The Missing Pieces: The Role of Secretion Systems in Campylobacter jejuni Virulence.

Campylobacter jejuni is likely the most common bacterial cause of gastroenteritis worldwide, responsible for millions of cases of inflammatory diarrhea characterized by severe abdominal cramps and blood in the stool. Further, C. jejuni infections are associated with post-infection sequelae in developed countries and malnutrition and growth-stunting in low- and middle-income countries. Despite the increasing prevalence of the disease, campylobacteriosis, and the recognition that this pathogen is a serious health threat, our understanding of C. jejuni pathogenesis remains incomplete. In this review, we focus on the Campylobacter secretion systems proposed to contribute to host-cell interactions and survival in the host. Moreover, we have applied a genomics approach to defining the structural and mechanistic features of C. jejuni type III, IV, and VI secretion systems. Special attention is focused on the flagellar type III secretion system and the prediction of putative effectors, given that the proteins exported via this system are essential for host cell invasion and the inflammatory response. We conclude that C. jejuni does not possess a type IV secretion system and relies on the type III and type VI secretion systems to establish a niche and potentiate disease.

Polar localization of CheO under hypoxia promotes Campylobacter jejuni chemotactic behavior within host.

Campylobacter jejuni is a food-borne zoonotic pathogen of worldwide concern and the leading cause of bacterial diarrheal disease. In contrast to other enteric pathogens, C. jejuni has strict growth and nutritional requirements but lacks many virulence factors that have evolved for pathogenesis or interactions with the host. It is unclear how this bacterium has adapted to an enteric lifestyle. Here, we discovered that the CheO protein (CJJ81176_1265) is required for C. jejuni colonization of mice gut through its role in chemotactic control of flagellar rotation in oxygen-limiting environments. CheO interacts with the chemotaxis signaling proteins CheA and CheZ, and also with the flagellar rotor components FliM and FliY. Under microaerobic conditions, CheO localizes at the cellular poles where the chemosensory array and flagellar machinery are located in C. jejuni and its polar localization depends on chemosensory array formation. Several chemoreceptors that mediate energy taxis coordinately determine the bipolar distribution of CheO. Suppressor screening for a ΔcheO mutant identified that a single residue variation in FliM can alleviate the phenotype caused by the absence of CheO, confirming its regulatory role in the flagellar rotor switch. CheO homologs are only found in species of the Campylobacterota phylum, mostly species of host-associated genera Campylobacter, Helicobacter and Wolinella. The CheO results provide insights into the complexity of chemotaxis signal transduction in C. jejuni and closely related species. Importantly, the recruitment of CheO into chemosensory array to promote chemotactic behavior under hypoxia represents a new adaptation strategy of C. jejuni to human and animal intestines.

Structure-based inhibitor design for reshaping bacterial morphology.

The spiral shape of intestinal pathogen Campylobacter jejuni is critical for invasion of intestinal mucosa epithelial cells. Insofar as this cell morphology plays a role in the pathology of C. jejuni infection, its restructuring by pharmacological intervention could be an unexplored means to prevention of infection. We recently described that peptidoglycan hydrolase 3 (Pgp3) is involved in the spiral-shape formation of C. jejuni. We report herein the design and synthesis of the hydroxamate-based inhibitors targeting Pgp3. C. jejuni cells exposed to these inhibitors changed from the helical- to rod-shaped morphology, comparable to the case of the pgp3-deletion mutant. Evidence for the mechanism of action was provided by crystal structures of Pgp3 in complex with inhibitors, shedding light into the binding modes of inhibitors within the active site, supported by kinetics and molecular-dynamics simulations. C. jejuni exposed to these inhibitors underwent the morphological change from helical- to rod-shaped bacteria, an event that reduce the ability for invasion of the host cells. This proof of concept suggests that alteration of morphology affects the interference with the bacterial infection.

The ALPK1 pathway drives the inflammatory response to Campylobacter jejuni in human intestinal epithelial cells.

