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Microbiology and Molecular Biology Reviews, March 2004, p. 132-153, Vol. 68, No. 1
1092-2172/04/$08.00+0 DOI: 10.1128/MMBR.68.1.132-153.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Laboratory of Sialobiology and Microbial Metabolomics, Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802
SUMMARY INTRODUCTION SIALIC ACID STRUCTURES EVOLUTION OF SIALIC ACID METABOLISM SIALIC ACID CATABOLISM Discovery of Sialic Acid Catabolism Diversity of nan Systems Relationships between NanAEK Proteins Sialic Acid Uptake by NanT Role of Sialidases in Sialic Acid Catabolism SIALIC ACID SIGNALING Regulation of nan Expression NanR as a Global Regulator Sialic Acid as a Regulator of Type 1 Fimbrial Phase Variation DECORATING THE CELL SURFACE WITH SIALIC ACID De Novo Synthesis Donor Scavenging trans-Sialidase Precursor Scavenging REGULATION OF CELL SURFACE SIALYLATION Regulation of PSA Capsule Expression in E. coli K1 Meningococcal PSA Phase Variation and LOS Sialylation Regulation of Sialylated LOS Glycoforms in H. influenzae Synthesis of PSA Chemotypes True Bacterial Protein Glycosylation CONCLUSIONS AND FUTURE DIRECTIONS ADDENDUM IN PROOF ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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The whimsically hyperbolic quote above, credited to Eric Sixmister, captures the excitement that many different researchers have experienced over the past 40 years while working on a class of tertiary gene products with manifold function in both eukaryotic and prokaryotic species (151). Sialic acid (less commonly called neuraminic acid) is the designation given to a family of over 40 naturally occurring nine-carbon keto sugars acids derived from the parent compound 2-keto-3-deoxy-5-acetamido-D-glycero-D-galacto-nonulosonic acid (N-acetylneuraminic acid [Neu5Ac]). The sialic acids and related nonulosonates are unique in nature by representing the only nine-carbon sugars found in prokaryotes. In eukaryotes, sialic acids have evolved to mediate a diverse range of cell-cell and cell-molecule interactions, including (i) stabilizing glycoconjugates and cell membranes due to charge-charge repulsion, (ii) mediating cell-cell regulation and acting as chemical messengers, (iii) regulating transmembrane receptor function, (iv) affecting membrane transport, (v) controlling the half-lives of circulating glycoproteins and cells, and (vi) contributing to the permselectivity of the glomerular endothelium and slit diaphragm (118). The relative biological importance of these processes to complex metazoan animals is demonstrated by early embryonic death of homozygous mouse mutants with a defect in sialic acid synthesis (123). Some bacteria have evolved a de novo pathway for sialic acid biosynthesis that differs from the eukaryotic method, whereas other microbes use truncated synthetic pathways utilizing sialyl precursors scavenged from animal hosts (148). Many commensal and pathogenic bacteria also use environmental (host) sialic acids as sources of carbon, nitrogen, energy, and amino sugars for cell wall synthesis (98). Microbial sialic acid metabolism has now been firmly established as a virulence determinant in a range of infectious diseases. In this review we argue that unraveling the molecular mechanisms of sialometabolism and its regulation provides a unifying theme for investigating the host-pathogen interactions in a wide range of invasive (extraintestinal) infectious diseases.
The name sialic acid comes from the Greek word sialon (saliva), consistent with the discovery of these carbohydrates in bovine submaxillary mucin and brain matter (neuroamine) by the German biochemists Blix in 1936 (21) and Klenk in 1941 (74), respectively. Gottschalk (55) was the first to propose a coherent structure of Neu5Ac and also provided a survey of the early history of sialobiology, as the field has since come to be known (110). The excellent short review by Hans Faillard on the early history of sialic acid research also is highly recommended (46). Any of the more recent monographs or review articles by Roland Schauer and his colleagues should be consulted for the methodological details for working with sialic acids (e.g., 118-120). Here we focus on the metabolism and signaling functions of microbial sialic acids after briefly discussing their structure and evolution.
