Contribution of Conserved ATP-Dependent Proteases of Campylobacter jejuni to Stress Tolerance and Virulence

In prokaryotic cells the ATP-dependent proteases Lon and ClpP (Clp proteolytic subunit) are involved in the turnover of misfolded proteins and the degradation of regulatory proteins, and depending on the organism, these proteases contribute variably to stress tolerance. We constructed mutants in the lon and clpP genes of the food-borne human pathogen Campylobacter jejuni and found that the growth of both mutants was impaired at high temperature, a condition known to increase the level of misfolded protein. Moreover, the amounts of misfolded protein aggregates were increased when both proteases were absent, and we propose that both ClpP and Lon are involved in eliminating misfolded proteins in C. jejuni. In order to bind misfolded protein, ClpP has to associate with one of several Clp ATPases. Following inactivation of the ATPase genes clpA and clpX, only the clpX mutant displayed the same heat sensitivity as the clpP mutant, indicating that the ClpXP proteolytic complex is responsible for the degradation of heat-damaged proteins in C. jejuni. Notably, ClpP and ClpX are required for growth at 42°C, which is the temperature of the intestinal tract of poultry, one of the primary carriers of C. jejuni. Thus, ClpP and ClpX may be suitable targets of new intervention strategies aimed at reducing C. jejuni in poultry production. Further characterization of the clpP and lon mutants revealed other altered phenotypes, such as reduced motility, less autoagglutination, and lower levels of invasion of INT407 epithelial cells, suggesting that the proteases may contribute to the virulence of C. jejuni.     

Heat Shock-Enhanced Conjugation Efficiency in Standard Campylobacter jejuni Strains

Campylobacter jejuni, the leading bacterial cause of human gastroenteritis in the United States, displays significant strain diversity due to horizontal gene transfer. Conjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance. It has been observed that heat shock could increase transformation efficiency in some bacteria. In this study, the effect of heat shock on C. jejuni conjugation efficiency and the underlying mechanisms were examined. With a modified Escherichia coli donor strain, different C. jejuni recipient strains displayed significant variation in conjugation efficiency ranging from 6.2 × 10⁻⁸ to 6.0 × 10⁻³ CFU per recipient cell. Despite reduced viability, heat shock of standard C. jejuni NCTC 11168 and 81-176 strains (e.g., 48 to 54°C for 30 to 60 min) could dramatically enhance C. jejuni conjugation efficiency up to 1,000-fold. The phenotype of the heat shock-enhanced conjugation in C. jejuni recipient cells could be sustained for at least 9 h. Filtered supernatant from the heat shock-treated C. jejuni cells could not enhance conjugation efficiency, which suggests that the enhanced conjugation efficiency is independent of secreted substances. Mutagenesis analysis indicated that the clustered regularly interspaced short palindromic repeats system and the selected restriction-modification systems (Cj0030/Cj0031, Cj0139/Cj0140, Cj0690c, and HsdR) were dispensable for heat shock-enhanced conjugation in C. jejuni. Taking all results together, this study demonstrated a heat shock-enhanced conjugation efficiency in standard C. jejuni strains, leading to an optimized conjugation protocol for molecular manipulation of this organism. The findings from this study also represent a significant step toward elucidation of the molecular mechanism of conjugative gene transfer in C. jejuni.     

Gene Expression Profiling of the Local Cecal Response of Genetic Chicken Lines That Differ in Their Susceptibility to Campylobacter jejuni Colonization

