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Protein glycosylation systems are widely recognized in bacteria, including members of the genus

This chapter discusses only the aspects of type IV piliation that are specific for N. gonorrhoeae. In particular, it reviews pilus-associated phenotypes, the regulation of pilin synthesis, pilus assembly, and pilus phase and antigenic variation. The major function of all bacterial pili is to express adhesive proteins away from the cell surface in order to mediate adherence to specific receptors. While piliation has been correlated with transformation competence, no direct role for pili in transformation has been discovered, and the molecular basis for the correlation remains unclear. There are some problems with the idea that PilA and PilB form a two-component system that regulates gonococcal pilin synthesis. The derived amino acid sequences of these proteins display little homology with those of known regulators and sensors. Using another approach, putative gonococcal pilus assembly genes have been identified by virtue of their DNA homology with the previously identified type IV pilus assembly genes of P.

Glycosylation of multiple proteins via O-linkage is well documented in bacterial species of Neisseria of import to human disease. Recent studies of protein glycosylation (pgl) gene distribution established that related protein glycosylation systems occur throughout the genus including nonpathogenic species. However, there are inconsistencies between pgl gene status and observed glycan structures. One of these relates to the widespread distribution of pglG, encoding a glycosyltransferase that in Neisseria elongata subsp. glycolytica is responsible for the addition of di-N-acetyl glucuronic acid at the third position of a tetrasaccharide. Despite pglG residing in strains of N. gonorrhoeae, N. meningitidis and N. lactamica, no glycan structures have been correlated with its presence in these backgrounds. Moreover, PglG function in N. elongata subsp. glycolytica minimally requires UDP-glucuronic acid (GlcNAcA), and yet N. gonorrhoeae, N. meningitidis and N. lactamica lack pglJ, the gene whose product is essential for UDP-GlcNAcA synthesis. We examined the functionality of pglG alleles from species spanning the Neisseria genus by genetic complementation in N. elongata subsp. glycolytica. The results indicate that select pglG alleles from N. meningitidis and N. lactamica are associated with incorporation of an N-acetyl-hexosamine at the third position and reveal the potential for an expanded glycan repertoire in those species. Similar experiments using pglG from N. gonorrhoeae failed to find any evidence of function suggesting that those alleles are missense pseudogenes. Taken together, the results are emblematic of how allelic polymorphisms can shape bacterial glycosyltransferase function and demonstrate that such alterations may be constrained to distinct phylogenetic lineages.

The gonococcal adenine methylases modA and modB, belonging to separate Type III restriction modification systems, are phase variable and could thus enable rapid adaptation to changing environments. However, the frequency of phase variation across transmission chains and the phenotypic impact of phase variation are largely unknown.Here we show that the repeat tracts enabling phase variation expand and contract at high rates in both modA and modB. For modB, multiple ON/OFF transition events were identified over the course of a single outbreak.A mixed effects model using population samples from Norway and a global meta-analysis collection indicates that modB in the OFF state is predictive of moderately decreased ceftriaxone susceptibility. Our findings suggest that modB orchestration of genome-wide 6-methyladenine modification controls the expression of genes modulating ceftriaxone susceptibility.ImportanceDespite significant progress, our current understanding of the genetic basis of antibiotic susceptibility remains incomplete. The gonococcal methylase modB is phase variable, meaning that it can be switched ON or OFF via contraction or expansion of a repeat tract in the gene during replication. We find that transitions between the ON and OFF state occur at high frequency. Furthermore, isolates harbouring modB in a configuration predicted to be inactive had decreased susceptibility to ceftriaxone, an antibiotic used to treat gonorrhea. This finding improves understanding of the genetic underpinnings of antibiotic resistance, but further work is needed to elucidate the mechanics and broader phenotypic effects of epigenetic modifications and transcription.

Bacterial type 4 pili (T4P) are extracellular polymers that initiate the formation of microcolonies and biofilms. T4P continuously elongate and retract. These pilus dynamics crucially affect the local order, shape, and fluidity of microcolonies. The major pilin subunit of the T4P bears multiple post-translational modifications. By interfering with different steps of the pilin glycosylation and phosphoform modification pathways, we investigated the effect of pilin post-translational modification on the shape and dynamics of microcolonies formed by Neisseria gonorrhoeae. Deleting the phosphotransferase responsible for phosphoethanolamine modification at residue serine 68 inhibits shape relaxations of microcolonies after perturbation and causes bacteria carrying the phosphoform modification to segregate to the surface of mixed colonies. We relate these mesoscopic phenotypes to increased attractive forces generated by T4P between cells. Moreover, by deleting genes responsible for the pilin glycan structure, we show that the number of saccharides attached at residue serine 63 affects the ratio between surface tension and viscosity and cause sorting between bacteria carrying different pilin glycoforms. We conclude that different pilin post-translational modifications moderately affect the attractive forces between bacteria but have severe effects on the material properties of microcolonies.

Bacterial protein glycosylation (BPG) comes in all variations of form and manifestation: N-versus O-linked, dedicated versus broad-spectrum, sequential versus en bloc glycan addition, cytoplasmic versus extracytoplasmic. Here, I evaluate and address recent advances in the field of O-linked BPG focusing on selected systems of notoriety and in which significant advances have occurred of late.