| SIALIC ACID STRUCTURES |
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The carboxylate group of sialic acids is deprotonated at physiological pH (pKa of 2.6) and confers the net negative charge that dominates the physiochemical properties of the family. Glycoketosidic linkages between sialic acids and sugars such as galactose, GlcNAc, or other sialyl residues (Sia) are synthesized by sialyltransferases linking the C-2 (reducing) donor hydroxyl to an appropriate acceptor hydroxyl group as follows (110): CMP-Sia + HO-acceptor
CMP + Sia-O-acceptor, where CMP-Sia is the obligate sialyl donor for all known sialyltransferases. In solution, free sialic acid exists predominantly in its ß conformation, as shown for Neu5Ac, with the carbohydrate's C-2 hydroxyl positioned axial to the ring (Fig. 1A). This form is in thermodynamic equilibrium with the minor 
isomer (151). Both forms are interconverted by mutarotation as the ring opens to the straight-chain conformation followed by ring closure. The ß configuration of the sialic acid substrate is maintained when coupled (activated) with CTP by CMP-sialic acid synthetase, which transfers the anomeric oxygen of ß-sialic acid to the -phosphate of CTP (3). Chemical reactivity of the acceptor hydroxyl is relatively low; therefore, all carbohydrate donors must be activated by first being coupled to a trinucleotide phosphate with removal of one or two phosphates. Sialic acid (and the octulosonate 2-keto-3-deoxy-D-manno-octulosonic acid [keto-deoxy octonate {KDO}] [Fig. 1F]) synthetases are unique enzymes in carbohydrate chemistry because they eliminate pyrophosphate instead of inorganic phosphate during the coupling reaction. Backside SN2 attack of the donor ß-linked hydroxyl group by an acceptor nonreducing hydroxyl yields -linked sialylated products (Sia-O-acceptor). Thus, all sialyltransferases operate by an inversion-of-configuration mechanism involving an activated CMP-Sia donor substrate and a suitable acceptor molecule. Although sialic acids may be linked to structures composed entirely of sugar (oligosaccharides and some polysaccharides), they are typically part of more complex structures involving lipids (glycolipids) or proteins (glycoproteins), which are collectively referred to as sialoglycoconjugates.
Dehydration (double-bond formation) at the sialic acid reducing end leads to formation of the planar structure shown for N-acetyl-2,3-didehydro-2-deoxyneuraminic acid (dideoxy-Neu5Ac [Neu5Ac2en]) in Fig. 1B. The flattened Neu5Ac2en ring mimics the transition state during hydrolysis of sialoglycoconjugates (Sia-O-acceptors) by glycosylhydrolases designated sialidases (synonymous with neuraminidases). Neu5Ac2en served as the lead compound for synthesis of the first rationally designed sialidase inhibitor, now marketed for clinical use as Relenza (zanamivir), an anti-influenza virus inhibitor that prevents spread of the virus during an infection and thus ameliorates flu symptoms without preventing infection (159). In another proposed use of sialidase inhibitors, a patent has been issued to the U.S. Army Medical Research and Materiel Command for the use of Neu5Ac2en to treat a variety of diseases involving inflammatory cells and mediators, including human immunodeficiency virus infection and AIDS (143a). Interestingly, Neu5Ac2en is formed spontaneously from CMP-Neu5Ac under physiological conditions (116) and as a by-product of sialidase action in vivo (16). Therefore, it seems that nature may have already co-opted Neu5Ac2en as a sialidase inhibitor. It is also tempting to speculate that Neu5Ac2en may be a component of the innate (antibody-independent) immune system, guarding us from certain pathogens that use sialidase for adhesion and cellular invasion. Alternatively (but not mutually exclusively), Neu5Ac2en could be part of an endogenous mammalian sialidase regulatory system, as suggested by one recent study (8).
2-Keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (keto-deoxy neuraminic acid [KDN]), 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic acid (legionaminic acid [Leg5Ac7Am]), and 5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid [Pse5,7Ac]) (Fig. 1C to E, respectively) are sialic acid-like nonulosonates found in eukaryotes and certain, generally nonpathogenic or opportunistic bacterial species such as Legionella pneumophila, Pseudomonas aeruginosa, and Campylobacter jejuni (53, 75, 76, 126, 137, 142). It is speculated that because neither Leg5Ac7Am nor Pse5,7Ac is found in eukaryotes, these sugars would offer no protection to the bacteria, as the host would recognize the carbohydrates as foreign by mounting an effective immune response (7). Angata and Varki (7) further speculated that polymers of Leg5Ac7Am or other sialic acids might serve to protect free-living bacteria from bacteriophage infection by blocking underlying receptors. However, it is difficult to reconcile this idea with the rapid modular evolution of phage genomes (95) and the presence of polysialic acids (PSA) as bona fide virulence factors and receptors for a variety of highly lytic phages (97). In other words, blocking of potential underlying receptors with new receptor species would seem to be an inefficient mechanism for dealing with the problem of phage infection. Thus, evocation of the "Red Queen effect" (145), named after the Red Queen's quote from Alice Through the Looking Glass, "it takes all the running you can do, to keep in the same place [and if] you want to get somewhere else, you must run twice as fast as that," to account for sialate structural diversity as a mechanism to thwart microbial infection (exogenous evolution) (7) may not be applicable for understanding bacterium-phage interactions. We may need to follow another white rabbit in order to understand how the diversity of microbial sialic acid structures has arisen.
