Capsaicin-sensitive vagal afferent neurons contribute to the detection of pathogenic bacterial colonization in the gut.

Vagal activation can reduce inflammation and disease activity in various animal models of intestinal inflammation via the cholinergic anti-inflammatory pathway. In the current model of this pathway, activation of descending vagal efferents is dependent on a signal initiated by stimulation of vagal afferents. However, little is known about how vagal afferents are activated, especially in the context of subclinical or clinical pathogenic bacterial infection. To address this question, we first determined if selective lesions of capsaicin-sensitive vagal afferents altered c-Fos expression in the nucleus of the solitary tract (nTS) after mice were inoculated with either Campylobacter jejuni or Salmonella typhimurium. Our results demonstrate that the activation of nTS neurons by intraluminal pathogenic bacteria is dependent on intact, capsaicin sensitive vagal afferents. We next determined if inflammatory mediators could cause the observed increase in c-Fos expression in the nTS by a direct action on vagal afferents. This was tested by the use of single-cell calcium measurements in cultured vagal afferent neurons. We found that tumor necrosis factor alpha (TNFα) and lipopolysaccharide (LPS) directly activate cultured vagal afferent neurons and that almost all TNFα and LPS responsive neurons were sensitive to capsaicin. We conclude that activation of the afferent arm of the parasympathetic neuroimmune reflex by pathogenic bacteria in the gut is dependent on capsaicin sensitive vagal afferent neurons and that the release of inflammatory mediators into intestinal tissue can be directly sensed by these neurons.

Examination of nanoparticle inactivation of Campylobacter jejuni biofilms using infrared and Raman spectroscopies.

AIMS: To investigate inactivation effect and mechanism of zinc oxide nanoparticles (ZnO NPs) activity against Campylobacter jejuni biofilms. METHODS AND RESULTS: ZnO NPs with concentrations of 0, 0·6, 1·2 and 6 mmol l(-1) were employed in antimicrobial tests against Camp. jejuni planktonic cells and biofilms. Campylobacter jejuni sessile cells in biofilms were more resistant to a low concentration of ZnO NPs when compared to planktonic cells. The ZnO NPs penetrated the extracellular polymeric substance (EPS) without damage to the EPS and directly interacted with the sessile bacterial cells, as determined using infrared spectroscopy and scanning electron microscopy. Raman spectroscopy shows alterations in quinone structures and damage to nucleic acids following Camp. jejuni treatment with ZnO NPs. The mechanism of DNA damage is most likely due to the generation of reactive oxygen species (ROS). Spectroscopic-based partial least squares regression (PLSR) models could predict the number of surviving sessile cell numbers within a bacterial biofilm (≥log 4 CFU, root mean square error of estimation <0·36) from Fourier transform infrared (FT-IR) spectral measurements. CONCLUSIONS: ZnO NPs were found to have antimicrobial activity against Camp. jejuni biofilms. ZnO NPs penetrated the biofilm EPS within 1 h without damaging it and interacted directly with sessile cells in biofilms. Alterations in the DNA/RNA bases, which are owing to the generation of ROS, appear to result in Camp. jejuni cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: ZnO NPs may offer a realistic strategy to eliminate Camp. jejuni biofilms in the environment.

The pathogenesis of Campylobacter jejuni-mediated enteritis.

Campylobacter jejuni, a gram-negative spiral shaped bacterium, is a frequent cause of gastrointestinal food-borne illness in humans throughout the world. Illness with C. jejuni ranges from mild to severe diarrheal disease. This article focuses on Campylobacter virulence determinants and their potential role in the development of C. jejuni-mediated enteritis. A model is presented that diagrams the interactions of C. jejuni with the intestinal epithelium. Additional work to identify and characterize C. jejuni virulence determinants is certain to provide novel insights into the diversity of strategies employed by bacterial pathogens to cause disease.

Role of Campylobacter jejuni potential virulence genes in cecal colonization.