The genus Neisseria includes three major species of importance to human health and disease ( Neisseria gonorrhoeae , Neisseria meningitidis , and Neisseria lactamica ) that express broad-spectrum O -linked protein glycosylation (Pgl) systems. The potential for related Pgl systems in other species in the genus, however, remains to be determined. Using a strain of Neisseria elongata subsp. glycolytica , a unique tetrasaccharide glycoform consisting of di- N -acetylbacillosamine and glucose as the first two sugars followed by a rare sugar whose mass spectrometric fragmentation profile was most consistent with di- N -acetyl hexuronic acid and a N -acetylhexosamine at the nonreducing end has been identified. Based on established mechanisms for UDP-di- N -acetyl hexuronic acid biosynthesis found in other microbes, we searched for genes encoding related pathway components in the N. elongata subsp. glycolytica genome. Here, we detail the identification of such genes and the ensuing glycosylation phenotypes engendered by their inactivation. While the findings extend the conservative nature of microbial UDP-di- N -acetyl hexuronic acid biosynthesis, mutant glycosylation phenotypes reveal unique, relaxed specificities of the glycosyltransferases and oligosaccharyltransferases to incorporate pathway intermediate UDP-sugars into mature glycoforms. IMPORTANCE Broad-spectrum protein glycosylation (Pgl) systems are well recognized in bacteria and archaea. Knowledge of how these systems relate structurally, biochemically, and evolutionarily to one another and to others associated with microbial surface glycoconjugate expression is still incomplete. Here, we detail reverse genetic efforts toward characterization of protein glycosylation mutants of N. elongata subsp. glycolytica that define the biosynthesis of a conserved but relatively rare UDP-sugar precursor. The results show both a significant degree of intra- and transkingdom conservation in the utilization of UDP-di- N -acetyl-glucuronic acid and singular properties related to the relaxed specificities of the N. elongata subsp. glycolytica system

Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model,Streptococcus sanguinis. In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology inS. sanguinis. We found that the two major pilins, PilE1 and PilE2, (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments; (ii) display only one of the post-translational modifications frequently found in pilins,i.e. a methylated N-terminus; (iii) are found in the same hetero-polymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved by NMR, revealed a classical pilin fold with a highly unusual flexible C-terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff thanbona fideTfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show thatS. sanguinisTfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines.

Bacterial type 4 pili (T4P) are extracellular polymers that initiate the formation of microcolonies and biofilms. T4P continuously elongate and retract. These pilus dynamics crucially affects the local order, shape, and fluidity of microcolonies. The major pilin subunit of the T4P bears multiple post-translational modifications. By interfering with different steps of the pilin glycosylation and phosphoform modification pathways, we investigated the effect of pilin post-translational modification on the shape and dynamics of microcolonies formed by Neisseria gonorrhoeae. Deleting the phosphotransferase responsible for phosphoethanolamine modification at residue serine 68 (S68) inhibits shape relaxations of microcolonies after pertubation and causes bacteria carrying the phosphoform modification to segregate to the surface of mixed colonies. We relate these mesoscopic phenotypes to increased attractive forces generated by T4P between cells. Moreover, by deleting genes responsible for the pilin glycan structure, we show that the number of saccharides attached at residue serine 63 (S63) affect the ratio between surface tension and viscosity and cause sorting between bacteria carrying different pilin glycoforms. We conclude that different pilin post-translational modifications moderately affect the attractive forces between bacteria but have severe effects on the materials properties of microcolonies.

We describe a novel genome integration system that enables the introduction of DNA fragments as large as 50 kbp into the chromosomes of recipient bacteria. This system, named BPI, comprises a bacterial artificial chromosome vector and phage-derived gene integration machinery. We introduced the wbm locus of Bordetella bronchiseptica, which is required for O antigen biosynthesis, into the chromosome of B. pertussis, which intrinsically lacks O antigen, using the BPI system. After the introduction of the wbm locus, B. pertussis presented an additional substance in the lipooligosaccharide fraction that was specifically recognized by the anti-B. bronchiseptica antibody but not the anti-B. pertussis antibody, indicating that B. pertussis expressed O antigen corresponding to that of B. bronchiseptica. O antigen-expressing B. pertussis was less sensitive to the bactericidal effects of serum and polymyxin B than the isogenic parental strain. In addition, an in vivo competitive infection assay showed that O antigen-expressing B. pertussis dominantly colonized the mouse respiratory tract over the parental strain. These results indicate that the BPI system provides a means to alter the phenotypes of bacteria by introducing large exogenous DNA fragments. IMPORTANCE Some bacterial phenotypes emerge through the cooperative functions of a number of genes residing within a large genetic locus. To transfer the phenotype of one bacterium to another, a means to introduce the large genetic locus into the recipient bacterium is needed. Therefore, we developed a novel system by combining the advantages of a bacterial artificial chromosome vector and phage-derived gene integration machinery. In this study, we succeeded for the first time in introducing a gene locus involved in O antigen biosynthesis of Bordetella bronchiseptica into the chromosome of B. pertussis, which intrinsically lacks O antigen, and using this system we analyzed phenotypic alterations in the resultant mutant strain of B. pertussis. The present results demonstrate that this system successfully accomplished the above-described purpose. We consider this system to be applicable to a number of bacteria other than Bordetella.

Summary The genus Neisseria contains two pathogenic species (N. meningitidis and N. gonorrhoeae) in addition to a number of commensal species that primarily colonize mucosal surfaces in man. Within the genus, there is considerable diversity and apparent redundancy in the components involved in respiration. Here, we identify a unique c-type cytochrome (cN) that is broadly distributed among commensal Neisseria, but absent in the pathogenic species. Specifically, cN supports nitrite reduction in N. gonorrhoeae strains lacking the cytochromes c5 and CcoP established to be critical to NirK nitrite reductase activity. The c-type cytochrome domain of cN shares high sequence identity with those localized c-terminally in c5 and CcoP and all three domains were shown to donate electrons directly to NirK. Thus, we identify three distinct but paralogous proteins that donate electrons to NirK. We also demonstrate functionality for a N. weaverii NirK variant with a C-terminal c-type heme extension. Taken together, modular domain distribution and gene rearrangement events related to these respiratory electron carriers within Neisseria are concordant with major transitions in the macroevolutionary history of the genus. This work emphasizes the importance of denitrification as a selectable trait that may influence speciation and adaptive diversification within this largely host-restricted bacterial genus.