Campylobacter jejuni (C. jejuni) is one of the most common causes of human bacterial enteritis worldwide primarily due to contaminated poultry products. Previously, we found a significant difference in C. jejuni colonization in the ceca between two genetically distinct broiler lines (Line A (resistant) has less colony than line B (susceptible) on day 7 post inoculation). We hypothesize that different mechanisms between these two genetic lines may affect their ability to resist C. jejuni colonization in chickens. The molecular mechanisms of the local host response to C. jejuni colonization in chickens have not been well understood. In the present study, to profile the cecal gene expression in the response to C. jejuni colonization and to compare differences between two lines at the molecular level, RNA of ceca from two genetic lines of chickens (A and B) were applied to a chicken whole genome microarray for a pair-comparison between inoculated (I) and non-inoculated (N) chickens within each line and between lines. Our results demonstrated that metabolism process and insulin receptor signaling pathways are key contributors to the different response to C. jejuni colonization between lines A and B. With C. jejuni inoculation, lymphocyte activation and lymphoid organ development functions are important for line A host defenses, while cell differentiation, communication and signaling pathways are important for line B. Interestingly, circadian rhythm appears play a critical role in host response of the more resistant A line to C. jejuni colonization. A dramatic differential host response was observed between these two lines of chickens. The more susceptible line B chickens responded to C. jejuni inoculation with a dramatic up-regulation in lipid, glucose, and amino acid metabolism, which is undoubtedly for use in the response to the colonization with little or no change in immune host defenses. However, in more resistant line A birds the host defense responses were characterized by an up-regulation lymphocyte activation, probably by regulatory T cells and an increased expression of the NLR recognition receptor NALP1. To our knowledge, this is the first time each of these responses has been observed in the avian response to an intestinal bacterial pathogen.     

Genomic Characterization of Campylobacter jejuni Strain M1

Campylobacter jejuni strain M1 (laboratory designation 99/308) is a rarely documented case of direct transmission of C. jejuni from chicken to a person, resulting in enteritis. We have sequenced the genome of C. jejuni strain M1, and compared this to 12 other C. jejuni sequenced genomes currently publicly available. Compared to these, M1 is closest to strain 81116. Based on the 13 genome sequences, we have identified the C. jejuni pan-genome, as well as the core genome, the auxiliary genes, and genes unique between strains M1 and 81116. The pan-genome contains 2,427 gene families, whilst the core genome comprised 1,295 gene families, or about two-thirds of the gene content of the average of the sequenced C. jejuni genomes. Various comparison and visualization tools were applied to the 13 C. jejuni genome sequences, including a species pan- and core genome plot, a BLAST Matrix and a BLAST Atlas. Trees based on 16S rRNA sequences and on the total gene families in each genome are presented. The findings are discussed in the background of the proven virulence potential of M1.     

Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis

Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.     

The Central, Surface-Exposed Region of the Flagellar Hook Protein FlgE of Campylobacter jejuni Shows Hypervariability among Strains

In a previous study, we observed that monoclonal antibodies raised against the hook protein FlgE of Campylobacter jejuni LIO 36, isolate 5226, bound exclusively to this strain. The aim of this study was to elucidate the molecular basis for these binding specificities. The hook protein-encoding gene flgE of C. jejuni was cloned in Escherichia coli and sequenced. The flgE genes of four additional C. jejuni strains were amplified by PCR and also sequenced. Comparison of the deduced amino acid sequences revealed a high degree of variability in the central parts of the FlgE proteins among the strains, including variable and hypervariable domains. These findings may indicate a selective pressure of C. jejuni hosts, forcing the bacteria to generate variations in surface-exposed antigenic determinants.     

Campylobacter jejuni Actively Invades the Amoeba Acanthamoeba polyphaga and Survives within Non Digestive Vacuoles

The Gram-negative bacterium Campylobacter jejuni is able to enter, survive and multiply within the free living amoeba Acanthamoeba polyphaga, but the molecular mechanisms behind these events are still unclear. We have studied the uptake and intracellular trafficking of viable and heat killed bacterial cells of the C. jejuni strain 81–176 in A. polyphaga. We found that viable bacteria associated with a substantially higher proportion of Acanthamoeba trophozoites than heat killed bacteria. Furthermore, the kinetics of internalization, the total number of internalized bacteria as well as the intracellular localization of internalized C. jejuni were dramatically influenced by bacterial viability. Viable bacteria were internalized at a high rate already after 1 h of co-incubation and were observed in small vacuoles tightly surrounding the bacteria. In contrast, internalization of heat killed C. jejuni was low at early time points and did not peak until 96 h. These cells were gathered in large spacious vacuoles that were part of the degradative pathway as determined by the uptake of fluorescently labeled dextran. The amount of heat killed bacteria internalized by A. polyphaga did never reach the maximal amount of internalized viable bacteria. These results suggest that the uptake and intracellular survival of C. jejuni in A. polyphaga is bacterially induced.     