Campylobacter jejuni, a common commensal in chickens, is one of the leading causes of bacterial gastroenteritis in humans worldwide. The aims of this investigation were twofold. First, we sought to determine whether mutations in the C. jejuni ciaB and pldA virulence-associated genes impaired the organism's ability to colonize chickens. Second, we sought to determine if inoculation of chicks with C. jejuni mutants could confer protection from subsequent challenge with the C. jejuni wild-type strain. The C. jejuni ciaB gene encodes a secreted protein necessary for the maximal invasion of C. jejuni into cultured epithelial cells, and the pldA gene encodes a protein with phospholipase activity. Also included in this study were two additional C. jejuni mutants, one harboring a mutation in cadF and the other in dnaJ, with which we have previously performed colonization studies. In contrast to results with the parental C. jejuni strain, viable organisms were not recovered from any of the chicks inoculated with the C. jejuni mutants. To determine if chicks inoculated with the C. jejuni mutants become resistant to colonization by the C. jejuni parental strain upon subsequent challenge, chicks were inoculated either intraperitoneally (i.p.) or both orally and i.p. with the C. jejuni mutants. Inoculated birds were then orally challenged with the parental strain. Inoculation with the C. jejuni mutants did not provide protection from subsequent challenge with the wild-type strain. In addition, neither the C. jejuni parental nor the mutant strains caused any apparent morbidity or mortality of the chicks. We conclude that mutations in genes cadF, dnaJ, pldA, and ciaB impair the ability of C. jejuni to colonize the cecum, that chicks tolerate massive inoculation with these mutant strains, and that such inoculations do not provide biologically significant protection against colonization by the parental strain.

Secretion of the virulence-associated Campylobacter invasion antigens from Campylobacter jejuni requires a stimulatory signal.

Campylobacter jejuni are a common cause of human diarrheal illness. Previous work has demonstrated that C. jejuni synthesize a novel set of proteins upon coculturing with epithelial cells, some of which are secreted. The secreted proteins have been collectively referred to as Campylobacter invasion antigens (Cia proteins). Metabolic labeling experiments revealed that Cia protein synthesis and secretion are separable and that secretion is the rate-limiting step of these processes. Additional work indicated that Cia protein synthesis is induced in response to bile salts and various eukaryotic host cell components. Host cell components also can induce Cia protein secretion. Culturing C. jejuni on plates supplemented with the bile salt deoxycholate retarded the inhibitory effect of chloramphenicol on C. jejuni invasion, as judged by the gentamicin-protection assay. These data suggest that the coordinate expression of the genes encoding the Cia proteins is subject to environmental regulation.

Identification of DT104 and U302 phage types among Salmonella enterica serotype typhimurium isolates by PCR.

A DNA sequence was identified in isolates of Salmonella enterica serotype Typhimurium definitive type 104 (DT104). The PCR amplification of an internal segment of this sequence identified DT104 and the closely related U302 phage type among 146 isolates of S. enterica serotype Typhimurium tested, thus providing a tool for rapid identification of DT104 and related isolates.

Temperature-regulated expression of bacterial virulence genes.

Virulence gene expression in most bacteria is a highly regulated phenomenon, affected by a variety of parameters including osmolarity, pH, ion concentration, iron levels, growth phase, and population density. Virulence genes are also regulated by temperature, which acts as an 'on-off' switch in a manner distinct from the more general heat-shock response. Here, we review temperature-responsive expression of virulence genes in four diverse pathogens.

Flow cytometric detection of host cell apoptosis induced by bacterial infection.

We detail two methods for detection of cell death induced by infection of a human monocytic cell line with invasive Campylobacter bacteria. Staining with a natural ligand for exposed phosphatidylserine residues coupled with propidum iodide discriminated between apoptosis and necrosis. Additionally, cells infected with a bacterial strain expressing green fluorescent protein stained with dye sensitive to mitochondrial membrane potential demonstrated a direct association of bacteria with dying cells. Analyses of cells stained by these methods employing flow cytometry enumerated proportions of cell populations undergoing either apoptosis or necrosis after bacterial infection in vitro.

