direct evidence that some component of the respiratory chain is an MccJ25 target by showing, for example, that MccJ25-resistant mutants encode a resistant protein. It might be added that throughout more than 20 years, thousands of MccJ25-resistant mutants have been generated and screened in Raúl Salomón's group, and in none of these the mutation affected a component of the respiratory chain.

E. coli has three respiratory oxygen reductases, which oxidize quinol in the cytoplasmic membrane and reduce O₂ to water; namely cytochrome bo₃ and two closely related bd cytochromes, bd-I and bd-II. Each of these enzymes directly contributes to generating the proton motive force, which is used in ATP synthesis. In search of some solid evidence that would support the idea of the MccJ25 double target mode of action, Galván et al.¹⁹⁸ examined the effect of MccJ25 in cells with mutations in one or more of the respiratory oxygen reductases, and in membranes prepared from these strains. They found that E. coli mutant strains lacking cytochromes bo₃ or bd-I are less sensitive to MccJ25 when transformed with the FhuA overexpressing plasmid pGC01.

MccJ25 was also shown to induce ROS production in membrane preparations, and this production was higher in membranes from strains that only expressed cytochrome bd-I. However, there was no detectable induction of ROS production by MccJ25 in vivo, when it was added to whole cells of these strains. The authors explained this contradicting result by assuming that the ROS production in vivo is not high enough to observe a significant difference between MccJ25-treated cells and the control assay, or by a low sensitivity of the fluorescent probe used. Therefore, the authors turned their attention to in vitro studies on isolated membranes.

Galván et al.¹⁹⁹ purified cytochromes bd-I and bo₃ from E. coli and demonstrated that MccJ25 weakly inhibited the enzymatic activity of the isolated cytochrome bd-I, inducing the production of ROS, but had no effect on purified cytochrome bo₃. Interestingly, MccJ25 was able to partially reduce cytochrome bd-I in the presence of cyanide, thus acting as a redox peptide. Although MccJ25 can thus trigger the generation of ROS, it is possible that the ROS generated in this way can be kept in check by the elaborate network of antioxidant enzymes of E. coli, including superoxide dismutase (SOD), catalase, and peroxidases.

It is furthermore to be expected that increased superoxide production by MccJ25 should lead to oxidative DNA lesions,²⁰⁰ and this, in turn, would induce the DNA repair system known as the SOS response. SOS induction leads to the overexpression of sulA (sfiA), one of the genes of the SOS regulon. However, when the strain E. coli AB1133 was modified to overexpress the FhuA receptor and harbor a sulA::lacZ fusion, it did not show any β-galactosidase activity when exposed to MccJ25. Moreover, E. coli AB1133 (pGC01) carrying a recA::kan deletion (recA mutants are unable to express the SOS system) was as sensitive to MccJ25 as the recA⁺ control (Raúl Salomón, unpublished results and ref. 1).

If oxidative stress does indeed contribute to the antibiotic effect of MccJ25, deletion mutants of antioxidant enzymes should be hypersusceptible to MccJ25. However, sodA, sodB, katG, katE, and ahpC knock-out mutants were not rendered more susceptible to MccJ25 (Raúl Salomón, unpublished results). On the other hand, although the respiratory rate is lowered in vivo, this may be a consequence of any action of MccJ25 that is affecting the growth rate of a targeted cell. MccJ25 arrests transcription, a major macromolecular synthetic process, and this could lead to lower metabolic rates and a deceleration of respiration. Supporting this idea, Lobritz et al.²⁰¹ found that rifampicin, an antibiotic that inhibits the transcription like MccJ25, also strongly depressed oxygen consumption in E. coli.

Finally, it must be kept in mind that superoxide generation and respiration inhibition by MccJ25 have only been observed for a single specific E. coli strain (E. coli AB1133) when it was transformed with a plasmid for overexpressing FhuA (i.e., no plasmid, no effects). The results cannot be replicated with other common E. coli laboratory strains. Therefore, for all the above reasons, the RNAP inhibition (which is not strain-dependent) appears as the central killing mechanism developed by MccJ25, while free radical formation and respiration inhibition appear not to be significant contributors to the antibacterial action of the peptide.

Nevertheless, free radical formation and respiration inhibition could still be events downstream of the RNAP inhibition that consolidate cell death. If so, ROS-production enhanced by MccJ25, although small, could be synergistic with the effects of other bactericidal agents used in therapy, ultimately killing by a vicious cycle where superoxides damage membranes and DNA, and such alterations (cellular and DNA stress respectively) would in turn lead to further increase of the ROS production.^191,202,203

MccJ25 also inhibits NADH dehydrogenases by reducing the availability of NAD⁺ through the flow of electrons to terminal oxidases with oxygen consumption,^193,204 which might be related with the beneficial impact of MccJ25 on inflammatory intestinal diseases, and, possibly, on colonic cancer.^205,206 These MccJ25-mediated effects could be synergistic with the use of NAD biosynthesis inhibitors developed to de-energize fast-growing types of cancers.²⁰⁷ Measurements of the inhibition of the NADH dehydrogenase in E. coli AB1133 by MccJ25 are variable and range from a relatively weak 21%¹⁹³ to no inhibition at all.¹⁹¹ Would these decreased rates of NADH oxidation, and the subsequent depletion of NAD⁺, be sufficient to trigger a metabolic shutdown? This remains to be clarified.

5.3.3 Effects of MccJ25 on mitochondria. The idea of testing the effect of MccJ25 on mitochondria was likely inspired by previous reports on the effect of MccJ25 on the respiratory chain of E. coli and the similarities between mitochondria and bacteria, according to the well-established endosymbiotic theory of the mitochondrial origin.²⁰⁸ Niklison Chirou et al.²⁰⁹ demonstrated for the first time that MccJ25 alters the permeability of rat heart mitochondrial membranes and dissipates the proton motive force, finally inducing the opening of the mitochondrial permeability transition pore (MTP) and cytochrome c release.

The following sequence of events was proposed to explain the effect of MccJ25 on mitochondria:²¹⁰ MccJ25 would insert itself into the hydrophobic region of the internal mitochondrial membrane in a receptor-independent manner, increasing its fluidity and provoking an increase of permeability, which would lead to a dissipation of the electrochemical gradient and a drastic reduction of ATP levels. This, in turn, should accelerate respiration in an attempt to reestablish the gradient, which would increase the rate of ROS formation. The increased permeability would also lead to a small increase in the matrix calcium concentration, which would subsequently activate the calcium uniporter, further stimulating the calcium influx and ROS production, with the concomitant peroxidation of lipids and the carbonylation of proteins. Protein damage, on the other hand, could lead to the inhibition of enzymes involved in the antioxidant defense, which would further exacerbate the oxidative stress. Both the increase of the intramitochondrial calcium concentration and the MTP opening would trigger mitochondrial swelling, with the concomitant release of the apoptotic inducer cytochrome c.

As the release of cytochrome c from intact mitochondria is an early critical event in the apoptotic cascade, Niklison-Chirou et al.²¹¹ examined whether MccJ25 would also be able to induce apoptosis in mammalian cells. However, MccJ25 had no effect on intact cells, even at a concentration as high as 400 μM; likely because MccJ25 is unable to cross the cytoplasmic membrane. Remarkably, the authors could observe that the MccJ25 variant MccJ25-Ga could inhibit the mitochondrial RNA polymerase (mitRNAP) in vitro by 80%, whereas the WT peptide inhibited the mitRNAP only by 25%. Furthermore, MccJ25-Ga was demonstrated to cause apoptosis in COS-7 cells. These results led to the hypothesis that apoptosis was the result of the combined action of MccJ25-Ga on both the mitochondrial membrane and the mitRNAP, and that the inhibition of the mitRNAP must be almost complete in order to cause cell apoptosis.