Fingerprinting of Campylobacter jejuni by Using Resolution-Optimized Binary Gene Targets Derived from Comparative Genome Hybridization Studies

The aim of this investigation was to exploit the vast comparative data generated by comparative genome hybridization (CGH) studies of Campylobacter jejuni in developing a genotyping method. We examined genes in C. jejuni that exhibit binary status (present or absent between strains) within known plasticity regions, in order to identify a minimal subset of gene targets that provide high-resolution genetic fingerprints. Using CGH data from three studies as input, binary gene sets were identified with “Minimum SNPs” software. “Minimum SNPs” selects for the minimum number of targets required to obtain a predefined resolution, based on Simpson's index of diversity (D). After implementation of stringent criteria for gene presence/absence, eight binary genes were found that provided 100% resolution (D = 1) of 20 C. jejuni strains. A real-time PCR assay was developed and tested on 181 C. jejuni and Campylobacter coli isolates, a subset of which have previously been characterized by multilocus sequence typing, flaA short variable region sequencing, and pulsed-field gel electrophoresis. In addition to the binary gene real-time PCR assay, we refined the seven-member single nucleotide polymorphism (SNP) real-time PCR assay previously described for C. jejuni and C. coli. By normalizing the SNP assay with the respective C. jejuni and C. coli ubiquitous genes, mapA and ceuE, the polymorphisms at each SNP could be determined without separate reactions for every polymorphism. We have developed and refined a rapid, highly discriminatory genotyping method for C. jejuni and C. coli that uses generic technology and is amenable to high-throughput analyses.     

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.     

Effects of disruption of heat shock genes on susceptibility of Escherichia coli to fluoroquinolones

Background

It is well known that expression of certain bacterial genes responds rapidly to such stimuli as exposure to toxic chemicals and physical agents. It is generally believed that the proteins encoded in these genes are important for successful survival of the organism under the hostile conditions. Analogously, the proteins induced in bacterial cells exposed to antibiotics are believed to affect the organisms' susceptibility to these agents.

Results

We demonstrated that Escherichia coli cells exposed to levofloxacin (LVFX), a fluoroquinolone (FQ), induce the syntheses of heat shock proteins and RecA. To examine whether the heat shock proteins affect the bactericidal action of FQs, we constructed E. coli strains with mutations in various heat shock genes and tested their susceptibility to FQs. Mutations in dnaK, groEL, and lon increased this susceptibility; the lon mutant exhibited the greatest effects. The increased susceptibility of the lon mutant was corroborated by experiments in which the gene encoding the cell division inhibitor, SulA, was subsequently disrupted. SulA is induced by the SOS response and degraded by the Lon protease. The findings suggest that the hypersusceptibility of the lon mutant to FQs could be due to abnormally high levels of SulA protein resulting from the depletion of Lon and the continuous induction of the SOS response in the presence of FQs.

Conclusion

The present results show that the bactericidal action of FQs is moderately affected by the DnaK and GroEL chaperones and strongly affected by the Lon protease. FQs have contributed successfully to the treatment of various bacterial infections, but their widespread use and often misuse, coupled with emerging resistance, have gradually compromised their utility. Our results suggest that agents capable of inhibiting the Lon protease have potential for combination therapy with FQs.     

