Jejuni Strain NCTC11168 Research Articles

Development of a Trivalent Construct Omp18/AhpC/FlgH Multi Epitope Peptide Vaccine Against Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is one of the major pathogens contributing to the enteritis in humans. Infection can lead to numerous complications, including but not limited to Guillain-Barre syndrome, reactive arthritis, and Reiter’s syndrome. Over the past two decades, joint efforts have been made toward developing a proper strategy of limiting the transmission of C. jejuni to humans. Nevertheless, except for biosecurity measures, no available vaccine has been developed so far. Judging from the research findings, Omp18, AhpC outer membrane protein, and FlgH flagellin subunits of C. jejuni could be adopted as surface protein antigens of C. jejuni for screening dominant epitope thanks to their strong antigenicity, expression of varying strains, and conservative sequence. In this study, bioinformatics technology was adopted to analyze the T-B antigenic epitopes of Omp18, AhpC, and FlgH in C. jejuni strain NCTC11168. Both ELISA and Western Blot methods were adopted to screen the dominant T-B combined epitope. GGS (GGCGGTAGC) sequence was adopted to connect the dominant T-B combined epitope peptides and to construct the prokaryotic expression system of tandem repeats of antigenic epitope peptides. The mouse infection model was adopted to assess the immunoprotective effect imposed by the trivalent T-B combined with antigen epitope peptide based on Omp18/AhpC/FlgH. In this study, a tandem epitope AhpC-2/Omp18-1/FlgH-1 was developed, which was composed of three epitopes and could effectively enhance the stability and antigenicity of the epitope while preserving its structure. The immunization of BALB/c mice with a tandem epitope could induce protective immunity accompanied by the generation of IgG2a antibody response through the in vitro synthesis of IFN-γ cytokines. Judging from the results of immune protection experiments, the colonization of C. jejuni declined to a significant extent, and it was expected that AhpC-2/Omp18-1/FlgH-1 could be adopted as a candidate antigen for genetic engineering vaccine of C. jejuni MAP.

Designing an efficient multi-epitope vaccine against Campylobacter jejuni using immunoinformatics and reverse vaccinology approach

Campylobacterjejuni causes acute diarrhea as a leading cause of morbidity and mortality in children especially in the developing countries of Asia and Africa. C.jejuni has been identified as a member of the priority pathogens category due to the sudden emergence of multidrug-resistant isolates. Therefore, it is important to develop a protective vaccine against this pathogen. In the present study, the Reverse vaccinology approach was used to identify vaccine targets from the proteome of diarrheagenic C. jejuni strain NCTC11168 for the development of chimeric vaccine candidates against C. jejuni. Pathogen proteins that have adhesin like properties and role in virulence but not present in the human host were selected for the design of a multi-epitope vaccine. MHC class I & II and B-cell epitopes present in the selected vaccine target proteins were screened using different immunoinformatics tools. The commonly predicted epitopes from their corresponding different servers were selected and further shortlisted based on their immunogenicity, antigenicity, and toxicity analysis. Shortlisted peptides were joined by GPGPG linkers to design a multi-epitope vaccine construct. Immune-modulating adjuvant monophosphoryl lipid sequence was added with the vaccine construct's N terminal using EAAAK linkers to enhance the immunogenicity. The designed vaccine construct was evaluated by antigenicity, allergenicity, solubility, and physicochemical analysis using various bioinformatics tools. A three-dimensional model of vaccine construct was modeled by the Phyre2 server and refinement by the GalaxyRefine tool. Constructed model quality was validated by the ProSA-web error-detection tool and the Ramachandran plot. After that vaccine model was docked with TLR-4 protein and complex stability confirmed by molecular dynamics simulation studies. Finally, In-silico cloning of vaccine constructs into a vector was performed to ensuring its effective expression in the microbial system.

HtrA‐dependent adherence and invasion of Campylobacter jejuni in human vs avian cells

The aim of this study was to investigate if HtrA is responsible for differences in adherence and invasion of Campylobacter jejuni towards human and chicken cell lines. Gentamicin protection assays were performed with either human Caco-2 or chicken 2G4 cells using C. jejuni strain NCTC11168 to compare the adhesion and invasion rates towards these two cell types. The results revealed significant differences in the adhesion and invasion rates between the human and avian cells. Deletion of the Campylobacter htrA gene, coding for the dual function of serine protease and chaperonin with a role in pathogenesis, led to a reduction of the rates in both cell lines. Using a single-amino acid substitution mutant (ΔhtrA/htrAS197A ) that lacked protease activity, but retained chaperonin activity, we show that the first is involved in the invasion of human Caco-2 and chicken 2G4 cells, whereas the latter mutant invaded at lower levels. Adherence towards the chicken cells is higher than towards Caco-2 cells and this is also dependent on HtrA. Together, this data suggests that the proteolytic activity of HtrA is involved in the difference in host response of C. jejuni towards human and chicken-derived cells.