Salmonella enterica remains a leading foodborne bacterial pathogen in the United States; infected livestock serve often as the source of contaminated food products. A study estimated that over a billion Salmonella gastroenteritis cases and up to 33 million typhoid cases occur annually worldwide, with 3.5 million deaths. Although many Salmonella strains with a broad host range present preferential associations with certain host species, it is not clear what determines the various levels of host adaptation. Here, causal properties of host associations were determined with allelic variants of three colonization factors of S. enterica serovar Newport, a most frequent zoonotic serovar. This is the first study that related not only individual but also a small group of host-associated gene variants with functional properties that cooperate to determine the level of host-adapted virulence. The detected associations should help to identify sources of Salmonella infections in both humans and animals., Allelic combinations and host specificities for three fimbrial adhesins, FimH, BcfD, and StfH, were compared for 262 strains of Salmonella enterica serovar Newport, a frequent human and livestock pathogen. Like FimH, BcfD had two major alleles (designated A and B), whereas StfH had two allelic groups, each with two alleles (subgroup A1 and A2 and subgroup B1 and B2). The most prevalent combinations of FimH/BcfD/StfH alleles in S. Newport were A/A/A1 and B/B/B1. The former set was most frequently found in bovine and porcine strains, whereas the latter combination was most frequently found in environmental and human isolates. Bacteria genetically engineered to express Fim, Bcf, or Stf fimbriae on their surface were tested with the different alleles for binding to human, porcine, and bovine intestinal epithelial cells. The major allelic combinations with bovine and porcine strains (A/A/A1) or with human isolates (B/B/B1) provided at least two alleles capable of binding significantly better than the other alleles to an intestinal epithelial cell line from the respective host(s). However, each combination of alleles kept at least one allele mediating binding to an intestinal epithelial cell from another host. These findings indicated that allelic variation in multiple adhesins of S. Newport contributes to bacterial adaptation to certain preferential hosts without losing the capacity to maintain a broad host range. IMPORTANCE Salmonella enterica remains a leading foodborne bacterial pathogen in the United States; infected livestock serve often as the source of contaminated food products. A study estimated that over a billion Salmonella gastroenteritis cases and up to 33 million typhoid cases occur annually worldwide, with 3.5 million deaths. Although many Salmonella strains with a broad host range present preferential associations with certain host species, it is not clear what determines the various levels of host adaptation. Here, causal properties of host associations were determined with allelic variants of three colonization factors of S. enterica serovar Newport, a most frequent zoonotic serovar. This is the first study that related not only individual but also a small group of host-associated gene variants with functional properties that cooperate to determine the level of host-adapted virulence. The detected associations should help to identify sources of Salmonella infections in both humans and animals.

Long-held to be a post-translational modification unique to Eukarya, it is now clear that both Bacteria and Archaea also perform protein glycosylation, namely the covalent attachment of mono- to polysaccharides to specific protein targets. At the same time, many of the roles assigned to this protein-processing event in eukaryotes, such as guiding protein folding/quality control, intracellular trafficking, dictating cellular recognition events and others, do not apply or are even irrelevant to prokaryotes. As such, protein glycosylation must serve novel functions in Bacteria and Archaea. Recent efforts have begun to elucidate some of these prokaryote-specific roles, which are addressed in this review.

O-acetylation is a common modification of bacterial glycoconjugates. By modifying oligosaccharide structure and chemistry, O-acetylation has important consequences for biotic and abiotic recognition events and thus bacterial fitness in general. Previous studies of the broad-spectrum O-linked protein glycosylation in pathogenic Neisseria species (including N. gonorrhoeae and N. meningitidis) have revealed O-acetylation of some of their diverse glycoforms and identified the committed acetylase, PglI. Herein, we extend these observations by using mass spectrometry to examine a complete set of all glycan variants identified to date. Regardless of composition, all glycoforms and all sugars in the oligosaccharide are subject to acetylation in a PglI-dependent fashion with the only exception of di-N-acetyl-bacillosamine. Moreover, multiple sugars in a single oligosaccharide could be simultaneously modified. Interestingly, O-acetylation status was found to be correlated with altered chain lengths of oligosaccharides expressed in otherwise isogenic backgrounds. Models for how this unprecedented phenomenon might arise are discussed with some having potentially important implications for the membrane topology of glycan O-acetylation. Together, the findings provide better insight into how O-acetylation can both directly and indirectly govern glycoform structure and diversity.

Summary Glycans manifest in conjunction with the broad spectrum O-linked protein glycosylation in species within the genus Neisseria display intra- and interstrain diversity. Variability in glycan structure and antigenicity are attributable to differences in the content and expression status of glycan synthesis genes. Given the high degree of standing allelic polymorphisms in these genes, the level of glycan diversity may exceed that currently defined. Here, we identify unique protein-associated disaccharide glycoforms that carry N-acetylglucosamine (GlcNAc) at their non-reducing end. This altered structure was correlated with allelic variants of pglH whose product was previously demonstrated to be responsible for the expression of glucose (Glc)-containing disaccharides. Allele comparisons and site-specific mutagenesis showed that the presence of a single residue, alanine at position 303 in place of a glutamine, was sufficient for GlcNAc versus Glc incorporation. Phylogenetic analyses revealed that GlcNAc-containing disaccharides may be widely distributed within the pgl systems of Neisseria particularly in strains of N. meningitidis. Although analogous minimal structural alterations in glycosyltransferases have been documented in association with lipopolysaccharide and capsular polysaccharide variability, this appears to be the first example in which such changes have been implicated in glycan diversification within a bacterial protein glycosylation system.

Bacterial protein glycosylation systems from varying species have been functionally reconstituted in Escherichia coli. Both N- and O-linked glycosylation pathways, in which the glycans are first assembled onto lipid carriers and subsequently transferred to acceptor proteins by an oligosaccharyltransferase (OTase), have been documented in bacteria. The identification and characterization of novel OTases with different properties may provide new tools for engineering glycoproteins of biotechnological interest. In the case of OTases involved in O-glycosylation (O-OTases), there is very low sequence homology between those from different bacterial species. The Wzy_C signature domain common to these enzymes is also present in WaaL ligases; enzymes involved in lipopolysaccharide biosynthesis. Therefore, the identification of O-OTases using solely bioinformatic methods is problematic. The hypothetical proteins BTH_I0650 from Burkholderia thailandensis E264 and VC0393 from Vibrio cholerae N16961 contain the Wzy_C domain. In this work, we demonstrate that both proteins have O-OTase activity and renamed them PglL(Bt) and PglL(Vc), respectively, similar to the Neisseria meningitidis counterpart (PglL(Nm)). In E. coli, PglL(Bt) and PglL(Vc) display relaxed glycan and protein specificity. However, effective glycosylation depends upon a specific combination of the protein acceptor, glycan and O-OTase analyzed. This knowledge has important implications in the design of glycoconjugates and provides novel tools for use in glycoengineering applications. The codification of enzymatically active O-OTase in the genomes of members of the Vibrio and Burkholderia genera suggests the presence of still unknown O-glycoproteins in these organisms, which might have a role in bacterial physiology or pathogenesis.

