Metals in infectious diseases and nutritional immunity

Almost 150 years ago, Trousseau discovered that the administration of iron-rich supplements to patients recovering from active tuberculosis increased the frequency of relapse. In the 1940s Schade and Caroline reported that iron-binding proteins in the blood and in the whites of eggs inhibit growth of bacteria through iron chelation. In the years since these foundational observations, it has become widely appreciated that the scarcity of nutrient metals within the vertebrate host poses a significant challenge to microbial pathogens. In fact, if one considers it from the standpoint of availability, the human body is one of the most metal-depleted environments on the planet.

There is, of course, significant metal within vertebrates, however this metal is sequestered and mobilized in an effort to make it unavailable to invading microbes. This process, known as “nutritional immunity”, is a critical host defense strategy against bacterial and fungal pathogens. So effective is nutritional immunity that in order to infect the host, microbes must be able to circumvent it. More recently, it has become appreciated that not only can vertebrates sequester metal from invading pathogens, but in certain instances, the toxic properties of metal can be used against the microbes. By harnessing the toxic properties of metal, vertebrates expand the repertoire of nutritional immunity to include metal sequestration and intoxication. Over time, this ferocious battle for and with nutrients has led to the evolution of exceptionally elegant host and microbial systems dedicated to the sequestration, uptake, and detoxification of metal. In this themed issue, a series of articles are presented that touch on numerous aspects of this arms race, investigating the struggle for nutritional metals from both the host and microbial perspectives.

The most studied metal ion at the host–pathogen interface is iron. This metal is critical for many cellular processes including respiration, DNA replication, and central metabolism. In vertebrates, iron is mainly intracellular, and extracellular iron is primarily bound to proteins. Zinc is also predominantly intracellular, where it serves as a co-factor for many proteins and enzymes. As the availability of both iron and zinc to microbes is limited, it is not surprising that many pathogens employ sophisticated mechanisms to acquire these metal ions in the vertebrate host. As discussed by Subashchandrabose and Mobley (C4MT00329B), one of the best examples of a pathogen that has evolved to acquire metal ions in the host is uropathogenic Escherichia coli (UPEC), the leading cause of urinary tract infections (UTI).

In the urinary tract, metals are scarce and are further limited by the up-regulation of metal-binding proteins, which are induced in response to infection, including the antimicrobial proteins lipocalin-2 and calprotectin. Lipocalin-2 sequesters a subset of siderophores; i.e. small iron chelators secreted by bacteria when iron is limited. One of the siderophores sequestered by lipocalin-2 is enterobactin, which is produced by many commensal and pathogenic Enterobacteriaceae. Similarly, calprotectin sequesters zinc and limits the availability of this metal to pathogens. To overcome these antimicrobial responses, UPEC and other UTI pathogens express multiple iron uptake systems and zinc transporters. Furthermore, these pathogens acquire nickel and detoxify copper as additional mechanisms to thrive in the host. In light of these findings, the authors conclude that, moving forward, the research community should capitalize on this knowledge and design new therapeutic interventions by targeting the metal transport systems employed by UTI pathogens.

Intracellular bacterial pathogens face additional challenges regarding the acquisition of nutrient metals. Indeed, diversion of cellular iron traffic is an important antimicrobial strategy for the host. In turn, organisms that reside within macrophages influence intracellular iron handling, which alters the outcome of the host–pathogen interaction. Haschka et al. (C4MT00328D) contribute a research article demonstrating that infection of macrophages by the intracellular pathogen Listeria monocytogenes leads to up-regulation of the major cellular iron exporter, ferroportin1. They then report that ferroportin1-mediated iron export is an active immune strategy to reduce the growth of wild-type L. monocytogenes inside the macrophage. In contrast, L. monocytogenes mutants that cannot produce listeriolysin O, and are thus restricted to the phagosome, are subjected to a distinct strategy, whereby iron remains in the macrophages contributing to the ROS-mediated intoxification of bacteria. The authors also present interesting data linking iron homeostasis and immune response pathways, emphasising the strong functional link between metal sequestration and immunity. Taken together, these results highlight the importance of host cellular iron trafficking during infection of macrophages, and demonstrate the impact a microbial invader's intracellular localization has on nutritional immunity.

Another example of a pathogen for which metal ions are essential for its replication within the mammalian host, and for eliciting disease, is Yersinia pestis, the causative agent of bubonic, septicemic, and pneumonic plague. Y. pestis has a number of iron, zinc, and manganese transporters, many of which have been shown to promote the pathogen's virulence. The transport mechanisms for these metal ions and their role in the three forms of plague are the subject of a review contributed by Perry et al. (C4MT00332B). The authors also discuss some of their recent findings, which identified a novel role for the siderophore yersiniabactin in zinc acquisition, independent of iron acquisition; as direct binding of yersiniabactin to zinc has not yet been demonstrated, it is possible that yersiniabactin plays an indirect role in this process. Nevertheless, yersiniabactin has recently been found by the Henderson lab to bind divalent copper ions, in addition to iron, during UTI. In this issue, Koh et al. (C4MT00341A) expand on this earlier work and use direct mass spectrometry to show that yersiniabactin also binds to nickel, cobalt, and chromium, and transports these metals via its cognate receptor, FyuA, in a TonB-dependent manner. These results further demonstrate that yersiniabactin is a promiscuous siderophore that can form stable complexes with several physiologically relevant metal ions, possibly supplying other essential trace elements to pathogens. These findings add considerably to the growing role of siderophores in circumventing nutritional immunity.

