Relationships of capsular polysaccharides belonging to Campylobacter jejuni HS1 serotype complex.

The Campylobacter jejuni capsule type HS1 complex is one of the most common serotypes identified worldwide, and consists of strains typing as HS1, HS1/44, HS44 and HS1/8. The capsule structure of the HS1 type strain was shown previously to be composed of teichoic-acid like glycerol-galactosyl phosphate repeats [4-)-α-D-Galp-(1-2)-Gro-(1-P-] with non-stoichiometric fructose branches at the C2 and C3 of Gal and non-stoichiometric methyl phosphoramidate (MeOPN) modifications on the C3 of the fructose. Here, we demonstrate that the capsule of an HS1/44 strain is identical to that of the type strain of HS1, and the capsule of HS1/8 is also identical to HS1, except for an additional site of MeOPN modification at C6 of Gal. The DNA sequence of the capsule locus of an HS44 strain included an insertion of 10 genes, and the strain expressed two capsules, one identical to the HS1 type strain, but with no fructose branches, and another composed of heptoses and MeOPN. We also characterize a HS1 capsule biosynthesis gene, HS1.08, as a fructose transferase responsible for the attachment of the ß-D-fructofuranoses residues at C2 and C3 of the Gal unit. In summary, the common component of all members of the HS1 complex is the teichoic-acid like backbone that is likely responsible for the observed sero-cross reactivity.

Evaluation of a conjugate vaccine platform against enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni and Shigella.

Enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni (CJ), and Shigella sp. are major causes of bacterial diarrhea worldwide, but there are no licensed vaccines against any of these pathogens. Most current approaches to ETEC vaccines are based on recombinant proteins that are involved in virulence, particularly adhesins. In contrast, approaches to Shigella and CJ vaccines have included conjugate vaccines in which Shigella lipopolysaccharides (LPS) or CJ capsule polysaccharides are chemically conjugated to proteins. We have explored the feasibility of developing a multi-pathogen vaccine by using ETEC proteins as conjugating partners for CJ and Shigella polysaccharides. We synthesized three vaccines in which two CJ polysaccharides were conjugated to two recombinant ETEC adhesins based on CFA/I (CfaEB) and CS6 (CssBA), and LPS from Shigella flexneri was also conjugated to CfaEB. The vaccines were immunogenic in mice as monovalent, bivalent and trivalent formulations. Importantly, functional antibodies capable of inducing hemaglutination inhibition (HAI) of a CFA/I expressing ETEC strain were induced in all vaccines containing CfaEB. These data suggest that conjugate vaccines could be a platform for a multi-pathogen, multi-serotype vaccine against the three major causes of diarrheal disease worldwide.

Campylobacter jejuni transcriptional and genetic adaptation during human infection.

Campylobacter jejuni infections are a leading cause of bacterial food-borne diarrhoeal illness worldwide, and Campylobacter infections in children are associated with stunted growth and therefore long-term deficits into adulthood. Despite this global impact on health and human capital, how zoonotic C. jejuni responds to the human host remains unclear. Unlike other intestinal pathogens, C. jejuni does not harbour pathogen-defining toxins that explicitly contribute to disease in humans. This makes understanding Campylobacter pathogenesis challenging and supports a broad examination of bacterial factors that contribute to C. jejuni infection. Here, we use a controlled human infection model to characterize C. jejuni transcriptional and genetic adaptations in vivo, along with a non-human primate infection model to validate our approach. We found that variation in 11 genes is associated with either acute or persistent human infections and includes products involved in host cell invasion, bile sensing and flagella modification, plus additional potential therapeutic targets. In particular, a functional version of the cell invasion protein A (cipA) gene product is strongly associated with persistently infecting bacteria and we identified its biochemical role in flagella modification. These data characterize the adaptive C. jejuni response to primate infections and suggest therapy design should consider the intrinsic differences between acute and persistently infecting bacteria. In addition, RNA sequencing revealed conserved responses during natural host commensalism and human infections. Thirty-nine genes were differentially regulated in vivo across hosts, lifestyles and C. jejuni strains. This conserved in vivo response highlights important C. jejuni survival mechanisms such as iron acquisition and evasion of the host mucosal immune response. These advances highlight pathogen adaptability across host species and demonstrate the utility of multidisciplinary collaborations in future clinical trials to study pathogens in vivo.

Phase-Variable Changes in the Position of O-Methyl Phosphoramidate Modifications on the Polysaccharide Capsule of Campylobacter jejuni Modulate Serum Resistance.