Another function of PSA and of microbial polysaccharides in general may be to conserve water. The dehydration steps prior to transmission electron microscopy of encapsulated bacteria produce amorphous blebs composed of collapsed polysaccharides (153). To visualize all but the most copious of capsules (which can be detected by particle exclusion under the light microscope), antipolysaccharide antibodies are necessary to cross-link sugar chains and stabilize the highly hydrated capsule structure (Fig. 2). Thus, bacteria, with their large surface area-to-volume ratios, are especially susceptible to desiccation. Sporulation is obviously one mechanism to combat environmental desert-like conditions, but carrying around a polysaccharide "canteen" might confer a similar survival advantage to encapsulated microorganisms. For example, mucoid strains of Escherichia coli, Acinetobacter calcoacetious, and Erwinia stewartii were found to be significantly more resistant to desiccation than corresponding nonmucoid variants (91).
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In conclusion, we suggest that the exact reason for sialic acid structural diversity in bacteria and their hosts is uncertain. However, the fact that such diversity exists is undeniable, and the number of new structures is expanding as analytical methods continue to improve. Given their usual terminal positions on host cell glycoconjugates, sialic acids are among the most prevalent molecules at the host-microbe interface. The analogy of sialic acid structural evolution seen as an arms race between and within species is thus likely to be an apt one (42).
| EVOLUTION OF SIALIC ACID METABOLISM |
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As described in the previous section on sialic acid structure, the identification of sialic acid-like molecules in a variety of bacterial species indicates that a rich diversity of nonulosonates exists in prokaryotes. The occurrence of sialic acid in certain larval stages during insect development further indicates that the basic biosynthetic machinery was intact prior to the divergence of the Coelomata (112). Adding to this discovery of increased distribution of sialic acids is the bewildering range of unicellular fungi in which sialic acids have been detected, with many apparently producing sialic acid by a de novo biosynthetic pathway (1). Sialic acids still have not been detected in plants or the Archaea, despite evidence of potential biosynthetic genes in the latter species (7). On the basis of a molecular phylogenetic approach, Angata and Varki (7) suggested that the evolution of both sialic acid and the related eight-carbon sugar KDO (Fig. 1F) diverged from enzymes involved in shikimic acid biosynthesis for aromatic amino acid production. In one intriguing scenario, the genes for sialic acid biosynthesis were transformed horizontally to a Coelomata ancestor (scenario 2 in reference 7). This suggestion is consistent with the observation that 20% of the 40 or so genes thought to have undergone horizontal transfer between bacteria and humans (117) are involved in sialic acid metabolism (7). Further analyses of these data provided strong evidence for horizontal transfer between the prokaryotic aldolase gene nanA and its orthologues in humans and other vertebrates, although the direction of this transfer could not be determined (4). These putative vertebrate aldolases strongly clustered with NanA from Yersinia pestis and Vibrio cholerae (4). Further understanding of sialic acid evolution should be aided greatly by investigating the genes and gene products involved in fungal sialic acid metabolism, a research area that currently remains almost completely unexplored.