The zwitterionic phospho-form moieties phosphoethanolamine (PE) and phosphocholine (PC) are important components of bacterial membranes and cell surfaces. The major type IV pilus subunit protein of Neisseria gonorrhoeae , PilE, undergoes posttranslational modifications with these moieties via the activity of the pilin phospho-form transferase PptA. A number of observations relating to colocalization of phospho-form and O -linked glycan attachment sites in PilE suggested that these modifications might be either functionally or mechanistically linked or interact directly or indirectly. Moreover, it was unknown whether the phenomenon of phospho-form modification was solely dedicated to PilE or if other neisserial protein targets might exist. In light of these concerns, we screened for evidence of phospho-form modification on other membrane glycoproteins targeted by the broad-spectrum O -linked glycosylation system. In this way, two periplasmic lipoproteins, NGO1043 and NGO1237, were identified as substrates for PE addition. As seen previously for PilE, sites of PE modifications were clustered with those of glycan attachment. In the case of NGO1043, evidence for at least six serine phospho-form attachment sites was found, and further analyses revealed that at least two of these serines were also attachment sites for glycan. Finally, mutations altering glycosylation status led to the presence of pptA -dependent PC modifications on both proteins. Together, these results reinforce the associations established in PilE and provide evidence for dynamic interplay between phospho-form modification and O -linked glycosylation. The observations also suggest that phospho-form modifications likely contribute biologically at both intracellular and extracellular levels.

Broad-spectrum O -linked protein glycosylation is well characterized in the major Neisseria species of importance to human health and disease. Within strains of Neisseria gonorrhoeae , N. meningitidis , and N. lactamica , protein glycosylation ( pgl ) gene content and the corresponding oligosaccharide structure are fairly well conserved, although intra- and interstrain variability occurs. The status of such systems in distantly related commensal species, however, remains largely unexplored. Using a strain of deeply branching Neisseria elongata subsp. glycolytica , a heretofore unrecognized tetrasaccharide glycoform consisting of di- N -acetylbacillosamine-glucose-di- N -acetyl hexuronic acid- N -acetylhexosamine (diNAcBac-Glc-diNAcHexA-HexNAc) was identified. Directed mutagenesis, mass spectrometric analysis, and glycan serotyping confirmed that the oligosaccharide is an extended version of the diNAcBac-Glc-based structure seen in N. gonorrhoeae and N. meningitidis generated by the successive actions of PglB, PglC, and PglD and glucosyltransferase PglH orthologues. In addition, a null mutation in the orthologue of the broadly conserved but enigmatic pglG gene precluded expression of the extended glycoform, providing the first evidence that its product is a functional glycosyltransferase. Despite clear evidence for a substantial number of glycoprotein substrates, the major pilin subunit of the endogenous type IV pilus was not glycosylated. The latter finding raises obvious questions as to the relative distribution of pilin glycosylation within the genus, how protein glycosylation substrates are selected, and the overall structure-function relationships of broad-spectrum protein glycosylation. Together, the results of this study provide a foundation upon which to assess neisserial O -linked protein glycosylation diversity at the genus level. IMPORTANCE Broad-spectrum protein glycosylation systems are well characterized in the pathogenic Neisseria species N. gonorrhoeae and N. meningitidis . A number of lines of evidence indicate that the glycan components in these systems are subject to diversifying selection and suggest that glycan variation may be driven in the context of glycosylation of the abundant and surface-localized pilin protein PilE, the major subunit of type IV pili. Here, we examined protein glycosylation in a distantly related, nonpathogenic neisserial species, Neisseria elongata subsp. glycolytica . This system has clear similarities to the systems found in pathogenic species but makes novel glycoforms utilizing a glycosyltransferase that is widely conserved at the genus level but whose function until now remained unknown. Remarkably, PilE pilin is not glycosylated in this species, a finding that raises important questions about the evolutionary trajectories and overall structure-function relationships of broad-spectrum protein glycosylation systems in bacteria.

Findings from a number of studies suggest that the PilA pilin proteins may play an important role in the pathogenesis of disease caused by species within the genus Francisella . As such, a thorough understanding of PilA structure and chemistry is warranted. Here, we definitively identified the PglA protein-targeting oligosaccharyltransferase by virtue of its necessity for PilA glycosylation in Francisella tularensis and its sufficiency for PilA glycosylation in Escherichia coli . In addition, we used mass spectrometry to examine PilA affinity purified from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica and demonstrated that the protein undergoes multisite, O -linked glycosylation with a pentasaccharide of the structure HexNac-Hex-Hex-HexNac-HexNac. Further analyses revealed microheterogeneity related to forms of the pentasaccharide carrying unusual moieties linked to the distal sugar via a phosphate bridge. Type A and type B strains of Francisella subspecies thus express an O -linked protein glycosylation system utilizing core biosynthetic and assembly pathways conserved in other members of the proteobacteria. As PglA appears to be highly conserved in Francisella species, O -linked protein glycosylation may be a feature common to members of this genus.

Although protein glycosylation systems are becoming widely recognized in bacteria, little is known about the mechanisms and evolutionary forces shaping glycan composition. Species within the genus Neisseria display remarkable glycoform variability associated with their O -linked protein glycosylation ( pgl ) systems and provide a well developed model system to study these phenomena. By examining the potential influence of two ORFs linked to the core pgl gene locus, we discovered that one of these, previously designated as pglH , encodes a glucosyltransferase that generates unique disaccharide products by using polyprenyl diphosphate-linked monosaccharide substrates. By defining the function of PglH in the glycosylation pathway, we identified a metabolic conflict related to competition for a shared substrate between the opposing glycosyltransferases PglA and PglH. Accordingly, we propose that the presence of a stereotypic, conserved deletion mutation inactivating pglH in strains of Neisseria gonorrhoeae , Neisseria meningitidis , and related commensals, reflects a resolution of this conflict with the consequence of reduced glycan diversity. This model of genetic détente is supported by the characterization of pglH “missense” alleles encoding proteins devoid of activity or reduced in activity such that they cannot exert their effect in the presence of PglA. Thus, glucose-containing glycans appear to be a trait undergoing regression at the genus level. Together, these findings document a role for intrinsic genetic interactions in shaping glycan evolution in protein glycosylation systems.