Campylobacter jejuni polysaccharide capsules (CPS) are characterized by the presence of nonstoichiometric O-methyl phosphoramidate (MeOPN) modifications. The lack of stoichiometry is due to phase variation at homopolymeric tracts within the MeOPN transferase genes. C. jejuni strain 81-176 contains two MeOPN transferase genes and has been shown previously to contain MeOPN modifications at the 2 and 6 positions of the galactose (Gal) moiety in the CPS. We demonstrate here that one of the two MeOPN transferases, encoded by CJJ81176_1435, is bifunctional and is responsible for the addition of MeOPN to both the 2 and the 6 positions of Gal. A new MeOPN at the 4 position of Gal was observed in a mutant lacking the CJJ81176_1435 transferase and this was encoded by the CJJ81176_1420 transferase. During routine growth of 81-176, the CJJ81176_1420 transferase was predominantly in an off configuration, while the CJJ81176_1435 transferase was primarily on. However, exposure to normal human serum selected for cells expressing the CJJ81176_1420 transferase. MeOPN modifications appear to block binding of naturally occurring antibodies to the 81-176 CPS. The absence of MeOPN-4-Gal resulted in enhanced sensitivity to serum killing, whereas the loss of MeOPN-2-Gal and MeOPN-6-Gal resulted in enhanced resistance to serum killing, perhaps by allowing more MeOPN to be put onto the 4 position of Gal.IMPORTANCECampylobacter jejuni undergoes phase variation in genes encoding surface antigens, leading to the concept that a strain of this organism consists of multiple genotypes that are selected for fitness in various environments. Methyl phosphoramidate modifications on the capsule of C. jejuni block access of preexisting antibodies in normal human sera to the polysaccharide chain, thus preventing activation of the classical arm of the complement cascade. We show that the capsule of strain 81-176 contains more sites of MeOPN modifications than previously recognized and that one site, on the 4 position of galactose, is more critical to complement resistance than the others. Exposure to normal human serum selects for variants in the population expressing this MeOPN modification.

Synthesis and immunodetection of 6-O-methyl-phosphoramidyl-α-D-galactose: a Campylobacter jejuni antigenic determinant.

Campylobacter jejuni is a leading cause of traveler's diarrhea. Previously, we have shown that a C. jejuni capsule polysaccharide (CPS) conjugate vaccine can fully prevent C.jejuni diarrhea in non-human primates. C.jejuni CPSs are decorated with non-stoichiometric amounts of O-methyl phosphoramidate (MeOPN) units that are key serospecific markers. In the case of C.jejuni serotype complex HS23/36, the MeOPN are at positions 2 and 6 of the CPS galactose (Gal). We describe here the synthesis of the p-methoxyphenyl glycoside of MeOPN→6-α-D-Galp, and its immunodetection by antisera raised by C.jejuni CPS conjugates with MeOPN at primary positions. The synthetic approach in this work served as the foundation for a similar MeOPN→6-Gal construct used in a conjugate vaccine, whose synthesis, immunogenicity and efficacy will be described elsewhere.

The design of a capsule polysaccharide conjugate vaccine against Campylobacter jejuni serotype HS15.

Campylobacter jejuni infection is now the main cause of diarrhea-related illnesses in humans. An efficacious vaccine for the traveler and developing world market would be welcomed. We are engaged in the discovery and characterization of serotype-specific C. jejuni capsule polysaccharides (CPSs) to study their role in virulence and as protective vaccine antigens. Our prototype conjugate vaccine with serotype HS23 CPS (strain 81-176) has been shown to fully protect non-human primates against diarrhea inflicted by C. jejuni HS23, but ultimately, a useful CPS-based vaccine will have to be multivalent. To this end, we describe here the creation of a CPS-conjugate vaccine against C. jejuni serotype HS15. Structural analysis revealed that a repeating block consisting of L-α-arabinofuranose (Ara) and 6-deoxy-L-α-gulo-heptopyranose (6d-gulo-Hep) comprised the CPS of serotype HS15 type strain ATCC 43442 [→3)-α-L-Araf-(1→3)-6d-L-α-gulo-Hepp(1→](n). Strategically, the non-reducing end of the CPS was activated and used in the attachment of CPS to CRM197 to yield a conjugate vaccine. A serological assessment of the CPS(HS15)-CRM197 conjugate with an anti-HS15 polyclonal antibody confirmed the conservation of antigenic epitopes, and subsequent inoculation of mice with CPS(HS15)-CRM197 revealed that this conjugate was indeed capable of raising anti-CPS(HS15) antibodies.

