Convergent evolution in the supercoiling of prokaryotic flagellar filaments.

The supercoiling of bacterial and archaeal flagellar filaments is required for motility. Archaeal flagellar filaments have no homology to their bacterial counterparts and are instead homologs of bacterial type IV pili. How these prokaryotic flagellar filaments, each composed of thousands of copies of identical subunits, can form stable supercoils under torsional stress is a fascinating puzzle for which structural insights have been elusive. Advances in cryoelectron microscopy (cryo-EM) make it now possible to directly visualize the basis for supercoiling, and here, we show the atomic structures of supercoiled bacterial and archaeal flagellar filaments. For the bacterial flagellar filament, we identify 11 distinct protofilament conformations with three broad classes of inter-protomer interface. For the archaeal flagellar filament, 10 protofilaments form a supercoil geometry supported by 10 distinct conformations, with one inter-protomer discontinuity creating a seam inside of the curve. Our results suggest that convergent evolution has yielded stable superhelical geometries that enable microbial locomotion.

Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as volunteers challenged with ETEC, diarrheal severity is significantly increased in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design.

Atomic structure of the Campylobacter jejuni flagellar filament reveals how ε Proteobacteria escaped Toll-like receptor 5 surveillance.

Vertebrates, from zebra fish to humans, have an innate immune recognition of many bacterial flagellins. This involves a conserved eight-amino acid epitope in flagellin recognized by the Toll-like receptor 5 (TLR5). Several important human pathogens, such as Helicobacter pylori and Campylobacter jejuni, have escaped TLR5 activation by mutations in this epitope. When such mutations were introduced into Salmonella flagellin, motility was abolished. It was previously argued, using very low-resolution cryoelectron microscopy (cryo-EM), that C. jejuni accommodated these mutations by forming filaments with 7 protofilaments, rather than the 11 found in other bacteria. We have now determined the atomic structure of the C. jejuni G508A flagellar filament from a 3.5-Å-resolution cryo-EM reconstruction, and show that it has 11 protofilaments. The residues in the C. jejuni TLR5 epitope have reduced contacts with the adjacent subunit compared to other bacterial flagellar filament structures. The weakening of the subunit-subunit interface introduced by the mutations in the TLR5 epitope is compensated for by extensive interactions between the outer domains of the flagellin subunits. In other bacteria, these outer domains can be nearly absent or removed without affecting motility. Furthermore, we provide evidence for the stabilization of these outer domain interactions through glycosylation of key residues. These results explain the essential role of glycosylation in C. jejuni motility, and show how the outer domains have evolved to play a role not previously found in other bacteria.

Enterotoxigenic E. coli virulence gene regulation in human infections.

Enterotoxigenic Escherichia coli (ETEC) is a global diarrheal pathogen that utilizes adhesins and secreted enterotoxins to cause disease in mammalian hosts. Decades of research on virulence factor regulation in ETEC has revealed a variety of environmental factors that influence gene expression, including bile, pH, bicarbonate, osmolarity, and glucose. However, other hallmarks of the intestinal tract, such as low oxygen availability, have not been examined. Further, determining how ETEC integrates these signals in the complex host environment is challenging. To address this, we characterized ETEC's response to the human host using samples from a controlled human infection model. We found ETEC senses environmental oxygen to globally influence virulence factor expression via the oxygen-sensitive transcriptional regulator fumarate and nitrate reduction (FNR) regulator. In vitro anaerobic growth replicates the in vivo virulence factor expression profile, and deletion of fnr in ETEC strain H10407 results in a significant increase in expression of all classical virulence factors, including the colonization factor antigen I (CFA/I) adhesin operon and both heat-stable and heat-labile enterotoxins. These data depict a model of ETEC infection where FNR activity can globally influence virulence gene expression, and therefore proximity to the oxygenated zone bordering intestinal epithelial cells likely influences ETEC virulence gene expression in vivo. Outside of the host, ETEC biofilms are associated with seasonal ETEC epidemics, and we find FNR is a regulator of biofilm production. Together these data suggest FNR-dependent oxygen sensing in ETEC has implications for human infection inside and outside of the host.

Campylobacter jejuni capsule types in a Peruvian birth cohort and associations with diarrhoeal disease severity.