The Gram-negative bacterium Campylobacter jejuni is a major cause of foodborne disease in humans. After infection, C. jejuni rapidly colonizes the mucus layer of the small and large intestine and induces a potent pro-inflammatory response characterized by the production of a large repertoire of cytokines, chemokines, and innate effector molecules, resulting in (bloody) diarrhea. The virulence mechanisms by which C. jejuni causes this intestinal response are still largely unknown. Here we show that C. jejuni releases a potent pro-inflammatory compound into its environment, which activates an NF-κB-mediated pro-inflammatory response including the induction of CXCL8, CXCL2, TNFAIP2 and PTGS2. This response was dependent on a functional ALPK1 receptor and independent of Toll-like Receptor and Nod-like Receptor signaling. Chemical characterization, inactivation of the heptose-biosynthesis pathway by the deletion of the hldE gene and in vitro engineering identified the released factor as the LOS-intermediate ADP-heptose and/or related heptose phosphates. During C. jejuni infection of intestinal cells, the ALPK1-NF-κB axis was potently activated by released heptose metabolites without the need for a type III or type IV injection machinery. Our results classify ADP-heptose and/or related heptose phosphates as a major virulence factor of C. jejuni that may play an important role during Campylobacter infection in humans.

The Campylobacter jejuni CiaD effector co-opts the host cell protein IQGAP1 to promote cell entry.

Campylobacter jejuni is a foodborne pathogen that binds to and invades the epithelial cells lining the human intestinal tract. Maximal invasion of host cells by C. jejuni requires cell binding as well as delivery of the Cia proteins (Campylobacter invasion antigens) to the host cell cytosol via the flagellum. Here, we show that CiaD binds to the host cell protein IQGAP1 (a Ras GTPase-activating-like protein), thus displacing RacGAP1 from the IQGAP1 complex. This, in turn, leads to the unconstrained activity of the small GTPase Rac1, which is known to have roles in actin reorganization and internalization of C. jejuni. Our results represent the identification of a host cell protein targeted by a flagellar secreted effector protein and demonstrate that C. jejuni-stimulated Rac signaling is dependent on IQGAP1.

Microbiota attenuates chicken transmission-exacerbated campylobacteriosis in Il10-/- mice.

Campylobacter jejuni is a prevalent foodborne pathogen mainly transmitting through poultry. It remains unknown how chicken-transmitted C. jejuni and microbiota impact on human campylobacteriosis. Campylobacter jejuni AR101 (Cj-P0) was introduced to chickens and isolated as passage 1 (Cj-P1). Campylobacter jejuni Cj-P1-DCA-Anaero was isolated from Cj-P0-infected birds transplanted with DCA-modulated anaerobic microbiota. Specific pathogen free Il10-/- mice were gavaged with antibiotic clindamycin and then infected with Cj-P0, Cj-P1, or Cj-P1-DCA-Anaero, respectively. After 8 days post infection, Il10-/- mice infected with Cj-P1 demonstrated severe morbidity and bloody diarrhea and the experiment had to be terminated. Cj-P1 induced more severe histopathology compared to Cj-P0, suggesting that chicken transmission increased C. jejuni virulence. Importantly, mice infected with Cj-P1-DCA-Anaero showed attenuation of intestinal inflammation compared to Cj-P1. At the cellular level, Cj-P1 induced more C. jejuni invasion and neutrophil infiltration into the Il10-/- mouse colon tissue compared to Cj-P0, which was attenuated with Cj-P1-DCA-Anaero. At the molecular level, Cj-P1 induced elevated inflammatory mediator mRNA accumulation of Il17a, Il1ß, and Cxcl1 in the colon compared to Cj-P0, while Cj-P1-DCA-Anaero showed reduction of the inflammatory gene expression. In conclusion, our data suggest that DCA-modulated anaerobes attenuate chicken-transmitted campylobacteriosis in mice and it is important to control the elevation of C. jejuni virulence during chicken transmission process.

A genomic concept in cellular interaction of clinical Campylobacter spp. with human epithelial colorectal adenocarcinoma cells.