The O-linked protein glycosylation pathway in Neisseria gonorrhoeae is responsible for the synthesis of a complex oligosaccharide on undecaprenyl diphosphate and subsequent en bloc transfer of the glycan to serine residues of select periplasmic proteins. Protein glycosylation (pgl) genes have been annotated on the basis of bioinformatics and top-down mass spectrometry analysis of protein modifications in pgl-null strains [Aas, F. E., et al. (2007) Mol. Microbiol. 65, 607-624; Vik, A., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 4447-4452], but relatively little biochemical analysis has been performed to date. In this report, we present the expression, purification, and functional characterization of seven Pgl enzymes. Specifically, the enzymes studied are responsible for synthesis of an uncommon uridine diphosphate (UDP)-sugar (PglD, PglC, and PglB-acetyltransferase domain), glycan assembly (PglB-phospho-glycosyltransferase domain, PglA, PglE, and PglH), and final oligosaccharide transfer (PglO). UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-hexose (DATDH), which is the first sugar in glycan biosynthesis, was produced enzymatically, and the stereochemistry was assigned as uridine diphosphate N'-diacetylbacillosamine (UDP-diNAcBac) by nuclear magnetic resonance characterization. In addition, the substrate specificities of the phospho-glycosyltransferase, glycosyltransferases, and oligosaccharyltransferase (OTase) were analyzed in vitro, and in most cases, these enzymes exhibited strong preferences for the native substrates relative to closely related glycans. In particular, PglO, the O-linked OTase, and PglB(Cj), the N-linked OTase from Campylobacter jejuni, preferred the native N. gonorrhoeae and C. jejuni substrates, respectively. This study represents the first comprehensive biochemical characterization of this important O-linked glycosylation pathway and provides the basis for further investigations of these enzymes as antibacterial targets.

Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation ( pgl ) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro . These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution.

Tissue damage predisposes humans to life-threatening disseminating infection by the opportunistic pathogen Pseudomonas aeruginosa. Bacterial adherence to host tissue is a critical first step in this infection process. It is well established that P. aeruginosa attachment to host cells involves type IV pili (TFP), which are retractile surface fibres. The molecular details of attachment and the identity of the bacterial adhesin and host receptor remain controversial. Using a mucosal epithelium model system derived from primary human tissue, we show that the pilus-associated protein PilY1 is required for bacterial adherence. We establish that P. aeruginosa preferentially binds to exposed basolateral host cell surfaces, providing a mechanistic explanation for opportunistic infection of damaged tissue. Further, we demonstrate that invasion and fulminant infection of intact host tissue requires the coordinated and mutually dependent action of multiple bacterial factors, including pilus fibre retraction and the host cell intoxication system, termed type III secretion. Our findings offer new and important insights into the complex interactions between a pathogen and its human host and provide compelling evidence that PilY1 serves as the principal P. aeruginosa adhesin for human tissue and that it specifically recognizes a host receptor localized or enriched on basolateral epithelial cell surfaces.

Three c-type cytochromes were identified in Neisseria meningitidis, based on predictions from genome sequences, that were hypothesized to be involved in electron transport to terminal electron acceptor reductases for oxygen (the cytochrome cbb(3) oxidase) and nitrite (the nitrite reductase, AniA). Mutants were generated by allelic exchange with disrupted copies of the genes encoding these cytochromes and the phenotypes of the resultant mutants analysed. It was found that cytochrome c(5) is required for in vivo nitrite reductase activity, whereas cytochromes c(x) and c(4) are both required for efficient growth using oxygen as an electron acceptor. Mutants in c(x), c(4), and c(x)+c(4) have a decreased capacity to reduce oxygen, but there is a background oxygen-reduction activity, indicating that there may be other routes for electron transfer from the cytochrome bc(1) complex to the cytochrome cbb(3) oxidase, whereas cytochrome c(5) appears to be the sole route of electrons to the nitrite reductase in N. meningitidis. Interestingly, cytochrome c(x) is highly similar to a domain of copper nitrite reductases from various proteobacteria, whereas cytochrome c(5) has high identity with a domain of the cytochrome cbb(3) oxidase of Neisseria gonorrhoeae, yet these two proteins function in oxygen respiration and nitrite respiration, respectively. This highlights a limitation of predicting protein function from similarity to known proteins, i.e. very closely related protein domains in different organisms can have different redox partners.

The PilE pilin subunit protein of Neisseria gonorrhoeae undergoes unique covalent modifications with phosphoethanolamine (PE) and phosphocholine (PC). The p ilin p hospho-form t ransferase A (PptA) protein, required for these modifications, shows sequence relatedness with and architectural similarities to lipopolysaccharide PE transferases. Here, we used regulated expression and mutagenesis as means to better define the relationships between PptA structure and function, as well as to probe the mechanisms by which other factors impact the system. We show here that pptA expression is coupled at the level of transcription to its distal gene, murF , in a division/cell wall gene operon and that PptA can act in a dose-dependent fashion in PilE phospho-form modification. Molecular modeling and site-directed mutagenesis provided the first direct evidence that PptA is a member of the alkaline phosphatase superfamily of metalloenzymes with similar metal-binding sites and conserved structural folds. Through phylogenetic analyses and sequence alignments, these conclusions were extended to include the lipopolysaccharide PE transferases, including members of the disparate Lpt6 subfamily, and the MdoB family of phosphoglycerol transferases. Each of these enzymes thus likely acts as a phospholipid head group transferase whose catalytic mechanism involves a trans -esterification step generating a protein-phospho-form ester intermediate. Coexpression of PptA with PilE in Pseudomonas aeruginosa resulted in high levels of PE modification but was not sufficient for PC modification. This and other findings show that PptA-associated PC modification is governed by as-yet-undefined ancillary factors unique to N. gonorrhoeae .