The polysaccharide capsule of Campylobacter jejuni modulates the host immune response.

Campylobacter jejuni is a major cause of bacterial diarrheal disease worldwide. The organism is characterized by a diversity of polysaccharide structures, including a polysaccharide capsule. Most C. jejuni capsules are known to be decorated nonstoichiometrically with methyl phosphoramidate (MeOPN). The capsule of C. jejuni 81-176 has been shown to be required for serum resistance, but here we show that an encapsulated mutant lacking the MeOPN modification, an mpnC mutant, was equally as sensitive to serum killing as the nonencapsulated mutant. A nonencapsulated mutant, a kpsM mutant, exhibited significantly reduced colonization compared to that of wild-type 81-176 in a mouse intestinal colonization model, and the mpnC mutant showed an intermediate level of colonization. Both mutants were associated with higher levels of interleukin 17 (IL-17) expression from lamina propria CD4(+) cells than from cells from animals infected with 81-176. In addition, reduced levels of Toll-like receptor 4 (TLR4) and TLR2 activation were observed following in vitro stimulation of human reporter cell lines with the kpsM and mpnC mutants compared to those with wild-type 81-176. The data suggest that the capsule polysaccharide of C. jejuni and the MeOPN modification modulate the host immune response.

Functional characterization of flagellin glycosylation in Campylobacter jejuni 81-176.

The major flagellin of Campylobacter jejuni strain 81-176, FlaA, has been shown to be glycosylated at 19 serine or threonine sites, and this glycosylation is required for flagellar filament formation. Some enzymatic components of the glycosylation machinery of C. jejuni 81-176 are localized to the poles of the cell in an FlhF-independent manner. Flagellin glycosylation could be detected in flagellar mutants at multiple levels of the regulatory hierarchy, indicating that glycosylation occurs independently of the flagellar regulon. Mutants were constructed in which each of the 19 serine or threonines that are glycosylated in FlaA was converted to an alanine. Eleven of the 19 mutants displayed no observable phenotype, but the remaining 8 mutants had two distinct phenotypes. Five mutants (mutations S417A, S436A, S440A, S457A, and T481A) were fully motile but defective in autoagglutination (AAG). Three other mutants (mutations S425A, S454A, and S460A) were reduced in motility and synthesized truncated flagellar filaments. The data implicate certain glycans in mediating filament-filament interactions resulting in AAG and other glycans appear to be critical for structural subunit-subunit interactions within the filament.

Structure of a sigma28-regulated nonflagellar virulence protein from Campylobacter jejuni.

Campylobacter jejuni, a Gram-negative motile bacterium, is a leading cause of human gastrointestinal infections. Although the mechanism of C.jejuni-mediated enteritis appears to be multifactorial, flagella play complex roles in the virulence of this human pathogen. Cj0977 is a recently identified virulence factor in C. jejuni and is expressed by a sigma(28) promoter that controls late genes in the flagellar regulon. A Cj0977 mutant strain is fully motile but significantly reduced in the invasion of intestinal epithelial cells in vitro. Here, we report the crystal structure of the major structural domain of Cj0977, which reveals a homodimeric "hot-dog" fold architecture. Of note, the characteristic hot-dog fold has been found in various coenzyme A (CoA) compound binding proteins with numerous oligomeric states. Structural comparison with other known hot-dog fold proteins locates a putative binding site for an acyl-CoA compound in the Cj0977 protein. Structure-based site-directed mutagenesis followed by invasion assays indicates that key residues in the putative binding site are indeed essential for the Cj0977 virulence function, suggesting a possible function of Cj0977 as an acyl-CoA binding regulatory protein.

Divergence of quaternary structures among bacterial flagellar filaments.

It has been widely assumed that the atomic structure of the flagellar filament from Salmonella typhimurium serves as a model for all bacterial flagellar filaments given the sequence conservation in the coiled-coil regions responsible for polymerization. On the basis of electron microscopic images, we show that the flagellar filaments from Campylobacter jejuni have seven protofilaments rather than the 11 in S. typhimurium. The vertebrate Toll-like receptor 5 (TLR5) recognizes a region of bacterial flagellin that is involved in subunit-subunit assembly in Salmonella and many other pathogenic bacteria, and this short region has diverged in Campylobacter and related bacteria, such as Helicobacter pylori, which are not recognized by TLR5. The driving force in the change of quaternary structure between Salmonella and Campylobacter may have been the evasion of TLR5.