It is surprising that such a slight modification at the C-terminus of MccJ25 not only allows the peptide to cross the cytoplasmic membrane, but also confers binding affinity towards an RNAP totally unrelated to the bacterial enzyme. In fact, the mitRNAP is a single-subunit enzyme that displays a significant degree of homology to the T7 and the T3 bacteriophage RNAPs.^212,213 As the assays for the mitRNAP inhibition were performed using crude sonicated mitochondria, it would be necessary to repeat these experiments with at least partially purified mitRNAP to really confirm, or disprove, these findings. Also, these results warrant a systematic revision of the cell-penetrating and the proapoptotic abilities of WT MccJ25 as this would be critical issues that need to be addressed should this lasso peptide ever be considered for utilization in food preservation or for medicinal purposes.

Soudy et al.²¹⁵ developed a synthetic decapeptide (Fig. 16A), named 18-4 (WxEAAYQrFL, where x stands for D-norleucine or D-Nle), which is proteolytically stable in biological fluids and selectively binds to the keratin 1 protein, a surface receptor overexpressed in the cytoplasmic membrane of breast cancer cells, yet scarcely present on the surface of normal cells.

Fig. 16 (A) Chemical structure of the synthetic decapeptide 18-4 that selectively binds to the keratin 1 protein and in turn mediates cellular uptake by triggering an endocytosis event. D-Amino acids are highlighted in orange. (B) Schematic representation of the MccJ25-18-4 conjugate as reported in the literature.²¹⁴

Upon binding, the peptide 18–4 is rapidly internalized, mostly by endocytosis. Based on the proapoptotic activity of MccJ25 on isolated mitochondria, Soudy et al.²¹⁴ conjugated peptide 18-4 to the C-terminus of WT MccJ25 (Fig. 16B) in a manner similar to the glycine methyl ester in MccJ25-Ga (see Fig. 9).

WT MccJ25 did not exhibit any cytotoxic activity against various cancer cell lines at concentrations up to 100 μM. In contrast, the MccJ25−18-4 conjugate entered the cancer cells through receptor-mediated endocytosis and, according to the authors, was most likely hydrolyzed in the cytoplasm, thereby releasing WT MccJ25. However, concrete experimental evidence supporting the hydrolytic release of WT MccJ25 was not presented in the study. It was further speculated that the released MccJ25 would then interact with the mitochondrial membrane and thereby would induce the series of events described above, which would eventually promote apoptosis and breast cancer cell death. The MccJ25−18-4 conjugate showed minimal cytotoxicity against normal endothelial HUVEC cells at concentration of 100 μM. Taken together, this study is an important first step in the investigation of MccJ25 and other lasso peptides as potential anticancer agents.

Finally, the production of superoxides in mitochondria triggered by MccJ25 would damage the mitochondrial DNA and the activity of complex I, thus leading to a deficiency in NAD⁺. The expected decrease in overall bioenergy of the cell might have consequences in different pathologies, from muscular to neuronal diseases, and possibly aging.^216–218

6 The further study of MccJ25 activity using in vitro and in vivo models and conceiving applications

Since the 2000's, MccJ25 received increasing attention amongst the microcins and bacteriocins for the development of applications. Two of its specific characteristics are likely responsible for the high interest it raises in the context of the growing crisis of bacterial resistances: (i) the potent and narrow spectrum of antibacterial activity of MccJ25 that is directed essentially against Escherichia, Salmonella, and Shigella at concentrations in the nanomolar to micromolar range²¹⁹ combined with only few resistances or cross-resistances with conventional antibiotics being observed for MccJ25 so far;^3,220 (ii) the good stability of MccJ25 under various harsh conditions (pH, temperature, chaotropic agents) and in the presence of many proteases, which stems from its lassoed structure (see Section 3.2.2).

These features spurred the development of both in vitro and in vivo models to better delineate the potential of MccJ25 not only for veterinary and medical applications but also for changing animal farming practices, since the passage of foodborne pathogens, such as Salmonella, from animals to humans through the food chain comprises a serious threat to human health.

6.1 Activity of MccJ25 in in vitro and in vivo models

In the 1990s, it was already demonstrated in several countries that the competitive exclusion of Salmonella in poultry could be accomplished under field conditions using diverse preparations, including complex consortia of bacterial strains.²²¹ Since then, many studies have been conducted to on the one hand evidence the potential involvement of MccJ25 in these properties and to on the other hand permit the transfer of the in vitro antibacterial activity of MccJ25 to in vivo settings.

A first pioneering study in 1999 established that MccJ25-producing strains isolated from poultry intestinal contents exhibited a strong activity against Salmonella enterica subsp. enterica serovar Enteritidis in mixed cultures.²²² It was further shown that this activity was due to MccJ25 and that this feature could be maintained in conditions mimicking the chicken gastric and intestinal tract environments.²²² This study demonstrated that this activity was not alleviated in the presence of bile, pancreatic enzymes, or acidic conditions.

In 2007, MccJ25 was further evidenced to retain its potent activity against Salmonella enterica serovar Newport both in complex biological matrices, such as whole blood, plasma and serum, but also in a mouse infection model.²¹⁹ Furthermore, the ability of MccJ25 to contribute to the competitive exclusion of Salmonella spp. under human or animal gut conditions was evidenced using in vitro static (PolyfermS system) and dynamic (TIM1 system) simulators of the gastrointestinal tract (GI).^63,223

Its stability in the different GI tract compartments was also determined in these models.^63,223,224 Indeed, the analysis of the MccJ25 degradome in the different GI compartments using LC-MS/MS and molecular networking analyses showed that degradation occurred mostly in the duodenum, while the stability of MccJ25 was much better under the acidic stomach conditions and in the colon. It was shown that the degradation in the duodenum is due to the presence of the pancreatin.

The pancreatin is a mixture of five proteases (i.e., trypsin, chymotrypsin, elastase, carboxypeptidases A/B) and mostly its elastase component with some minor contribution of the chymotrypsin component mediate the proteolysis of the lasso peptide loop region (Fig. 17).

Fig. 17 Schematic representation of the MccJ25 degradome as observed under artificial gastrointestinal conditions.⁶³ Note, that the numbers shown refer to the numbering of intact WT MccJ25.

Numerous studies identified a strong in vitro activity of MccJ25 against enterohemorrhagic E. coli O157:H7,^111,225 enterotoxigenic E. coli (ETEC),^205,226,227S. enterica,²²⁵ and clinically relevant veterinary multidrug resistant (MDR) E. coli and Salmonella strains. No cytotoxicity,^224,226,227 as well as no increase in drug resistance²²⁷ were observed in different cell models. This highly specific antibacterial activity was also demonstrated in vivo in mice models^205,228 and in broiler chickens.²²⁹

In another study, the already probiotic strain E. coli Nissle 1917, with the capability of producing microcin M and microcin H47, was engineered to also produce MccJ25 (Fig. 18) and was then administered to turkeys challenged with pathogenic S. enterica Enteritidis.²³⁰ The treatment reduced Salmonella counts in turkey caeca compared with the WT E. coli Nissle 1917 strain, indicating that the lasso peptide is probably able to contribute to controlling the pathogen growth under these conditions.

Fig. 18 Comparison of (A) the native MccJ25 BGC^31,32 with (B) an engineered BGC for the production of MccJ25 by E. coli Nissle 1917 in a turkey model setting.²³⁰ ProTeOn²³² is a synthetic activator protein consisting of the DNA binding domain of the reverse tetracycline repressor protein fused to the C-terminal, constitutive transcription activation domain of the LuxR transcription factor from Vibrio fischeri. Here, the mcjA and proteon genes were placed after a DNA promoter site that is recognized by the ProTeOn activator protein, yielding a positive feedback loop that was meant to further increase expression levels.