Regardless of the exact evolutionary details of sialic acid metabolism, the widespread use of these sugars for regulating immunity and the central nervous system (5, 73, 119, 120, 125), two organs unique to deuterostomes, is consistent with the apotheosis of sialic acids in this lineage. In the context of organ system function, it will be interesting to determine what role sialic acids play in insect physiology or development. While the occurrence of sialic acid synthesis has been increasingly detected in prokaryotes and unicellular eukaryotes (see above), many more microorganisms have the metabolic machinery to degrade sialic acids as sources of carbon, nitrogen, and energy and as precursors to cell wall biosynthesis (148). The preponderance of sialic acids in animals also makes the sialic acids attractive as potential signals that could be exploited for microbial environmental (host) sensing. A description of sialic acid catabolism logically follows.
| SIALIC ACID CATABOLISM |
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E. coli K1 synthesizes a homopolymer of sialic acid known as PSA. While working on the genetics of PSA biosynthesis, Vimr and Troy (156, 157) noted that only about 10% of radiolabeled sialic acid added to the growth medium was incorporated into PSA despite quantitative uptake of the label into cells. On the assumption that the bacteria expressed an efficient transporter and degradative system for sialic acid dissimilation, mutants that failed to use sialic acid as the sole carbon source were isolated. The genetic defects in two of these nan (for N-acylneuraminate) mutants were subsequently located in genes for sialate transport (nanT) and the aldolase (nanA) (156). The nanAT genes were part of an operon that responded to apparent induction based on sialic acid availability. Upon completion of the E. coli K-12 genomic DNA sequencing project (19), the nan operon was seen to potentially include, in addition to nanAT, open reading frames yhcHIJ and a putative upstream regulator, yhcK (98). The results of the genetic and physiological studies indicated that exogenous sialic acid is transported by a secondary transporter (NanT) of the major facilitator superfamily and degraded intracellularly by NanA to yield pyruvate and the amino sugar ManNAc (156, 157). The products of yhcI (renamed nanK) and yhcJ (renamed nanE) were suggested to function by first phosphorylating ManNAc and then epimerizing the ManNAc-6-P generated by the kinase reaction to GlcNAc-6-P (98). Recent biochemical analyses confirmed that NanK is an ATP-dependent kinase specific for ManNAc and that NanE is a reversible 2-epimerase (104). The upstream regulatory open reading frame yhcK was renamed nanR and shown to repress the nan operon in the absence of sialic acid (98). The function of yhcH is unknown, and a null mutation in this open reading frame conferred no detectable growth phenotype on sialic acid (70). The GlcNAc-6-P produced by NanE was shown to enter the amino sugar degradative pathway encoded by nagBA (98), converting GlcNAc-6-P ultimately to fructose-6-P by GlcNAc-6-P deacetylase (NagA) and glucosamine-6-P deaminase (NagB). Sialic acid thus can serve as the sole carbon or nitrogen source in E. coli and as a source of amino sugars (GlcNAc and glucosamine) for cell wall biosynthesis (82, 98). NanA-catalyzed degradation of sialic acid yields a triose phosphate (pyruvate) and hexosamine (ManNAc), which makes sialic acid an attractive nutritional source for microbes that associate with vertebrates.
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The nanATEK-yhcH system in E. coli is derepressed over 200-fold by mutations in nanR or during growth on sialic acid as the sole carbon source, suggesting that E. coli may not usually exist in a sialic acid-rich natural environment (98). Tight nan regulation appears to result from tandem binding of two or more NanR homodimers to an operator that overlaps the nanA promoter, thus blocking RNA polymerase binding. Homologues of NanR are present in close relatives of E. coli, such as Salmonella enterica and Shigella spp., but not in any of the other species shown in Fig. 3 (70). However, most of these species may encode their own linked nan regulators (shown for various open reading frames by the magenta color), which is further evidence for the individual evolution of the various nan systems. Unraveling the genetic rationale for such apparent regulatory diversity offers great promise for better understanding the details of a variety of host-bacterium interactions.
In addition to NanAEK and NagBA, a complete nan system also should include a permease of some type for sialic acid uptake. As in the previous case of NanR, we found few obvious orthologues of NanT in the bacterial nan systems shown in Fig. 3. However, the Y. pestis nan system (Fig. 3E) includes an open reading frame (ypo3016) encoding a putative transporter with homology to NanT, while other transporters of this general type were detected in the gram-positive species S. pneumoniae (Fig. 3F), C. perfringens (Fig. 3H), Staphylococcus aureus (Fig. 3J), and L. plantarum (Fig. 3K), suggesting that these organisms rely on a mechanism of sialic acid uptake similar to that of E. coli, namely, a proton symporter or antiporter (secondary transporter of the major facilitator superfamily) (93, 146).
