The Intestinal Microbiota Influences Campylobacter jejuni Colonization and Extraintestinal Dissemination in Mice.

Campylobacter jejuni is a leading cause of human foodborne gastroenteritis worldwide. The interactions between this pathogen and the intestinal microbiome within a host are of interest as endogenous intestinal microbiota mediates a form of resistance to the pathogen. This resistance, termed colonization resistance, is the ability of commensal microbiota to prevent colonization by exogenous pathogens or opportunistic commensals. Although mice normally demonstrate colonization resistance to C. jejuni, we found that mice treated with ampicillin are colonized by C. jejuni, with recovery of Campylobacter from the colon, mesenteric lymph nodes, and spleen. Furthermore, there was a significant reduction in recovery of C. jejuni from ampicillin-treated mice inoculated with a C. jejuni virulence mutant (ΔflgL strain) compared to recovery of mice inoculated with the C. jejuni wild-type strain or the C. jejuni complemented isolate (ΔflgL/flgL). Comparative analysis of the microbiota from nontreated and ampicillin-treated CBA/J mice led to the identification of a lactic acid-fermenting isolate of Enterococcus faecalis that prevented C. jejuni growth in vitro and limited C. jejuni colonization of mice. Next-generation sequencing of DNA from fecal pellets that were collected from ampicillin-treated CBA/J mice revealed a significant decrease in diversity of operational taxonomic units (OTUs) compared to that in control (nontreated) mice. Taken together, we have demonstrated that treatment of mice with ampicillin alters the intestinal microbiota and permits C. jejuni colonization. These findings provide valuable insights for researchers using mice to investigate C. jejuni colonization factors, virulence determinants, or the mechanistic basis of probiotics.

Analysis of the Campylobacter jejuni genome by SMRT DNA sequencing identifies restriction-modification motifs.

Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. The goal of this study was to analyze the C. jejuni F38011 strain, recovered from an individual with severe enteritis, at a genomic and proteomic level to gain insight into microbial processes. The C. jejuni F38011 genome is comprised of 1,691,939 bp, with a mol.% (G+C) content of 30.5%. PacBio sequencing coupled with REBASE analysis was used to predict C. jejuni F38011 genomic sites and enzymes that may be involved in DNA restriction-modification. A total of five putative methylation motifs were identified as well as the C. jejuni enzymes that could be responsible for the modifications. Peptides corresponding to the deduced amino acid sequence of the C. jejuni enzymes were identified using proteomics. This work sets the stage for studies to dissect the precise functions of the C. jejuni putative restriction-modification enzymes. Taken together, the data generated in this study contributes to our knowledge of the genomic content, methylation profile, and encoding capacity of C. jejuni.

Reducing Campylobacter jejuni colonization of poultry via vaccination.

Campylobacter jejuni is a leading bacterial cause of human gastrointestinal disease worldwide. While C. jejuni is a commensal organism in chickens, case-studies have demonstrated a link between infection with C. jejuni and the consumption of foods that have been cross-contaminated with raw or undercooked poultry. We hypothesized that vaccination of chickens with C. jejuni surface-exposed colonization proteins (SECPs) would reduce the ability of C. jejuni to colonize chickens, thereby reducing the contamination of poultry products at the retail level and potentially providing a safer food product for consumers. To test our hypothesis, we injected chickens with recombinant C. jejuni peptides from CadF, FlaA, FlpA, CmeC, and a CadF-FlaA-FlpA fusion protein. Seven days following challenge, chickens were necropsied and cecal contents were serially diluted and plated to determine the number of C. jejuni per gram of material. The sera from the chickens were also analyzed to determine the concentration and specificity of antibodies reactive against the C. jejuni SECPs. Vaccination of chickens with the CadF, FlaA, and FlpA peptides resulted in a reduction in the number of C. jejuni in the ceca compared to the non-vaccinated C. jejuni-challenged group. The greatest reduction in C. jejuni colonization was observed in chickens injected with the FlaA, FlpA, or CadF-FlaA-FlpA fusion proteins. Vaccination of chickens with different SECPs resulted in the production of C. jejuni-specific IgY antibodies. In summary, we show that the vaccination of poultry with individual C. jejuni SECPs or a combination of SECPs provides protection of chickens from C. jejuni colonization.

The focal complex of epithelial cells provides a signalling platform for interleukin-8 induction in response to bacterial pathogens.

