The occurrence and biological significance of the α-keto-sugars pseudaminic acid and legionaminic acid within pathogenic bacteria

Abstract

This article describes the occurrence of the acidic sugars based on pseudaminic acid and legionaminic acid within pathogenic Gram-negative bacteria. In addition to presenting the structural variations found in these sugars, this article also discusses the potential biological role of these unusual sugars.

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Matthew Zunk

Matthew Zunk graduated from Griffith University with a Bachelor of Pharmaceutical Science, first class honours in 2008; being awarded academic excellence in 2004, 2006 and 2008 and receiving multiple scholarships for his academic achievements. Currently he is finalising his PhD at the Institute for Glycomics (Griffith University) under the supervision of Dr Milton J. Kiefel. Since 2012, Mr Zunk has held an academic position within the School of Pharmacy at Griffith University, Gold Coast Campus, teaching undergraduate Pharmaceutical Science and Drug Discovery and Development. His main research interests cover synthetic chemistry and the synthesis of bacterial carbohydrates.

Milton J. Kiefel

Milton J. Kiefel completed his PhD in organic chemistry at the University of Melbourne in 1990, before undertaking a postdoctoral position with Professor Gerald Pattenden (University of Nottingham, UK). In 1993 he joined the carbohydrate chemistry group of Prof Mark von Itzstein at Monash University, and in 2000 moved to Griffith University to take up a lecturing position within the Institute for Glycomics. In 2005 he was promoted to Senior Lecturer. His research interests involve the synthesis of novel carbohydrates that can be used as probes to elucidate the function of various carbohydrate-utilising proteins that are related to human diseases.

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Nonulosonic acids

Nonulosonic acids are a structural class of 9-carbon carbohydrate monomers that are α-keto acids. The most abundant naturally occurring nonulosonic acids are the sialic acids (or neuraminic acids) and their derivatives (Fig. 1).^1,2 The sialic acid family is widely distributed throughout nature with over 50 naturally occurring derivatives having been identified to date within all domains of cellular organisms.^1,3 Sialic acids have been found in nearly all mammalian tissues including embryonic tissues, and play an important role in a number of diseases. For example, overexpression of terminal sialylation mechanisms is directly associated with cancer malignancies.⁴ The natural occurrence of sialic acids is not limited to mammalian cells, with this structurally diverse family of compounds having been isolated from invertebrate and non-invertebrate species alike. Sialic acids have been isolated from insects, found in fish eggs, and they are increasingly being discovered within pathogenic and non-pathogenic bacteria and archaeal organisms.^2,5,6 The importance of sialic acids means they have been discussed in detail elsewhere. For a detailed review of the diversity of sialic acids see articles by Varki,^2,5 for an account of the structure, function and metabolism of sialic acids see Traving & Schauer,¹ whilst the structural diversity and phylogenetic relationship of organisms encoding for sialic acids has been reviewed by Angata & Varki.³

Fig. 1 Naturally occurring sialic acids N-acetylneuraminic acid (Neu5Ac (1)) and keto-deoxynonulosonic acid (KDN (2)).

Our increased understanding of the crucial role that sialic acids play in the lifecycle of various organisms has driven the search for new nonulosonic acid derivatives. Through this process an important sub-class of nonulosonic acid derivative has been found that appear to be unique to bacterial species. These recently identified nonulosonic acids are structurally characterised as 5,7-diamino-3,5,7,9-tetradeoxy-nonulosonate derivatives of which two important parent compounds are known, pseudaminic acid (3) and legionaminic acid (4) (Fig. 2).

Fig. 2 Pseudaminic acid (3) and Legionaminic acid (4).

The biosynthesis of all nonulosonic acids has a number of unique evolutionary aspects.^3,5,6 The first is that a nucleotide-activated hexose sugar serves as a precursor to nine carbon α-keto acids. In the case of sialic acid derivatives, this hexose sugar is a D-mannose derivative, whilst compounds such as the pseudaminic and legionaminic acids are synthesised from a nucleotide activated N-acetylglucosamine. Secondly all known nine carbon α-keto acids are synthesised by an enzyme catalysed condensation reaction, whereby a six carbon unit is condensed with a 3 carbon unit (typically a pyruvate derivative) to form the desired nine carbon acidic sugar. Biosynthetically, certain chemical transformations must occur to the nucleotide-activated hexose sugar in order for the appropriate 6 carbon unit to be obtained, since it is the stereochemical and functional group detail that this hexose possesses that directly determines the structural characteristics of the nine carbon nonulosonate that will be generated. Lastly, all nonulosonic acids must be nucleotide activated (usually CMP-activated) in order to be incorporated into the varying structural glycans where they are found.⁷