Bacterial transporters can be divided into four major types (a fifth type, not shown, includes group translocation catalyzed by the PTS): facilitated diffusion (Fig. 4A), ATP-binding cassette (ABC) transporters (Fig. 4B), secondary (ion- or proton-coupled) symporters or antiporters (Fig. 4C), and tripartite ATP-independent periplasmic (TRAP) transporters (Fig. 4D). The systems analyzed in Fig. 3 indicate that TRAP transporters are either closely linked or part of the nan operons of H. influenzae (Fig. 3B), Pasteurella multocida (Fig. 3C), V. cholerae (Fig. 3D), and Fusobacterium nucleatum (Fig. 3I). TRAP transporters include a periplasmic binding component that is thought to increase uptake affinity, a membrane-spanning polypeptide analogous to secondary transporters, and a second membrane polypeptide of unknown function (49, 72, 101). The substrates of TRAP transporters appear to be carboxylic acids such as malate, succinate, or fumarate, among which sialic acid may be considered a monocarboxylate (Fig. 1A). In some cases the two membrane TRAP transporter components may be fused into a single polypeptide (72), as in HI0147, pm1708, or fn1473 (Fig. 3B, C, and I, respectively). These TRAP transporter systems invariably include linked genes (shown in bronze-yellow) of unknown function that in at least one case were shown to be dispensable for dicarboxylic acid uptake (49). A bovine isolate of P. multocida that was attenuated for systemic pasteurellosis was isolated with an insertion mutation in pm1709 (50), which is predicted to encode a periplasmic TRAP component. The defect in this strain has been shown to prevent sialic acid transport, thereby providing direct evidence for the proposed function of TRAP transporters in sialic acid uptake (E. R. Vimr, S. M. Steenbergen, R. Caughlan, J. Garfinkle, and T. E. Fuller, unpublished data). To the extent that sialic acid uptake is required for the virulence of diverse pathogenic microorganisms, new therapeutic agents aimed at blocking transport offer a new class of nonantibiotic drugs that could have utility for treating a wide range of human or animal infectious diseases.
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Although the concentration of total sialic acid in humans serum is quite high (ca. 2 mM), almost all of it (99.9%) is bound to proteins or lipid acceptors under normal physiological conditions, making it unavailable to microbial metabolism (128). However, the low free sialic acid concentration in serum may simply reflect the rapid clearance of low-molecular-weight substances by the glomerular filtration barrier of the vertebrate kidney (51). Indeed, the concentration of free sialic acid in certain vertebrate tissues is dramatically greater than that of the bound nonulosonates, suggesting that a given animal may consist of subenvironments containing variable amounts of free sialic acids (6). In the colon of a vertebrate animal existing on a meat-rich diet, the high concentration of sialidase-positive commensals presumably allows sialidase-negative E. coli to successfully compete for free sialic acid (156). In contrast, an organism like V. cholerae colonizing a more rarefied intestinal environment presumably finds it advantageous to excrete its own sialidase for scavenging host sialoglycoconjugates from the small intestinal lumen (151). To compensate for loss of environmental sialic acid by diffusion, V. cholerae may need a high-affinity TRAP transporter to scavenge sialic acids released by its excreted sialidase (Fig. 3D). Similarly, H. influenzae (sialidase negative) and P. multocida (sialidase positive) are obligate commensals of the nasopharynx, an environment where free sialic acid concentrations may be low. A high-affinity TRAP transporter under these growth conditions may be beneficial to persistence. Other potential functions of microbial sialidases unrelated to nutrition, such as modulation of host innate immunity, have been discussed previously (148), as has the potential use of sialidases for interspecies competition between sialidase-positive and sialidase-negative bacteria occupying the same niche (124).
Although, as discussed above, the systemic concentration of free sialic acid is low, localized increases caused by inflammatory cues could create foci of highly concentrated free sialic acids independently of microbial sialidase action. This suggestion follows from observations that inflammatory neutrophils undergo an interleukin-8-inducible recruitment of intracellular sialidase(s) to the cell surface, where release of bound sialic acids from surface molecules and the surfaces of cells in the surrounding environment has the potential to raise local sialic acid concentrations (39). Thus, inflammation triggered by microbial products such as endotoxin (lipopolysaccharide [LPS]) may trigger events resulting in increased free sialic acids during an infection. Effective scavenging of these "endogenous" free sialates may involve the high-affinity uptake provided by TRAP or ABC transporters.