Campylobacter jejuni is a leading cause of bacterial diarrhoea worldwide. The objective of this study was to examine the association between C. jejuni capsule types and clinical signs and symptoms of diarrhoeal disease in a well-defined birth cohort in Peru. Children were enrolled in the study at birth and followed until 2 years of age as part of the Malnutrition and Enteric Infections birth cohort. Associations between capsule type and clinical outcomes were assessed using the Pearson's χ2 and the Kruskal-Wallis test statistics. A total of 318 C. jejuni samples (30% from symptomatic cases) were included in this analysis. There were 22 different C. jejuni capsule types identified with five accounting for 49.1% of all isolates. The most common capsule types among the total number of isolates were HS4 complex (n = 52, 14.8%), HS5/31 complex (n = 42, 11.9%), HS15 (n = 29, 8.2%), HS2 (n = 26, 7.4%) and HS10 (n = 24, 6.8%). These five capsule types accounted for the majority of C. jejuni infections; however, there was no significant difference in prevalence between symptomatic and asymptomatic infection (all p > 0.05). The majority of isolates (n = 291, 82.7%) were predicted to express a heptose-containing capsule. The predicted presence of methyl phosphoramidate, heptose or deoxyheptose on the capsule was common.

Rifaximin Fails to Prevent Campylobacteriosis in the Human Challenge Model: A Randomized, Double-Blind, Placebo-Controlled Trial.

Background: Campylobacter species are a leading cause of diarrheal disease globally with significant morbidity. Primary prevention efforts have yielded limited results. Rifaximin chemoprophylaxis decreases rates of travelers' diarrhea and may be suitable for high-risk persons. We assessed the efficacy of rifaximin in the controlled human infection model for Campylobacter jejuni. Methods: Twenty-eight subjects were admitted to an inpatient facility and randomized to a twice-daily dose of 550 mg rifaximin or placebo. The following day, subjects ingested 1.7 × 105 colony-forming units of C. jejuni strain CG8421. Subjects continued prophylaxis for 3 additional days, were followed for campylobacteriosis for 144 hours, and were subsequently treated with azithromycin and ciprofloxacin. Samples were collected to assess immunologic responses to CG8421. Results: There was no difference (P = 1.0) in the frequency of campylobacteriosis in those receiving rifaximin (86.7%) or placebo (84.6%). Additionally, there were no differences in the clinical signs and symptoms of C. jejuni infection to include abdominal pain/cramps (P = 1.0), nausea (P = 1.0), vomiting (P = .2), or fever (P = 1.0) across study groups. Immune responses to the CG8421 strain were comparable across treatment groups. Conclusions: Rifaximin did not prevent campylobacteriosis in this controlled human infection model. Given the morbidity associated with Campylobacter infection, primary prevention efforts remain a significant need. Clinical Trials Registration: NCT02280044.

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.

Campylobacter jejuni extracellular vesicles harboring cytolethal distending toxin bind host cell glycans and induce cell cycle arrest in host cells.

Cytolethal distending toxins (CDTs) are released by Gram-negative pathogens into the extracellular medium as free toxin or associated with extracellular vesicles (EVs), commonly known as outer membrane vesicles (OMVs). CDT production by the gastrointestinal pathogen Campylobacter jejuni has been implicated in colorectal tumorigenesis. Despite CDT being a major virulence factor for C. jejuni, little is known about the EV-associated form of this toxin. To address this point, C. jejuni mutants lacking each of the three CDT subunits (A, B, and C) were generated. C. jejuni cdtA, cdtB, and cdtC bacteria released EVs in similar numbers and sizes to wild-type bacteria, ranging from 5 to 530 nm (mean ± SEM = 118 ±6.9 nm). As the CdtAC subunits mediate toxin binding to host cells, we performed "surface shearing" experiments, in which EVs were treated with proteinase K and incubated with host cells. These experiments indicated that CDT subunits are internal to EVs and that surface proteins are probably not involved in EV-host cell interactions. Furthermore, glycan array studies demonstrated that EVs bind complex host cell glycans and share receptor binding specificities with C. jejuni bacteria for fucosyl GM1 ganglioside, P1 blood group antigen, sialyl, and sulfated Lewisx. Finally, we show that EVs from C. jejuni WT but not mutant bacteria induce cell cycle arrest in epithelial cells. In conclusion, we propose that EVs are an important mechanism for CDT release by C. jejuni and are likely to play a significant role in toxin delivery to host cells. IMPORTANCE: Campylobacter jejuni is the leading cause of foodborne gastroenteritis in humans worldwide and a significant cause of childhood mortality due to diarrheal disease in developing countries. A major factor by which C. jejuni causes disease is a toxin, called cytolethal distending toxin (CDT). The biology of this toxin, however, is poorly understood. In this study, we report that C. jejuni CDT is protected within membrane blebs, known as extracellular vesicles (EVs), released by the bacterium. We showed that proteins on the surfaces of EVs are not required for EV uptake by host cells. Furthermore, we identified several sugar receptors that may be required for EV binding to host cells. By studying the EV-associated form of C. jejuni CDT, we will gain a greater understanding of how C. jejuni intoxicates host cells and how EV-associated CDT may be used in various therapeutic applications, including as anti-tumor therapies.