This study aimed to realize the genomic concept of cellular interaction of clinical Campylobacter spp. with human epithelial colorectal adenocarcinoma cells. It was indicated that the mean adherence and invasion rate of C.jejuni isolates was significantly higher than C.coli and the highest adhesion rate among the C.jejuni and C.coli belonged to strains harboring 4 (flaA, cadF, peb1A, and flpA) and 3 (flaA, cadF, and peb1A) adherence genes, respectively, which indicates that the adhesion potential of C.coli and C.jejuni strains is associated with the coordinate function and cumulative effect of selected virulence-associated genes. The highest invasion rate in C.jejuni (10.3%) and C.coli (8.4%) isolates belonged to strains which concomitantly contained 3 (ciaB, iamA, and tlp1) and 2 (ciaB and iamA) invasion-associated genes which emphasizes on the cooperative roles of these genes in C.jejuni and C.coli invasion to Caco-2 cells. The toxicity of C.jejuni for Caco-2 cells was proved higher than that of C.coli. There was a positive correlation between adherence, invasion and toxicity of both C.jejuni and C.coli isolates. Moreover, the expression levels of CDT-producing genes in C.jejuni strains was significantly higher than that of C.coli. The average cytotoxicity of the strains with all three CDT-encoding genes (cdtA, cdtB and cdtC) was statistically higher than those lacking one or more CDT subunits. A crucial contribution of CdtB to the cytotoxicity of Campylobacter strains was detected. Following the treatment of epithelial cells with C.jejuni or C.coli, IL-8 and TNF-α were significantly increased compared to untreated Caco-2 cells, and the highest IL-8 expression was observed in both C.jejuni and C.coli expressing all CDTs (cdtA, cdtB, and cdtC). We, for the first time, indicated the major contribution of TLR2 and TLR4 in campylobacter initiation of pathogenesis, while increased invasiveness and cytotoxicity was significantly associated with the increased expression of TLR4 in C.jejuni isolates.

Campylobacter jejuni Cas9 Modulates the Transcriptome in Caco-2 Intestinal Epithelial Cells.

The zoonotic human pathogen Campylobacter jejuni is known for its ability to induce DNA-damage and cell death pathology in humans. The molecular mechanism behind this phenomenon involves nuclear translocation by Cas9, a nuclease in C. jejuni (CjeCas9) that is the molecular marker of the Type II CRISPR-Cas system. However, it is unknown via which cellular pathways CjeCas9 drives human intestinal epithelial cells into cell death. Here, we show that CjeCas9 released by C. jejuni during the infection of Caco-2 human intestinal epithelial cells directly modulates Caco-2 transcriptomes during the first four hours of infection. Specifically, our results reveal that CjeCas9 activates DNA damage (p53, ATM (Ataxia Telangiectasia Mutated Protein)), pro-inflammatory (NF-κB (Nuclear factor-κB)) signaling and cell death pathways, driving Caco-2 cells infected by wild-type C. jejuni, but not when infected by a cas9 deletion mutant, towards programmed cell death. This work corroborates our previous finding that CjeCas9 is cytotoxic and highlights on a RNA level the basal cellular pathways that are modulated.

Proteomics of Campylobacter jejuni Growth in Deoxycholate Reveals Cj0025c as a Cystine Transport Protein Required for Wild-type Human Infection Phenotypes.

Campylobacter jejuni is a major cause of food-borne gastroenteritis. Proteomics by label-based two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) identified proteins associated with growth in 0.1% sodium deoxycholate (DOC, a component of gut bile salts), and system-wide validation was performed by data-independent acquisition (DIA-SWATH-MS). LC-MS/MS quantified 1326 proteins (∼82% of the predicted C. jejuni proteome), of which 1104 were validated in additional biological replicates by DIA-SWATH-MS. DOC resulted in a profound proteome shift with 512 proteins showing significantly altered abundance. Induced proteins were associated with flagellar motility and antibiotic resistance; and these correlated with increased DOC motility and resistance to polymyxin B and ciprofloxacin. DOC also increased human Caco-2 cell adherence and invasion. Abundances of proteins involved in nutrient transport were altered by DOC and aligned with intracellular changes to their respective carbon sources. DOC increased intracellular levels of sulfur-containing amino acids (cysteine and methionine) and the dipeptide cystine (Cys-Cys), which also correlated with reduced resistance to oxidative stress. A DOC induced transport protein was Cj0025c, which has sequence similarity to bacterial Cys-Cys transporters. Deletion of cj0025c (Δcj0025c) resulted in proteome changes consistent with sulfur starvation, as well as attenuated invasion, reduced motility, atypical morphology, increased antimicrobial susceptibility and poor biofilm formation. Targeted metabolomics showed Δcj0025c could use known C. jejuni amino and organic acid substrates commensurate with wild-type. Medium Cys-Cys levels however, were maintained in Δcj0025c relative to wild-type. A toxic Cys-Cys mimic (selenocystine) inhibited wild-type growth, but not Δcj0025c Provision of an alternate sulfur source (2 mm thiosulfate) restored Δcj0025c motility. Our data confirm that Cj0025c is a Cys-Cys transporter that we have named TcyP consistent with the nomenclature of homologous proteins in other species.

