An atypical lipoteichoic acid from Clostridium perfringens elicits a broadly cross-reactive and protective immune response.

Clostridium perfringens is a leading cause of food-poisoning and causes avian necrotic enteritis, posing a significant problem to both the poultry industry and human health. No effective vaccine against C. perfringens is currently available. Using an antiserum screen of mutants generated from a C. perfringens transposon-mutant library, here we identified an immunoreactive antigen that was lost in a putative glycosyltransferase mutant, suggesting that this antigen is likely a glycoconjugate. Following injection of formalin-fixed whole cells of C. perfringens HN13 (a laboratory strain) and JGS4143 (chicken isolate) intramuscularly into chickens, the HN13-derived antiserum was cross-reactive in immunoblots with all tested 32 field isolates, whereas only 5 of 32 isolates were recognized by JGS4143-derived antiserum. The immunoreactive antigens from both HN13 and JGS4143 were isolated, and structural analysis by MALDI-TOF-MS, GC-MS, and 2D NMR revealed that both were atypical lipoteichoic acids (LTAs) with poly-(ß1→4)-ManNAc backbones substituted with phosphoethanolamine. However, although the ManNAc residues in JGS4143 LTA were phosphoethanolamine-modified, a few of these residues were instead modified with phosphoglycerol in the HN13 LTA. The JGS4143 LTA also had a terminal ribose and ManNAc instead of ManN in the core region, suggesting that these differences may contribute to the broadly cross-reactive response elicited by HN13. In a passive-protection chicken experiment, oral challenge with C. perfringens JGS4143 lead to 22% survival, whereas co-gavage with JGS4143 and α-HN13 antiserum resulted in 89% survival. This serum also induced bacterial killing in opsonophagocytosis assays, suggesting that HN13 LTA is an attractive target for future vaccine-development studies.

Bacterial AB5 toxins inhibit the growth of gut bacteria by targeting ganglioside-like glycoconjugates.

The AB5 toxins cholera toxin (CT) from Vibrio cholerae and heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli are notorious for their roles in diarrheal disease, but their effect on other intestinal bacteria remains unexplored. Another foodborne pathogen, Campylobacter jejuni, can mimic the GM1 ganglioside receptor of CT and LT. Here we demonstrate that the toxin B-subunits (CTB and LTB) inhibit C. jejuni growth by binding to GM1-mimicking lipooligosaccharides and increasing permeability of the cell membrane. Furthermore, incubation of CTB or LTB with a C. jejuni isolate capable of altering its lipooligosaccharide structure selects for variants lacking the GM1 mimic. Examining the chicken GI tract with immunofluorescence microscopy demonstrates that GM1 reactive structures are abundant on epithelial cells and commensal bacteria, further emphasizing the relevance of this mimicry. Exposure of chickens to CTB or LTB causes shifts in the gut microbial composition, providing evidence for new toxin functions in bacterial gut competition.

Phase Variable Expression of a Single Phage Receptor in Campylobacter jejuni NCTC12662 Influences Sensitivity Toward Several Diverse CPS-Dependent Phages.

Campylobacter jejuni NCTC12662 is sensitive to infection by many Campylobacter bacteriophages. Here we used this strain to investigate the molecular mechanism behind phage resistance development when exposed to a single phage and demonstrate how phase variable expression of one surface component influences phage sensitivity against many diverse C. jejuni phages. When C. jejuni NCTC12662 was exposed to phage F207 overnight, 25% of the bacterial cells were able to grow on a lawn of phage F207, suggesting that resistance develops at a high frequency. One resistant variant, 12662R, was further characterized and shown to be an adsorption mutant. Plaque assays using our large phage collection showed that seven out of 36 diverse capsular polysaccharide (CPS)-dependent phages could not infect 12662R, whereas the remaining phages formed plaques on 12662R with reduced efficiencies. Analysis of the CPS composition of 12662R by high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) showed a diminished signal for O-methyl phosphoramidate (MeOPN), a phase variable modification of the CPS. This suggested that the majority of the 12662R population did not express this phase variable modification in the CPS, indicating that MeOPN serves as a phage receptor in NCTC12662. Whole genome analysis of 12662R showed a switch in the length of the phase variable homopolymeric G tract of gene 06810, encoding a putative MeOPN-transferase located in the CPS locus, resulting in a non-functional protein. To confirm the role of 06810 in phage resistance development of NCTC12662, a 06810 knockout mutant in NCTC12662 was constructed and analyzed by HR-MAS NMR demonstrating the absence of MeOPN in the CPS of the mutant. Plaque assays using NCTC12662Δ06810 demonstrated that seven of our CPS-dependent Campylobacter phages are dependent on the presence of MeOPN for successful infection of C. jejuni, whereas the remaining 29 phages infect independently of MeOPN, although with reduced efficiencies. Our data indicate that CPS-dependent phages uses diverse mechanisms for their initial interaction with their C. jejuni host.