The 2022 Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference: Summary of breakout workshops.

The global public health nonprofit organization PATH hosted the third Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference in Washington, DC, from November 29 to December 1, 2022. This international gathering focused on cutting-edge research related to the development of vaccines against neglected diarrheal pathogens including Shigella, enterotoxigenic Escherichia coli (ETEC), Campylobacter, and non-typhoidal Salmonella. In addition to the conference's plenary content, the agenda featured ten breakout workshops on topics of importance to the enteric vaccine field. This unique aspect of VASE Conferences allows focused groups of attendees to engage in in-depth discussions on subjects of interest to the enteric vaccine development community. In 2022, the workshops covered a range of topics. Two focused on the public health value of enteric vaccines, with one examining how to translate evidence into policy and the other on the value proposition of potential combination vaccines against bacterial enteric pathogens. Two more workshops explored new tools for the development and evaluation of vaccines, with the first on integrating antigen/antibody technologies for mucosal vaccine and immunoprophylactic development, and the second on adjuvants specifically for Shigella vaccines for children in low- and middle-income countries. Another pair of workshops covered the status of vaccines against two emerging enteric pathogens, Campylobacter and invasive non-typhoidal Salmonella. The remaining four workshops examined the assessment of vaccine impact on acute and long-term morbidity. These included discussions on the nature and severity of intestinal inflammation; cellular immunity and immunological memory in ETEC and Shigella infections; clinical and microbiologic endpoints for Shigella vaccine efficacy studies in children; and intricacies of protective immunity to enteric pathogens. This article provides a brief summary of the presentations and discussions at each workshop in order to share these sessions with the broader enteric vaccine field.

Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as human volunteers challenged with ETEC, diarrheal severity is significantly increased severity in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of human volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected human volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design.

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.

Lack of homologous protection against Campylobacter jejuni CG8421 in a human challenge model.

BACKGROUND: Campylobacter jejuni is a common cause of diarrhea and is associated with serious postinfectious sequelae. Although symptomatic and asymptomatic infections are recognized, protective immunity is not well understood. Previous data suggests that interferon γ (IFN-γ) may be associated with protection. To better define the clinical and immunologic development of protective immunity to C. jejuni, we assessed the ability of an initial infection to prevent clinical illness after a second experimental infection. METHODS: Subjects with no clinical or immunologic evidence of prior infection with C. jejuni received an initial challenge with C. jejuni CG8421 with rechallenge 3 months later. The primary endpoint was campylobacteriosis, as defined by diarrhea and/or systemic signs. Close inpatient monitoring was performed. Serum immunoglobulin A (IgA) and immunoglobulin G (IgG), fecal IgA, IgA antibody-secreting cells (ASCs), and IFN-γ production were evaluated. All subjects were treated with antibiotics and were clinically well at discharge. RESULTS: Fifteen subjects underwent a primary infection with C. jejuni CG8421; 14 (93.3%) experienced campylobacteriosis. Eight subjects received the second challenge, and all experienced campylobacteriosis with similar severity. Immune responses after primary infection included serum IgA, IgG, ASC, and IFN-γ production. Responses were less robust after secondary infection. CONCLUSIONS: In naive healthy adults, a single infection with CG8421 did not protect against campylobacteriosis. Although protection has been demonstrated with other strains and after continuous environmental exposure, our work highlights the importance of prior immunity, repeated exposures, and strain differences in protective immunity to C. jejuni. CLINICAL TRIALS REGISTRATION: NCT01048112.

Description of novel capsule biosynthesis loci of Campylobacter jejuni clinical isolates from South and South-East Asia.