Structural diversity and occurrence of 5,7-diamino-3,5,7,9-tetradeoxy-nonulosonates

In 1984, Knirel and colleagues characterised a new type of “sialic acid-like” sugar, which was extracted and purified from the O-chain polysaccharide of the LPS of Pseudomonas aeruginosa O7 and O9 and Shigella boydii type 7.⁸ This new compound was identified at the time as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid, which Knirel generically named pseudaminic acid.⁸ Since 1984 there have been a number of naturally occurring pseudaminic acid derivatives discovered, which all have an L-glycero-L-manno configuration but vary in the types of N-acyl substitution they contain at C-5 and C-7. Following from this initial discovery, these derivatives are proving to be wide spread within bacterial glycan structures having been so far identified and isolated from a growing number of bacterial species (Table 1).

Table 1 Structures of naturally occurring 3,5,7,9-tetradeoxy-nonulosonic acids found in pathogenic bacteria

Nonulosonic acid	Bacterial source	Reference
L-glycero-L-manno (pseudaminic acid)
	Escherichia coli O136	62
	Proteus vulgaris O39	63
	Campylobacter jejuni 81-176	13 and 33
	Campylobacter jejuni 11168	19
	Campylobacter coli VC167	34
	Pseudoateromonas atlantica T9	9
	Helicobacter pylori 1061	12 and 33
	Pseudoalteromonas atlantica IAM 14165	64
	Aeromonas caviae UU51	33
	Rhizobium sp. NGR234	65
	Sinorhizobium meliloti Rm1021	66 and 67
	Cellulophaga fucicola	68
	Vibrio vulnificus 27562	69
	Piscirickettsia salmonis	70
	Pseudomonas aeruginosa O10a	8
	Shigella boydii type 7	8, 71 and 72
	Pseudomonas aeruginosa O9a, 9b	21 and 71
	Sinorhizobium fredii HH103	73
	Kribbella spp. VKM	74 and 75
	Actinoplanes utahensis VKM Ac-674	74 and 76
	Pseudomonas aeruginosa O7a, 7b, 7d and O7a, 7d (immunotype 6)	77 and 78
	Pseudoalteromonas distincta KMM 638	79
	Vibrio cholera O:2	80
	Campylobacter jejuni 81-176	13 and 33
	Campylobacter coli VC167	34
R′ = Ac; R′′ = glycerate	Vibrio vulnificus 27562	69
R′ = C(=O)CH2CH(OH)CH2; R′′ = C(=O)H	Pseudomonas aeruginosa O7a, 7b, 7c	78
R′ = R′′ = C(=O)CH(OH)CH2OH	Campylobacter jejuni 81-176	13
R′ = Ac; R′′ = C(=NH)CH3	Campylobacter jejuni 11168	19
R′ = C(=O)CH(OCH3)CH2OCH3; R′′ = C(=NH)CH3	Campylobacter jejuni 11168	19
R′ = C(=O)CH(OCH3)CH2OCH3; R′′ = Ac	Campylobacter jejuni 11168	19
D-glycero-D-galacto (legionaminic acid)
	Vibrio alginolyticus 945-80	22 and 24
	Acinetobacter baumannii O24	22 and 28
	Vibrio parahaemolyticus O2	26 and 27
	Campylobacter jejuni 11168	19
	Escherichia coli O161	29
	Legionella pneumophila serogroup 1	17 and 22
	Pseudomonas fluorescens ATCC 49271	22 and 23
	Vibrio salmonicida NCMB 2262	22 and 25
	Campylobacter coli VC167	20
	Campylobacter jejuni 11168	19
R′ = C(=O)CH2CH(OH)CH3; R′′ = Ac	Acinetobacter baumannii O24	22 and 28
R′ = Ac; R′′ = C(=O)CH(NHAc)CH3	Vibrio parahaemolyticus KX-V212	26
	Escherichia coli O161	29
R′ = CH(NHCH3)CH3; R′′ = Ac	Campylobacter coli VC167	20
	Campylobacter jejuni 11168	19
R′ = C(=O)CH2CH2CH(CO2H)NHCH3; R′′ = Ac	Clostridium botulinum	81
D-glycero-D-talo (4-epi-legionaminic acid)
R′ = Ac	Legionella pneumophila serogroup 1	22 and 39
R′ = Ac	Legionella pneumophila serogroups 3, 4, 5,6,8,9,10,11,12,14, Lansing 3, 16453-92	40
R′ = Ac	Shewanella japonica KMM 3601	41
R′ = C(=NH)CH3	Legionella pneumophila serogroup 2	40
L-glycero-D-galacto (8-epi-legionaminic acid)
	Pseudomonas aeruginosa O12	21
	Providencia stuartii O20	38
	Escherichia coli O108	30
R′ = C(=NH)CH3; R′′ = Ac	Morganella morganii KF 1676 (RK 4222)	36
R′ = Ac; R′′ = C(=NH)CH3	Shewanella putrefaciens A6	37
R′ = Ac; R′′ = C(=O)CH(NHAc)CH3	Escherichia coli O108	31
R′ = C(=O)CH2CH(OH)CH3; R′′ = Ac	Salmonella arizonae O61	32
R′ = C(=O)CH2CH2CH2OH; R′′ = Ac	Yersinia ruckeri O1	35