Discovery of a Glutamine Kinase Required for the Biosynthesis of the O-Methyl Phosphoramidate Modifications Found in the Capsular Polysaccharides of Campylobacter jejuni.

Bacterial capsular polysaccharides (CPS) are complex carbohydrate structures that play a role in the overall fitness of the organism. Campylobacter jejuni, known for being a major cause of bacterial gastroenteritis worldwide, produces a CPS with a unique O-methyl phosphoramidate (MeOPN) modification on specific sugar residues. The formation of P-N bonds in nature is relatively rare, and the pathway for the assembly of the phosphoramidate moiety in the CPS of C. jejuni is unknown. In this investigation we discovered that the initial transformation in the biosynthetic pathway for the MeOPN modification of the CPS involves the direct phosphorylation of the amide nitrogen of l-glutamine with ATP by the catalytic activity of Cj1418. The other two products are AMP and inorganic phosphate. The l-glutamine-phosphate product was characterized using 31P NMR spectroscopy and mass spectrometry. We suggest that this newly discovered enzyme be named l-glutamine kinase.

Experimental Evaluation of Host Adaptation of Lactobacillus reuteri to Different Vertebrate Species.

The species Lactobacillus reuteri has diversified into host-specific lineages, implying a long-term association with different vertebrates. Strains from rodent lineages show specific adaptations to mice, but the processes underlying the evolution of L. reuteri in other hosts remain unknown. We administered three standardized inocula composed of strains from different host-confined lineages to mice, pigs, chickens, and humans. The ecological performance of each strain in the gastrointestinal tract of each host was determined by typing random colonies recovered from fecal samples collected over five consecutive days postadministration. Results revealed that rodent strains were predominant in mice, confirming previous findings of host adaptation. In chickens, poultry strains of the lineage VI (poultry VI) and human isolates from the same lineage (human VI) were recovered at the highest and second highest rates, respectively. Interestingly, human VI strains were virtually undetected in human feces. These findings, together with ancestral state reconstructions, indicate poultry VI and human VI strains share an evolutionary history with chickens. Genomic analysis revealed that poultry VI strains possess a large and variable accessory genome, whereas human VI strains display low genetic diversity and possess genes encoding antibiotic resistance and capsular polysaccharide synthesis, which might have allowed temporal colonization of humans. Experiments in pigs and humans did not provide evidence of host adaptation of L. reuteri to these hosts. Overall, our findings demonstrate host adaptation of L. reuteri to rodents and chickens, supporting a joint evolution of this bacterial species with several vertebrate hosts, although questions remain about its natural history in humans and pigs.IMPORTANCE Gut microbes are often hypothesized to have coevolved with their vertebrate hosts. However, the evidence is sparse and the evolutionary mechanisms have not been identified. We developed and applied an experimental approach to determine host adaptation of L. reuteri to different hosts. Our findings confirmed adaptation to rodents and provided evidence of adaptation to poultry, suggesting that L. reuteri evolved via natural selection in different hosts. By complementing phylogenetic analyses with experimental evidence, this study provides novel information about the mechanisms driving host-microbe coevolution with vertebrates and serve as a basis to inform the application of L. reuteri as a probiotic for different host species.

Biological roles of the O-methyl phosphoramidate capsule modification in Campylobacter jejuni.

