Molecular characterization of a cytotoxic porin protein from Campylobacter jejuni and its role in campylobacteriosis

Campylobacter jejuni is a major cause of enteritis in developed and developing countries. A cytotoxic complex was isolated from organism-free filtrates of broth grown organisms by screening for activity in HEp-2 cells following high performance liquid chromatography using a combination of size exclusion and DEAE column chromatography. The toxin was found to coincide with a 45 kDa protein possessing an N-terminus sequence indicative of a bacterial outer membrane porin protein together with a high molecular weight carbohydrate which was determined to be lipopolysaccharide (LPS). The carbohydrate portion of the LPS had reactivity for the lectins Galanthus nivalis agglutinin, Maakia amurensis agglutinin and Datura stromonium agglutinin. The cytotoxicity of the porin-LPS complex was heat-labile at 70\sp\circC within 30 min. It was also found to be resistant to trypsin and to degradation by the enzymes neuriminidase and glycosidase F as well as to oxidation with sodium meta-periodate. The complex induced DNA fragmentation, cytoplasmic blebbing and nuclear condensation in HEp-2 cells after 24 h of intoxication possibly indicating that the cells were becoming apoptotic. Oligonucleotides were generated from the N-terminus of the porin protein. Using a combination of vectorette and inverse polymerase chain reaction (PCR) and cloning, a 1.45 kb fragment was sequenced and found to contain a 1275 base pair (bp) open reading frame (ORF). The ORF, designated porA encoded a 424 amino acid protein with a 22 residue leader sequence and had a calculated molecular weight of 45.6 kDa and pI of 4.44. The mature protein was 402 amino acids in length and had a molecular weight of 43.5 kDa and a pI of 4.35. The translated PorA protein had a 50% sequence homology with Haemophilis influenzae major outer membrane protein P2. Thirty-two strains of Campylobacter sp. and closely related organisms were screened for the porA gene and expression of cytotoxin in cell culture. Although all strains examined produced a cytotoxin, 21 of the 32 strains (66%) were genotypically positive for porA and of these 21 of 23 (91%) were C. jejuni strains. It is postulated that this cytotoxic protein is a major virulence factor for C. jejuni and may be responsible for the clinical symptoms usually associated with campylobacteriosis

Intramuscular immunization of mice with KatA (catalase) generates functional antibodies against Campylobacter jejuni invasion of gut epithelial cells in vitro

The high incidence of Campylobacter jejuni-associated diarrhea, increase in the frequency of drug resistance in clinical isolates, and the recent association of C. jejuni infections and Guillain-Barré syndrome has heightened the need to develop effective anti-Campylobacter vaccines. Due to the risk of auto-immunity, vaccination using inactivated whole-cells or attenuated cells cannot be considered. Subunit vaccines however are a viable alternative and have been shown to be effective against similar pathogens. The main goal of this research is to test efficacy of the catalase A (KatA) protein as vaccine candidate against this pathogen. It was determined that KatA co-fractionates with both the soluble and insoluble cell fractions and a balanced IgG1/IgG2a response could be produced against it following immunization in mice. It was demonstrated that these antibodies can mediate complement-specific bactericidal activity and reduced adhesion and invasion of human epithelial (Caco-2) cells by C. jejuni using established in vitro assays.Master's These