The efficacy of MccJ25 in the GI tract was also evaluated as a strategy to control the negative effects of postweaning stress in pig husbandry.²³¹ When MccJ25 was used as a feed additive, it reduced inflammation, attenuated diarrhea, and improved growth performance for weaned pigs, which are particularly susceptible to pathogen infections.

6.2 MccJ25 effects on the intestinal barrier function and the gut microbiota

It is tempting to consider the translation of the MccJ25 in vivo properties described above into veterinary applications or farming practices. Yet, this requires a better understanding of the underlying mechanisms, which are connected to anti-inflammatory and immunomodulatory properties on one side, and the modulation of the gut microbiota on the other side. These two properties are tightly linked to each other and were both evidenced in cell cultures and in vivo.

MccJ25 was shown to be able to relieve the inflammatory response of IPEC-J2 cells to infection by ETEC K88, a main pathogen associated with acute diarrhea in human infants and young animals. This effect involves the modulation of the pro-inflammatory interleukins IL-6 and IL-8 as well as of the tumor necrosis factor-α (TNF-α) levels,²²⁶ which are markers of the systemic inflammatory response.

In a mouse model of intestinal inflammation caused by an ETEC K88 infection, the inflammatory responses of the intestinal epithelial cells are activated. In this model, the therapeutic administration of MccJ25 attenuated the diarrheic clinical symptoms, reduced the pathogen colonization, improved the intestinal morphology, and decreased the inflammatory pathologies as well as the intestinal permeability, thereby improving the global host health.

The effects of MccJ25 in inflammation were found to be similar to what has been observed for gentamicin, but with an even higher efficiency against ETEC infections. These MccJ25 properties could be correlated with greater expression of IL-6, IL-8, and TNF-α, and with a lower expression of IL-10,²⁰⁵ which in turn modulates NF-kB activity; a key regulator of the immune response to infection. Furthermore, the MccJ25 treatment restored the expression levels of claudin-1 and occludin in the ileum and the colon after these were affected by the ETEC K88 infection, thus improving the intestinal barrier function by acting on the tight junctional complexes between enterocytes.^205,228 It was hypothesized that the inhibition of the ETEC-caused expression of inflammatory cytokines by MccJ25 could be due to a downregulation of the MAP-kinase and NF-kB pathways in the jejunum.²²⁸

In other models or other conditions, similar effects were observed. Upon gavage of mice with MccJ25, lower inflammation levels accompanied with a higher body weight, an ameliorated mucosal morphology, a reduced intestinal permeability, and a tighter intestinal barrier were seen.²³³ In a lipopolysaccharide-treated mouse septicemia model, MccJ25 treatment alleviated the inflammatory response by inhibiting the expression and the secretion of pro-inflammatory factors.²²⁷ In an E. coli mouse infection model, MccJ25 downregulated both the NF-kB and the mitogen-activated protein kinases. Thereby, the production levels of essential signaling proteins in the Toll-like receptor/interleukin-1 receptor-induced activation of the NF-kB pathway and key pro-inflammatory cytokines were reduced. These findings indicate that MccJ25 acts as both an antibacterial and an anti-endotoxin agent.²²⁷

Similar trends were observed in a poultry model. When the diet of broiler chickens challenged with pathogenic E. coli and Salmonella was supplemented with MccJ25, the broilers showed an increase in growth performance and gut health, which was correlated with decreased concentrations of TNF-α, IL-1b and IL-6.²²⁹ Supplementation of the drinking water with MccJ25 in combination with reuterin, an antibacterial aldehyde produced by probiotic strains of Lactobacillus reuteri, increased the growth performances of broiler chickens to a similar extent than the use of conventional antibiotics as growth promoters.²³⁴

Concomitantly, the fecal microbiota composition of broilers was modulated and improved.²³⁴ Here, especially the abundance of beneficial bacteria belonging to Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae families were increased as well as the concentrations of short-chain fatty acids.²³⁴ Similarly, the fecal microbiota composition of mice treated with MccJ25 upon gavage was positively modified with an increase in Bifidobacterium counts and a decrease in coliform bacteria, which was again accompanied with an increase in short-chain fatty acids.²³³

MccJ25 was also able to reduce the non-infectious intestinal inflammation in a mouse model of ulcerative colitis (DSS-induced gut inflammation) by regulating the gut microbiota in vivo.²⁰⁶ In this model, MccJ25 significantly attenuated the inflammation and improved the intestinal barrier function disrupted by the inflammatory process. Moreover, the gut microbiota modulated by MccJ25 appears to play a central role in this process.

It is now widely recognized that broad-spectrum antibiotics are no panacea, since they increase the probability of developing resistant or persistent populations of bacteria and as they can cause a dysbiosis of the gut microbiota. In contrast, the potent and selective antagonistic activity of MccJ25 against S. enterica Newport in the in vitro colon model was not accompanied with a strong perturbation of the microbiota composition, which is in agreement with its restricted spectrum of antibacterial activity.²³⁵

6.3 Occurrence of MccJ25 producing strains in the genus Escherichia in diverse microbiota

Genome mining in the APD3 antimicrobial peptide database²³⁶ for BGCs similar to the one producing MccJ25 revealed that MccJ25-like clusters are found among Escherichia marmotae genomes (33.33% out of 15 genomes) with a much higher propensity than in E. coli (0.95%/14 017 genomes), Shigella sonnei (0.16%/1275 genomes), or Salmonella enterica (0.01%/9722 genomes) genomes. Such clusters were further completely absent from the genomes of several species of the Klebsiella genus (K. pneumoniae, K. variicola, K. quasipneumoniae, K. michiganensis; 6634, 247, 237, and 77 genomes, respectively), Enterobacter cloacae (516 genomes), E. hormachei (644 genomes), and Citrobacter freundii (516 genomes) that were analyzed.

Amongst the E. coli genomes, BGCs for the production of MccJ25 were more frequent (0.95%) than clusters for the production of MccL (0.69%), MccPDI (0.25%), Mcc24 (0.01%), or MccE492 (0%), but less frequent than BGCs for the production of MccB17 (3.64%), MccI47 (4.26%), MccM (7.43%), MccH47 (7.98%), or MccV (8.58%).⁴

The apparent abundance of MccJ25-type BGCs observed in E. marmotae rather than in E. coli genomes is noteworthy. However, it should also be taken into account that this observation is based on a much smaller number of E. marmotae than E. coli genomes (15 versus 14 017). Furthermore, the species E. marmotae was only recently proposed²³⁷ as E. marmotae and E. coli are phenotypically indistinguishable. The known E. marmotae strains were therefore previously classified within the E. coli cryptic clade V,²³⁸ which was considered an environmental species with a low pathogenic potential. The E. marmotae type strain itself was isolated from a faecal sample of the wild marmot Marmota himalayana, which lives on the Qinghai-Tibetan plateau.²³⁷

In vitro infection assays and the characterization of several virulence genes suggested that E. marmotae might be a human pathogen.²³⁹ This hypothesis is corroborated by both the ability of E. marmotae to acquire resistance genes and the description of clinical cases of invasive human infections (septic shock, blood, bone, urinary tract, and bile duct infections) where E. marmotae was identified as the likely pathogen.²⁴⁰

The possible roles that MccJ25 might play in the microbiomes of the animals that are the wild reservoir of E. marmotae can only be speculated upon. In the NCBI databank, 45 E. marmotae genome sequences can be found. These stem from samples taken from marmots, wild bears, birds, and less frequently from freshwater, human, and farm animal (e.g., sheep and cows) samples. As both squirrels and marmots are closely related members of the taxonomic family of the Sciuridae, it could be of interest to also investigate squirrel microbiota in this context.