Campylobacter jejuni is a major cause of bacterial gastroenteritis worldwide, and the capsular polysaccharide (CPS) of this organism is required for persistence and disease. C. jejuni produces over 47 different capsular structures, including a unique O-methyl phosphoramidate (MeOPN) modification present on most C. jejuni isolates. Although the MeOPN structure is rare in nature it has structural similarity to some synthetic pesticides. In this study, we have demonstrated, by whole genome comparisons and high resolution magic angle spinning NMR, that MeOPN modifications are common to several Campylobacter species. Using MeOPN biosynthesis and transferase mutants generated in C. jejuni strain 81-176, we observed that loss of MeOPN from the cell surface correlated with increased invasion of Caco-2 epithelial cells and reduced resistance to killing by human serum. In C. jejuni, the observed serum mediated killing was determined to result primarily from activation of the classical complement pathway. The C. jejuni MeOPN transferase mutant showed similar levels of colonization relative to the wild-type in chickens, but showed a five-fold drop in colonization when co-infected with the wild-type in piglets. In Galleria mellonella waxmoth larvae, the MeOPN transferase mutant was able to kill the insects at wild-type levels. Furthermore, injection of the larvae with MeOPN-linked monosaccharides or CPS purified from the wild-type strain did not result in larval killing, indicating that MeOPN does not have inherent insecticidal activity.

Investigating the potential of conserved inner core oligosaccharide regions of Moraxella catarrhalis lipopolysaccharide as vaccine antigens: accessibility and functional activity of monoclonal antibodies and glycoconjugate derived sera.

We investigated the conservation and antibody accessibility of inner core epitopes of Moraxella catarrhalis lipopolysaccharide (LPS) in order to assess their potential as vaccine candidates. Two LPS mutants, a single mutant designated lgt2 and a double mutant termed lgt2/lgt4, elaborating truncated inner core structures were generated in order to preclude expression of host-like outer core structures and to create an inner core structure that was shared by all three serotypes A, B and C of M. catarrhalis. Murine monoclonal antibodies (mAbs), designated MC2-1 and MC2-10 were obtained by immunising mice with the lgt2 mutant of M. catarrhalis serotype A strain. We showed that mAb MC2-1 can bind to the core LPS of wild-type (wt) serotype A, B and C organisms and concluded that mAb MC2-1 defines an immunogenic inner core epitope of M. catarrhalis LPS. We were unsuccessful in obtaining mAbs to the lgt2/lgt4 mutant. MAb MC2-10 only recognised the lgt2 mutant and the wt serotype A strain, and exhibited a strong requirement for the terminal N-acetyl-glucosamine residue of the lgt2 mutant core oligosaccharide, suggesting that this residue was immunodominant. Subsequently, we showed that both mAbs MC2-1 and MC2-10 could facilitate bactericidal killing of the lgt2 mutant, however neither mAb could facilitate bactericidal killing of the wt serotype A strain. We then confirmed and extended the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against M. catarrhalis wt strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes three conjugation strategies that either uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule, or a strong base treatment to remove all fatty acids from the LPS, thus creating amino functionalities in the lipid A region to conjugate via maleimide-thiol linker strategies targeting the carboxyl residues of the carrier protein and the free amino functionalities of the derived lipid A region of the carbohydrate resulted in a high loading of carbohydrates per carrier protein from these carbohydrate preparations. Immunisation derived antisera from rabbits recognised fully extended M. catarrhalis LPS and whole cells. Moreover, bactericidal activity was demonstrated to both the immunising carbohydrate antigen and importantly to wt cells, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by M. catarrhalis.

Functional characterization of Lpt3 and Lpt6, the inner-core lipooligosaccharide phosphoethanolamine transferases from Neisseria meningitidis.