The sialidase gene, nanH, of S. enterica was the first chromosomal gene in this organism to be ascribed an origin by cross-kingdom horizontal gene transfer (63). Although the complete physiological function of nanH in the salmonellae is unknown, the evidence indicated that it was part of an active bacteriophage or phage remnant with homology to the lambdoid group (108). Subsequent studies showed that nanH is part of an intact prophage, Fels-1, that also encodes a novel superoxide dismutase (SodCIII), neither of which seems to be a component of the phage itself or involved in phage maturation (48). However, expression of nanH in S. enterica is high (ca. 2% of the total soluble protein), indicating that it is probably not involved in the prophage life cycle per se, because high expression continues during the lysogenic phase of the host-phage relationship (64). If nanH has no direct function in Fels-1 propagation, its persistent expression must confer some selective advantage to nanH-positive salmonellae. Possible functions include scavenging sialic acids for nutrition during colonization, systemic infection, or, possibly, the intracellular phase of infection and modulation of host cell surfaces after enzyme release following prophage induction. Indeed, enzyme release could be tied to the ordinary phage life cycle, whereby spontaneous prophage induction destroys a portion of the bacterial population but provides a benefit to the remaining population as a whole, perhaps by modifying effector responses of host inflammatory cells or molecules. Note that whereas nanH has been found only in LT2 isolates of S. enterica serovar Typhimurium, it is common (>50%) among S. enterica serovar Arizonae isolates (63).
S. enterica NanH was the first bacterial sialidase to have its structure solved in three dimensions (36), thereby supporting the sialidase superfamily hypothesis identifying the conserved active sites and overall architecture of the sialidase catalytic domain in other bacterial, viral, protozoan, and mammalian sialidases (63, 151). The enzyme is easy to purify in large amounts in its native form and is marketed by several commercial interests. The ease of genetic manipulation offered by S. enterica together with the availability of pure NanH and a three-dimensional structure should make it straightforward to determine its function in the host-bacterium interaction. Although the in vivo function of bacterial sialidase has been a topic of considerable speculation (54, 88, 139, 140), no definitive studies exist. From an evolutionary perspective, it is fascinating to speculate that phages are general mediators of horizontal gene transfer, a process that may be proceeding to this day at levels that are currently unknown but could be high (108). Thus, at the tertiary structural level S. enterica NanH may be more closely related to mammalian sialidases than it is to its bacterial counterparts, which would be consistent with cross-kingdom gene transfer (63). This putative relationship between a bacterial sialidase and a mammalian sialidase is not indicated by simple phylogenetic reconstruction (7), but the primary structures of most sialidases are poorly conserved, thus potentially blurring the true relationships among this group of enzymes.
| SIALIC ACID SIGNALING |
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Evidence that NanR responds to sialic acid binding comes from observations that sialic acid induces the nan operon in an aldolase-deficient mutant which is unable to initiate sialic acid breakdown, thereby preventing accumulation of the potential inducers ManNAc, pyruvate, and ManNAc-6-P, and endogenous induction by sialic acid produced biosynthetically in an E. coli K1 nanA mutant (156, 157). In addition, among a variety of sugars tested as potential inducers, only sialic acid was effective in vivo (70). In vitro evidence was equivocal but suggested that the thermodynamically disfavored -isomer of sialic acid may bind NanR to cause the conformational change associated with derepression (70). Preliminary results suggest that sialic acid influences the equilibrium between NanR homodimers (active) and monomers (inactive) instead of simply perturbing the structure of the intact homodimer.
In addition to the function of NanR in regulating nan expression, the intergenic region between nanR and nanA includes a functional CAP site as noted above, a potential nitrogen-regulated site for binding the nitrogen assimilation control (NAC) protein, and two GATC Dam methylation sites, one overlapping the CAP site and another located immediately 5' to the first tandem GGTATA repeat. Expression of nac is controlled by the alternative sigma factor NtrC, and in turn NAC either represses or activates genes involved in acquisition of nitrogen sources other than the preferred source, ammonia. NAC thus links the regulation of genes under control of the housekeeping sigma factor with NtrC. Operators activated by NAC have the consensus sequence ATA-N6-TNGTAT, while those repressed have a slightly different sequence, ATAA-N8-GAT (65). The potential nan NAC site, ATAAGCTTTCTGTAT, thus most closely resembles an activating operator, ATA-N6-CTGTAT, with one mismatch (underlined). Activation of nan transcription by NAC would make sense physiologically because sialic acid can serve as a sole nitrogen source in E. coli (82), suggesting that the NAC site in nan may be functional. However, microarray transcriptional analysis of NtrC-regulated genes did not provide evidence of nan induction or repression by nitrogen limitation (167).