Rational design of vaccines for the control of Campylobacter in chickens

Campylobacter is the leading cause of bacterial food-borne diarrhoeal disease in the developed world and a significant cause of infant morbidity and mortality in developing countries. Epidemiological studies implicate poultry as a key source of infection, with up to 80% of human cases being attributable to the avian reservoir. An effective vaccine for broilers is predicted to limit the incidence of human campylobacteriosis. Vaccination of chickens with CjaA, either in recombinant form or vectored in live-attenuated Salmonella, has been reported to significantly reduce caecal colonisation by C. jejuni, with more invasive carriers eliciting greater protection. However, protection remains modest and is slow to develop. I therefore sought to improve such vaccines, first by vectoring codon-optimised CjaA in a licensed avian pathogenic E. coli ΔaroA vaccine. In two independent trials, White Leghorn birds were vaccinated subcutaneously on the day of hatch and 14 days later then challenged with C. jejuni M1 at 28 days post-hatch. No protection was observed despite significant induction of CjaA-specific serum IgY, however, a previously described S. Typhimurium ΔaroA vaccine vectoring CjaA also failed to protect. Owing to the variability observed with live CjaA-based vaccines in these and previous studies, other candidate antigens were sought and evaluated as subunits. Twenty-one candidate C. jejuni antigens were cloned and expressed as glutathione-S-transferase (GST) fusions. Nine of these could be purified in adequate soluble quantities to be tested in vivo. The intervals of vaccination and challenge were as above, with GST alone or GSTCjaA acting as negative and positive controls, respectively. Each antigen was administered subcutaneously in TiterMax Gold® adjuvant at the molar equivalent of the doses of GSTCjaA. Repeated testing of initially promising candidates revealed that, when averaged across three independent trials, GST-SodB and GST-FliD induced statistically significant reductions in caecal colonisation of 1-2 log10 colony-forming units of C. jejuni at 48 and 56 days post-hatch compared to negative controls. Induction of antigen-specific serum IgY was measured by enzyme linked-immunosorbent assays using maltose-binding protein fusions to each antigen. This revealed significant induction of antigen specific serum IgY for the majority of the antigens tested, even when no protection was observed. In the SodB- and FliD-vaccinated groups, the peak of antigen-specific serum IgY was not coincident with the onset of protection and the fold-change in specific IgY levels in individual birds did not correlate with caecal Campylobacter numbers. Furthermore, sera from SodB-vaccinated birds failed to detect SodB in the outer membrane or surface of Campylobacter cells, indicating that SodB-specific antibodies are unlikely to be neutralising. Taken together, these studies identified two novel protective antigens that, with further optimisation, could form part of an anti-Campylobacter vaccine for broilers. However further studies are required to define the nature and consequences of immune responses required for protection

Identification of host-pathogen interacting molecules of Campylobacter jejuni using phage display technology and in silico sequence analysis

Campylobacter jejuni is the leading cause of human gastroenteritis world-wide, and the antecedent infection in Guillain-Barré syndrome. Although much progress has been made regarding its virulence determinants,our understanding of the molecular basis of C. jejuni pathogenesis still lags behind that of other enteric pathogens. Bacterial pathogenesis is often dependent on proteinaceous virulence factors transported to the bacterial cell surface or released into the external environment. Understanding the molecular basis of interactions between these proteins and host cells is necessary in understanding and controlling infections. Background: Previously identified adhesins of C. jejuni NCTC11168 have still not provided us with a total understanding of the pathogenesis of campylobacteriosis. It is hypothesized that as yet unidentified C. jejuni surface proteins interact with host proteins contributing to colonisation and pathogenesis. This study sought to screen the genome of C. jejuni NCTC11168 to identify additional genes that may code for other adhesins using phage display technology and in silico sequence analysis. Methods: A phage display library was constructed by the insertion of randomly fragmented chromosomal DNA of C. jejuni NCTC11168 into the phagemid vector pG8SAET. Following affinity panning of the library against holo- and apo-lactoferrin, enriched clones were screened with ELISA to identify affinity-binding clones. Several phage clones were randomly selected and their C. jejuni DNA inserts sequenced and analysed with bio-informatic tools. In order to identify novel autotransporter proteins of C. jejuni, the amino acid sequences of previously described adhesion-associated autotransporters were employed in BLAST searches of the predicted coding sequences of C. jejuni NCTC11168 genome database. Results: Screening of the phage display library resulted in the identification of C. jejuni NCTC11168 gene, Cj0609c, encoding a putative periplasmic protein, designated LimC, predicted to be a member of the SGNH-family of hydrolases, a diverse family of lipases and esterases. Searching the genome of C. jejuni NCTC11168, Cj0628 was identified as coding a protein with characteristics of autotransporters, designated CapA. CapA was demonstrated to be surface-exposed, mutants of which had a lowered ability to associate with or invade Caco-2 cells and failed to colonize chicken guts, indicating that CapA plays a role in host association and colonization by Campylobacter. Conclusion: LimC may be involved in binding of C. jejuni to lactoferrin for iron acquisition in vivo, and/or play further role in adhesion, colonization and internalisation of C. jejuni into host cells. CapA also plays a role in adhesion. Further characterization of these proteins should contribute to our understanding of the pathogenesis of C. jejuni