The absence of cecal colonization of chicks by a mutant of Campylobacter jejuni not expressing bacterial fibronectin-binding protein.

Campylobacter jejuni is a common cause of human gastrointestinal illness throughout the world. Infections with C. jejuni and Campylobacter coli are frequently acquired by eating undercooked chicken. The ability of C. jejuni to become established in the gastrointestinal tract of chickens is believed to involve binding of the bacterium to the gastrointestinal surface. A 37-kD outer membrane protein, termed CadF, has been described that facilitates the binding of Campylobacter to fibronectin. This study was conducted to determine whether the CadF protein is required for C. jejuni to colonize the cecum of newly hatched chicks. Day-of-hatch chicks were orally challenged with C. jejuni F38011, a human clinical isolate, or challenged with a mutant in which the cadF gene was disrupted via homologous recombination with a suicide vector. This method of mutagenesis targets a predetermined DNA sequence and does not produce random mutations in unrelated genes. The parental C. jejuni F38011 readily colonized the cecum of newly hatched chicks. In contrast, the cadF mutant was not recovered from any of 60 chicks challenged, indicating that disruption of the cadF gene renders C. jejuni incapable of colonizing the cecum. CadF protein appears to be required for the colonization of newly hatched leghorn chickens.

Bacterial secreted proteins are required for the internaliztion of Campylobacter jejuni into cultured mammalian cells.

Presented here is the first evidence that Campylobacter jejuni secrete proteins upon co-cultivation with host cells and in INT 407 cell-conditioned medium. A C. jejuni gene designated ciaB for Campylobacter invasion antigen B was identified, using a differential screening technique, which is required for this secretion process and the efficient entry of this bacterium into a host cell. The C. jejuni ciaB gene encodes a protein of 610 amino acids with a calculated molecular mass of 73 154 Da. The deduced amino acid sequence of the CiaB protein shares similarity with type III secreted proteins associated with the invasion of host cells from other more extensively characterized bacterial pathogens. In vitro binding and internalization assays revealed that the binding of C. jejuni ciaB null mutants was indistinguishable from that of the parental isolate, whereas a significant reduction was noted in internalization. Confocal microscopic examination of C. jejuni-infected cells revealed that CiaB was translocated into the cytoplasm of the host cells. Culturing C. jejuni with INT 407 cells or in INT 407-conditioned medium resulted in the secretion of at least eight proteins, ranging in size from 12.8 to 108 kDa, into the culture medium. C. jejuni ciaB null mutants were deficient in the secretion of all eight proteins, indicating that CiaB is required for the secretion process. The identification of the C. jejuni ciaB gene represents a significant advance in understanding the molecular mechanism of C. jejuni internalization and the pathogenesis of C. jejuni-mediated enteritis.

Identification of the enteropathogens Campylobacter jejuni and Campylobacter coli based on the cadF virulence gene and its product.

Campylobacter jejuni and Campylobacter coli are common causes of gastroenteritis in humans. Infection with C. jejuni or C. coli is commonly acquired by eating undercooked chicken. The goal of this study was to develop specific detection assays for C. jejuni and C. coli isolates based on the cadF virulence gene and its product. The cadF gene from C. jejuni and C. coli encodes a 37-kDa outer membrane protein that promotes the binding of these pathogens to intestinal epithelial cells. A fragment of approximately 400 bp was amplified from 38 of 40 (95%) C. jejuni isolates and 5 of 6 (83.3%) C. coli isolates with primers designed to amplify an internal fragment of the cadF gene. PCR was then used to amplify Campylobacter DNA from store-bought chickens. A 400-bp band was amplified from 26 of the 27 chicken carcasses tested by the PCR-based assay. The CadF protein was detected in every C. jejuni and C. coli isolate tested, as judged by immunoblot analysis with a rabbit anti-C. jejuni 37-kDa serum. In addition, methanol-fixed samples of whole-cell C. jejuni and C. coli were detected with the rabbit anti-37-kDa serum by using an indirect-immunofluorescence microscopy assay. These findings indicate that the cadF gene and its product are conserved among C. jejuni and C. coli isolates and that a PCR assay based on the cadF gene may be useful for the detection of Campylobacter organisms in food products.