Phenotypes of Campylobacter jejuni luxS Mutants Are Depending on Strain Background,Kind of Mutation and Experimental Conditions

Since the discovery that Campylobacter (C.) jejuni produces Autoinducer 2 (AI-2), various studies have been conducted to explore the function and role of AI-2 in C. jejuni. However, the interpretation of these analyses has been complicated by differences in strain backgrounds, kind of mutation and culture conditions used. Furthermore, all research on AI-2 dependent phenotypes has been conducted with AI-2 synthase (luxS) mutants. This mutation also leads to a disruption of the activated-methyl-cycle. Most studies lack sufficient complementation resulting in not knowing whether phenotypes of luxS mutants depend on disrupted metabolism or lack of AI-2. Additionally, no AI-2 receptor has been found yet. All this contributes to an intensive discussion about the exact role of AI-2 in C. jejuni. Therefore, we examined the impact of different experiment settings on three different C. jejuni luxS mutants on growth and motility (37°C and 42°C). Our study showed that differing phenotypes of C. jejuni luxS mutants depend on strain background, mutation strategy and culture conditions. Furthermore, we complemented experiments with synthetic AI-2 or homocysteine as well as the combination of both. Complementation with AI-2 and AI-2+homocysteine significantly increased the cell number of C. jejuni NCTC 11168ΔluxS in stationary phase compared to the non-complemented C. jejuni NCTC 11168ΔluxS mutant. Genetic complementation of both C. jejuni 81-176 luxS mutants resulted in wild type comparable growth curves. Also swarming ability could be partially complemented. While genetic complementation restored swarming abilities of C. jejuni 81-176ΔluxS, it did not fully restore the phenotype of C. jejuni 81-176::luxS, which indicates that compensatory mutations in other parts of the chromosome and/or potential polar effects may appear in this mutant strain. Also with neither synthetic complementation, the phenotype of the wild type-strains was achieved, suggesting yet another reason for differing phenotypes other than communication and methionine metabolism for C. jejuni luxS mutants.     

Campylobacter jejuni FlpA Binds Fibronectin and Is Required for Maximal Host Cell Adherence