Molecular Typing of Campylobacter jejuni and Campylobacter coli of Human Strains Isolated in Turkey Over an Eight-Year Period.

BACKGROUND: Campylobacter spp. is one of the leading causes of bacterial foodborne infections worldwide. In this study, we aimed to investigate the genetic diversity of 341 Campylobacter strains isolated in Turkey. METHODS: Campylobacter spp. was identified by phenotypical methods and PCR. Species level identification was carried out by the hippurate hydrolysis test and PCR. C. jejuni and C. coli strains were typed by using flaA-RFLP and PFGE. RESULTS: Of 341 strains, 300 (88%), 37 (10.8%), and four were identified as C. jejuni, C. coli, and non-jejuni/non-coli, respectively. The hippurate hydrolysis test misidentified 12% of 341 strains. The typeabilities of flaA-RFLP and PFGE were 100% for C. coli, whereas those of flaA-RFLP and PFGE for C. jejuni were 99.3% and 99%, respectively. The discriminatory power of the combination of PFGE and flaA-RFLP was determined to be higher than either method alone for both C. jejuni and C. coli. Both of the strains were so diverse that 80% and 64% of C. jejuni and C. coli genotypes included only one strain, respectively. In two patients, Campylobacter strains that were isolated from the first stool samples were C. jejuni where as those isolated from the second samples, collected eight and 20 days after the collection of the first samples, were C. coli. C. jejuni strains that were recovered from two different stool samples of two patients, collected 1 - 2 days apart, were found to be genetically different. CONCLUSIONS: Species identification of Campylobacter strains should be done using molecular methods. Combination of two methods is prerequisite for increasing the accuracy of molecular typing. Mixed or subsequent infection by different Campylobacter species and C. jejuni of different genotypes should not be underestimated.

Campylobacter concisus Impairs Sodium Absorption in Colonic Epithelium via ENaC Dysfunction and Claudin-8 Disruption.

The epithelial sodium channel (ENaC) can increase the colonic absorptive capacity for salt and water. Campylobacter concisus is a common pathogenic epsilonproteobacterium, causing enteritis and diarrhea. It can induce barrier dysfunction in the intestine, but its influence on intestinal transport function is still unknown. Therefore, our study aimed to characterize C. concisus effects on ENaC using the HT-29/B6-GR/MR (epithelial cell line HT-29/B6 transfected with glucocorticoid and mineralocorticoid receptors) cell model and mouse colon. In Ussing chambers, C. concisus infection inhibited ENaC-dependent Na+ transport as indicated by a reduction in amiloride-sensitive short circuit current (-55%, n = 15, p < 0.001). This occurred via down-regulation of ß- and γ-ENaC mRNA expression and ENaC ubiquitination due to extracellular signal-regulated kinase (ERK)1/2 activation, predicted by Ingenuity Pathway Analysis (IPA). In parallel, C. concisus reduced the expression of the sealing tight junction (TJ) protein claudin-8 and induced claudin-8 redistribution off the TJ domain of the enterocytes, which facilitates the back leakage of Na+ ions into the intestinal lumen. In conclusion, C. concisus caused ENaC dysfunction via interleukin-32-regulated ERK1/2, as well as claudin-8-dependent barrier dysfunction-both of which contribute to Na+ malabsorption and diarrhea.

N-glycosylation of the CmeABC multidrug efflux pump is needed for optimal function in Campylobacter jejuni.