The presence of overlapping GATC methylation sites is observed in a variety of different CAP sites, indicating that hemimethylation controls gene expression, as shown by microarray analysis of an E. coli dam mutant (92). In that study, nan expression was decreased in the dam mutant background, as expected from the need for positive control exerted by CAP binding (70). The presence of another GATC site located immediately upstream of the first tandem GGTATA repeat of the nan operator could affect NanR binding either positively or negatively. Expression of the nan operon thus is negatively regulated by NanR in response to sialic acid and positively regulated by CAP with hemimethylation functioning as an antiactivator. Additionally, NAC may still regulate nan under an unknown set of conditions, and potential hemimethylation of the GATC site proximal to the nan operator could affect the affinity of NanR binding.
Sialic acid also could act as an environmental signal through its immediate breakdown products, pyruvate and ManNAc. Thus, pyruvate by binding to the GntR-FadR family member PdhR would derepress aceEF (pyruvate dehydrogenase) expression and that of any other genes regulated by PdhR or other pyruvate response elements (100). Indirect regulation by sialic acid breakdown products would be NanR independent and likely to be observed only in cultures containing an active nan system exposed to an exogenous supply of sialic acid. Bacterial responses to sialic acid are therefore expected to be complex and to involve direct regulatory effects mediated by NanR and indirect effects exerted by PdhR and other regulators that may recognize ManNAc, ManNAc-6-P, or further-downstream products of sialic acid dissimilation such as the nagBA products (98). Results of microarray expression profiling indicate that expression of four dozen E. coli genes responds (either up or down) at least fourfold when cells are grown with sialic acid compared to glycerol. The use of cDNA derived from a glycerol-grown culture for these comparisons should exclude CAP as a major source of the observed differences in relative gene expression. Although we expect most of these changes in gene expression to be exerted through sialic acid breakdown products, it is possible that regulators other than NanR bind sialic acid. Even by limiting consideration to E. coli, the physiological responses to environmental sialic acid are proving to be highly complex, involving changes in expression of multiple genes either through the direct action of NanR or through indirect effects of sialic acid-derived metabolism on other regulatory proteins. Given the diversity of nan systems shown in Fig. 3, each organism is likely to maximize its responses to host sialic acid as a mechanism for colonization and persistence in vivo. Our initial results and the results of others (see below) are likely to be of general significance for understanding a range of host-pathogen-commensal interactions involving microbial responses to host-derived sialic acid. As an ubiquitous and frequently terminal sugar unit on most mammalian cell surfaces, sialic acid is one of the first and most prevalent molecules that a microbe would encounter when entering virtually any host subenvironment. The signaling functions of sialic acids in the host-microbe interaction are thus likely to have profound implications for microbial colonization, persistence, and, in certain cases, disease progression.
Communication between the host and resident microbiota mediated by monosaccharides is not unprecedented (62), suggesting a rich lexicon of chemical signals derived from the metabolism of host glycoconjugates. Furthermore, as discussed below, the host need not be a deaf bystander to the communication. Some bacteria scavenge host sialic acids and represent them in various oligosaccharide structural contexts at the cell surface, where host sialic acid-binding proteins can interpret these signals. This type of recognition by the host could ultimately prove inimical to the bacterium (67), but it is equally possible that bacteria can create a type of molecular cognitive dissonance in the host's ability to respond accordingly to the microbe (148).
Deletion of a region located >500 bp upstream from the fimB promoter removes the NanR-like binding site. This region appears to function as an antirepressor of fimB expression as measured by reporter fusion assays and effects of mutations in the region on fimbrial phase variation (45). Type 1 fimbriation is a known virulence factor in uropathogenesis (9, 34), a potential factor in Crohn's disease (23), and both necessary and sufficient for E. coli invasion of epithelial cells (83). El-Labany et al. (45) showed that sialic acid added exogenously to the growth medium reduced fimB expression, perhaps through an effect on hemimethylation of the cis-acting site mediated through one or more DNA-binding proteins. The effect of sialic acid in suppressing off-to-on phase variation of type 1 fimbriation in E. coli occurred at a concentration of sialic acid that is normally present in urine (45), suggesting a physiological role of sialic acid in controlling fimbrial phase variation during infection. Our results indicate that the effect of sialic acid on fimbrial phase variation could be mediated at least in part by NanR (70; Steenbergen et al., unpublished data). However, the control of type 1 fimbrial phase variation appears to involve more than just sialic acid, suggesting a potentially complex mechanism requiring other cis- and trans-acting factors (45).