The lipooligosaccharide (LOS) of Neisseria meningitidis contains heptose (Hep) residues that are modified with phosphoethanolamine (PEtn) at the 3 (3-PEtn) and/or 6 (6-PEtn) position. The lpt3 (NMB2010) and lpt6 (NMA0408) genes of N. meningitidis, which are proposed to encode the required HepII 3- and 6-PEtn transferases, respectively, were cloned and overexpressed as C-terminally polyhistidine-tagged fusion proteins in Escherichia coli and found to localize to the inner membrane, based on sucrose density gradient centrifugation. Lpt3-His(6) and Lpt6-His(6) were purified from Triton X-100-solubilized membranes by nickel chelation chromatography, and dot blot analysis of enzymatic reactions with 3-PEtn- and 6-PEtn-specific monoclonal antibodies demonstrated conclusively that Lpt3 and Lpt6 are phosphatidylethanolamine-dependent LOS HepII 3- and 6-PEtn transferases, respectively, and that both enzymes are capable of transferring PEtn to both fully acylated LOS and de-O-acylated (de-O-Ac) LOS. Further enzymatic studies using capillary electrophoresis-mass spectrometry (MS) demonstrated that both Lpt3 and Lpt6 are capable of transferring PEtn to de-O-Ac LOS molecules already containing PEtn at the 6 and 3 positions of HepII, respectively, demonstrating that there is no obligate order of PEtn addition in the generation of 3,6-di-PEtn LOS moieties in vitro.

Phosphoethanolamine is located at the 6-position and not at the 7-position of the distal heptose residue in the lipopolysaccharide from Neisseria meningitidis.

Previous studies on LPS from Neisseria meningitidis strains M992B, the immunotype L6 strain, NMB, the type strain, a candidate LPS vaccine strain 6275z, and an extensively used clinical strain M986 had suggested that the location of the phosphoethanolamine (PEtn) residue was the 7-position of the distal heptose residue (HepII) of the inner-core oligosaccharide (OS). In all cases, this was only established by chemical methods, methylation linkage analyses. In this study, we have used standard NMR techniques to unequivocally show that the PEtn residue is actually located at the 6-position and not at the 7-position of the HepII residue in all of these strains. The 6-PEtn transferase genes were sequenced and their translated amino acid sequences were shown to be greater than 96% identical to that of the Lpt6 transferase from the L4 immunotype strain, which has been shown to transfer PEtn to the 6-position of the distal heptose residue. We discuss the implications of these findings with respect to the immunotyping scheme for the meningococci and in the context of LPS-based vaccine development.

Direct oxidation of sugar nucleotides to the corresponding uronic acids: TEMPO and platinum-based procedures.

The direct oxidation of UDP-alpha-d-glucose and UDP-N-acetyl-alpha-d-glucosamine to the corresponding uronic acids was explored using either TEMPO or platinum-catalysed oxidation with molecular oxygen. Whilst TEMPO-based procedures gave rise to substantial over-oxidation and/or degradation of UDP-glucose, oxidation of UDP-N-acetyl-glucosamine to UDP-N-acetyl-glucosaminuronic acid was achieved with >90% conversion and ca. 65% isolated yield using a platinum-catalysed procedure.

Evidence that WbpD is an N-acetyltransferase belonging to the hexapeptide acyltransferase superfamily and an important protein for O-antigen biosynthesis in Pseudomonas aeruginosa PAO1.