Bacterial pathogens can induce an inflammatory response from epithelial tissues due to secretion of the pro-inflammatory chemokine interleukin-8 (IL-8). Many bacterial pathogens manipulate components of the focal complex (FC) to induce signalling events in host cells. We examined the interaction of several bacterial pathogens with host cells, including Campylobacter jejuni, to determine if the FC is required for induction of chemokine signalling in response to bacterial pathogens. Our data indicate that secretion of IL-8 is triggered by C. jejuni, Helicobacter pylori and Salmonella enterica serovar Typhimurium in response to engagement of ß1 integrins. Additionally, we found that the secretion of IL-8 from C. jejuni infected epithelial cells requires FAK, Src and paxillin, which in turn are necessary for Erk 1/2 recruitment and activation. Targeting the FC component paxillin with siRNA prevented IL-8 secretion from cells infected with several bacterial pathogens, including C. jejuni, Helicobacter pylori, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio parahaemolyticus. Our findings indicate that maximal IL-8 secretion from epithelial cells in response to bacterial infection is dependent on the FC. Based on the commonality of the host response to bacterial pathogens, we propose that the FC is a signalling platform for an epithelial cell response to pathogenic organisms.

Investigating the responses of Cronobacter sakazakii to garlic-drived organosulfur compounds: a systematic study of pathogenic-bacterium injury by use of high-throughput whole-transcriptome sequencing and confocal micro-raman spectroscopy.

We present the results of a study using high-throughput whole-transcriptome sequencing (RNA-seq) and vibrational spectroscopy to characterize and fingerprint pathogenic-bacterium injury under conditions of unfavorable stress. Two garlic-derived organosulfur compounds were found to be highly effective antimicrobial compounds against Cronobacter sakazakii, a leading pathogen associated with invasive infection of infants and causing meningitis, necrotizing entercolitis, and bacteremia. RNA-seq shows changes in gene expression patterns and transcriptomic response, while confocal micro-Raman spectroscopy characterizes macromolecular changes in the bacterial cell resulting from this chemical stress. RNA-seq analyses showed that the bacterial response to ajoene differed from the response to diallyl sulfide. Specifically, ajoene caused downregulation of motility-related genes, while diallyl sulfide treatment caused an increased expression of cell wall synthesis genes. Confocal micro-Raman spectroscopy revealed that the two compounds appear to have the same phase I antimicrobial mechanism of binding to thiol-containing proteins/enzymes in bacterial cells generating a disulfide stretching band but different phase II antimicrobial mechanisms, showing alterations in the secondary structures of proteins in two different ways. Diallyl sulfide primarily altered the α-helix and ß-sheet, as reflected in changes in amide I, while ajoene altered the structures containing phenylalanine and tyrosine. Bayesian probability analysis validated the ability of principal component analysis to differentiate treated and control C. sakazakii cells. Scanning electron microscopy confirmed cell injury, showing significant morphological variations in cells following treatments by these two compounds. Findings from this study aid in the development of effective intervention strategies to reduce the risk of C. sakazakii contamination in the food production environment and on food contact surfaces, reducing the risks to susceptible consumers.

Invasion of epithelial cells by Campylobacter jejuni is independent of caveolae.

Caveolae are 25-100 nm flask-like membrane structures enriched in cholesterol and glycosphingolipids. Researchers have proposed that Campylobacter jejuni require caveolae for cell invasion based on the finding that treatment of cells with the cholesterol-depleting compounds filipin III or methyl-ß-cyclodextrin (MßCD) block bacterial internalization in a dose-dependent manner. The purpose of this study was to determine the role of caveolae and caveolin-1, a principal component of caveolae, in C. jejuni internalization. Consistent with previous work, we found that the treatment of HeLa cells with MßCD inhibited C. jejuni internalization. However, we also found that the treatment of HeLa cells with caveolin-1 siRNA, which resulted in greater than a 90% knockdown in caveolin-1 protein levels, had no effect on C. jejuni internalization. Based on this observation we performed a series of experiments that demonstrate that MßCD acts broadly, disrupting host cell lipid rafts and C. jejuni-induced cell signaling. More specifically, we found that MßCD inhibits the cellular events necessary for C. jejuni internalization, including membrane ruffling and Rac1 GTPase activation. We also demonstrate that MßCD disrupted the association of the ß1 integrin and EGF receptor, which are required for the maximal invasion of epithelial cells. In agreement with these findings, C. jejuni were able to invade human Caco-2 cells, which are devoid of caveolae, at a level equal to that of HeLa cells. Taken together, the results of our study demonstrate that C. jejuni internalization occurs in a caveolae-independent manner.

The Campylobacter jejuni CiaD effector protein activates MAP kinase signaling pathways and is required for the development of disease.