Campylobacter jejuni is one of the most frequent bacterial causes of food-borne gastrointestinal disease in developed countries. Previous work indicates that the binding of C. jejuni to human intestinal cells is crucial for host colonization and disease. Fibronectin (Fn), a major constituent of the extracellular matrix, is a ∼250-kDa glycoprotein present at regions of cell-to-cell contact in the intestinal epithelium. Fn is composed of three types of repeating units: type I (∼45 amino acids), type II (∼60 amino acids), and type III (∼90 amino acids). The deduced amino acid sequence of C. jejuni flpA (Cj1279c) contains at least three Fn type III domains. Based on the presence of the Fn type III domains, we hypothesized that FlpA contributes to the binding of C. jejuni to human INT 407 epithelial cells and Fn. We assessed the contribution of FlpA in C. jejuni binding to host cells by in vitro adherence assays with a C. jejuni wild-type strain and a C. jejuni flpA mutant and binding of purified FlpA protein to Fn by enzyme-linked immunosorbent assay (ELISA). Adherence assays revealed the binding of the C. jejuni flpA mutant to INT 407 epithelial cells was significantly reduced compared with that for a wild-type strain. In addition, rabbit polyclonal serum generated against FlpA blocked C. jejuni adherence to INT 407 cells in a concentration-dependent manner. Binding of FlpA to Fn was found to be dose dependent and saturable by ELISA, demonstrating the specificity of the interaction. Based on these data, we conclude that FlpA mediates C. jejuni attachment to host epithelial cells via Fn binding.Members of the genus Campylobacter are gram-negative, asaccharolytic, motile bacteria, which grow optimally in the laboratory at temperatures between 37 and 42°C under microaerophilic conditions. Although members of Campylobacter spp. were initially recognized to cause disease in sheep and cattle, Campylobacter jejuni was not recognized as a human pathogen until much later (25). Infection of humans with C. jejuni is characterized by a rapid onset of fever, abdominal cramps, and diarrhea. C. jejuni is now recognized as one of the leading bacterial causes of gastroenteritis in the world. In spite of the incidence of campylobacteriosis, relatively few C. jejuni virulence genes have been characterized, and our understanding of the virulence properties of C. jejuni is limited compared with that of other enteric pathogens, including Salmonella, Shigella, and Yersinia spp.The ability of C. jejuni to cause disease is a complex, multifactorial process. Virulence factors that contribute to the pathogenesis of C. jejuni are associated with motility, host (target) cell adherence, host cell invasion, protein secretion, alteration of host cell signaling pathways, induction of host cell death, evasion of host immune defenses, iron acquisition, and drug/detergent resistance (14, 18). The binding of C. jejuni to specific host cell ligands is hypothesized to play a fundamental role in host colonization and disease progression, since it prevents the organism''s clearance from the intestine by peristalsis and fluid flow. Fauchere et al. (5) reported that C. jejuni isolates recovered from individuals with fever and diarrhea adhered to cultured cells in greater numbers than isolates recovered from asymptomatic individuals. While there is no evidence indicating that C. jejuni produces fimbriae that assist in host colonization (7), a number of constitutively synthesized proteins have been proposed to act as adhesins. Bacterial adhesins are surface-exposed macromolecules that facilitate an organism''s binding to the host cell receptors. Known and putative C. jejuni adhesins include CadF, CapA, FlpA, and PorA (MOMP) (6).An emerging theme among pathogenic microorganisms is their ability to utilize host cell molecules during the infectious process to facilitate their binding and entry into host cells (27). More specifically, many bacterial pathogens have been found to bind to fibronectin (Fn), which in turn modifies host cell signaling pathways to the pathogen''s advantage. Fn exists as a dimer of nearly identical 250-kDa subunits that are linked by a pair of disulfide bonds near their C termini. Each Fn monomer is composed of three types of repeating units: type I (∼45 amino acids), type II (∼60 amino acids), and type III (∼90 amino acids) (22). In total, each monomer contains 12 type I repeats, two type II repeats, and 15 to 17 type III repeats. Fn participates in many cellular interactions, including tissue repair, embryogenesis, blood clotting, and cell migration/adhesion. Plasma Fn, which is synthesized by hepatocytes, is soluble (22). In contrast, Fn involved in host cell-extracellular matrix (ECM) interaction, which is synthesized by chondrocytes, fibroblasts, endothelial cells, macrophages, and certain epithelial cells, is present in an insoluble form (22). Fn serves as an adhesion molecule that anchors cells to ECM components, including collagen and other proteoglycan substrates.The bacterial proteins that bind to ECM components have been termed microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) (23). The C. jejuni CadF protein is a member of the MSCRAMM family and one of the most extensively characterized C. jejuni virulence determinants (10-12, 15, 16, 19-21, 24, 28). CadF mediates the binding of C. jejuni to Fn, promotes bacterium-host cell interactions, and facilitates the organism''s colonization of chickens (10, 11, 15, 16, 20, 21, 28). In addition to CadF, we recently reported that a mutation in Cj1279c resulted in a C. jejuni mutant that poorly colonized broiler chickens compared with a C. jejuni wild-type strain. The product encoded by the Cj1279c gene was termed Fibronectin-like protein A (FlpA) because the protein harbors Fn type III domains (6). The goal of this study was to characterize the binding properties of FlpA and to determine if this protein is a member of the MSCRAMM family. Here we provide experimental evidence that C. jejuni FlpA is surface exposed, promotes the bacterium''s attachment to host epithelial cells, and has Fn binding activity. Assays were also performed to determine if CadF and FlpA act cooperatively to promote binding of C. jejuni to host cells and Fn. We submit that the identification of a second MSCRAMM in C. jejuni highlights the importance of Fn binding in host colonization and disease.     