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Within these bacterial species pseudaminic acid and its derivatives are found as components of important cell surface glycans (oligosaccharides and glycoproteins), such as LPS O-antigens,⁹ capsular polysaccharides,¹⁰ pili¹¹ and flagellin^12,13 all of which are essential for bacterial virulence.^14,15 Interestingly, unlike the sialic acids, pseudaminic acids are found within bacterial glycans as both α and β linked carbohydrates, being less commonly found at the terminal end of glycans. To date most of the glycans that have been characterised contain pseudaminic acid, and its naturally occurring derivatives, as internally linked sugars, often including linkages through the Nitrogen-based functionalities at C-5 and C-7, such as the C-7 linked pseudaminic acid derivative found in Kribbella spp. (Fig. 3).¹⁶

Fig. 3 An example of a teichulosonic acid fragment containing C-4 linked and C-7 nitrogen linked pseudaminic acid residue from Kribbella spp.¹⁶

Legionaminic acid (4) (5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid) was first discovered in 1994 as α-(2,4)-linked homopolymer component of the Legionella pneumophila serotype 1, O-chain LPS.¹⁷ L. pneumophila is the causative agent of Legionnaires' disease, which replicates within the alveolar macrophages within human lung tissues, generally leading to a chronic and often fatal pneumonia.¹⁸ Studies have shown that legionaminic acid appears to be important in the virulence capabilities of L. pneumophila as it is believed to be the LPS of serotype 1 that is the key determinant for the development of the disease.¹⁸ Thus far legionaminic acid and its naturally occurring derivatives have been isolated from other pathogenic species, including Campylobacter,^19,20 Pseudomonas,^21–23 Vibrio,^22,24–27 Acinetobacter,^22,28 Escherichia^29–31 and Salmonella³² (Table 1). Most notably Campylobacter strains which are gastrointestinal pathogens in numerous mammalian species heavily decorate their flagellin via O-linked serine and threonine residues with this important carbohydrate moiety and also utilise pseudaminic acid pathways in the same manner.^13,19,33,34

To date, as with the pseudaminic acids numerous naturally occurring derivatives of legionaminic acid have been identified, possessing various N-substitutions at C-5 and C-7 (Table 1) and numerous linkages to other sugars. Interestingly, there have been two natural structurally distinct isomers of legionaminic acid (4) identified to date.²² The 4-epi (5) and 8-epi (6) isomers of legionaminic acid (Fig. 4) have been fully characterised and isolated from a number of important Gram-negative pathogens (Table 1).²² The 8-epi-legionaminic acid (6) isomer which has an L-glycero-D-galacto configuration has been identified in a number of Gram-negative bacterial strains (Table 1).^{21,30–32,35–38} The 4-epi-legionaminic acid (5) isomer having a D-glycero-D-talo configuration has so far been isolated from L. pneumophila strains,^22,39,40 and only one other species, the marine anaerobic Proteobacteria Shewanella japonica⁴¹ (Table 1).