The intestinal microbiota is known to play an important role in the process of hibernation for animals like bears and marmots.^241,242 Due to the low temperatures during the winter season, some birds also have a mild-type of hibernation called torpor. In general, hibernation is made possible by an increase in body weight prior to the process and, interestingly, MccJ25 administration has been shown to increase the body weight of chickens; likely by acting on villus height/crypt depth of the duodenum and jejunum.²²⁹ Moreover, the MccJ25-triggered decrease in NAD⁺ concentrations (see Section 4.2.2) could potentially reduce the overall host metabolism, thereby acting as a bioenergetic inhibitor, which in combination with increased nutritional reserves could eventually influence the hibernation and torpor phenotypes.

Finally, the possibility of MccJ25 acting on the central neural system as a neuropeptide-like molecule and hence contributing to hibernation/torpor cannot be discarded, as intestinal neuropeptides, similar to microcins, often also exhibit antibacterial activities.²⁴³ The hypothesis about such putative functions of MccJ25 is currently under investigation by certain co-authors of this review.

Taken all of this information and the origin of the original MccJ25 producing strain into account, it therefore becomes apparent that future research needs to address how the production of MccJ25-like lasso peptides can shape a host microbiota and how it affects the human or animal host of the producing strain, especially since studies have shown how MccJ25 can also act on mitochondria (see Section 5.3.3).

6.4 Design of applications of MccJ25: consortia, formulations, synergies

Many studies conducted from the 2000's and onwards not only enabled the transfer of the properties of MccJ25 from in vitro to in vivo, as exposed above, but also facilitated the development of improved modes of administration via different approaches, such as the use of nanoparticles, or employing MccJ25 in specific combinations with other bacteriocins and antimicrobial agents.

Synergetic preparations composed of multiple antimicrobial molecules (which can include both bacteriocins and conventional antibiotics) with different mechanisms of action constitute an interesting approach to fight antibiotic resistances and their further development. For example, it was demonstrated how mixtures of MccJ25 with reuterin and lactic acid reduce the viability of Salmonella enterica on broiler chicken carcasses as efficiently as peracetic acid, which is currently used for this purpose, despite being recognized as toxic and hazardous to human health.²⁴⁴ Spraying raw chicken legs with an MccJ25/reuterin mixture before storage at 4 °C for 10 days maintained the viable counts of S. enterica below the allowed thresholds and thereby extended the shelf-life by 7 days, which is comparable to the results accomplished by the usual peracetic acid treatment.²⁴⁵

Synergistic consortia of MccJ25 or other bacteriocins with reuterin or organic acids (citric or lactic acid) increase membrane permeabilization and thereby enhance bacteriocin uptake into the cytoplasm. Such consortia were conceived and applied against a large panel of spoilage, pathogenic and MDR bacteria.²⁴⁶ The MccJ25/reuterin/lactic acid, MccJ25/reuterin/citric acid, and MccJ25/citric acid/lactic acid mixtures showed a good synergetic activity against Klebsiella pneumoniae and Salmonella enterica Enteritidis. The skin toxicity and skin sensitization of reuterin and various bacteriocins, including MccJ25, were assessed on human cell lines in view of topical applications and hand sanitizers.²⁴⁷ Here, it was shown how concentrations of MccJ25 up to 400 mg mL⁻¹ and 100 mg mL⁻¹ did not exhibit toxicity to NHEK cells or skin sensitization, respectively.

In vitro screens against a panel of Enterobacteriaceae using various combinations of different microcins or of microcins with conventional antibiotics pointed towards synergetic interactions of MccJ25 with chloramphenicol and with the antimicrobial peptide colistin (Fig. 19).²²⁰ The synergy between MccJ25 and colistin results from the membrane permeabilizing effect of colistin that in turn increases the MccJ25 uptake. This mechanism falls in the so-called “access enabling mode” of synergy, while other synergetic combinations fall in the “mechanism cooperation mode”.²⁴⁸

Fig. 19 Chemical structures and cellular targets of the conventional antibiotics that were shown to act synergistically with MccJ25.^220,248

Indeed, the synergy of MccJ25 and chloramphenicol arises from their combined effects: MccJ25 blocks the transcription via RNAP inhibition, whereas chloramphenicol blocks the translation by inhibiting the peptide bond formation via an interaction with the 50S ribosomal protein.²²⁰ MccJ25 and sulfamonomethoxine (SMM) also show a significant synergetic effect. SMM is a sulfonamide antibiotic (Fig. 19) that disrupts the folate pathway by inhibiting the dihydropteroate synthase.²⁴⁸ Therefore, the MccJ25/SMM synergy also arises from mechanism cooperation. Moreover, MccJ25 exhibited synergetic effects with a series of other antibiotics (namely, sulfamethoxazole, sulfadiazine, cerulenin, chloramphenicol, clarithromycin, ofloxacin, and tobramycin), but to a lesser extent than with SMM.

Due to the narrow spectrum of activity of MccJ25, the observed synergies hold potential for developing novel treatments in the future. Here, it should be investigated if MccJ25 could be used to selectively sensitize pathogenic Enterobacteria against otherwise non-lethal concentrations of a synergizing antibiotic, which could result in a treatment regimen with the potential to spare beneficial commensal bacteria. In this context, it will also be interesting to determine the actual mechanisms of synergy between MccJ25 and other antibiotics.

Nanomaterial-based formulations are attracting high interest in the domain of antibiotic therapy, both as delivery systems and as direct antimicrobial agents.²⁴⁹ Conjugates of chitosan nanoparticles and MccJ25 (CNM) form a class of polymeric nanomaterials (Fig. 20)^250–253 that have good bactericidal activity against foodborne pathogens and antibiotic resistant bacteria.²⁵³ They bind lipopolysaccharides, neutralize endotoxins without exhibiting cytotoxicity to eukaryotic cells (mouse RAW264.7 cells), and decrease the lipopolysaccharide-induced inflammatory response by reducing the levels of nitric oxide and pro-inflammatory cytokines,²⁵³ like it was observed before in vivo with free MccJ25. CNMs show promise in the food industry and for veterinary medicine, as they protect against the foodborne pathogen E. coli O157:H7 thanks to their bactericidal activity without inducing cytotoxicity or genotoxicity in IPEC-J2 cells.²⁵¹ Oral administration of CNMs in mice enhances the host defense against E. coli O157:H7 infections by improving the host immune functions and gut health, through enhancing the intestinal barrier, and via immune modulation.²⁵² However, too high CNM concentrations can also induce adverse effects, showing the necessity to subtly control between beneficial dosages and toxicity risks.²⁵²

Fig. 20 (A) Chemical structure of the chitosan polymer and (B) schematic representation of the conjugation of MccJ25 with chitosan nanoparticles for the generation of CNMs.^250–253

A possible utilization of MccJ25 for the development of novel biomarkers or molecular imaging probes for the early detection of diseases such as cancer^214,254 or infections²⁵⁵ could also be imagined. A positron emission tomography (PET) probe for integrin αvβ3 positive tumors was generated based on the MccJ25 variant MccJ25(G12R/I13G/G14D/T15F/G21K) that was carrying the RGD integrin binding motif in its loop (see also Section 6.2) and had a lysine residue at its C-terminus for conjugation.²⁵⁴ For probe generation, the C-terminal lysine was labelled with a bifunctional chelator binding the positron emitter ⁶⁴Cu. The resulting conjugate (Fig. 21) showed good stability in mouse plasma. PET imaging of integrin αvβ3 positive tumor bearing mice was performed using this probe and an analogous compound based on MccJ25(G21K) as negative control. The probe featuring the RGD motif could clearly distinguish the αvβ3 positive tumor and showed a significantly higher accumulation in tumor cells than the probe based on MccJ25(G21K). Moreover, the probe carrying the RGD motif was selective to U87MG tumors compared to MCF-7 tumors which have low expression levels of the αvβ3 integrin receptor. These results exemplify the potential of MccJ25 and its derivatives in the domain of cancer research, both for treatment and early detection.