Induction of neutrophil extracellular traps by Campylobacter jejuni

Campylobacter jejuni is the leading cause of bacterial‐derived gastroenteritis worldwide and can lead to several post‐infectious inflammatory disorders. Despite the prevalence and health impacts of the bacterium, interactions between the host innate immune system and C. jejuni remain poorly understood. To expand on earlier work demonstrating that neutrophils traffic to the site of infection in an animal model of campylobacteriosis, we identified significant increases in several predominantly neutrophil‐derived proteins in the faeces of C. jejuni‐infected patients, including lipocalin‐2, myeloperoxidase and neutrophil elastase. In addition to demonstrating that these proteins significantly inhibited C. jejuni growth, we determined they are released during formation of C. jejuni‐induced neutrophil extracellular traps (NETs). Using quantitative and qualitative methods, we found that purified human neutrophils are activated by C. jejuni and exhibit signatures of NET generation, including presence of protein arginine deiminase‐4, histone citrullination, myeloperoxidase, neutrophil elastase release and DNA extrusion. Production of NETs correlated with C. jejuni phagocytosis/endocytosis and invasion of neutrophils suggesting that host‐ and bacterial‐mediated activities are responsible for NET induction. Further, NET‐like structures were observed within intestinal tissue of C. jejuni‐infected ferrets. Finally, induction of NETs significantly increased human colonocyte cytotoxicity, indicating that NET formation during C. jejuni infection may contribute to observed tissue pathology. These findings provide further understanding of C. jejuni–neutrophil interactions and inflammatory responses during campylobacteriosis.     

Isolation and Characterization of Campylobacter jejuni subsp. jejuni from Macaroni Penguins (Eudyptes chrysolophus) in the Subantarctic Region

On Bird Island, South Georgia, albatrosses (n = 140), penguins (n = 100), and fur seals (n = 206) were sampled for Campylobacter jejuni. C. jejuni subsp. jejuni was recovered from three macaroni penguins (Eudyptes chrysolophus). These isolates, the first reported for the subantarctic region, showed low genetic diversity and high similarity to Northern Hemisphere C. jejuni isolates, possibly suggesting recent introduction to the area.     

Characterization of Campylobacter jejuni Biofilms under Defined Growth Conditions

Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries and is a source of public health and economic burden. C. jejuni, present as normal flora in the intestinal tract of commercial broiler chickens and other livestock, is probably the main source of human infections. The presence of C. jejuni in biofilms found in animal production watering systems may play a role in the colonization of these animals. We have determined that C. jejuni can form biofilms on a variety of abiotic surfaces commonly used in watering systems, such as acrylonitrile butadiene styrene and polyvinyl chloride plastics. Furthermore, C. jejuni biofilm formation was inhibited by growth in nutrient-rich media or high osmolarity, and thermophilic and microaerophilic conditions enhanced biofilm formation. Thus, nutritional and environmental conditions affect the formation of C. jejuni biofilms. Both flagella and quorum sensing appear to be required for maximal biofilm formation, as C. jejuni flaAB and luxS mutants were significantly reduced in their ability to form biofilms compared to the wild-type strain.     

Detection of Campylobacter jejuni and Salmonella typhimurium in chicken using PCR for virulence factor hipO and invA genes (Saudi Arabia)

Campylobacter jejuni and Salmonella typhimurium are the leading causes of bacterial food contamination in chicken carcasses. Contamination is particularly associated with the slaughtering process. The present study isolated C. jejuni and S. typhimurim from fifty chicken carcass samples, all of which were acquired from different companies in Riyadh, Saudi Arabia. The identification of C. jejuni was performed phenotypically by using a hippurate test and genetically using a polymerase chain reaction with primers for 16S rRNA and hippurate hydrolase (hipO gene). For the dentification of S. typhimurim, a serological Widal test was carried out using serum anti-S. typhimurium antibodies. Strains were genetically detected using invA gene primers. The positive isolates for C. jejuni showed a specific molecular size of 1448 bp for 16S rRNA and 1148 bp for hipO genes. However, the positive isolates of the invA gene exhibited a specific molecular size at 244 bp using polymerase chain reaction (PCR). Comparing sequencing was performed with respect to the invA gene and the BLAST nucleotide isolates that were identified as Salmonella enterica subsp. enterica serovar typhimurium strain ST45, thereby producing a similarity of 100%. The testing identified C. jejuni for hippuricase, GenBank: {"type":"entrez-nucleotide","attrs":{"text":"Z36940.1","term_id":"535805"}}Z36940.1. While many isolates of Salmonella spp. that contained the invA gene were not necessarily identified as S. typhimurim, the limiting factor for the Widal test used antiS. typhimurum antibodies. The multidrug resistance (MDR) of C. jejuni isolates in chickens was compared with the standard C. jejuni strain ATCC 22931. Similarly, S. typhimurium isolates were compared with the standard S. typhimurium strain ATCC 14028.     