Protein glycosylation in Campylobacter jejuni: partial suppression of pglF by mutation of pseC.

Campylobacter jejuni has systems for N- and O-linked protein glycosylation. Although biochemical evidence demonstrated that a pseC mutant in the O-linked pathway accumulated the product of pglF in the N-linked pathway, analyses of transformation frequencies and glycosylation statuses of N-glycosylated proteins indicated a partial suppression of pglF by pseC.

Targeted metabolomics analysis of Campylobacter coli VC167 reveals legionaminic acid derivatives as novel flagellar glycans.

Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic acid biosynthetic enzymes. This is the first report of legionaminic acid in Campylobacter sp. and the first report of legionaminic acid derivatives as modifications on a protein.

Changes in flagellin glycosylation affect Campylobacter autoagglutination and virulence.

Analysis of the complete flagellin glycosylation locus of Campylobacter jejuni strain 81-176 revealed a less complex genomic organization than the corresponding region in the genome strain, C. jejuni NCTC 11168. Twenty-four of the 45 genes found between Cj1293 and Cj1337 in NCTC 11168 are missing in 81-176. Mutation of six new genes, in addition to three previously reported, resulted in a non-motile phenotype, consistent with a role in synthesis of pseudaminic acid (PseAc) or transfer of PseAc to flagellin. Mutation of Cj1316c or pseA had been shown to result in loss of the acetamidino form of pseudaminic acid (PseAm). Mutation of a second gene also resulted in loss of PseAm, as well as a minor modification that appears to be PseAm extended with N-acetyl-glutamic acid. Previously described mutants in C. jejuni 81-176 and Campylobacter coli VC167 that produced flagella lacking PseAm or PseAc failed to autoagglutinate. This suggests that interactions between modifications on adjacent flagella filaments are required for autoagglutination. Mutants (81-176) defective in autoagglutination showed a modest reduction in adherence and invasion of INT407 cells. However, there was a qualitative difference in binding patterns to INT407 cells using GFP-labelled 81-176 and mutants lacking PseAm. A mutant lacking PseAm was attenuated in the ferret diarrhoeal disease model.

A sigma28-regulated nonflagella gene contributes to virulence of Campylobacter jejuni 81-176.

A Campylobacter jejuni 81-176 mutant in Cj0977 was fully motile but reduced >3 logs compared to the parent in invasion of intestinal epithelial cells in vitro. The mutant was also attenuated in a ferret diarrheal disease model. Expression of Cj0977 protein was dependent on a minimal flagella structure.

Pseudaminic acid, the major modification on Campylobacter flagellin, is synthesized via the Cj1293 gene.

Flagellins from Campylobacter jejuni 81-176 and Campylobacter coli VC167 are heavily glycosylated. The major modifications on both flagellins are pseudaminic acid (Pse5Ac7Ac), a nine carbon sugar that is similar to sialic acid, and an acetamidino-substituted analogue of pseudaminic acid (PseAm). Previous data have indicated that PseAm is synthesized via Pse5Ac7Ac in C. jejuni 81-176, but that the two sugars are synthesized using independent pathways in C. coli VC167. The Cj1293 gene of C. jejuni encodes a putative UDP-GlcNAc C6-dehydratase/C4-reductase that is similar to a protein required for glycosylation of Caulobacter crescentus flagellin. The Cj1293 gene is expressed either under the control of a sigma 54 promoter that overlaps the coding region of Cj1292 or as a polycistronic message under the control of a sigma 70 promoter upstream of Cj1292. A mutant in gene Cj1293 in C. jejuni 81-176 was non-motile and non-flagellated and accumulated unglycosylated flagellin intracellularly. This mutant was complemented in trans with the homologous C. jejuni gene, as well as the Helicobacter pylori homologue, HP0840, which has been shown to encode a protein with UDP-GlcNAc C6-dehydratase/C4-reductase activity. Mutation of Cj1293 in C. coli VC167 resulted in a fully motile strain that synthesized a flagella filament composed of flagellin in which Pse5Ac7Ac was replaced by PseAm. The filament from the C. coli Cj1293 mutant displayed increased solubility in SDS compared with the wild-type filament. A double mutant in C. coli VC167, defective in both Cj1293 and ptmD, encoding part of the independent PseAm pathway, was also non-motile and non-flagellated and accumulated unglycosylated flagellin intracellularly. Collectively, the data indicate that Cj1293 is essential for Pse5Ac7Ac biosynthesis from UDP-GlcNAc, and that glycosylation is required for flagella biogenesis in campylobacters.