Identification of proteins required for the internalization of Campylobacter jejuni into cultured mammalian cells.

Clinical and in vitro experimental data suggest that invasion of intestinal epithelial cells is an essential step in the pathogenesis of Campylobacter jejuni-mediated enteritis. However, the molecular mechanism of C. jejuni internalization remains poorly defined. The goal of this study was to identify a gene that encodes a protein required for the internalization of C. jejuni into host cells. A C. jejuni gene, designated ciaB, was identified upon immunoscreening C. jejuni genomic DNA-phage libraries with an antiserum generated against C. jejuni co-cultivated with INT 407 cells. The C. jejuni ciaB gene encodes a protein of 610 amino acids with a calculated molecular mass of 73,154 Da. The deduced amino acid sequence of the CiaB protein shares similarity with type III secreted proteins, associated with invasion of host cells, from other more extensively characterized bacterial pathogens. In vitro binding and internalization assays revealed that the binding of C. jejuni ciaB null mutants was indistinguishable from that of the parental isolate, whereas a significant reduction was noted in internalization. Immunoblot analysis using an anti-CiaB specific antibody revealed that CiaB is secreted into the supernatant fluids upon co-cultivation of C. jejuni with INT 407 cell conditioned medium. Metabolic labeling experiments revealed that at least eight C. jejuni proteins, ranging in size from 12.8 to 108 kDa, are secreted into the culture medium. C. jejuni ciaB null mutants were deficient in the secretion of all proteins, indicating that CiaB is required for the secretion process. Identification of the C. jejuni ciaB gene represents a significant advance in understanding the molecular mechanism of C. jejuni internalization.

Secretion of Campylobacter jejuni Cia proteins is contact dependent.

Campylobacter jejuni is a common cause of human gastrointestinal disease worldwide. Despite the prevalence of C. jejuni infections, the mechanisms of C. jejuni pathogenesis remain ill-defined. Invasion of the cells lining the intestinal tract is hypothesized to be essential for the development of C. jejuni-mediated enteritis. Recent studies in our laboratory have revealed that C. jejuni secrete proteins, termed Cia for Campylobacter invasion antigens, upon incubation with human intestinal cells. A mutation in one of the genes encoding a secreted protein resulted in an invasion-deficient phenotype. The purpose of this study was to identify a component capable of stimulating the synthesis and secretion of the Cia proteins from C. jejuni. Here, we report that these processes can be induced upon incubating C. jejuni in medium supplemented with fetal bovine serum. The synthesis and secretion of the Cia proteins were not affected by heat-treatment of the fetal bovine serum, indicating that the stimulating molecule in serum is heat stable. The stimulatory molecule was not unique to fetal bovine serum as sera from other sources including human, pig, sheep, goat, rabbit, mouse, and chicken also induced the synthesis and release of the Cia proteins. These findings indicate that the synthesis and secretion of the Cia proteins can be induced in a cell-free system by incubating C. jejuni in serum-supplemented tissue culture medium.

Codon usage in the A/T-rich bacterium Campylobacter jejuni.

Campylobacter jejuni is a Gram negative, microaerophilic pathogen that causes gastroenteritis in humans. The genome of C. jejuni is AT-rich, with a mol% G + C of 30.4. This high AT content was hypothesized to result in unique codon usage. In the present study, we analyzed the codon usage of sixty-seven C. jejuni genes and generated a codon frequency table. As predicted, the codon usage of C. jejuni revealed a strong bias towards codons ending in A or U. In addition to determining codon usage frequencies, the relative synonymous codon usage values were calculated to identify rare and optimal codons. Seventeen codons were identified as optimal and twelve codons as rare. Thirty-two codons exhibited little or no bias. A plot of the effective number of codons versus the third position %G + C values for the sixty-seven genes revealed that C. jejuni uses an average of 39 of the 61 codons to encode proteins. These data will be useful for various molecular analyses including selection of degenerate primers to screen C. jejuni-genomic DNA libraries.