The HtrA Protease of Campylobacter jejuni Is Required for Heat and Oxygen Tolerance and for Optimal Interaction with Human Epithelial Cells

Campylobacter jejuni is a predominant cause of food-borne bacterial gastroenteritis in the developed world. We have investigated the importance of a homologue of the periplasmic HtrA protease in C. jejuni stress tolerance. A C. jejuni htrA mutant was constructed and compared to the parental strain, and we found that growth of the mutant was severely impaired both at 44°C and in the presence of the tRNA analogue puromycin. Under both conditions, the level of misfolded protein is known to increase, and we propose that the heat-sensitive phenotype of the htrA mutant is caused by an accumulation of misfolded protein in the periplasm. Interestingly, we observed that the level of the molecular chaperones DnaK and ClpB was increased in the htrA mutant, suggesting that accumulation of nonnative proteins in the periplasm induces the expression of cytoplasmic chaperones. While lack of HtrA reduces the oxygen tolerance of C. jejuni, the htrA mutant was not sensitive to compounds that increase the formation of oxygen radicals, such as paraquat, cumene hydroperoxide, and H₂O₂. Using tissue cultures of human epithelial cells (INT407), we found that the htrA mutant adhered to and invaded human epithelial cells with a decreased frequency compared to the wild-type strain. This defect may be a consequence of the observed altered morphology of the htrA mutant. Thus, our results suggest that in C. jejuni, HtrA is important for growth during stressful conditions and has an impact on virulence.     

Effects of amino acids on polysaccharide synthesis ofEscherichia coli K-12lon + andlon − strains

Ultraviolet-sensitivelon ^? mutant ofEscherichia coli K-12 produced abundant polysaccharide when grown in a minimal medium at 37 C, but not when grown in a broth medium. The repression of polysaccharide synthesis in the broth-grownlon ^? andlon ⁺ cells was studied. The effects were largely dependent on the amino acid concentrations and on the requirements of the strain used. At 200 μg per ml of each of the essential amino acids, histidine, proline, and threonine, there was complete inhibition of polysaccharide synthesis. At 200 μg per ml the required amino acids, tryptophane and tyrosine promoted polysaccharide synthesis. Most amino acids inhibited cell growth at 200 μg per ml but the inhibiting effect was smaller at 400 μg per ml. Polysaccharide synthesis of cells was not correlated with the growth rate, and occurred even under non-growing conditions.     

Campylobacter jejuni pdxA Affects Flagellum-Mediated Motility to Alter Host Colonization

Vitamin B6 (pyridoxal-5''-phosphate, PLP) is linked to a variety of biological functions in prokaryotes. Here, we report that the pdxA (putative 4-hydroxy-L-threonine phosphate dehydrogenase) gene plays a pivotal role in the PLP-dependent regulation of flagellar motility, thereby altering host colonization in a leading foodborne pathogen, Campylobacter jejuni. A C. jejuni pdxA mutant failed to produce PLP and exhibited a coincident loss of flagellar motility. Mass spectrometric analyses showed a 3-fold reduction in the main flagellar glycan pseudaminic acid (Pse) associated with the disruption of pdxA. The pdxA mutant also exhibited reduced growth rates compared with the WT strain. Comparative metabolomic analyses revealed differences in respiratory/energy metabolism between WT C. jejuni and the pdxA mutant, providing a possible explanation for the differential growth fitness between the two strains. Consistent with the lack of flagellar motility, the pdxA mutant showed impaired motility-mediated responses (bacterial adhesion, ERK1/2 activation, and IL-8 production) in INT407 cells and reduced colonization of chickens compared with the WT strain. Overall, this study demonstrated that the pdxA gene affects the PLP-mediated flagellar motility function, mainly through alteration of Pse modification, and the disruption of this gene also alters the respiratory/energy metabolisms to potentially affect host colonization. Our data therefore present novel implications regarding the utility of PLP and its dependent enzymes as potent target(s) for the control of this pathogen in the poultry host.