The increasing evidence indicating that siderophore functions may be more complex than previously thought, is also the topic of a critical review authored by Holden and Bachman (C4MT00333K). In addition to their ability to chelate iron and – as discussed above – other metals, siderophores can also perturb host cell homeostasis. For example, some siderophores have been shown to stabilize the master transcription factor hypoxia inducible factor-1α, and to induce the expression of the pro-inflammatory cytokine interleukin-6. Another newly identified feature of siderophores is their non-redundant effects during infection. Consistent with this idea, strains of Klebsiella pneumoniae carrying the siderophore aerobactin are associated with more invasive disease. Furthermore, during K. pneumoniae lung infection, yersiniabactin promotes pneumonia but does not promote the pathogen's perivascular growth. In contrast, enterobactin augments K. pneumoniae infiltration of the perivascular space in lipocalin-2 deficient mice. In summary, besides being a strategy for nutrient acquisition, siderophores can also help a pathogen to colonize specific niches within the host.

Expansion of the field of nutritional immunity has led to an increased focus on the subcellular destination and function of metals acquired by pathogens during infection. With regard to iron, a significant percentage of the metal inside the bacterial cell is in the form of iron–sulfur clusters. Iron–sulfur clusters are important co-factors of critical cellular processes including respiratory and photosynthetic electron transport. Miller and Auerbuch (C5MT00012B) review existing literature describing the role of bacterial iron–sulfur clusters in mammalian pathogens. The authors provide a description of the host factors involved in sequestration of iron during infection before focusing on bacterial regulatory proteins that contain iron–sulfur clusters; these proteins include the [4Fe–4S]-containing proteins FNR, Wbl, and aconitase, as well as the [2Fe–2S]-containing bacterial proteins IscR, NsrR, SoxR, and AirSR. Taken together, this timely review provides an excellent summary of the importance of Fe–S cluster coordinating regulators in bacterial pathogenesis.

Fungal pathogens are a significant and underappreciated cause of human disease and in a perspective authored by Duncan Wilson (C4MT00331D), the evolution of zinc uptake systems in fungal pathogens is discussed. The author compares and contrast zinc importers in distinct fungi, with an emphasis on the genetic events that lead to the acquisition or loss of these systems. This is followed by a short discussion on the regulatory factors that control zinc importers and, finally, the state of knowledge on the conservation of fungal-secreted zinc binding proteins, known as “zincophores”, is provided. The perspective ends with an emphasis on the role of fungal zinc acquisition systems in pathogenesis, setting the stage for a research article by D'Orazio et al. (C5MT00017C), in which the authors investigate the role of the ZnuABC zinc transport system in Pseudomonas aeruginosa pathogenicity. The authors determine that efficient zinc acquisition is critical for the expression of various virulence features typical of P. aeruginosa, and that ZnuABC also plays an important role in zinc homeostasis in this microorganism. Together, these two articles highlight the increasing appreciation for the importance of microbial zinc acquisition on the outcome of the host–pathogen interaction.

The explosion in the body of literature in the area of nutritional immunity has been expanded to include host-directed metal intoxication strategies that inhibit microbial growth. In particular, copper intoxication is now appreciated as a major defense strategy against microbial pathogens. The antimicrobial properties of copper have been known for millennia, however, as pointed out by Ladomersky and Petris (C4MT00327F), the realization that copper is mobilized by the innate immune system is a fairly recent discovery. They also discuss how it has become appreciated that bacterial pathogens encode numerous copper tolerance genes that help protect them against this innate defense. The authors subdivide these bacterial copper resistance strategies into systems involved in transmembrane copper export, copper sequestration factors, and multi-copper-oxidases. They provide an excellent review of the current knowledge regarding these systems, and the mechanisms by which they enable bacterial survival in the presence of toxic levels of copper. Their review concludes with an update regarding the regulation of copper tolerance genes, and the contribution of these systems to virulence in numerous bacterial pathogens. This conclusion sets the stage for a focused review by Shi and Darwin (C4MT00305E) on copper homeostasis in Mycobacterium tuberculosis, which is a very significant threat to global public health. In this review, the authors discuss the observation that dietary copper supplementation leads to increased copper in M. tuberculosis granulomas and reduced bacterial load at these sites of infection. M. tuberculosis has three pathways that combat this copper intoxication, including copper-responsive copper efflux systems, a bacterial encoded metallothionein, and a copper-responsive multi-copper oxidase. The observation that inactivation of most of these systems alone does not lead to attenuation of M. tuberculosis in murine models of infection highlights the importance of encoding redundant systems to protect the pathogen against copper overload.

As the field of nutritional immunity continues to mature, new technologies are being applied to investigations focused on the struggle for metal between host and pathogen. In this regard, Niemiec et al. (C4MT00346B) present an exciting research article describing their application of a combination of SR-XRF and ICP-MS to define the trace element properties of human neutrophils. Neutrophils were chosen for this analysis based on their critical role as innate immune cells that protect against microbial invaders, and their considerable armamentarium of antimicrobial proteins that work through metal chelation. The authors investigated both neutrophil supernatants after NET formation as well as neutrophil whole cell lysates for total metal content. The combination of SR-XRF, ICP-MS, and live cell imaging enabled the analysis of trace elements in human neutrophils down to nanoscopic levels. This approach has considerable applications in the field of nutritional immunity, and this work provides an excellent road map for any investigator interested in adapting this integrated technique to their own research programs.

In summary, this themed issue provides a snapshot of the current state of the field of nutritional immunity and microbial countermeasures, and emphasises the tremendous amount of activity in this area. As with any active area of research, the more we learn about these processes, the more it becomes clear how much more there is to know.

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