DNA sequence and mutational analyses of the pVir plasmid of Campylobacter jejuni 81-176.

The circular pVir plasmid of Campylobacter jejuni strain 81-176 was determined to be 37,468 nucleotides in length with a G+C content of 26%. A total of 83% of the plasmid represented coding information, and all but 2 of the 54 predicted open reading frames were encoded on the same DNA strand. There were seven genes on the plasmid in a continguous region of 8.9 kb that encoded orthologs of type IV secretion proteins found in Helicobacter pylori, including four that have been described previously (D. J. Bacon, R. A. Alm, D. H. Burr, L. Hu, D. J. Kopecko, C. P. Ewing, T. J. Trust, and P. Guerry, Infect. Immun. 68:4384-4390, 2000). There were seven other pVir-encoded proteins that showed significant similarities to proteins encoded by the plasticity zones of either H. pylori J99 or 26695. Mutational analyses of 19 plasmid genes identified 5 additional genes that affect in vitro invasion of intestinal epithelial cells. These included one additional gene encoding a component of a type IV secretion system, an ortholog of Cj0041 from the chromosome of C. jejuni NCTC 11168, two Campylobacter plasmid-specific genes, and an ortholog of HP0996 from the plasticity zone of H. pylori 26695.

Structural heterogeneity of carbohydrate modifications affects serospecificity of Campylobacter flagellins.

Flagellin from Campylobacter coli VC167 is post-translationally modified at > or = 16 amino acid residues with pseudaminic acid and three related derivatives. The predominant modification was 5,7-diacetamido-3,5,7,9 - tetradeoxy - l - glycero - l - manno - nonulosonic acid (pseudaminic acid, Pse5Ac7Ac), a modification that has been described previously on flagellin from Campylobacter jejuni 81-176. VC167 lacked two modi-fications present in 81-176 and instead had two unique modifications of masses 431 and 432 Da. Flagellins from both C. jejuni 81-176 and C. coli VC167 were also modified with an acetamidino form of pseudaminic acid (PseAm), but tandem mass spectrometry indicated that the structure of PseAm differed in the two strains. Synthesis of PseAm in C. coli VC167 requires a minimum of six ptm genes. In contrast, PseAm is synthesized in C. jejuni 81-176 via an alternative pathway using the product of the pseA gene. Mutation of the ptm genes in C. coli VC167 can be detected by changes in apparent Mr of flagellin in SDS-PAGE gels, changes in isoelectric focusing (IEF) patterns and loss of immunoreactivity with antiserum LAH2. These changes corresponded to loss of both 315 Da and 431 Da modifications from flagellin. Complementation of the VC167 ptm mutants with the 81-176 pseA gene in trans resulted in flagellins containing both 315 and 431 Da modifications, but these flagellins remained unreactive in LAH2 antibody, suggesting that the unique form of PseAm encoded by the ptm genes contributes to the serospecificity of the flagellar filament.

Phase variation of Campylobacter jejuni 81-176 lipooligosaccharide affects ganglioside mimicry and invasiveness in vitro.

The outer cores of the lipooligosaccharides (LOS) of many strains of Campylobacter jejuni mimic human gangliosides in structure. A population of cells of C. jejuni strain 81-176 produced a mixture of LOS cores which consisted primarily of structures mimicking GM(2) and GM(3) gangliosides, with minor amounts of structures mimicking GD(1b) and GD(2). Genetic analyses of genes involved in the biosynthesis of the outer core of C. jejuni 81-176 revealed the presence of a homopolymeric tract of G residues within a gene encoding CgtA, an N-acetylgalactosaminyltransferase. Variation in the number of G residues within cgtA affected the length of the open reading frame, and these changes in cgtA corresponded to a change in LOS structure from GM(2) to GM(3) ganglioside mimicry. Site-specific mutation of cgtA in 81-176 resulted in a major LOS core structure that lacked GalNAc and resembled GM(3) ganglioside. Compared to wild-type 81-176, the cgtA mutant showed a significant increase in invasion of INT407 cells. In comparison, a site-specific mutation of the neuC1 gene resulted in the loss of sialic acid in the LOS core and reduced resistance to normal human serum but had no affect on invasion of INT407 cells.