Campylobacter jejuni is a prevalent gastrointestinal pathogen associated with increasing rates of antimicrobial resistance development. It was also the first bacterium demonstrated to possess a general N-linked protein glycosylation pathway capable of modifying > 80 different proteins, including the primary Campylobacter multidrug efflux pump, CmeABC. Here we demonstrate that N-glycosylation is necessary for the function of the efflux pump and may, in part, explain the evolutionary pressure to maintain this protein modification system. Mutants of cmeA in two common wildtype (WT) strains are highly susceptible to erythromycin (EM), ciprofloxacin and bile salts when compared to the isogenic parental strains. Complementation of the cmeA mutants with the native cmeA allele restores the WT phenotype, whereas expression of a cmeA allele with point mutations in both N-glycosylation sites is comparable to the cmeA mutants. Moreover, loss of CmeA glycosylation leads to reduced chicken colonization levels similar to the cmeA knock-out strain, while complementation fully restores colonization. Reconstitution of C. jejuni CmeABC into Escherichia coli together with the C. jejuni N-glycosylation pathway increases the EM minimum inhibitory concentration and decreases ethidium bromide accumulation when compared to cells lacking the pathway. Molecular dynamics simulations reveal that the protein structures of the glycosylated and non-glycosylated CmeA models do not vary from one another, and in vitro studies show no change in CmeA multimerization or peptidoglycan association. Therefore, we conclude that N-glycosylation has a broader influence on CmeABC function most likely playing a role in complex stability.

In vitro spleen cell cytokine responses of adult mice immunized with a recombinant PorA (major outer membrane protein [MOMP]) from Campylobacter jejuni.

There is no information on cytokine profiles for use as markers of protection in Campylobacter jejuni infection. To study this, we used outer membrane protein (MOMP [PorA]) as the vaccine for protection and spleen cell cytokines as markers of protection. We cloned and expressed porA from C. jejuni111 and immunized mice by the intraperitoneal route. Subsequently, mice were orally challenged with live C. jejuni 111. The vaccine induced protection as evidenced by reduced fecal excretion of C. jejuni111. Cytokines were measured in vitro after stimulation of spleen cells with MOMP. The levels of pro-inflammatory cytokines, IL-12, TNF-α, IL-17A and IL-17F were similar in control and test mice. The levels of pro-inflammatory cytokines, IL-2 and IFN-γ were higher in control mice than in test mice, and the levels of pro-inflammatory cytokines, IL-8 and IL-1ß were higher in test mice than in control mice. Among the two anti-inflammatory cytokines, the levels were similar for IL-10 but higher for IL-4 in test mice than in control mice. Ratios of pro-inflammatory to anti-inflammatory cytokines showed a bias towards an anti-inflammatory response in favor of antibody production reflecting the role of antibodies in immunity. Cytokine production patterns by spleen cells may be used as markers of protection in the mouse model.

Microbial metabolite deoxycholic acid shapes microbiota against Campylobacter jejuni chicken colonization.

Despite reducing the prevalent foodborne pathogen Campylobacter jejuni in chickens decreases campylobacteriosis, few effective approaches are available. The aim of this study was to use microbial metabolic product bile acids to reduce C. jejuni chicken colonization. Broiler chicks were fed with deoxycholic acid (DCA), lithocholic acid (LCA), or ursodeoxycholic acid (UDCA). The birds were also transplanted with DCA modulated anaerobes (DCA-Anaero) or aerobes (DCA-Aero). The birds were infected with human clinical isolate C. jejuni 81-176 or chicken isolate C. jejuni AR101. Notably, C. jejuni 81-176 was readily colonized intestinal tract at d16 and reached an almost plateau at d21. Remarkably, DCA excluded C. jejuni cecal colonization below the limit of detection at 16 and 28 days of age. Neither chicken ages of infection nor LCA or UDCA altered C. jejuni AR101 chicken colonization level, while DCA reduced 91% of the bacterium in chickens at d28. Notably, DCA diet reduced phylum Firmicutes but increased Bacteroidetes compared to infected control birds. Importantly, DCA-Anaero attenuated 93% of C. jejuni colonization at d28 compared to control infected birds. In conclusion, DCA shapes microbiota composition against C. jejuni colonization in chickens, suggesting a bidirectional interaction between microbiota and microbial metabolites.