Neisseria gonorrhoeae expresses an O-linked protein glycosylation pathway that targets PilE, the major pilin subunit protein of the Type IV pilus colonization factor. Efforts to define glycan structure and thus the functions of pilin glycosylation (Pgl) components at the molecular level have been hindered by the lack of sensitive methodologies. Here, we utilized a ‘top-down’ mass spectrometric approach to characterize glycan status using intact pilin protein from isogenic mutants. These structural data enabled us to directly infer the function of six components required for pilin glycosylation and to define the glycan repertoire of strain N400. Additionally, we found that the N. gonorrhoeae pilin glycan is O-acetylated, and identified an enzyme essential for this unique modification. We also identified the N. gonorrhoeae pilin oligosaccharyltransferase using bioinformatics and confirmed its role in pilin glycosylation by directed mutagenesis. Finally, we examined the effects of expressing the PglA glycosyltransferase from the Campylobacter jejuni N-linked glycosylation system that adds N-acetylgalactosamine onto undecaprenylpyrophosphate-linked bacillosamine. The results indicate that the C. jejuni and N. gonorrhoeae pathways can interact in the synthesis of O-linked di- and trisaccharides, and therefore provide the first experimental evidence that biosynthesis of the N. gonorrhoeae pilin glycan involves a lipid-linked oligosaccharide precursor. Together, these findings underpin more detailed studies of pilin glycosylation biology in both N. gonorrhoeae and N. meningitidis, and demonstrate how components of bacterial O- and N-linked pathways can be combined in novel glycoengineering strategies.

The zwitterionic phospho-forms phosphoethanolamine and phosphocholine are recognized as influential and important substituents of pathogen cell surfaces. PilE, the major pilin subunit protein of the type IV pilus (Tfp) colonization factor of Neisseria gonorrhoeae undergoes unique, post-translational modifications with these moieties. These phospho-form modifications have been shown to be O-linked alternately to a specific, conserved serine residue of PilE. However, the enzymes and precursors involved in their addition are unknown, and the full spectrum of PilE post-translational modifications has yet to be defined. Here, an intact protein-based mass spectrometric approach was integrated with bioinformatics and reverse genetics to address these matters. Specifically we show that a protein limited in its distribution to pathogenic Neisseria species and structurally related to enzymes implicated in phosphoethanolamine modification of lipopolysaccharide is necessary for PilE covalent modification with phosphoethanolamine and phosphocholine. These findings strongly suggest that protein phospho-form modification is mechanistically similar to processes underlying analogous modifications of prokaryotic saccharolipid glycans. We also show that PilE undergoes multisite and hierarchical phospho-form modifications and that the stoichiometries of site occupancy can be influenced by PilE primary structure and the abundance of the pilin-like protein PilV. Together, these findings have important implications for the structure and antigenicity of PilE.

Summary Following attachment of Neisseria gonorrhoeae to human epithelial cell lines, the cellular pilus receptor CD46 is shed from the cell and accumulates in the media. In this report, we assess Neisseria -induced alterations in CD46 surface distribution and charac- terize this complement regulatory protein following its release from the infected cell. Within 3 h of attach- ment of gonococci to human epithelial cell lines, CD46 is enriched beneath sites of microcolony adhe- sion. By 6 h post infection, differential ultracentrifu- gation of culture media from ME-180 monolayers resulted in sedimentation of structurally and function- ally intact CD46. Electron microscopy of these 100 000 g pellets revealed 30-200 nm vesicles. These vesicles likely originated from the host cell as they contained additional host cell surface proteins includ- ing CD55 and the epidermal growth factor receptor. Further, these vesicles were visualized by quick- freeze, deep-etch electron microscopy in association with the surface of infected ME-180 cells and with pili of adherent gonococci. Like CD46 shedding, CD46 redistribution and vesicle release were insensitive to colchicine and cytochalasin-D but dependent on expression of the pilus retraction protein PilT. This vesiculation may represent a host cell defence response in which surface proteins that are com- monly exploited by pathogens, such as CD46, are removed from the cell.

Type IV pili (Tfp) play central roles in prokaryotic cell biology and disease pathogenesis. As dynamic filamentous polymers, they undergo rounds of extension and retraction modelled as pilin subunit polymerization and depolymerization events. Currently, the molecular mechanisms and components influencing Tfp dynamics remain poorly understood. Using Neisseria gonorrhoeae as a model system, we show that mutants lacking any one of a set of five proteins sharing structural similarity to the pilus subunit are dramatically reduced in Tfp expression and that these defects are suppressed in the absence of the PilT pilus retraction protein. Thus, these molecules are not canonical assembly factors but rather act as effectors of pilus homeostasis by promoting extension/polymerization events in the presence of PilT. Furthermore, localization studies support the conclusion that these molecules form a Tfp-associated complex and influence levels of PilC, the epithelial cell adhesin, in Tfp-enriched shear fractions. This is the first time that the step at which individual pilin-like proteins impact on Tfp expression has been defined. The findings have important implications for understanding Tfp dynamics and fundamental Tfp structure/function relationships.