Fig. 21 Schematic representation of the generation of a PET probe for αvβ3 integrin receptor positive cancer cell lines based on the lasso peptide variant MccJ25(G12R/I13G/G14D/T15F/G21K) as reported in the literature.²⁵⁴

In addition, a radiotracer derived from the primary structure of MccJ25 was assayed in a nuclear medicine approach for E. coli infection imaging.²⁵⁵ Here, a synthetic 14-residue peptide (Fig. 22) was based on the ring sequence of MccJ25 by replacing Gly1 and Glu8 with Cys residues to form a macrocycle through disulfide bond formation. This macrocycle was followed by a linear sequence of L-Tyr, D-Tyr, γ-aminobutyric acid (GABA), D-Phe, L-Tyr, L-Gly. The resulting synthetic peptide exhibited antibiotic properties, particularly against E. coli,²⁵⁶ and could be conjugated to specific ligands able to chelate a metastable nuclear isomer of the technetium-99 isotope (^99mTc),²⁵⁵ which is one of the most commonly used medical radioisotopes. Imaging using the resulting radioprobe showed the accumulation of the radiotracer in the infection zone and a major renal excretory pathway.

Fig. 22 Comparison of the primary structure of MccJ25 with a MccJ25-inspired, synthetic 14-residue peptide exhibiting antibacterial properties.^255,256

7 Bioengineering utilizing the MccJ25 lasso peptide scaffold

7.1 Library screens of MccJ25

A common exercise in the study of RiPPs is the generation of a set of precursor variants to evaluate the effects of amino acid substitutions on the biosynthesis and/or the activity of a RiPP. In the lasso peptide field, the first examples of this type of mutational analysis were carried out on MccJ25. The plasmid-borne nature of the MccJ25 BGC facilitates the generation of amino acid substitution variants since mutants of the mcjA gene can be readily generated using PCR techniques and can then be reintroduced into a plasmid rather than having to recombine the mutant gene into the genome of the producing organism.

The first example of extensive mutagenesis of mcjA was carried out by Pavlova et al.,¹⁸⁷ where the modified mcjA genes were being reintroduced into the pTUC202 plasmid and transformed into the E. coli strain DH5α for production. Here, all positions except the isopeptide bond-forming Glu8 were exchanged with every other canonical amino acid and an E8D exchange was also tested.

The resulting library of variants was then assessed via MALDI-TOF-MS of the culture supernatants, showing that 242 out of the 381 tested MccJ25 variants were produced and exported by E. coli DH5α (Fig. 23), thereby also providing the first example of genetic engineering of a lasso peptide. The variants detected via mass spectrometry were next tested for in vitro RNAP inhibition using a fluorescence-detected abortive initiation assay. These experiments revealed that a remarkable fraction of MccJ25 single amino acid substitution variants, 155 of the 242 variants whose production was confirmed by MALDI-TOF-MS, could still inhibit the RNAP (Fig. 23). The majority of the variants that retained the RNAP inhibition activity carried substitutions in the loop, whereas residues in the lasso peptide ring and tail were shown to be more important for the RNAP inhibition and hence less susceptible for exchanges. These insights were borne out later in structural studies of the MccJ25-RNAP complex.¹¹⁴

Fig. 23 Overview of the results obtained from an extensive mutational analysis study of the MccJ25 scaffold spanning a total of 381 tested variants.¹⁸⁷

Finally, in a third stage of evaluation, the subset of MccJ25 variants that inhibited the RNAP in vitro was tested for antimicrobial activity against E. coli DH5α and Shigella flexneri clinical isolate BK 12440. Of the 381 variants constructed in this study, 70 retained antimicrobial activity (Fig. 23). This was a remarkable finding; despite the requirement for the MccJ25 variant to productively interact with the outer membrane transporter FhuA, the inner membrane transporter SbmA, and its cytoplasmic target in form of the RNAP, the MccJ25 scaffold could “absorb” a wide variety of amino acid substitutions while still retaining its antimicrobial activity.

Another early example of mutagenesis on the MccJ25 scaffold was carried out by Pan et al. using a computational protein design algorithm.²⁵⁷ Briefly, this algorithm generated a set of possible triple amino acid substitution variants in silico. The Gly and Pro positions were fixed in this in silico library as was the Glu8 position that forms the isopeptide bond with Gly1. Those amino acids with hydrophobic side chains in WT MccJ25 were only allowed to vary to other hydrophobic amino acids, while polar amino acids were allowed to vary to the entire set of amino acids. Finally, given its role in the uptake, the His5 residue was only allowed to vary to Lys or Arg.

The in silico library, comprising 3.2 × 10¹¹ variants, was evaluated in two stages. In the first stage, the sequence selection, a ranked list of sequences predicted to fold into the lasso shape was generated by solving a global optimization problem.²⁵⁸ In the second stage, the fold specificity of the selected sequences was computed and a ranked list of the variants was generated. The computational design approach was validated by choosing eight of the top predictions for experimental evaluation (Fig. 24). Six of the eight variants were successfully produced and exported to the culture medium at yields of ∼50 to ∼150% of the WT peptide as judged by HPLC. Three of the six variants that were produced also had measurable activity against the hypersensitive strain Salmonella Newport, though the activity was significantly weaker than that of the WT peptide. Nonetheless, this study took the important step of demonstrating that MccJ25 could tolerate multiple amino acid substitutions while still retaining its structure and function.

Fig. 24 Schematic overview of the primary structures of the MccJ25 variants that were generated for experimental validation of a computational design approach.²⁵⁷ Positions carrying amino acid substitutions are highlighted in red.

Another study by Pan and Link further built on this idea by generating libraries of triple amino acid substitution variants of MccJ25.²⁵⁹ Two libraries were generated; one targeting the MccJ25 ring (positions Ala3, His5, and Val6) and one targeting the MccJ25 loop (positions Gly12, Ile13, and Thr15). In both libraries, the codons corresponding to each targeted position were mutagenized using a reduced codon set encoded by the degenerate codon NNT, where N is any of the four bases. This degenerate codon covers 15 amino acids in 16 codons, reducing the screening effort required to cover the entire library.

With this encoding scheme, each of the libraries contained 4096 distinct mutants at the gene level corresponding to 3375 distinct triple amino acid substitution variants. Since screening each of these library members one-by-one as was done in Pavlova et al.¹⁸⁷ was not feasible, the libraries were instead screened en masse in a high-throughput fashion (Fig. 25). To facilitate screening, the MccJ25 BGC was refactored to put the mcjA gene under the control of an IPTG-inducible T5 promoter. The mcjBC operon was left under its native constitutive promoter p_mcjBC, while the transporter gene mcjD was placed under the arabinose-inducible p_araBAD promoter. In this way, the production and export of MccJ25 could be controlled independently. The libraries were cloned into the refactored BGC in glucose-rich conditions to repress any leaky expression of the mcjA gene.

Fig. 25 Schematic showing the underlying principle of the screening approach developed for the analysis of MccJ25 libraries.²⁵⁹ (A) The genes mcjA and mcjBC are located on the plasmid pWC8 that also encodes the LacI repressor. The T5 promoter controls the mcjA expression, while the native mcjBCD promoter is controlling mcjBC expression. (B) A second plasmid, pJP31, is expressing mcjD under control of the BAD promoter. This plasmid also encodes the arabinose repressor AraC. (C) When cells carrying both plasmids are cultivated on LB agar supplemented with glucose and arabinose, mcjA transcription is repressed, while mcjD expression is induced. Colonies from this plate can then be transferred via replica plating to (D) another LB agar plate containing glucose and IPTG. Under these conditions, mcjA expression is induced, while expression of the McjD transporter is suppressed. Cells producing MccJ25 variants able to inhibit the RNAP will not survive under these conditions, as they lack the means of self-immunity. Thus, colonies observed on the first plate (C) that are absent from the replica plate (D) harbor the potential to produce bioactive MccJ25 variants.