Campylobacter jejuni bile exposure influences outer membrane vesicles protein content and bacterial interaction with epithelial cells.

Campylobacter jejuni is a prevalent human pathogen and a major cause of bacterial gastroenteritis in the world. In humans, C. jejuni colonizes the intestinal tract and its tolerance to bile is crucial for bacteria to survive and establish infection. C. jejuni produces outer membrane vesicles (OMVs) which have been suggested to be involved in virulence. In this study, the proteome composition of C. jejuni OMVs in response to low concentration of bile was investigated. We showed that exposure of C. jejuni to low concentrations of bile, similar to the concentration in cecum, induced significant changes in the protein profile of OMVs released during growth without affecting the protein profile of the bacteria. This suggests that bile influences a selective packing of the OMVs after bacterial exposure to low bile. A low concentration of bile was found to increase bacterial adhesion to intestinal epithelial cells, likely by an enhanced hydrophobicity of the cell membrane following exposure to bile. The increased bacterial adhesiveness was not associated with increased invasion, instead bile exposure decreased C. jejuni invasion. OMVs released from bacteria upon exposure to low bile showed to increase both adhesion and invasion of non-bile-exposed bacteria into intestinal epithelial cells. These findings suggest that C. jejuni in environments with low concentrations of bile produce OMVs that facilitates colonization of the bacteria, and this could potentially contribute to virulence of C. jejuni in the gut.

Pathway based therapeutic targets identification and development of an interactive database CampyNIBase of Campylobacter jejuni RM1221 through non-redundant protein dataset.

The bacterial species Campylobacter jejuni RM1221 (CjR) is the primary cause of campylobacteriosis which poses a global threat for human health. Over the years the efficacy of antibiotic treatment is becoming more fruitless due to the development of multiple drug resistant strains. Therefore, identification of new drug targets is a valuable tool for the development of new treatments for affected patients and can be obtained by targeting essential protein(s) of CjR. We conducted this in silico study in order to identify therapeutic targets by subtractive CjR proteome analysis. The most important proteins of the CjR proteome, which includes chokepoint enzymes, plasmid, virulence and antibiotic resistant proteins were annotated and subjected to subtractive analyses to filter out the CjR essential proteins from duplicate or human homologous proteins. Through the subtractive and characterization analysis we have identified 38 eligible therapeutic targets including 1 potential vaccine target. Also, 12 potential targets were found in interactive network, 5 targets to be dealt with FDA approved drugs and one pathway as potential pathway based drug target. In addition, a comprehensive database 'CampyNIBase' has also been developed. Besides the results of this study, the database is enriched with other information such as 3D models of the identified targets, experimental structures and Expressed Sequence Tag (EST) sequences. This study, including the database might be exploited for future research and the identification of effective therapeutics against campylobacteriosis. URL: (http://nib.portal.gov.bd/site/page/4516e965-8935-4129-8c3f-df95e754c562#Banner).

A novel mouse model of Campylobacter jejuni enteropathy and diarrhea.

Campylobacter infections are among the leading bacterial causes of diarrhea and of 'environmental enteropathy' (EE) and growth failure worldwide. However, the lack of an inexpensive small animal model of enteric disease with Campylobacter has been a major limitation for understanding its pathogenesis, interventions or vaccine development. We describe a robust standard mouse model that can exhibit reproducible bloody diarrhea or growth failure, depending on the zinc or protein deficient diet and on antibiotic alteration of normal microbiota prior to infection. Zinc deficiency and the use of antibiotics create a niche for Campylobacter infection to establish by narrowing the metabolic flexibility of these mice for pathogen clearance and by promoting intestinal and systemic inflammation. Several biomarkers and intestinal pathology in this model also mimic those seen in human disease. This model provides a novel tool to test specific hypotheses regarding disease pathogenesis as well as vaccine development that is currently in progress.

Cellular Tight Junctions Prevent Effective Campylobacter jejuni Invasion and Inflammatory Barrier Disruption Promoting Bacterial Invasion from Lateral Membrane in Polarized Intestinal Epithelial Cells.