Type IV pilus dynamics is important for virulence, motility, and DNA transfer in a wide variety of prokaryotes. The type IV pilus system constitutes a very robust and powerful molecular machine that transports pilus polymers as well as DNA through the bacterial cell envelope. In Neisseria gonorrhoeae , pilus retraction is a highly irreversible process that depends on PilT, an AAA ATPase family member. However, when levels of PilT are reduced, the application of high external forces ( F = 110 ± 10 pN) induces processive pilus elongation. At forces of >50 pN, single pili elongate at a rate of v = 350 ± 50 nm/s. For forces of

Although natural genetic transformation is a widely disseminated form of genetic exchange in prokaryotic species, the proficiencies with which DNA recognition, uptake and processing occur in nature vary greatly. However, the molecular factors and interactions underlying intra- and interspecies diversity in levels of competence for natural genetic transformation are poorly understood. In Neisseria gonorrhoeae, the Gram-negative aetiologic agent of gonorrhoea, DNA binding and uptake involve components required for Type IV pilus (Tfp) biogenesis as well as those which are structurally related to Tfp biogenesis components but dispensable for organelle expression. We demonstrate here that the gonococcal PilV protein, structurally related to Tfp pilin subunits, is an intrinsic inhibitor of natural genetic transformation which acts ultimately by reducing the levels of sequence-specific DNA uptake into the cell. Specifically, we show that DNA uptake is enhanced in strains bearing pilV mutations and reduced in strains overexpressing PilV. Furthermore, we show that PilV exerts its effect by acting as an antagonist of ComP, a positive effector of sequence-specific DNA binding. As it prevents the accumulation of ComP at a site where it can be purified by shear extraction of intact cells, the data are most consistent with PilV either obstructing ComP trafficking or altering ComP stability. In addition, we report that ComP and PilV play overlapping and partially redundant roles in Tfp biogenesis and document other genetic interactions between comP and pilV together with the pilE and pilT genes required for the expression of retractile Tfp. Together, the results reveal a novel mechanism by which the levels of competence are governed in prokaryotic species and suggest unique ways by which competence might be modulated.

The mechanisms by which DNA is taken up into the bacterial cell during natural genetic transformation are poorly understood. Although related components essential to the uptake of DNA during transformation have been defined in Gram-negative species, it remains unclear whether DNA binding and uptake are dissociable events. Therefore, DNA uptake has been the earliest definable step in any Gram-negative transformation pathway. In the human pathogen Neisseria gonorrhoeae, sequence-specific DNA uptake requires an intact type IV pili (Tfp) biogenesis machinery along with three molecules that are dispensable for Tfp expression: ComP (a pilin subunit-like molecule), PilT (a cytoplasmic protein involved in pilus retraction) and ComE (a periplasmic protein with intrinsic DNA-binding activity). By conditionally altering the levels of ComP and PilT expression, we show here that DNA binding and uptake are resolvable events. Consequently, we are able to demonstrate that PilT is largely dispensable for functional DNA binding and, therefore, contributes specifically to uptake. Furthermore, sequence specificity in this system is imposed at the level of DNA binding, a process that is influenced by both ComP and PilE. However, sequence-specific DNA binding is not attributable to an intrinsic property of the Tfp subunit protein. Finally, we demonstrate the existence of a robust, non-specific DNA-binding activity associated with the expression of both Tfp and PilT, which is unrelated to transformation but obscures the observation of specific binding events.

Expression of Type IV pili (Tfp), multifunctional surface appendages expressed by Gram-negative species of medical and environmental significance, has previously been shown to be regulated by consensus two-component systems. Elucidation of their unique biogenesis pathway and the dynamics of pilus growth and retraction involved in motility have revealed a novel step at which regulation might be imposed. Studies of Tfp expression following adherence to host tissue clearly demonstrate regulation by modulation of the retraction process. In addition, a large set of components related to flagellar chemosensory pathways has been shown to influence Tfp expression levels in many species. Like their flagellar counterparts, the Tfp-dedicated homologues are proposed to function by regulating motor function. Rather than dictating the switch frequencies of organelle rotation, however, they are hypothesized to control the rates of fiber extrusion and retraction.

Glycans manifest in conjunction with the broad spectrum O-linked protein glycosylation in species within the genus Neisseria display intra- and interstrain diversity. Variability in glycan structure and antigenicity are attributable to differences in the content and expression status of glycan synthesis genes. Given the high degree of standing allelic polymorphisms in these genes, the level of glycan diversity may exceed that currently defined. Here, we identify unique protein-associated disaccharide glycoforms that carry N-acetylglucosamine (GlcNAc) at their non-reducing end. This altered structure was correlated with allelic variants of pglH whose product was previously demonstrated to be responsible for the expression of glucose (Glc)-containing disaccharides. Allele comparisons and site-specific mutagenesis showed that the presence of a single residue, alanine at position 303 in place of a glutamine, was sufficient for GlcNAc versus Glc incorporation. Phylogenetic analyses revealed that GlcNAc-containing disaccharides may be widely distributed within the pgl systems of Neisseria particularly in strains of N. meningitidis. Although analogous minimal structural alterations in glycosyltransferases have been documented in association with lipopolysaccharide and capsular polysaccharide variability, this appears to be the first example in which such changes have been implicated in glycan diversification within a bacterial protein glycosylation system.

The alteration of gene expression by DNA rearrangement appears to be an essential part of most biological systems. When an extremely high level of diversification for a set of gene products with a common function is advantageous, these rearrangements are achieved by the reassortment and recombination of repeated gene segments. The generation of the variable domains in avian immunoglobulins, antigenic variation of surface proteins in African trypanosomes and Borrelia hermsii, and pilus antigenic variation in Neisseria gonorrhoeae each results from the transfer of genetic information from nonexpressed donor alleles or pseudogenes to an active expression locus, a process likened to gene conversion occurring in spore-forming fungi. In this chapter, the author concentrates primarily on recent studies that have influenced understanding of variable pilus expression in gonococci. The vast majority of recombination events between double-stranded DNA forms of the filamentous bacteriophage f1 appear to be a consequence of gene conversion, and the mechanism appears to be the formation of asymmetric heteroduplex molecules. It may also seem that reciprocal exchange and gene conversion demand fundamentally distinct recombination reactions, but mechanistically, these two outcomes may simply be alternative resolutions of the same basic event. The identification of equivalent gonococcal genes by interspecies complementation or by virtue of their DNA homologies and the construction of mutants altered in their expression should make it possible to define their role in the chemistry of the exchange reaction.