Although we saw no evidence of changes in fimB expression (or of fimAICDFGH expression) in our microarray analyses (Steenbergen et al., unpublished data), the absolute effect of sialic acid on fimB expression was relatively low (45) and may not have been detected by our methods. In contrast, the effect of sialic acid or nanR deletion on expression of the divergently transcribed yjhATS operon was dramatic, confirming the importance of NanR to its expression. NanR thus appears to act as a classical repressor of nan, functioning in an antirepressor mechanism regulating fimB expression from a distance and as a repressor of yjhATS (and yjhBC) also by acting at a distance. The precise mechanism of NanR binding to its nanA operator and how it regulates promoters hundreds of base pairs from its binding site await investigation. Likely possibilities with precedent in other systems include DNA bending or looping to bring proteins into contact with other regulatory elements, supercoiling effects at downstream sites, or even oligomerization of bound effector molecules through cooperative binding that may mimic certain nucleoprotein structures.
We stress that NanR is found only in E. coli and very closely related bacteria such as S. enterica and Shigella spp. (70). Despite this limited distribution of NanR, potential regulatory molecules abound in the nan systems of less closely related gram-negative and gram-positive bacteria (Fig. 3), suggesting that there may be diverse mechanisms for sensing the host sialic acid concentration and responding accordingly. Many important regulatory phenomena involving sialic acids clearly remain to be elucidated in these organisms. The results of these studies should be of manifold importance to pathogenesis and for understanding the host-microbe interaction.
| DECORATING THE CELL SURFACE WITH SIALIC ACID |
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The synthesis and activation of Leg5Ac7Am and Pse5,7Ac appear to depend on orthologues of NeuABC. L. pneumophila neuA and neuB were shown to complement synthetase- and synthase-defective E. coli K1 mutants, respectively (80), while three separate neuB orthologues from C. jejuni were shown to complement the K1 synthase mutant (71). These complementation results are unusual because the hexosamine precursors of legionaminic and pseudaminic acids would appear to be structurally distinct from ManNAc, suggesting that orthologues of NeuC synthesize the hexosamine precursors, which can then serve as substrates for the NeuA and NeuB homologues. The differences between mammalian and bacterial de novo sialic acid synthesis noted above may reflect the varied uses of sialic acids in microbes for nutrition, for environmental signaling, and as sources of amino sugars for cell wall biosynthesis (98). It will now be interesting, and presumably straightforward, to determine the mechanism(s) of de novo sialic acid synthesis in unicellular fungi, where some studies have correlated the presence of sialic acid with a given strain's relative pathogenicity (1, 105).
Expression of the PSA capsule by E. coli K1 also depends on an intact neuE gene (154). Although the exact function of NeuE is unknown, the polypeptide was shown to include a C-terminal putative polyprenol-binding domain that would anchor NeuE to the inner membrane (133), where one of its functions could be to initiate PSA biosynthesis. However, transmission electron microscopy of thin sections from a neuE null mutant (Fig. 9) indicated intracellular accumulation of PSA, seen as lacunae of the type detected previously in capsule mutants with defects in PSA export (30). This translocation-defective phenotype of a neuE mutant would seem to exclude NeuE as a sialyltransferase, suggesting instead that it may function by coupling PSA synthesis to export. McGowen et al. also found no evidence for the proposed sialyltransferase function of NeuE (85). Alternatively, if the small amount of residual sialyltransferase activity in a neuS (polysialyltransferase) null mutant is carried out by NeuE, then it might function to initiate PSA biosynthesis (perhaps by transferring sialic acid to a lipid acceptor) in a translocation-competent form (133). Therefore, in the absence of NeuE, PSA may be synthesized but fail to engage the export apparatus, accumulating intracellularly as shown in Fig. 9. Zhou and Troy (166) have recently carried out an extensive in vitro study of the polyprenol-binding domain of NeuE,