BACKGROUND: Enteric pathogens utilize a distinct set of proteins to modulate host cell signaling events that promote host cell invasion, induction of the inflammatory response, and intracellular survival. Human infection with Campylobacter jejuni, the causative agent of campylobacteriosis, is characterized by diarrhea containing blood and leukocytes. The clinical presentation of acute disease, which is consistent with cellular invasion, requires the delivery of the Campylobacter invasion antigens (Cia) to the cytosol of host cells via a flagellar Type III Secretion System (T3SS). We identified a novel T3SS effector protein, which we termed CiaD that is exported from the C. jejuni flagellum and delivered to the cytosol of host cells. RESULTS: We show that the host cell kinases p38 and Erk 1/2 are activated by CiaD, resulting in the secretion of interleukin-8 (IL-8) from host cells. Additional experiments revealed that CiaD-mediated activation of p38 and Erk 1/2 are required for maximal invasion of host cells by C. jejuni. CiaD contributes to disease, as evidenced by infection of IL-10 knockout mice. Noteworthy is that CiaD contains a Mitogen-activated protein (MAP) kinase-docking site that is found within effector proteins produced by other enteric pathogens. These findings indicate that C. jejuni activates the MAP kinase signaling pathways Erk 1/2 and p38 to promote cellular invasion and the release of the IL-8 pro-inflammatory chemokine. CONCLUSIONS: The identification of a novel T3SS effector protein from C. jejuni significantly expands the knowledge of virulence proteins associated with C. jejuni pathogenesis and provides greater insight into the mechanism utilized by C. jejuni to invade host cells.

The fibronectin-binding motif within FlpA facilitates Campylobacter jejuni adherence to host cell and activation of host cell signaling.

Campylobacter jejuni is a gram-negative, curved and rod-shaped bacterium that causes human gastroenteritis. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Epithelial cells infected with C. jejuni strains containing mutations in the FlpA and CadF fibronectin (Fn)-binding proteins exhibit reduced invasion of host cells and a C. jejuni CadF FlpA double mutant is impaired in the activation of epidermal growth factor receptor (EGFR) and Rho GTPase Rac1. Although these observations establish a role for Fn-binding proteins during C. jejuni invasion, their mechanistic contributions to invasion-associated signaling are unclear. We examined FlpA, a C. jejuni Fn-binding protein composed of three FNIII-like repeats D1, D2 and D3, to identify the interactions required for cellular adherence on pathogen-induced host cell signaling. We report that FlpA binds the Fn gelatin-binding domain via a motif within the D2 repeat. Epithelial cells infected with a flpA mutant exhibited decreased Rac1 activation and reduced membrane ruffling that coincided with impaired delivery of the secreted Cia proteins and reduced cell association. Phosphorylation of the Erk1/2 kinase, a downstream effector of EGFR signaling, was specifically associated with FlpA-mediated activation of ß1-integrin and EGFR signaling. In vivo experiments revealed that FlpA is necessary for C. jejuni disease based on bacterial dissemination to the spleen of IL-10(-/-) germ-free mice. Thus, a novel Fn-binding motif within FlpA potentiates activation of Erk1/2 signaling via ß1-integrin during C. jejuni infection.

The cooperative action of bacterial fibronectin-binding proteins and secreted proteins promote maximal Campylobacter jejuni invasion of host cells by stimulating membrane ruffling.

This study was performed to elucidate the host cell scaffolding and signalling molecules that Campylobacter jejuni utilizes to invade epithelial cells. We hypothesized that the C. jejuni fibronectin-binding proteins and secreted proteins are required for cell signalling and maximal invasion of host cells. C. jejuni binding to host cells via the CadF and FlpA fibronectin-binding proteins activated the epidermal growth factor (EGF) pathway, as evidenced by inhibitor studies and immunoprecipitation coupled with immunoblot analysis using antibodies reactive against total and active EGF receptor. Inhibitor studies revealed maximal C. jejuni host cell invasion was dependent upon PI3-Kinase, c-Src and focal adhesion kinase (FAK), all of which are known to participate in cytoskeletal rearrangements. Knockdown of endogenous Dock180, which is a Rac1-specific guanine nucleotide exchange factor, using siRNA revealed that C. jejuni invasion was significantly reduced compared with cells treated with scrambled siRNA. We further demonstrated that the C. jejuni Cia proteins are, in part, responsible for Rho GTPase Rac1 recruitment and activation, as judged by immunofluorescence microscopy and Rac1 activation. Based on these data, we present a model that illustrates that C. jejuni utilizes a coordinated mechanism involving both adhesins and secreted proteins to promote membrane ruffling and host cell invasion.