Protease Activity of Campylobacter jejuni HtrA Modulates Distinct Intestinal and Systemic Immune Responses in Infected Secondary Abiotic IL-10 Deficient Mice.

Even though human Campylobacter jejuni infections are progressively increasing worldwide, the underlying molecular mechanisms of pathogen-host-interactions are still not fully understood. We have recently shown that the secreted serine protease HtrA plays a key role in C. jejuni cellular invasion and transepithelial migration in vitro, and is involved in the onset of intestinal pathology in murine infection models in vivo. In the present study, we investigated whether the protease activity of HtrA had an impact in C. jejuni induced acute enterocolitis. For this purpose, we perorally infected secondary abiotic IL-10−/− mice with wildtype C. jejuni strain NCTC11168 (11168WT) or isogenic bacteria carrying protease-inactive HtrA with a single point mutation at S197A in the active center (11168HtrA−S197A). Irrespective of the applied pathogenic strain, mice harbored similar C. jejuni loads in their feces and exhibited comparably severe macroscopic signs of acute enterocolitis at day 6 postinfection (p.i.). Interestingly, the 11168HtrA−S197A infected mice displayed less pronounced colonic apoptosis and immune cell responses, but enhanced epithelial proliferation as compared to the 11168WT strain infected controls. Furthermore, less distinct microscopic sequelae in 11168HtrA−S197A as compared to parental strain infected mice were accompanied by less distinct colonic secretion of pro-inflammatory cytokines such as MCP-1, IL-6, TNF, and IFN-γ in the former as compared to the latter. Strikingly, the S197A point mutation was additionally associated with less pronounced systemic pro-inflammatory immune responses as assessed in serum samples. In conclusion, HtrA is a remarkable novel virulence determinant of C. jejuni, whose protease activity is not required for intestinal colonization and establishment of disease, but aggravates campylobacteriosis by triggering apoptosis and pro-inflammatory immune responses.

High Throughput Method for Analysis of Repeat Number for 28 Phase Variable Loci of Campylobacter jejuni Strain NCTC11168.

Mutations in simple sequence repeat tracts are a major mechanism of phase variation in several bacterial species including Campylobacter jejuni. Changes in repeat number of tracts located within the reading frame can produce a high frequency of reversible switches in gene expression between ON and OFF states. The genome of C. jejuni strain NCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats. This protocol outlines a method—the 28-locus-CJ11168 PV-analysis assay—for rapidly determining ON/OFF states of 28 of these phase-variable loci in a large number of individual colonies from C. jejuni strain NCTC11168. The method combines a series of multiplex PCR assays with a fragment analysis assay and automated extraction of fragment length, repeat number and expression state. This high throughput, multiplex assay has utility for detecting shifts in phase variation states within and between populations over time and for exploring the effects of phase variation on adaptation to differing selective pressures. Application of this method to analysis of the 28 polyG/polyC tracts in 90 C. jejuni colonies detected a 2.5-fold increase in slippage products as tracts lengthened from G8 to G11 but no difference between tracts of similar length indicating that flanking sequence does not influence slippage rates. Comparison of this observed slippage to previously measured mutation rates for G8 and G11 tracts in C. jejuni indicates that PCR amplification of a DNA sample will over-estimate phase variation frequencies by 20-35-fold. An important output of the 28-locus-CJ11168 PV-analysis assay is combinatorial expression states that cannot be determined by other methods. This method can be adapted to analysis of phase variation in other C. jejuni strains and in a diverse range of bacterial species.

Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168.

Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically methylate adenine in 5΄CCCGA and 5΄CCTGA sequences. Alterations in the levels of specific transcripts were detected using RNA-Seq in phase-variants and mutants of cj0031c but these changes did not correlate with observed differences in phenotypic behaviour. Alterations in restriction of phage growth were also associated with phase variation (PV) of cj0031c and correlated with presence of sites in the genomes of these phages. We conclude that PV of a Type IIG restriction-modification system causes changes in site-specific methylation patterns and gene expression patterns that may indirectly change adaptive traits.

Microarray transposon tracking for the mapping of conditionally essential genes in Campylobacter jejuni.

Although whole genome approaches to the study of bacteria have grown substantially in the past decade, there is still a need for quick and easy methods for the determination of which genes are essential for the growth of these bacteria under specific growth conditions. There are numerous methods to accomplish this depending on the resources and equipment available, each with their own advantages and disadvantages. Here we describe a method we successfully employed to map the essential genes of Campylobacter jejuni using a microarray transposon tracking approach where we constructed a saturated transposon mutant library in the C. jejuni strain NCTC11168 and used a genomic microarray approach to identify genes lacking transposon insertions under standard laboratory growth conditions. With a fully saturated library, the absence of transposon insertions can be used as an indicator of a gene essential for the survival and growth for the conditions used for the mutant library.