Campylobacter jejuni invasion is closely related to C. jejuni pathogenicity. The intestinal epithelium contains polarized epithelial cells that form tight junctions (TJs) to provide a physical barrier against bacterial invasion. Previous studies indicated that C. jejuni invasion of non-polarized cells involves several cellular features, including lipid rafts. However, the dynamics of C. jejuni invasion of polarized epithelial cells are not fully understood. Here we investigated the interaction between C. jejuni invasion and TJ formation to characterize the mechanism of C. jejuni invasion in polarized epithelial cells. In contrast to non-polarized epithelial cells, C. jejuni invasion was not affected by depletion of lipid rafts in polarized epithelial cells. However, depletion of lipid rafts significantly decreased C. jejuni invasion in TJ disrupted cells or basolateral infection and repair of cellular TJs suppressed lipid raft-mediated C. jejuni invasion in polarized epithelial cells. In addition, pro-inflammatory cytokine, TNF-α treatment that induce TJ disruption promote C. jejuni invasion and lipid rafts depletion significantly reduced C. jejuni invasion in TNF-α treated cells. These data demonstrated that TJs prevent C. jejuni invasion from the lateral side of epithelial cells, where they play a main part in bacterial invasion and suggest that C. jejuni invasion could be increased in inflammatory condition. Therefore, maintenance of TJs integrity should be considered important in the development of novel therapies for C. jejuni infection.

Lactobacillus fermentum 3872 as a potential tool for combatting Campylobacter jejuni infections.

Due to the global spread of multidrug resistant pathogenic bacteria, alternative approaches in combating infectious diseases are required. One such approach is the use of probiotics. Lactobacillus fermentum 3872 is a promising probiotic bacterium producing a range of antimicrobial compounds, such as hydrogen peroxide and lactic acid. In addition, previous studies involving genome sequencing and analysis of L. fermentum 3872 allowed the identification of a gene encoding a cell surface protein referred to as collagen binding protein (CBP) (not found in other strains of the species, according to the GenBank database), consisting of a C-terminal cell wall anchor domain (LPXT), multiple repeats of 'B domains' that form stalks presenting an "A domain" required for adhesion. In this study, we found that the CBP of L. fermentum 3872 binds to collagen I present on the surface of the epithelial cells lining the gastrointestinal tract. Moreover, we found that this host receptor is also used for attachment by the major gastrointestinal pathogen, Campylobacter jejuni. Furthermore, we identified an adhesin involved in such interaction and demonstrated that both L. fermentum 3872 and its CBP can inhibit binding of this pathogen to collagen I. Combined with the observation that C. jejuni growth is affected in the acidic environment produced by L. fermentum 3872, the finding provides a good basis for further investigation of this strain as a potential tool for fighting Campylobacter infections.

Key Role of Capsular Polysaccharide in the Induction of Systemic Infection and Abortion by Hypervirulent Campylobacter jejuni.

Campylobacter jejuni is a zoonotic pathogen, and a hypervirulent clone, named clone SA, has recently emerged as the predominant cause of ovine abortion in the United States. To induce abortion, orally ingested Campylobacter must translocate across the intestinal epithelium, spread systemically in the circulation, and reach the fetoplacental tissue. Bacterial factors involved in these steps are not well understood. C. jejuni is known to produce capsular polysaccharide (CPS), but the specific role that CPS plays in systemic infection and particularly abortion in animals remains to be determined. In this study, we evaluated the role of CPS in bacteremia using a mouse model and in abortion using a pregnant guinea pig model following oral challenge. Compared with C. jejuni NCTC 11168 and 81-176, a clone SA isolate (IA3902) resulted in significantly higher bacterial counts and a significantly longer duration of bacteremia in mice. The loss of capsule production via gene-specific mutagenesis in IA3902 led to the complete abolishment of bacteremia in mice and abortion in pregnant guinea pigs, while complementation of capsule expression almost fully restored these phenotypes. The capsule mutant strain was also impaired for survival in guinea pig sera and sheep blood. Sequence-based analyses revealed that clone SA possesses a unique CPS locus with a mosaic structure, which has been stably maintained in all clone SA isolates derived from various hosts and times. These findings establish CPS as a key virulence factor for the induction of systemic infection and abortion in pregnant animals and provide a viable candidate for the development of vaccines against hypervirulent C. jejuni.