Cloning, sequencing, and characterization of the lipopolysaccharide biosynthetic enzyme heptosyltransferase I gene (waaC) from Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni and Campylobacter coli are common causes of gastrointestinal disease and a proportion of C. jejuni infections have been shown to be associated with the Guillain-Barré syndrome. The waaC gene from Campylobacter coli, involved in lipopolysaccharide core biosynthesis, was cloned by complementation of a heptose-deficient strain of Salmonella typhimurium, as judged by novobiocin sensitivity, lipopolysaccharide (LPS)-specific phage sensitivity, and polyacrylamide-resolved lipopolysaccharide profiles. The C. jejuni waaC gene was subsequently cloned using the waaC gene isolated from C. coli as a probe. The C. jejuni and C. coli waaC genes are capable of encoding proteins of 342 amino acids with calculated molecular masses of 39381Da and 39317Da, respectively. Sequence and in-vitro analyses suggested that the C. coli waaC gene may be transcribed from its own promoter. Translation of the C. coli waaC gene in a cell-free system yielded a protein with a Mr of 39000. The waaC gene was detected in every C. jejuni and C. coli isolate tested as judged by dot-blot hybridization analysis. Southern hybridization analysis indicated that both Campylobacter species contain a single copy of the waaC gene. Unlike Escherichia coli and S. typhimurium isolates, the waaC gene in C. jejuni and C. coli isolates does not appear to be linked to the waaF (rfaF) gene.

Characterization of the thermal stress response of Campylobacter jejuni.

Campylobacter jejuni, a microaerophilic, gram-negative bacterium, is a common cause of gastrointestinal disease in humans. Heat shock proteins are a group of highly conserved, coregulated proteins that play important roles in enabling organisms to cope with physiological stresses. The primary aim of this study was to characterize the heat shock response of C. jejuni. Twenty-four proteins were preferentially synthesized by C. jejuni immediately following heat shock. Upon immunoscreening of Escherichia coli transformants harboring a Campylobacter genomic DNA library, one recombinant plasmid that encoded a heat shock protein was isolated. The recombinant plasmid, designated pMEK20, contained an open reading frame of 1,119 bp that was capable of encoding a protein of 372 amino acids with a calculated molecular mass of 41,436 Da. The deduced amino acid sequence of the open reading frame shared similarity with that of DnaJ, which belongs to the Hsp-40 family of molecular chaperones, from a number of bacteria. An E. coli dnaJ mutant was successfully complemented with the pMEK20 recombinant plasmid, as judged by the ability of bacteriophage lambda to form plaques, indicating that the C. jejuni gene encoding the 41-kDa protein is a functional homolog of the dnaJ gene from E. coli. The ability of each of two C. jejuni dnaJ mutants to form colonies at 46 degreesC was severely retarded, indicating that DnaJ plays an important role in C. jejuni thermotolerance. Experiments revealed that a C. jejuni DnaJ mutant was unable to colonize newly hatched Leghorn chickens, suggesting that heat shock proteins play a role in vivo.

Identification and molecular cloning of a gene encoding a fibronectin-binding protein (CadF) from Campylobacter jejuni.