Partial Proteome Map of Campylobacter Jejuni Strain Nctc11168 by Gel-Free Proteomics Analysis

A proteome map of the foodborne pathogen Campylobacter jejuni NCTC11168 was analyzed using a state-of-the-art gel-free proteomic approach for the first time. A whole cell protein extract was prepared from the C. jejuni strain NCTC11168 grown in brain heart infusion (BHI) broth at 42°C under microaerobic conditions. A gel-free technique using isobaric tags for relative and absolute quantitation (iTRAQ) was employed to create a protein expression profile of the strain. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to identify the proteins. Protein functionalities were searched to classify them. A total of 235 proteins were identified in the whole cell protein fraction of C. jejuni NCTC11168 cells using iTRAQ analysis. Functional grouping of the identified proteins showed that forty percent of these proteins were associated with energy metabolism, protein synthesis and genetic information processing. iTRAQ was faster, easier and proved more sensitive than two-dimensional gel-based proteomics approaches previously applied to C. jejuni, making it an attractive tool for further studies of cellular physiological response. DOI: http://dx.doi.org/10.3126/ijasbt.v2i4.11253 Int J Appl Sci Biotechnol, Vol. 2(4): 464-477

In Vitro Protein Expression Profile of Campylobacter jejuni Strain NCTC11168 by Two-dimensional Gel Electrophoresis and Mass Spectrometry

ObjectiveTo investigate the protein expression profiles of the major food-borne pathogen Campylobacter jejuni NCTC11168. MethodsMembrane and soluble cellular proteins were extracted from the genome-sequenced C. jejuni strain NCTC11168. Protein expression profiles were determined using two-dimensional gel electrophoresis (2-DE). All the detected spots on the 2-DE map were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF) analysis. ResultsA total of 537 and 333 spots were detected from the whole cell and membrane-associated proteins of C. jejuni NCTC11168 cultured on Columbia agar medium at 42 °C by 2-DE and Coomassie Brilliant Blue staining, respectively. Analyses of whole cell and membrane-associated proteins included 399 and 133 spots, respectively, which included 182 and 53 functional proteins identified by MALDI-TOF/TOF analysis. ConclusionThe comprehensive expression protein profiles of C. jeuni NCTC11168 obtained in this study will be useful for elucidating the roles of these proteins in further pathogenesis investigation.

Role of fliY gene in pathogenicity-associated chemotaxis and colonization of Campylobacter jejuni

To construct a knockout fliY gene mutant strain of Campylobacter jejuni for determining the role of FliY protein in flagellar movement related to bacterial motility, chemotaxis and colonization. The plasmid pBluescript-II-SK was used to construct the suicide plasmid; according to homologous exchange principle, the suicide plasmid was utilized to generate fliY gene knockout mutant(fliY) in Campylobacter jejuni strain NCTC11168. The fliY mutant strain was identified by PCR, sequencing and Western blotting. The chemotactic and colonizing abilities of fliY mutant were determined by colony migration test and bacterial chemotactic test in vitro, and colonization test in jejunum of mice. The fliY(-)mutant strain showed a growth curve in medium similar to that of wild-type strain. PCR, sequencing and Western blotting assay confirmed that the fliY gene in fliY(-)mutant was deleted. Compared to the wild-type strain, the colonies of fliY-mutant on semisolid plate were much smaller (P <0.05), the chemotactic ability of fliY mutant towards sodium deoxycholate and bovine bile was significantly attenuated (P <0.05), and the number of fliY mutant (CFU) in jejunal tissue specimens of the infected mice was significantly decreased (P<0.05). The function of C.jejuni fliY gene refers to controlling flagellar movement, which is involved in bacterial chemotaxis and colonization.

Assessment of chicken protection against Campylobacter jejuni infection by immunization with avirulent Salmonella enterica sv. Typhimurium strain producing Campylobacter CjaD/Pal protein