Di-N-acetylated uronic acid residues are unique sugar moieties observed in the lipopolysaccharides (LPS) of respiratory pathogens including several serotypes of Pseudomonas aeruginosa and several species of Bordetella. WbpD of P. aeruginosa PAO1 (serotype O5) is a putative 3-N-acetyltransferase that has been implicated in the biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid [UDP-d-Man(2NAc3NAc)A], a precursor for the d-Man(2NAc3NAc)A residues in the B-band O antigen of this bacterium. A chromosomal knockout mutant of wbpD is incapable of producing either long-chain B-band O antigen (> or = 2 repeating units) or semi-rough LPS (lipid A-core + one repeat). Adding wbpD in trans restored LPS production to the wild-type level; this indicates that wbpD is important for biosynthesis of individual B-band O-antigen repeating units. WbpD contains left-handed beta-helical (LbetaH) structure as observed by Conserved Domain analysis and in silico secondary and tertiary structure predictions. This feature suggested that WbpD belongs to the hexapeptide acyltransferase (HexAT) superfamily of enzymes. WbpD was overexpressed as an N-terminally histidine-tagged fusion protein (His6-WbpD) and purified to > 95% purity. The protein was subjected to Far-UV circular dichroism spectroscopy, and the data revealed that WbpD contains left-handed helical structure, which substantiated in silico predictions made earlier. Results from SDS-PAGE, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS), and gel filtration analyses indicated that His6-WbpD has trimeric organization, consistent with the quaternary structure of HexATs. The binding of acetyl-CoA by WbpD was demonstrated by MALDI-TOF MS, suggesting that WbpD is an acetyltransferase that utilizes a direct-transfer reaction mechanism. Incubation of WbpD with acetyl-CoA significantly enhanced the stability of the protein and prevented precipitation over a course of 14 days. As a substrate for studying the enzymatic activity of WbpD is unavailable at present, a structure-based model for the LbetaH domain of WbpD was generated. Comparisons between this model and the LbetaH domains of known HexATs suggested that Lys136 plays a role in acetyl-CoA binding. A K136A site-directed mutant construct could only partially complement the wbpD knockout, and this mutation also reduced the stabilizing effects of acetyl-CoA, while a K136R mutation showed no discernible effect on complementation of the wbpD mutant or the stabilizing effects of acetyl-CoA on the purified mutant protein. A modified pathway was proposed for the biosynthesis of UDP-d-Man(2NAc3NAc)A, in which WbpD is involved in the catalysis of the fourth step by acting as a UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid 3-N-acetyltransferase.

Biochemical characterization of WbpA, a UDP-N-acetyl-D-glucosamine 6-dehydrogenase involved in O-antigen biosynthesis in Pseudomonas aeruginosa PAO1.

WbpA (PA3159) is an enzyme involved in the biosynthesis of unusual di-N-acetyl-d-mannosaminuronic acid-derived sugar nucleotides found in the O antigen of Pseudomonas aeruginosa PAO1 (serotype O5). The wbpA gene that encodes this enzyme was cloned into pET-28a, overexpressed as a histidine-tagged fusion protein, and purified by nickel chelation chromatography. Capillary electrophoresis was used to examine substrate conversion by WbpA, and the data revealed that WbpA is a UDP-N-acetyl-D-glucosamine 6-dehydrogenase (EC 1.1.1.136), which uses NAD(+) as a coenzyme. The enzyme reaction product was purified by HPLC and analyzed using NMR spectroscopy. Our results showed unequivocally that the product of the WbpA reaction is UDP-N-acetyl-d-glucosaminuronic acid. WbpA requires either NH(4)(+) or K(+) for activity and the accompanying anions exert secondary effects on activity consistent with their ranking in the Hofmeister series. Kinetic analysis showed positive cooperativity with respect to UDP-N-acetyl-d-glucosamine binding with a K(0.5) of 94 microM, a k(cat) of 86 min(-1), and a Hill coefficient of 1.8. In addition, WbpA has a K(0.5) for NAD(+) of 220 microM, a k(cat) of 86 min(-1), and a Hill coefficient of 1.1. The oligomerization state of WbpA was analyzed by gel filtration, dynamic light scattering, and analytical ultracentrifugation, with all three techniques indicating that WbpA exists as a trimer in solution. However, tertiary structure predictions suggested a tetramer, which was supported by data from transmission electron microscopy. The electron micrograph of negatively stained WbpA samples revealed structures with 4-fold symmetry.

Nonradiolabeling assay for WaaP, an essential sugar kinase involved in biosynthesis of core lipopolysaccharide of Pseudomonas aeruginosa.

waaP is present in the lipopolysaccharide (LPS) core gene clusters of a wide range of gram-negative bacteria, and is an essential gene in Pseudomonas aeruginosa. The WaaP protein is a sugar kinase that adds phosphate to heptose I in the core oligosaccharide. This study describes the standardization and utility of a chemiluminescence-based enzyme-linked immunosorbent assay for the detection of WaaP kinase activity. Important features of the assay include high sensitivity, the preparation of dephosphorylated LPS as a substrate, and the use of monoclonal antibody 7-4 that specifically recognizes phosphate substituents in the LPS core.