Response of Campylobacter Jejuni to Iron and Hydrogen Peroxide

Iron affects the physiology of bacteria in two different ways: as a micronutrient for bacterial growth and as a catalyst for the formation of hydroxyl radicals. The present study reports the response of Campylobacter jejuni/ to iron and oxidative stresses using DNA microarrays. C. jejuni/ response to hydrogen peroxide (HP), cumene hydroperoxide (CHP) and menadione was investigated. The response to hydrogen peroxide was investigated both in the presence and the absence of iron. Characterization of the ferric uptake regulator (Fur) and peroxidase regulator (PerR) mutants by transcriptome profiling allowed the identification of Fur and PerR regulated genes, respectively. Chicken colonization model was used to test different C. jejuni/ mutants for their ability to colonize chicken ceca. C. jejuni/ responded to iron availability by differential expression of genes belonging to several functional groups, ranging from energy metabolism, to cell surface structures, to iron acquisition, and to oxidative stress defenVeterinary Pathobiolog

Identification of an immunogenic 18 kDa protein of Helicobacter pylori using alkaline phosphatase gene fusions : a thesis presented in partial fulfillment of the requirement for the Doctor of Philosophy in Molecular Microbiology, Massey University, Palmerston North, NZ

Secreted or surface-associated proteins play an important role in the immunopathogenesis of Helicobacter pylori infection. The aim of this study was to identify, using a genetic approach, H. pylori exported proteins and assess their role in the host immune response to infection. As part of this work, an H. pylori expression library was constructed and screened with a monoclonal antibody raised to a component of outer membrane vesicles from H. pylori, identified and characterised in a separate study. The screening strategy identified a locus of the genome containing two genes encoding exported proteins. Subsequent expression studies identified the gene product detected by the antibody as Lpp20, which encodes a well characterised lipoprotein from H. pylori. In addition, the use of alkaline phosphatase (AP) gene fusion methodology enabled the identification of a large number of other H. pylori exported proteins. Immunoscreening of a selection of enzymatically active H. pylori AP fusion proteins was carried out by Western blot analysis with patient sera and lymphocyte proliferation assays using peripheral blood mononuclear cells from H. pylori infected individuals. These assays identified a novel H. pylori exported antigen which was recognised by antibody derived from H. pylori infected individuals. Southern blot analysis revealed that the gene encoding the protein was absent in other Helicobacter species tested and sequence analysis of the gene demonstrated that it is highly conserved among H. pylori isolates. In order to obtain pure recombinant protein, the gene encoding the protein was cloned and expressed as a Beta-galactosidase (β-gal) fusion in Escherichia coli and the protein purified by affinity chromatography. The size of the recombinant protein released (18 kDa) was consistent with the calculated molecular mass of the polypeptide deduced from the DNA sequence. In Western blot assays, the purified protein was recognised by 71% of sera taken from patients infected with H. pylori, but by only 16% of sera taken from patients with unrelated or with no gastrointestinal disease. These results indicated that the 18 kDa protein from H. pylori was immunogenic and expressed in vivo. In other experiments, it was found that oral administration of this antigen did not protect mice against H. pylori colonisation following challenge with H. pylori

Regulating Campylobacter jejuni flagellar gene expression: transcriptional and post-transcriptional mechanisms of control

The bacterial pathogen Campylobacter jejuni is the leading cause of foodborne gastroenteritis in the developed world. C. jejuni flagella are crucial virulence determinants, but the regulation of these complex organelles within different environments is not fully understood. Moreover, regulatory RNAs are important for virulence and flagellar gene expression in many prokaryotes, but their role in C. jejuni biology is unknown. The first aim was to understand flagellar regulation in acidic conditions and what effect this has on virulence. The most acidic pH C. jejuni was able to survive was pH 3.6 and acid-shock at this pH and pH 5 increased expression of a subset of flagellar genes and increased invasion of intestinal epithelial cells. The second aim of this study was to characterise the function of two paralogous small non-coding RNAs (less than 50 nucleotides), NC1 and NC4, which were identified in the C. jejuni NCTC11168 transcriptome and are predicted to regulate flagella gene expression. NC1 and NC4 expression was dependent on the flagellar sigma factor, sigma28, and post-transcriptionally regulated expression of predicted sigma54-dependent C. jejuni flagellar gene targets in an E. coli based GFP reporter system. However, microarray and phenotypic analysis showed no clear differences in gene expression between NC1/NC4 deletion and over-expression mutants compared to the wild-type strain. The conclusions are that flagellar gene expression is regulated by acidic conditions and C. jejuni invasion of intestinal epithelial cells may be primed in response to acid. In addition, the transcription of NC1 and NC4 is linked to flagella expression and they may function to post-transcriptionally regulate sigma54-dependent flagella genes in C. jejuni. Although the biological significance of NC1 and NC4 remains unknown, this is the first study to show that non-coding RNAs are potential regulators of gene expression in Campylobacter