Assessment of chicken protection against Campylobacter jejuni infection by immunization with avirulent Salmonella enterica sv. Typhimurium strain producing Campylobacter CjaD/Pal protein Pawel Laniewski,1 Malgorzata Lis,2 Agnieszka Wyszynska,1 Pawel Majewski,3 Renata Godlewska,1 Elzbieta Katarzyna Jagusztyn-Krynicka11Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland; 2Biowet Pulawy Ltd, Pulawy, Poland; 3Department of Vertebrate Physiology, Institute of Zoology, Faculty of Biology, University of Warsaw, Warsaw, PolandAbstract: Campylobacter jejuni is a major food-borne pathogen, causing gastroenteritis worldwide. Chickens are considered to be one of the most common sources of human C. jejuni infection in developed countries. Campylobacter CjaD/Pal protein (annotated as Cj0113 in C. jejuni strain NCTC11168) is a highly immunogenic, membrane-located antigen, conserved among different strains, with the potential to provide broad protection against C. jejuni colonization. The present study examines the immunogenicity and the general efficacy of avirulent S. enterica sv. Typhimurium Δcrp Δcya expressing C. jejuni CjaD as a chicken vaccine against Campylobacter colonization. The high copy number plasmid pYA3341 Asd+ was used as a cloning vector. Here, 1- and 14-day old chickens were orally immunized with a delivery vector strain, expressing C. jejuni CjaD. Two weeks later, they were challenged with a wild-type C. jejuni strain isolated from chicken carcasses. This schedule of immunization induced significant levels of serum-specific IgG as well as mucosal intestinal sIgA as measured by ELISA tests using Campylobacter membrane proteins as a coating antigen. Nevertheless, protection experiments did not result in significant reduction of colonization of vaccinated birds relative to nonvaccinated birds.Keywords: Campylobacter, cjaD, immunization, Pal

CheA/CheY signaling systemresponsible for chemotaxis in vitro and colonization in vivo of Campylobacter jejuni

Objective To determine the effects of Che A and CheY proteins ofCampylobacter jejuni regulating the bacterial chemotaxis in vitro and colonization invivo. Methods By using pET42a plasmid and E. coli BL21DE3 as expression vector andexpression strain, respectively, prokaryotic expression systems of cheA and cheY genes ofC. jejuni strain NCTC11168 was constructed. Rabbits were immunized with the targetrecombinant expression proteins, rCheA and rCheY, to prepare the antisera. rCheA-IgG andrCheY-IgG in the antisera were separated using DEAE-52 ion exchange column. pBluescript-II -SK was applied to construct suicide plasmid which used to generate cheA gene knock-outmutant (cheA-). A chemotaxis model in vitro of C. jejuni based on DOC-HAP, the chemotacticability of cheA' mutant as well as the effect of rCheA-IgG and closantel inhibiting thebacterial chemotaxis were demonstrated. The phosphorylation levels of CheA and CheY afterDOC treatment were examined by using either rCheA-IgG or CheY-IgG capture method. Thedifference of colonization ability between cheA- mutant and wild-type of C. jejuni in micewere checked and then compared. Results The constructed prokaryotic expression systemscould efficiently express rCheA and rCheY. The data from PCR and sequencing confirmed thecheA gene knock out from cheA- mutant chromosome. cheA- mutant lost its chemotacticability towards DOC. Both the rCheA-IgG and closantel could inhibit the chemotaxis ofwild-type of C. jejuni (P < 0.05 ). When treatment of DOC, the phosphorylation levelsof CheA and CheY in wild-type of C. jejuni rapidly decreased (P < 0. 05 ). Thecolonization ability in murine jejunum of cheA- mutant was also lower than that of thewild-type ( P<0.05 ) . Conclusion Chemotaxis-associated two-component signaling system(Che-TCSS) of C. jejuni are composed of CheA and CheY, and the two proteins are activatedby dephosphorylation. CheA in the Che-TCSS play a critical role in chemotaxis in vitro andcolonization in vivo of C. jejuni, and this protein can be used as a target for developingnovel anti- C. jejuni drugs.

Identification and characterization of the aspartate chemosensory receptor of Campylobacter jejuni

Summary Campylobacter jejuni is a highly motile bacterium that responds via chemotaxis to environmental stimuli to migrate towards favourable conditions. Previous in silico analysis of the C. jejuni strain NCTC11168 genome sequence identified 10 open reading frames, tlp1‐10, that encode putative chemosensory receptors. We describe the characterization of the role and specificity of the Tlp1 chemoreceptor (Cj1506c). In vitro and in vivo models were used to determine if Tlp1 had a role in host colonization. The tlp1‐ isogenic mutant was more adherent in cell culture, however, showed reduced colonization ability in chickens. Specific interactions between the purified sensory domain of Tlp1 and l‐aspartate were identified using an amino acid array and saturation transfer difference nuclear magnetic resonance spectroscopy. Chemotaxis assays showed differences between migration of wild‐type C. jejuni cells and that of a tlp1‐ isogenic mutant, specifically towards aspartate. Furthermore, using yeast two‐hybrid and three‐hybrid systems for analysis of protein–protein interactions, the cytoplasmic signalling domain of Tlp1 was found to preferentially interact with CheV, rather than the CheW homologue of the chemotaxis signalling pathway; this interaction was confirmed using immune precipitation assays. This is the first identification of an aspartate receptor in bacteria other than Escherichia coli and Salmonella enterica serovar Typhimurium.