Fig. 4 The D-glycero-D-talo isomer 4-epi-legionaminic acid (5), and the L-glycero-D-galacto isomer 8-epi-legionaminic acid (6).

More recently Kandiba et al.⁴² have identified a 5-N-formyl derivative of legionaminic acid as a terminal residue within the N-glycosylated VP4-derived (structural protein) pentasaccharide glycan of the haloarchaeal pleomorphic virus (HRPV-1).⁴² This discovery is the first known occurrence of this typically O-linked sugar being isolated from an N-linked glycoprotein and also the first time a legionaminic acid derivative has been identified in an archaeal-derived glycan structure. This group has subsequently analysed the putative nonulosonic acid biosynthetic pathway in Archaea.⁶ This work highlights the ever increasing knowledge that the legionaminic acids are widespread within nature and have important metabolic and virulent capabilities.

Biosynthesis of 5,7-diamino-3,5,7,9-tetradeoxy-nonulosonates

Biosynthetically both pseudaminic acid (3) and legionaminic acid (4) are synthesised from a nucleotide activated N-acetyl-glucosamine. The complete biosynthesis of pseudaminic acid has been characterised within Campylobacter jejuni and Helicobacter pylori (Scheme 1).^20,43–45 Within these bacteria pseudaminic acid is generated from UDP-N-acetylglucosamine (7) in an efficient five step pathway (Scheme 1).^20,43–45 The first step in the pathway is catalysed by PseB an enzyme that has a dual function as a NAD(P)-dependant dehydratase and C-5 epimerase, converting UDP-α-D-GlcNAc (7) to UDP-2-acetamido-2,6-dideoxy-β-D-arabino-hexos-4-ulose, 8.^45–48 Compound 8 is converted by PseC into UDP-4-amino-4,6-dideoxy-β-L-AltNAc 9 which, as can be seen in Scheme 1, rearranges the entire molecule converting 8 from a D-sugar to an L-sugar.^44,45,49 The intermediate 9 is then shuttled through a further two enzyme catalysed reactions, involving PseH and PseG,^50,51 being converted into 2,4-diacetamindo-2,4,6-trideoxy-β-L-altropyranose 10. Compound 10 is the substrate for PseI, the enzyme that converts it into pseudaminic acid (3) via an aldol condensation with phosphoenolpyruvate (PEP) (Scheme 1).^45,46,52 In order to be incorporated into O-antigens, or utilised in O-linked protein glycosylation pathways, pseudaminic acid (3) must be activated as its CMP-linked derivative.^45,46 This reaction is catalysed by PseF, a CMP-pseudaminic acid synthetase, which utilises CTP, producing CMP-pseudaminic acid (11) and pyrophosphate (Scheme 1).^45,46

Scheme 1 Biosynthetic pathways of pseudaminic acid (3) and legionaminic acid (4).

The characterisation of this pathway was made possible largely by the fact that within nature all nonulosonates such as pseudaminic acid (3) and sialic acids are synthesised via enzymes that have a highly conserved phylogenetic ancestry.^3,5 To date genetic studies have shown that all bacteria that encode for nonulosonic acids share a high degree of common genetic homology in regards to the enzymes they produce in order to create the varying 9 carbon sugars they utilise within their life cycles.^3,44

Many of the enzymes involved in the biosynthesis of pseudaminic acid (3) have now been extensively studied and characterised. Interestingly, PseB (first enzyme in the pathway) has been found to be inhibited at a micromolar level by CMP-pseudaminic acid (11) (K_i(app) = 18.7μM). This suggests a possible negative feedback mechanism operating within the pathway controlling the forward direction of the biosynthesis, since even high concentrations of the natural substrate UDP-GlcNAc (7) were unable to competitively override the inhibition by 11.^45,46 PseG the fourth enzyme in the biosynthetic pathway was fully characterised in 2009 (ref. 51) via X-ray crystallography with both the apo-form and with a UDP-product bound to the active site being published at 1.8 Å resolution.⁵¹ This data provided the proof that PseG was indeed a UDP-hydrolase enzyme which belongs to a superfamily of inverting glycosyltransferases known as the metal-independent GT-B family (Family 28).^50,51 However, even though PseG belonged to this glycosyltransferase superfamily, Liu and Tanner⁵⁰ ruled out the possibility that PseG could be the missing CMP-pseudaminyltransferase that would be responsible for incorporating CMP-activated pseudaminic acid (11) into glycan structures.⁵⁰