Molecular evolutionary analyses and epidemiology of Vibrio parahaemolyticus in Thailand

Vibrio parahaemolyticus is a seafood-borne pathogenic bacterium which is a major cause of gastroenteritis worldwide. In the present study, the genetic relationships and population structure of isolates originating from clinical and seafood production sources in Thailand were investigated by multilocus sequence typing (MLST). Nucleotide sequence variation of virulence-related genes including haemolysin and TTSS1 genes among Thai and worldwide isolates was also analyzed. The outer membrane proteome of V. parahaemolyticus isolate RIMD2210633 was predicted using bioinformatic approaches, and the outer membrane proteomes of eight isolates from different sources and representing different MLST sequence type characterized using proteomics. The 101 Thai V. parahaemolyticus isolates examined were recovered from clinical samples (n=15), healthy human carriers (n=18), various fresh seafood (n=18), frozen shrimps (n=16), fresh-farmed shrimp tissue (n=18) and shrimp-farm water (n=16). Phylogenetic analysis revealed a high degree of genetic diversity within the V. parahaemolyticus population, although isolates recovered from clinical samples, farmed shrimp and water samples represented five distinct clusters. The majority of clinical isolates were resolved into two genetic clusters and none of these isolates were found to share sequence types (STs) with strains isolated from human carriers, seafood, or water. Similarly, STs representing human carrier isolates differed from those of clinical, seafood and water isolates. The limited genetic diversity of the clinical isolates suggested non-random selection for pathogenic strains, but the absence of such strains in local seafood raises questions about the likely source of infection. Extensive serotypic diversity occurred among isolates representing the same STs and recovered from the same source at the same time point. Furthermore, evidence of interspecies horizontal gene transfer and intragenic recombination was observed at the recA locus in a large proportion of isolates; this has a substantial effect on the apparent phylogenetic relationships of the isolates. Notably, the majority of these recombinational exchanges occurred among clinical and carrier isolates, suggesting that the human intestinal tract is serving as a reservoir that is driving evolutionary change and leading to the emergence of new, potentially pathogenic strains. MLST was also applied to study genetic relationships between V. parahaemolyticus isolates from Thailand (n=101) and those from European countries (n=9). With the exception of the pandemic ST3 which was resolved from two isolates from Thai human carriers, two clinical isolates from England and a clinical isolate from Norway, none of the other European isolates examined in this study shared the same ST with the Thai isolates. This study demonstrated that Thai human carrier isolates are capable of harbouring virulence-related genes including the haemolysin-encoding genes tdhA, tdhS, trh1 and trh2, and the TTSS1-related genes vcrD1, vscC2 and VP1680, that are present in clinical isolates. In particular, the Thai human carrier isolate VP132 shared identical TTSS1-related gene fragments with the pandemic V. parahaemolyticus serotype O3:K6 (RIMD2210633) and related strains (AQ3810, AQ4037, Peru466, AN5034 and K5030) of worldwide distribution. A total of 117 outer membrane proteins (OMPs) were predicted from the genome of V. parahaemolyticus isolate RIMD2210633. A total 73 OMPs proteins were identified from eight V. parahaemolyticus isolates recovered from clinical samples, human carriers, oyster, shrimp tissue and water in Thailand. Of the 117 predicted OMPS, 32 were identified in eight strains by proteomic analysis. OmpU, a non-specific porin protein, represents the most abundantly expressed protein in all eight isolates. OMPs involved in TTSSs (YscW, YscJ, YscC, PopN and VscC2) and iron uptake (IrgA, putative 83 Da decaheme outer membrane cytochrome C, PvuA1, PvuA2, LutA, FhuE, HutA and putative-regulated protein B) were predicted from the genome of V. parahaemolyticus isolate RIMD2210633, but were not recovered from any of the eight Thai isolates. The absence of TTSS and iron uptake related OMPs in the eight representative strains that were grown under in vitro conditions may suggest an important requirement for in vivo growth conditions to induce expression of important virulence factor-related OMPs in V. parahaemolyticus. There was no clear association between OMP profile and the source of isolation, ST or serotype. However, a high degree of variation of OMP profiles was observed in isolates from different sources as well as in the isolates representing the same ST. This study demonstrated the usefulness of a multidisciplinary approach that includes MLST, virulence-related gene DNA sequence analysis, bioinformatic prediction and gel-based proteomic analyses for the study of molecular evolutionary relationships and the epidemiology of V. parahaemolyticus isolates from clinical and seafood production sources. The outcomes of this study highlight the role of human carriers as a reservoir of potentially pathogenic V. parahaemolyticus and this should be considered as one of the possible contamination sources in the surveillance of seafood safety