Screening of the library was carried out using replica plating. The library was plated on media containing glucose to repress leaky mcjA expression and arabinose to ensure that any lasso peptide that was produced could be exported (the initial plate). These colonies were stamped onto plates with the restrictive condition of IPTG/glucose (the replica plate). On the replica plate, MccJ25 variant expression is induced while expression of the McjD transporter is repressed. As a consequence, any colonies that produce a functional, antimicrobial MccJ25 variant will not grow on the replica plate (Fig. 25) since the immunity factor McjD is not produced.

Ultimately, an excess of 20 000 colonies were screened in both the ring and loop libraries (i.e., 5× oversampling) to ensure coverage of the entire libraries. In this initial screening stage, roughly 50% of the colonies were growth inhibited on the replica plate, demonstrating that a large fraction of the triple amino acids substitution variants putatively retained some RNAP inhibition activity. Those colonies that resulted in the most potent growth inhibition on the replica plate were picked from the initial plate, sequenced, and tested for inhibition of Salmonella Newport and E. coli DH5α. Over 100 colonies were analyzed in this way, with the vast majority of the potent triple amino acid substitution variants containing exchanges in the loop of MccJ25.

This result suggested that while many MccJ25 variants with ring substitutions were produced and competent at inhibiting the RNAP, these same variants were unable to be efficiently taken up into the cytosol of susceptible cells. In contrast, many of the produced MccJ25 variants with substitutions in the loop were competent both at RNAP inhibition and import. Twelve of the most potent loop triple amino acid substitution variants (Fig. 26) were more closely assessed for their production levels and their antimicrobial activity against E. coli DH5α and S. Newport. All of these variants exhibited improved efficacy against E. coli DH5α, and six of them even showed increased activity against the already hypersensitive S. Newport. This study thereby provided further insight into the structure–activity relationship of MccJ25 while also demonstrating that directed evolution and high-throughput screening approaches could be applied to lasso peptides.

Fig. 26 Overview of the primary structures of the twelve most potent MccJ25 variants that were selected from a library of MccJ25 variants and assessed for their antimicrobial activity against E. coli DH5α and S. Newport.²⁵⁹ Positions carrying amino acid substitutions are highlighted in red.

Given its gene encoded nature, a lasso peptide precursor gene can also be modified in vivo via genetic code expansion to insert non-canonical amino acids (ncAAs) into the lasso peptide. Piscotta et al. demonstrated this on MccJ25 by mutagenizing positions both within the ring (Val6) and the loop (Phe10, Ile13, and Phe19) with the amber stop codon.²⁶⁰ Using an engineered pyrrollysyl-tRNA synthetase (PylRS), a set of four meta-substituted Phe analogs were introduced at each of these four positions, generating a set of 16 possible ncAA-substituted MccJ25 variants. Fifteen out of the 16 possible variants (Fig. 27) were produced and detectable by HPLC, and two of the variants with m-bromoPhe and m-chloroPhe substitutions retained near WT activity. The ability to generate ncAA-substituted variants of MccJ25 was also a key linchpin to solving the X-ray crystal structure of the MccJ25-RNAP complex.¹¹⁴ Initial co-crystal structures between the RNAP and MccJ25 had clearly detectable electron density in the secondary channel of the RNAP corresponding to MccJ25, but not at atomic resolution. The m-bromoPhe variants of MccJ25 discussed above were initially evaluated to see whether the bromine heavy atom and its anomalous diffraction could improve the resolution of MccJ25 within the RNAP secondary channel. This was ultimately unsuccessful, presumably because of the delocalization of the bromine signal due to the rotation of the Phe side chain. It was reasoned that para-bromoPhe would solve this problem as rotation of the phenyl ring would not delocalize the bromine signal. This was borne out, and co-crystal structures with exchanges of both His5 and Phe10 to p-bromoPhe MccJ25 were solved, allowing for precise positioning of the MccJ25 ligand within the RNAP.

Fig. 27 Scheme depicting the various ncAAs that were incorporated at different positions (6, 10, 13, and 19) of the MccJ25 scaffold using the amber codon (TAG) suppression technology. Names of variants that could be heterologously produced are highlighted in green, names of variants that could not be detected in red.²⁶⁰

Finally, another route to generate MccJ25 variants would be the chemical modification of the isolated lasso peptide. So far, this route has only scarcely been explored and reported chemical modifications (Fig. 7) included glycine amidation of the C-terminus, carbethoxylation of the His5 residue, and the simultaneous conversion of both Tyr9 and Tyr20 into 3-nitrotyrosine residues. It can however be imagined how this approach could broaden the scope of what variations can be introduced into the MccJ25 scaffold by combining chemical modification with normal mutagenesis of the core peptide and the introduction of ncAAs via amber codon suppression strategies or the use of auxotrophic production strains.

In the subsequent subchapters, we will discuss how mutagenesis of mcjA enabled access to MccJ25 variants harboring novel biological activities.

7.2 MccJ25-derived αvβ3 integrin antagonists

Epitope grafting describes the process of transplanting a small, bioactive (peptide) epitope onto a stable (peptidic) scaffold, thereby combining the activity derived from the epitope with the enhanced stability of the scaffold molecule (Fig. 28A).

Fig. 28 (A) Schematic representation of the concept of epitope grafting. (B) Comparison of the primary structure of WT MccJ25 with the bioengineered αvβ3 integrin receptor antagonists that have been reported.^266,267 The bioactive RGD peptide epitope is shown in red, the Phe residues introduced for mediating additional hydrophobic interactions with the receptor binding pocket in green.

RiPPs are predestined for such applications as the genetic origin of their precursors enables the incorporation of small peptide sequences by simple mutagenesis of the core peptide-encoding regions of their precursor genes. Indeed, there have been several examples of how RiPPs can be utilized in context of epitope grafting efforts, accomplishing the introduction of diverse new activities into the chosen scaffolds.^261–271

One of the RiPP scaffolds successfully employed in the past was MccJ25.^265–267 MccJ25 is the so far only lasso peptide used for such projects due to several reasons: (1) it can be readily produced with high titers in E. coli from a relatively simple plasmid-based system. (2) MccJ25 is renowned for its remarkable stability, even amongst lasso peptides, as discussed above. (3) MccJ25 has one of the longest known loop regions of lasso peptides, whose protruding nature is ideal to present the epitope to a potential interacting partner.

For the initial proof-of-concept that lasso peptides can be used for epitope grafting, the human αvβ3 integrin was chosen as target for the grafting efforts. This integrin receptor recognizes a simple tripeptide motif, RGD, and plays a key role in the process of angiogenesis, the generation of new blood vessels.^272,273 Therefore, it is an interesting target for suppressing the growth of certain tumors.^272–274 This is due to the fact that tumor growth is limited by the blood capillaries present in the tissue surrounding the growing tumor, as they are key for supplying the tumor with nutrients, but also for disposing waste products. Without the formation of new blood vessels, the tumor growth would be stunted, if not completely halted. Hence, it is not surprising that a lot of research went into the development of potent, high affinity αvβ3 integrin receptor antagonists, which in turn means that there is a manifold of assays established for evaluating potential candidate molecules with regards to binding affinity and antagonistic activity. The combination of the small size of the RGD epitope with the availability of suitable assays therefore made the αvβ3 integrin receptor a bona fide first target for establishing lasso peptide-based epitope grafting as a concept.