Synonymously the legionaminic acid biosynthetic pathway, which in the case of C. jejuni is an alternate pathway that operates at the same time as the pseudaminic acid pathway, has also been fully characterised.⁵³ In C. jejuni legionaminic acid (4) is generated from a nucleotide activated N-acetylglucosamine derivative, although the starting molecule is GDP-rather than UDP-N-acetylglucosamine (Scheme 1).⁵³ The first step in the biosynthesis of legionaminic acid (4) is catalysed by LegB an NAD-dependent 4,6-dehydratase, which converts GDP-N-acetyl-glucosamine (12) to GDP-2-acetamido-2,6-dideoxy-α-D-xylo-hexos-4-ulose (13).⁵³ LegC a PLP-dependent aminotransferase then catalysis the next step converting 13 into GDP-4-amino-4,6-dideoxy-α-D-GlcNAc, which is further processed by an N-acetyltransferase (LegH) to give GDP-2,4-diacetamido-2,4,6-trideoxy-α-D-glucopyranose (14). Intermediate 14 is then converted into 2,4-diacetamido-2,4,6-trideoxy-D-mannopyranose (15) via the catalysis of an enzyme known as LegG, which acts as an NDP-sugar hydrolase and 2-epimerase.⁵³ Compound 15 is a 6-deoxy-mannose derivative or D-rhamnose sugar which is then transformed into legionaminic acid (4) by condensation with a 3-carbon unit (PEP) by the enzyme LegI a legionaminic acid synthase (Scheme 1).⁵³ As within all nonulosonate biosynthetic pathways the resulting 9 carbon α-keto sugar must be nucleotide activated before being able to be incorporated into glycans.^3,44 In the case of legionaminic acid it is CMP-activated by the enzyme LegF a CMP-legionaminic acid synthetase producing CMP-legionaminic acid (16) (Scheme 1).⁵³

Remarkably, despite a detailed knowledge of the role of key enzymes in the biosynthesis of nucleotide-activated CMP-pseudaminic acid (11) and CMP-legionaminic acid (16), along with the isolation and characterisation of numerous naturally occurring derivatives (Table 1), the isolation and characterization of possible glycosyltransferase(s) responsible for the incorporation of these CMP-linked nonulosonates into glycan structures remains elusive. However, recently it has been reported that the glycosyltransferase responsible for the transfer of CMP-activated pseudaminic acid onto the flagellin of Aeromonas caviae has been found.⁵⁴ Whilst these authors did not show direct evidence that the motility-associated factor maf gene provides the enzyme responsible for adding pseudaminic acid onto the flagellin, their studies provide strong evidence that maf1 is indeed a pseudaminyl transferase.⁵⁴ This is the first report describing the identification of a probable pseudaminyl transferase, and will perhaps provide important information for further studies aimed at identifying this glycosyltransferase enzyme in other bacteria.

The task of finding all enzymes involved in the biosynthesis and incorporation of these nonulosonic acids into bacterial glycans is clearly an area of interest. Shoenhofen et al.⁴⁵ remarked that after the full characterisation of the pseudaminic acid biosynthetic pathway, the entire pathway could now be screened for inhibitors in vitro, rather than just screening specific enzymes. This screening could potentially be accomplished by combining all five of the biosynthetic enzymes, UDP-GlcNAc (7), and potential inhibitors, thus screening for phosphate release, which only occurs during the last biosynthetic step.⁴⁵

Biological significance of 5,7-diamino-3,5,7,9-tetradeoxy-nonulosonates in Gram-negative bacteria

Opportunistic bacterial species achieve their pathogenicity via virulence factors which are generally proteins or molecules that are associated with key structural components of the bacterium.^13,55 Whilst the outer membrane is the major component contributing to the virulence of all pathogenic Gram-negative bacteria, individual bacterial species also contain other components that contribute to their virulence.¹⁴ Structures such as flagellin and pili provide a bacterium motility, which aids in colonisation, invasive and evasive mechanisms.^13,56 Motility is a key factor in the adaptation and virulence capacity of many bacterial pathogens that colonize mucosal surfaces. For example the gastroenteritis causing Campylobacter and Helicobacter species demonstrate highly efficient flagellin motility under conditions of elevated viscosity such as that found in the gastric lumen.¹³ Studies have shown that although flagellin are a major structural determinate for bacterial virulence, it is in fact the glycosylated ends of these flagellin that facilitate bacterial host interactions and therefore largely confer immune responses.^12,13