Competence for transformation is widespread among bacterial species. In the case of Gram-negative systems, a key step to transformation is the import of DNA across the outer membrane. Although multiple factors are known to affect DNA transport, little is known about the dynamics of DNA import. Here, we characterized the spatio-temporal dynamics of DNA import into the periplasm of Neisseria gonorrhoeae. DNA was imported into the periplasm at random locations around the cell contour. Subsequently, it was recruited at the septum of diplococci at a time scale that increased with DNA length. We found using fluorescent DNA that the periplasm was saturable within minutes with ∼40 kbp DNA. The DNA-binding protein ComE quantitatively governed the carrying capacity of the periplasm in a gene-dosage-dependent fashion. As seen using a fluorescent-tagged derivative protein, ComE was homogeneously distributed in the periplasm in the absence of external DNA. Upon addition of external DNA, ComE was relocalized to form discrete foci colocalized with imported DNA. We conclude that the periplasm can act as a considerable reservoir for imported DNA with ComE governing the amount of DNA stored potentially for transport through the inner membrane., Author Summary Bacterial transformation is the import and inheritable integration of external DNA. As such, it is believed to be a major evolutionary force. A key step is the import of DNA through the outer membrane. Here, we have characterized the spatio-temporal dynamics of DNA during import and residence in the periplasm of the Gram-negative pathogen Neisseria gonorrhoeae. We found that the periplasm can serve as a reservoir for imported DNA that can fill within five minutes by importing DNA from the environment. The amount of imported DNA roughly corresponds to the size of a phage genome. The periplasmic DNA-binding protein ComE is homogeneously distributed in the periplasm in the absence of extracellular DNA. It relocates rapidly to imported DNA when external DNA is added to competent gonococci. As ComE governs the carrying capacity of the periplasm, we propose that it might condense DNA, thus linking DNA uptake to its compaction. Although the import through the outer membrane was localized all around the cell contour, the major part of the imported DNA relocated to the septum at the center of diplococci. Our findings strongly support the idea that the periplasm masses DNA independently of transport through the inner membrane.

Summary The ability of bacteria to establish complex communities on surfaces is believed to require both bacterial‐substratum and bacterial‐bacterial interactions, and type IV pili appear to play a critical but incompletely defined role in both these processes. Using the human pathogen Neisseria gonorrhoeae, spontaneous mutants defective in bacterial selfaggregative behaviour but quantitatively unaltered in pilus fibre expression were isolated by a unique selective scheme. The mutants, carrying single amino acid substitutions within the conserved aminoterminal domain of the pilus fibre subunit, were reduced in the ability to adhere to a human epithelial cell line. Co-expression of the altered alleles in the context of a wild-type pilE gene confirmed that they were dominant negative with respect to aggregation and human cell adherence. Strains expressing two copies of the altered alleles produced twice as much purifiable pili but retained the aggregative and adherence defects. Finally, the defects in aggregative behaviour and adherence of each of the mutants were suppressed by a loss-of-function mutation in the twitching motility gene pilT. The correlations between self-aggregation and the net capacity of the microbial population to adhere efficiently demonstrates the potential significance of bacterial cell ‐c ell interactions to colonization.

Neisseria gonorrhoeae employs diverse strategies with which to adhere to and invade host cells during the course of infection. These primary encounters provide means by which biologically active molecules can be efficiently targeted to disrupt or exploit normal host cell metabolism and immune response elements, which in turn leads to the pathological responses characteristic of gonococcal disease. Current studies have begun to elucidate in detail the molecular interactions orchestrating these processes and the signaling events that they provoke.

Type IV pili (Tfp) are a unique class of multifunctional surface organelles in Gram-negative bacteria, which play important roles in prokaryotic cell biology. Although components of the Tfp biogenesis machinery have been characterized, it is not clear how they function or interact. Using Neisseria gonorrhoeae as a model system, we report here that organelle biogenesis can be resolved into two discrete steps: fiber formation and translocation of the fiber to the cell surface. This conclusion is based on the capturing of an intermediate state in which the organelle is retained within the cell owing to the simultaneous absence of the secretin family member and biogenesis component PilQ and the twitching motility/pilus retraction protein PilT. This finding is the first demonstration of a specific translocation defect associated with loss of secretin function, and additionally confirms the role of PilT as a conditional antagonist of stable pilus fiber formation. These findings have important implications for Tfp structure and function and are pertinent to other membrane translocation systems that utilize a highly related set of components.

The expression of type IV pili (Tfp) by Neisseria gonorrhoeae has been shown to be essential for natural genetic transformation at the level of sequence-specific uptake of DNA. All previously characterized mutants defective in this step of transformation either lack Tfp or are altered in the expression of Tfp-associated properties, such as twitching motility, autoagglutination and the ability to bind to human epithelial cells. To examine the basis for this relationship, we identified potential genes encoding polypeptides sharing structural similarities to PilE, the Tfp subunit, within the N. gonorrhoeae genome sequence database. We found that disruption of one such gene, designated comP (for competence-associated prepilin), leads to a severe defect in the capacity to take up DNA in a sequence-specific manner, but does not alter Tfp biogenesis or expression of the Tfp-associated properties of auto-agglutination, twitching motility and human epithelial cell adherence. Indirect evidence based on immunodetection suggests that ComP is expressed at very low levels relative to that of PilE. The process of DNA uptake in gonococci, therefore, is now known to require the expression of at least three distinct components: Tfp, the recently identified PilT protein and ComP.

Understanding the structural biology of type IV pili, fibres responsible for the virulent attachment and motility of numerous bacterial pathogens, requires a detailed understanding of the three-dimensional structure and chemistry of the constituent pilin subunit. X-ray crystallographic refinement of Neisseria gonorrhoeae pilin against diffraction data to 2.6 A resolution, coupled with mass spectrometry of peptide fragments, reveals phosphoserine at residue 68. Phosphoserine is exposed on the surface of the modelled type IV pilus at the interface of neighbouring pilin molecules. The site-specific mutation of serine 68 to alanine showed that the loss of the phosphorylation alters the morphology of fibres examined by electron microscopy without a notable effect on adhesion, transformation, piliation or twitching motility. The structural and chemical characterization of protein phosphoserine in type IV pilin subunits is an important indication that this modification, key to numerous regulatory aspects of eukaryotic cell biology, exists in the virulence factor proteins of bacterial pathogens. These O-linked phosphate modifications, unusual in prokaryotes, thus merit study for possible roles in pilus biogenesis and modulation of pilin chemistry for optimal in vivo function.