Campylobacter jejuni, a Gram-negative bacterium, is a common cause of gastrointestinal disease. By analogy with other enteric pathogens such as Salmonella and Shigella, the ability of C. jejuni to bind to host cells is thought to be essential in the pathogenesis of enteritis. Scanning electron microscopy of infected INT407 cells suggested that C. jejuni bound to a component of the extracellular matrix. Binding assays using immobilized extracellular matrix proteins and soluble fibronectin showed specific and saturable binding of fibronectin to C. jejuni. Ligand immunoblot assays using 125I-labelled fibronectin revealed specific binding to an outer membrane protein with an apparent molecular mass of 37 kDa. A rabbit antiserum, raised against the gel-purified protein, reacted with a 37 kDa protein in all C. jejuni isolates (n = 15) as tested by immunoblot analysis. Antibodies present in convalescent serum from C. jejuni-infected individuals also recognized a 37 kDa protein. The gene encoding the immunoreactive 37kDa protein was cloned and sequenced. Sequencing of overlapping DNA fragments revealed an open reading frame (ORF) that encodes a protein of 326 amino acids with a calculated molecular mass of 36872Da. The deduced amino acid sequence of the ORF exhibited 52% similarity and 28% identity to the root adhesin protein from Pseudomonas fluorescens. Isogenic C. jejuni mutants which lack the 37 kDa outer membrane protein, which we have termed CadF, displayed significantly reduced binding to fibronectin. Biotinylated fibronectin bound to a protein with an apparent molecular mass of 37 kDa in the outer membrane protein extracts from wild-type C. jejuni as judged by ligand-binding blots. These results indicate that the binding of C. jejuni to fibronectin is mediated by the 37 kDa outer membrane protein which is conserved among C. jejuni isolates.

Identification of a functional homolog of the Escherichia coli and Salmonella typhimurium cysM gene encoding O-acetylserine sulfhydrylase B in Campylobacter jejuni.

The final step of L-cysteine biosynthesis in Escherichia coli and Salmonella typhimurium consists of the formation of L-cysteine from O-acetylserine and sulfide. This reaction can be catalyzed by two enzymes, O-acetylserine sulfhydrylase A and O-acetylserine sulfhydrylase B, the former of which has been more rigorously characterized. In contrast to O-acetylserine sulfhydrylase A, O-acetylserine sulfhydrylase B is preferentially used for cysteine biosynthesis during anaerobic growth and is able to utilize thiosulfate as a substrate. Campylobacter jejuni is a micro-aerophilic, Gram-negative bacterium, and a member of the epsilon subdivision of eubacteria. We have cloned, sequenced, and expressed a gene from C. jejuni that encodes a protein of 299 aa with a calculated molecular mass of 32,367 Da. Complementation analysis of an E. coli cysteine auxotroph with the pMEK34-14 recombinant plasmid containing a 1.2-kb insert of chromosomal DNA from C. jejuni revealed that transformants were capable of growth in medium containing either sulfide or thiosulfate as sole sulfur sources. These data indicate that the cloned C. jejuni gene is a functional homolog of the cysM gene that codes for O-acetylserine sulfhydrylase B in E. coli and S. typhimurium.

GlpQ: an antigen for serological discrimination between relapsing fever and Lyme borreliosis.

Tick-borne relapsing fever is caused by numerous Borrelia species maintained in nature by Ornithodoros tick-mammal cycles. Serological confirmation is based on either an immunofluorescence assay or an enzyme-linked immunosorbent assay using whole cells or sonicated Borrelia hermsii as the antigen. However, antigenic variability of this bacterium's outer surface proteins and antigens shared with the Lyme disease spirochete (B. burgdorferi), may cause both false-negative and false-positive results when testing sera of patients suspected to have either relapsing fever or Lyme disease. To develop a specific serological test for relapsing fever, we created a genomic DNA library of B. hermsii, screened transformed Escherichia coli cells for immunoreactivity with high-titered (> or = 1:2,048) human anti-B. hermsii antiserum, and selected an immunoreactive clone (pSPR75) expressing a 39-kDa protein. DNA sequencing, subcloning, and serum adsorption experiments identified the immunoreactive protein as a homolog of GlpQ, a glycerophosphodiester phosphodiesterase identified previously in E. coli, Haemophilus influenzae, and Bacillus subtilis. Serum samples from humans and mice infected with B. hermsii or other species of relapsing fever spirochetes contained antibodies recognizing GlpQ, whereas serum samples from Lyme disease and syphilis patients were nonreactive. Serologic tests based on this antigen will identify people exposed previously to relapsing fever spirochetes and help clarify the distribution of relapsing fever and Lyme disease in situations in which the occurrence of their causative agents is uncertain.