Within Campylobacter and Helicobacter strains the carbohydrate modifications of their flagellin glycans are largely based on pseudaminic acid (3) and legionaminic acid (4).^12,13,16 The major modifications identified on flagellin from C. jejuni are O-linked pseudaminic acid, or changes to the N-acyl groups (see examples in Table 1). Structural investigations of flagellin from isogenic mutants of Campylobacter and Helicobacter strains^{13,16,34,57–59} have provided sufficient proof that pseudaminic acid (3) is an essential glycosylation molecule involved in flagellin assembly and function, and therefore these carbohydrates have an important role in the pathogenicity of these bacteria.^34,56 Indeed knock out mutants of key pseudaminic acid biosynthetic genes in several strains of Campylobacter are non-motile and accumulate intracellular flagellin subunits of reduced molecular mass, which relates to the bacterium becoming non-viable pathogens.^34,59 Analysis of the flagellin from these knock out mutants by mass spectroscopy confirmed the lack of any glycosylation, providing specific proof of a functional role for the glycosylation process in flagellin assembly.^34,59 Flagellin glycosylation with pseudaminic acid in Aeromonas hydrophila AH-3 has also been shown to be essential for motility.⁶⁰

Further studies have shown that pseudaminic acid (3) is a potential substrate and signalling molecule for many proteins essential for flagellin assembly not only in C. jejuni but also H. pylori, with nonulosonates accounting for up to 10% of the total mass of flagellin glycoprotein in certain strains.^{13,16,34,57–59} Flagellin from C. jejuni and H. pylori strains have been shown to contain 19 amino acids (Ser/Thr residues) that are O-linked to the monosaccharides pseudaminic acid (3) and legionaminic acid (4) and several of their analogues, dependent on the strain.^{13,16,34,57–59} Discoveries such as this have produced great interest, since directly O-linked pseudaminic acid was the first report of an acidic monosaccharide being linked to a protein within a bacterium.⁶¹

Although the exact biological function of both pseudaminic acid (3) and legionaminic acid (4) and their naturally occurring derivatives is not fully understood,^34,54,56 it is known that the presence of these post-translational glycolytic modifications of flagellin subunits is absolutely necessary for the assembly of a functional flagellin. Since motility is required for colonisation, the presence of these nonulosonates can be considered a virulence factor in these bacteria.^{13,16,34,57–59} It has also been noted that due to the structural similarity of these bacterial nonulosonic acids to that of eukaryote sialic acids, this may contribute to bacterial virulence by dampening the immune response to invading bacterium that utilise these important carbohydrates.^9,16 Furthermore the relative structural similarities these sugars (especially the legionaminic acids) have with the sialic acids may also enable a potential interaction with host sialic acid-specific lectins, which could explain their possible influence in adhesion and infection.⁵³

Conclusions

To date, the exact biological significance of pseudaminic and legionaminic acid along with their naturally occurring derivatives remains to be fully determined. However, the presence of these complex sugars as components of cell surface-associated glycans in numerous clinically important pathogenic Gram-negative bacteria suggests a potential role in host–bacterial interactions and therefore virulence. The increasing discovery of these carbohydrates within important bacterial structural elements demonstrates that these unique higher sugars are more common to bacteria than previously believed. Moreover, at present due to the importance of their role in the pathogenic capabilities of many clinically important drug-resistant Gram-negative bacteria, there is an opportunity for exploring possible new therapeutic targets that focus on proteins or enzymes associated with the biosynthesis or incorporation of these complex sugars into bacterial glycans. It is expected that the coming decade will see a significant increase in research activity on this area, especially if new improved synthetic strategies emerge that allow the efficient synthesis of pseudaminic acid or legionaminic acid analogues.

Notes and references

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Footnote

† Current address: School of Pharmacy, Griffith University Gold Coast, Parklands Drive, Southport, Queensland, 4222, Australia. E-mail: E-mail: m.zunk@griffith.edu.au, Fax: +61 7 5552 8804, Tel: +61 7 5552 8344

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