Campylobacter jejuni colonization of broiler chickens

The pathogenesis of C. jejuni in broiler chickens is still poorly understood despite the importance of poultry meat as a source of infection in humans. The overall objective of this project was to understand the role of flagella and Campylobacter invasion antigens in mucosal and systemic colonization, and to evaluate the vaccine potential of C. jejuni paralyzed flagella mutants. As a first step to track C. jejuni in vivo, a Green Fluorescent Protein (GFP) reporter system that is constitutively expressed was constructed. The system was transformed into different C. jejuni strains and isolates, and their mucosal and systemic spreading was studied over the period of 7 days. C. jejuni NCTC11168V1 and V26 share the same background but differ in their ability to colonize chickens. C. jejuni 81-176 and K2-55 share the same genetic background but K2-55 has an insertion mutation in pflA gene that produced paralyzed flagella. Although the K2-55 flagella remained intact structurally, it did not secret Campylobacter invasion antigens (Cia). The reporter system was stable in all of these strains both in vitro and in vivo. Fluorescent bacteria were visualized successfully using fluorescent and confocal microscopes. C. jejuni NCTC11168V1 and 81-176 were detected in the intestinal tract and in the liver and spleen of more than 30% of the challenged birds, while V26 and K2-55 were only detected in the intestinal tract. C. jejuni 81-176 and K2-55 did not spread systemically to the spleen and liver of BALB/c mice challenged using the same approach, although they colonized the ceca. A live attenuated vaccine based on C. jejuni K2-55 protected broiler chickens from C. jejuni 81-176 challenge in chickens following streptomycin treatment of drinking water. The same vaccine had no significant protection against a heterolgous C. jejuni NCTC11168V1 strain challenge. The vaccine was a poor stimulator of secretory IgA. Macrophage-like HD11 cells inflammatory response to the presence of C. jejuni K2-55 was not significantly different from their response to wild-type 81-176 when measured by qRT-PCR. The lack of Cia secretion and motility had no effect on expression of IL-1â, IL-2, IL-6, IL-8, IL, IL-10, IL-12â, or TLR5. A flgK mutant expressing the flagella up to the hook had a significantly lower expression of these genes

Interactions between Campylobacters and their bacteriophages

Campylobacter jejuni is a leading cause of human bacterial enteritis worldwide. Consumption of contaminated poultry meat is considered a major source of infection. The use of virulent bacteriophages as a form of biocontrol to specifically reduce this pathogen in poultry (phage therapy) is a promising intervention that does not rely on antimicrobials and therefore circumvents the emergence of antibiotic-resistant Campylobacter strains. In order to achieve this, a better understanding of the mechanisms involved in phage-host interactions at the molecular level would assist in the development of the strategy and the selection of bacteriophages. The main objective of this study was to therefore examine such interactions between Campylobacter and its virulent phages. To achieve this, the transcriptional response of C. jejuni to phage infection was investigated, along with the role of a Type II restriction-modification system during phage infection of Campylobacter. These studies were conducted using the highly phage-sensitive Campylobacter strain, C. jejuni PT14, in conjunction with a number of group II and III bacteriophages (Eucampyvirinae). Transcriptome studies (RNA-Seq) revealed a phage-induced host response that included a demand for iron and oxygen. This was highlighted by the up-regulation of several siderophore-based iron acquisition genes and down-regulation of genes associated with a number of anaerobic electron transport pathways that utilise alternative electron acceptors to oxygen. In addition, the pattern of gene regulation also suggested apo-Fur regulation of the iron-responsive and flagellar biogenesis genes. This host response has been proposed to occur as a consequence of the reduction of ribonucleotides to form deoxyribonucleotides during phage DNA replication. This process is catalysed by the enzyme ribonucleotide reductase and requires iron and oxygen during the formation of a reactive di-iron centre within the β-subunit of the enzyme. Unusually knock-out mutants of a Type II restriction-modification system had a negative impact on phage replication. The A911_00150 mutant displayed pleiotropic changes in motility, cell based invasion and the ability to colonise chickens. Transcriptome analysis highlighted down-regulation of the genes required for the synthesis of the bacterial flagellum