Initially, the RGD epitope was introduced in the center of the loop region, replacing the residues at position 12–14 of WT MccJ25, yielding the variant MccJ25(G12R/I13G/G14D), or MccJ25(RGD) for short (Fig. 28B).²⁶⁷

MccJ25(RGD) already possessed a decent mid-nM binding affinity for the target receptor (IC₅₀ = 12 ± 1.9 nM) and was able to inhibit capillary formation in an in vitro assay employing human umbilical vein endothelial cells (HUVECs).²⁶⁷ However, the selectivity of the interaction with the target receptor versus off target receptors was unsatisfying.

Improvement of both the affinity and the selectivity of the target binding was accomplished through rational means. From studies using small cyclic peptide-based binders,²⁷³ it was already known that placing a phenylalanine adjacent to the RGD binding epitope can improve both of these parameters through enabling additional hydrophobic interactions with the binding pocket. Whereas introduction of a Phe residue before the RGD motif (V11F/G12R/I13G/G14D; MccJ25(FRGD), Fig. 28B) did not improve the affinity to the target receptor (IC₅₀ = 21 ± 0.9 nM) and only slightly improved the selectivity of the compound,²⁶⁶ the introduction of the Phe after the RGD motif (G12R/I13G/G14D/T15F; MccJ25(RGDF), Fig. 28B) had a positive impact on the binding affinity (IC₅₀ = 4.1 ± 0.8 nM) and improved the selectivity of this interaction by an order of magnitude.²⁶⁶ In addition, MccJ25(RGDF) was able to inhibit capillary formation in HUVEC assays like MccJ25(RGD) did.²⁶⁶

A PET probe for αvβ3 integrin receptor positive tumor cancer cell lines was developed²⁵⁴ based on MccJ25(RGDF) (see also Section 6.4).

7.3 MccJ25-derived ClpP binders

The prokaryotic Clp protease complexes can play important roles in protein homeostasis, stress management, virulence regulation, cell differentiation, and protein quality control, making them, for example, promising targets for the development of new antimycobacterial drugs.^275–278 Clp complexes consist of two important elements^{275,277,279,280} (Fig. 29A): (1) the central tetradecameric ClpP barrel that holds the proteolytic active sites in its center. (2) Specific Clp–ATPase hexamers (e.g., ClpA, ClpC, or ClpX), which recognize proteins marked for degradation and catalyze the unfolding of the protein structures in an ATP-dependent manner, while funneling the resulting linear peptide chains into the entry pores of the ClpP tetradecamers for degradation. Clp-ATPases therefore determine the specificity of the Clp protease complex, whereas lone ClpP tetradecamers only accept substrates small enough to unselectively diffuse through their entry pores.

Fig. 29 (A) Schematic representation of a ClpP tetradecamer, a Clp–ATPase hexamer, and the active Clp protease complex. Note, that the Clp–ATPase hexamers can dock to the H-pockets on both the bottom and the top of the ClpP barrel. (B) Schematic depiction of the MccJ25 variants generated²⁶⁵ with the goal of obtaining ClpP-binding lasso peptides. Residues exchanged compared to the WT sequence are highlighted in red. (C) Chemical structures of the IGF-motif, ADEP1, and ADEP2. (D) Co-crystal structure of the E. coli ClpP tetradecamer with bound ADEP1 (PDB code 3MT6).²⁸² On the left, a top view of the ClpP complex with bound ADEP1 is given and one of the bound ADEP1 molecules is highlighted and zoomed-in upon. On the far right, a side view of the H-pocket with bound ADEP1 is shown with a transparent protein surface to highlight which part of the ADEP1 molecule is binding into the H-pocket.

The interactions between the Clp–ATPase hexamers and the ClpP tetradecamers is mediated through specific loops on the Clp–ATPase surface (Fig. 29A).^277,279,280 These loops are carrying conserved, hydrophobic tripeptide sequences, e.g., IGF, IGL, or VGF, which bind into corresponding hydrophobic binding sites (H-pockets) on the surface of the ClpP barrel.

The combination of a loop structure with a small tripeptide binding motif inspired another MccJ25-based epitope grafting campaign.²⁶⁵ Here, the goal was to use the lasso peptide fold as a structural mimic of the Clp–ATPase binding loops through the incorporation of the tripeptide binding motifs. For this purpose, the three aforementioned binding epitopes were introduced into the loop of MccJ25 (Fig. 28B) starting with the incorporation either from position 11 (MccJ25(11IGL), MccJ25(11IGF), MccJ25(11VGF); thereby replacing Val11/Gly12/Ile13), position 12 (MccJ25(12IGL), MccJ25(12IGF), MccJ25(12VGF); thereby replacing Gly12/Ile13/Gly14), or position 13 (MccJ25(13IGL), MccJ25(13IGF), MccJ25(13VGF); thereby replacing Ile13/Gly14/Thr15).²⁶⁵

This approach has precedent in the form of acyldepsipeptides (ADEPs).^{276,278,281,282} For example, ADEP1 (Fig. 29C and D) is a natural product that is structurally mimicking the IGF binding motif, thereby becoming a high affinity ClpP H-pocket binder.

Upon binding, ADEP1 is not only inhibiting the ClpP/Clp–ATPase interactions, it is also triggering the opening of the ClpP barrel entry pores, which in turn allows for the unregulated degradation of smaller essential cytosolic proteins resulting in cell death. More potent synthetic derivates of this natural product have been developed as well, e.g., ADEP2 (Fig. 29C). It therefore made sense to assess if any of the nine engineered MccJ25 variants would exhibit similar properties than ADEP2. Hence, it was investigated if any of the engineered lasso peptides had an effect on the stability, the peptidase activity, and the Clp–ATPase interactions of the model ClpPs from Bacillus subtilis (BsClpP) and Staphylococcus aureus (SaClpP).²⁶⁵

The purified SaClpP tetradecamer is not stable in Tris buffers at pH 8.0 and will dissociate into smaller subunits. As it was known that the addition of ADEP2 can stabilize the protein complex under these conditions and thereby prevent its disintegration, it was tested if any of the nine MccJ25 variants would have comparable stabilizing effects on SaClpP. Indeed, the MccJ25 variants containing either of the three conserved Clp–ATPase binding motifs starting from position 12 onwards were able to stabilize the complex.²⁶⁵ This property was most pronounced for MccJ25(12IGF). The stabilizing effect could be observed both by gel filtration experiments and by assessment of the retained peptidase activity of SaClpP in a Tris buffer at pH 8.0. It was also shown that the peptidase activity observed was dependent on the MccJ25(12IGF) concentration employed.²⁶⁵ Unlike with ADEP2, the MccJ25(12IGF) interaction with SaClpP was too weak to significantly interfere with the interactions between SaClpP and the SaClpX ATPase, as was shown in a proteolysis assay using ssrA-tagged enhanced green fluorescence protein (eGFP) as substrate.²⁶⁵

In contrast to SaClpP, the BsClpP tetradecamer cannot be stabilized after purification by use of any specific buffer conditions and will eventually dissociate into its monomeric units. While ADEP2 was shown to be able to trigger the formation of the active, tetradecameric BsClpP complex from the monomers in solution, none of the tested MccJ25 variants was able to accomplish the same feat.²⁶⁵ However, it was demonstrated that once a low ADEP2 concentration had already triggered the BsClpP complex assembly, some of the MccJ25 variants were able to further increase the BsClpP peptidase activity upon addition.²⁶⁵ Again, this effect was most pronounced for MccJ25(12IGF). It was furthermore demonstrated how MccJ25(12IGF) was able to increase the peptidase activity of tetradecameric BsClpP, when it was freshly obtained from size exclusion chromatography and before it had time to dissociate into its monomers.²⁶⁵ Furthermore, the increase of the BsClpP peptidase activity was shown to be dependent on the used concentrations of MccJ25(12IGF), no matter if complex assembly was accomplished by ADEP2 addition or if freshly eluted BsClpP complex was used in the absence of additional ADEP2.²⁶⁵

When it was tested if ADEP2 could displace MccJ25(12IGF), it was however observed that even at high concentrations, where all H-pockets of BsClpP should be saturated with ADEP2, addition of MccJ25(12IGF) still caused an additional synergistic effect on the BsClpP peptidase activity.²⁶⁵ This strongly suggests that MccJ25(12IGF) does not bind into the H-pocket but must instead interact with a different binding site that is independent of ADEP2 binding. While this is in stark contrast with the initial working hypothesis this lasso peptide-based ClpP-binder was generated on, it provides an interesting perspective with regards to the identification and further targeting of this potential novel allosteric ClpP binding site in the future.