Campylobacter jejuni is a major cause of bacterial diarrhea worldwide and associated with numerous sequela, including Guillain-Barré Syndrome, inflammatory bowel disease, reactive arthritis, and irritable bowel syndrome. C. jejuni is unusual for an intestinal pathogen in its ability to coat its surface with a polysaccharide capsule (CPS). The genes responsible for the biosynthesis of the phase variable CPS is located in the hypervariable region of C. jejuni genome which has been used to develop multiplex PCR to classify CPS types based on the Penner serotypes. However, there still are non-typable CPS C. jejuni by the current multiplex PCR scheme. The application of the next generation sequencing and whole genome analysis software were used for the identification of novel capsule biosynthesis of C. jejuni isolates. Unique PCR primers were designed to identify these new capsule biosynthesis loci. The designed primers sets were combined in a new multiplex mix called epsilon. The unique sequences provide an additional information of the biosynthesis loci responsible for some of the common CPS sugars/residues such as heptose, deoxtyheptose and MeOPN among C. jejuni in this new group of CPS multiplex assay. This new primer complements the current C. jejuni multiplex capsule typing system and will help in identifying previously untypeable capsule locus of C. jejuni isolates.

Bioactivity and efficacy of a hyperimmune bovine colostrum product- Travelan, against shigellosis in a non-Human primate model (Macaca mulatta).

Infectious diarrhea is a World Health Organization public health priority area due to the lack of effective vaccines and an accelerating global antimicrobial resistance crisis. New strategies are urgently needed such as immunoprophylactic for prevention of diarrheal diseases. Hyperimmune bovine colostrum (HBC) is an established and effective prophylactic for infectious diarrhea. The commercial HBC product, Travelan® (Immuron Ltd, Australia) targets multiple strains of enterotoxigenic Escherichia coli (ETEC) is highly effective in preventing diarrhea in human clinical studies. Although Travelan® targets ETEC, preliminary studies suggested cross-reactivity with other Gram-negative enteric pathogens including Shigella and Salmonella species. For this study we selected an invasive diarrheal/dysentery-causing enteric pathogen, Shigella, to evaluate the effectiveness of Travelan®, both in vitro and in vivo. Here we demonstrate broad cross-reactivity of Travelan® with all four Shigella spp. (S. flexneri, S. sonnei, S. dysenteriae and S. boydii) and important virulence factor Shigella antigens. Naïve juvenile rhesus macaques (NJRM) were randomized, 8 dosed with Travelan® and 4 with a placebo intragastrically twice daily over 6 days. All NJRM were challenged with S. flexneri 2a strain 2457T on the 4th day of treatment and monitored for diarrheal symptoms. All placebo-treated NJRM displayed acute dysentery symptoms within 24-36 hours of challenge. Two Travelan®-treated NJRM displayed dysentery symptoms and six animals remained healthy and symptom-free post challenge; resulting in 75% efficacy of prevention of shigellosis (p = 0.014). These results strongly indicate that Travelan® is functionally cross-reactive and an effective prophylactic for shigellosis. This has positive implications for the prophylactic use of Travelan® for protection against both ETEC and Shigella spp. diarrheal infections. Future refinement and expansion of pathogens recognized by HBC including Travelan® could revolutionize current management of gastrointestinal infections and outbreaks in travelers' including military, peacekeepers, humanitarian workers and in populations living in endemic regions of the world.

Route and antigen shape immunity to dmLT-adjuvanted vaccines to a greater extent than biochemical stress or formulation excipients.

A key aspect to vaccine efficacy is formulation stability. Biochemical evaluations provide information on optimal compositions or thermal stability but are routinely validated by ex vivo analysis and not efficacy in animal models. Here we assessed formulations identified to improve or reduce stability of the mucosal adjuvant dmLT being investigated in polio and enterotoxigenic E. coli (ETEC) clinical vaccines. We observed biochemical changes to dmLT protein with formulation or thermal stress, including aggregation or subunit dissociation or alternatively resistance against these changes with specific buffer compositions. However, upon injection or mucosal vaccination with ETEC fimbriae adhesin proteins or inactivated polio virus, experimental findings indicated immunization route and co-administered antigen impacted vaccine immunogenicity more so than dmLT formulation stability (or instability). These results indicate the importance of both biochemical and vaccine-derived immunity assessment in formulation optimization. In addition, these studies have implications for use of dmLT in clinical settings and for delivery in resource poor settings.

Discrimination of major capsular types of Campylobacter jejuni by multiplex PCR.

The polysaccharide capsule (CPS) of Campylobacter jejuni is the major serodeterminant of the Penner serotyping scheme. There are 47 Penner serotypes of C. jejuni, 22 of which fall into complexes of related serotypes. A multiplex PCR method for determination of capsule types of Campylobacter jejuni which is simpler and more affordable than classical Penner typing was developed. Primers specific for each capsule type were designed on the basis of a database of gene sequences from the variable capsule loci of 8 strains of major serotypes sequenced in this study and 10 published sequences of other serotypes. DNA sequence analysis revealed a mosaic nature of the capsule loci, suggesting reassortment of genes by horizontal transfer, and demonstrated a high degree of conservation of genes within Penner complexes. The multiplex PCR can distinguish 17 individual serotypes in two PCRs with sensitivities and specificities ranging from 90 to 100% using 244 strains of known Penner type.

A systematic review and meta-analysis of Penner serotype prevalence of Campylobacter jejuni in low- and middle-income countries.

INTRODUCTION: While Campylobacter jejuni is a leading foodborne bacterial pathogen worldwide, it poses a particular risk to susceptible populations in low- and middle-income countries (LMICs). A capsule-conjugate vaccine approach has been proposed as a potential solution, but little information exists on circulating C. jejuni capsule types in LMICs. The capsule is the major serodeterminant of the Penner typing scheme, which is based on serum recognition of Campylobacter heat-stable antigens. We conducted a systematic review and meta-analysis to estimate the distribution of Penner serotypes associated with C. jejuni enteritis in LMICs. Vaccine coverage assessments for hypothetical regional and global C. jejuni vaccines were also estimated. METHODS: A systematic review of the literature published from 1980 to 2019 was performed using PubMed, Scopus, and Web of Science databases. Articles were assessed for eligibility and data were abstracted. Pooled C. jejuni serotype prevalence in LMICs was estimated by region and globally using random-effects models. RESULTS: A total of 36 studies were included, capturing 4,434 isolates from LMICs. Fifteen serotypes were present in a sufficient number of studies to be included in analyses. Among these, HS4c was the most common serotype globally (12.6%), though leading capsule types varied among regions. HS2, HS3c, HS4c, HS5/31, HS8/17, and HS10 were all among the 10 most common region-specific serotypes. CONCLUSIONS: The results of this review suggest that an octavalent vaccine could provide up to 66.9% coverage of typable strains worldwide, and 56.8-69.0% regionally. This review also highlights the paucity of available data on capsules in LMICs; more testing is needed to inform vaccine development efforts.

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.

Exploring Changes in the Host Gut Microbiota During a Controlled Human Infection Model for Campylobacter jejuni.

Campylobacter jejuni infection is a leading cause of foodborne disease, common to children, adult travelers, and military populations in low- to middle-income countries. In the absence of a licensed vaccine, efforts to evaluate prophylactic agents are underway. The prophylactic efficacy of a twice-daily, 550 mg dose of the antibiotic rifaximin demonstrated no efficacy against campylobacteriosis in a controlled human infection model (CHIM); however, samples from the CHIM study were utilized to assess how the human gut microbiome responds to C. jejuni infection, and if a 'protective' microbiota exists in study participants not developing campylobacteriosis. Statistically significant, but minor, differences in study participant beta diversity were identified during the challenge period (p = 0.002, R2 = 0.042), but no significant differences were otherwise observed. Pre-challenge alpha diversity was elevated in study participants who did not develop campylobacteriosis compared to those who did (p < 0.001), but alpha diversity declined in all study participants from the pre-challenge period to post-discharge. Our work provides insight into gut microbiome shifts observed during a C. jejuni CHIM and following antibiotic treatment. This study utilized a high dose of 1.7 x 105 colony-forming units of C. jejuni; future work could include CHIM studies performed with inocula more closely mimicking natural exposure as well as field studies involving naturally-occurring enteric infections.

Examination of 189 Campylobacter Species Isolates from the Global Enteric Multicenter Study.

We have examined the draft genomes of 189 Campylobacter species isolates from the Global Enteric Multicenter Study, in which Campylobacter species were identified as significant pathogens.