7.4 Topological engineering with MccJ25

In the parlance of supramolecular chemists, MccJ25 and other lasso peptides are natural examples of [1]rotaxanes, or threaded molecules with a single free end. As discussed elsewhere in this review, MccJ25 can be cleaved within its loop by, e.g., thermolysin to generate a [2]rotaxane, a threaded molecule with two free ends.

While there are many natural examples of slipknotted and other entangled proteins, most notably the hormone leptin,²⁸³ lasso peptides are unique among these because the size of the interlocking moieties are similar to the size of chemically synthesized rotaxanes and other mechanically interlocked molecules (MIMs),²⁸⁴ the topic of the 2016 Nobel Prize in Chemistry.

Allen and Link envisioned that loop-cleaved MccJ25 could be a useful building block for generating more complex MIMs generated solely from peptidic building blocks.⁵⁵ To realize this, the MccJ25 precursor peptide was mutagenized to introduce two cysteine residues, one at the C-terminus, replacing Gly21, and one in the loop substituting Ile13. This MccJ25 variant further included a G12R substitution to generate a trypsin cleavage site.

Upon treatment with trypsin, MccJ25(G12R/I13C/G21C) was efficiently cleaved into a [2]rotaxane with Cys residues at either end of the linear peptide threaded through the isopeptide bonded ring (Fig. 30A). Self-assembly of this [2]rotaxane by oxidation in an aerobic environment led to the formation of a mixture of [3] and [4]catenanes (Fig. 30B and C), molecules comprised of two or three isopeptide bonded macrocycles threaded around the central ring. This work represented the first demonstration of MIMs similar in size to synthetic examples but made entirely from peptides.

Fig. 30 (A) The loop of the variant MccJ25(G12R/I13C/G21C) can be selectively opened by trypsin treatment. After cleavage, oxidation of the resulting [2]rotaxane can yield novel (B) [3]catenane and (C) [4]catenane species through the formation of disulfide bridges. The three-dimensional structure of the thereby obtained [3]catenane (PDB code 5T56) was determined by NMR spectroscopy and is shown in the upper right corner.

Schröder et al.⁵⁴ greatly expanded on this idea by generating a larger set of Cys-decorated MccJ25 building blocks that could be forged into a wide range of MIM structures including a lasso peptide with inverted stereochemistry relative to the native peptide, a [2]catenane (two interlocking rings), molecular daisy chains, and exotic structures like a double lasso macrocycle, all made purely from peptides.Whereas synthetic MIMs require sophisticated templating approaches to be carried out in organic solvents to generate threading, the MccJ25 biosynthetic enzymes do the hard work of generating a pre-threaded building block for the construction of any number of biocompatible peptidic MIMs. Such structures can bring the promise of MIM-based molecular machines into the biological realm with potential applications in catalysis and biomaterials.

Through access to the processing enzymes of MccJ25 (or other lasso peptides) for in vitro use, bioengineering of lasso peptide scaffolds could not only be simplified but also be extended to utilize substrates featuring non-canonical amino acids. As the general applicability of the MccJ25 lasso peptide for bioengineering efforts has already been demonstrated, the potential here seems significant.

Yet, one should also not forget to consider the benefit an efficient and completely synthetic route to access MccJ25 and other lasso peptides would have. Despite this remaining a very daunting task for organic chemists, achieving such a synthesis would be viewed as an important breakthrough that would be much lauded for any ambitious chemists up for a challenge.

8 Conclusion and outlook

Thirty years is a long time for researching a single natural product. Nonetheless, an amazing breadth of data, insights, and innovations have launched from the work on MccJ25, making it the most well studied and probably also the most remarkable lasso peptide investigated so far.

Looking back, it is interesting to acknowledge how MccJ25 was the “first” in many respects considering lasso peptide research: the first proteobacterial lasso peptide, the first antimicrobially active lasso peptide, the first lasso peptide with a known biosynthetic gene cluster, the first lasso peptide with an identified mode-of-action, the first lasso peptide for which its biosynthesis was investigated, and probably many more “firsts” that more scrutiny can uncover. Yet, MccJ25 moved far beyond just being a first example for many things in the field of lasso peptides and remains crucial for further developing this field and for pioneering new innovations and technologies.

Until now, MccJ25 is the only lasso peptide used for epitope grafting and topological engineering studies. It is also blazing the trail for utilizing lasso peptides in a drug development context. New formulations and modes of administration are being developed utilizing MccJ25, synergies of conventional antibiotics with MccJ25 have been discovered that might facilitate new applications, and more and more animal studies reveal the potential, yet also the limitations, that lasso peptide scaffolds carry with regards to therapeutical uses (both for the treatment of human diseases, but also in a veterinary medicine context).

The reasons why MccJ25 has played such an important role in lasso peptide research probably include its early discovery and the ready availability of MccJ25 (and variants thereof) via heterologous production. However, MccJ25 also has many unique features, which were highlighted in this review, that facilitated research projects that would have not been possible with other lasso peptides.

Despite the efforts of many research groups from different backgrounds, countries, and research interests for over thirty years now, it does not seem that the research work on and with MccJ25 will end anytime soon. Amongst the many unresolved questions in front of us, the ones seeming the most interesting (and maybe also the most daunting) are: (i) elucidating the final steps of the underlying mechanisms of lasso peptide biosynthesis, (ii) determining the potential of lasso peptides as new drug leads for human and veterinary medicine, (iii) assessing the potential use of non-pathogenic bacteria producing antimicrobial lasso peptides as novel probiotics, (iv) uncovering the impact lasso peptides (both antimicrobial and otherwise) produced by commensal and pathogenic bacteria have on the host but also on the surrounding microbiota.

For all of these aspects, MccJ25 still remains a good model system that will help to further develop this field, will contribute to get a better understanding of lasso peptides in general, and will assist in evaluating what applications we might eventually be able to utilize this fascinating group of natural products for. Yet, the continuously increasing interest in lasso peptides and the fact that more and more research groups are getting involved in this field demonstrate that there is also much potential beyond MccJ25. Therefore, we are looking forward to what new insights and discoveries the next thirty years will bring; both for MccJ25, but also for lasso peptides in general.

9 Author contributions

All authors participated in the writing of this review article. Every author contributed segments relating to their own previous work on this topic and on other subjects relating to their own fields of expertise. J. D. H. further compiled and edited all contributions into a final draft and reviewed it together with the other authors.

10 Conflicts of interest

There are no conflicts to declare.

11 Acknowledgements

We would like to thank Roberto Kolter and Victor de Lorenzo for their help with identifying all the participants of the 1983 microcin kick-off meeting in La Alhambra, Granada (Spain).

12 Notes and references

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