Voon Loong Chan

JlpA, a novel surface-exposed lipoprotein specific to Campylobacter jejuni, mediates adherence to host epithelial cells.

A 1116 bp open reading frame (ORF), designated jlpA, encoding a novel species‐specific lipoprotein of Campylobacter jejuni TGH9011, was identified from recombinant plasmid pHIP‐O. The jlpA gene encodes a polypeptide (JlpA) of 372 amino acid residues with a molecular mass of 42.3 kDa. JlpA contains a typical signal peptide and lipoprotein processing site at the N‐terminus. The presence of a lipid moiety on the JlpA molecule was confirmed by the incorporation of [3H]‐palmitic acid. Immunoblotting analysis of cell surface extracts prepared using glycine–acid buffer (pH 2.2) and proteinase K digestion of whole cells indicated that JlpA is a surface‐exposed lipoprotein in C. jejuni. JlpA is loosely associated with the cell surface, as it is easily extracted from the C. jejuni outer membrane by detergents, such as sarcosyl and Triton X‐100. JlpA is released to the culture medium, and its concentration increases in a time‐dependent fashion. The adherence of both insertion and deletion mutants of jlpA to HEp‐2 epithelial cells was reduced compared with that of parental C. jejuni TGH9011. Adherence of C. jejuni to HEp‐2 cells was inhibited in a dose‐dependent manner when the bacterium was preincubated with anti‐GST–JlpA antibodies or when HEp‐2 cells were preincubated with JlpA protein. A ligand‐binding immunoblotting assay showed that JlpA binds to HEp‐2 cells, which suggests that JlpA is C. jejuni adhesin.

FlaC, a protein of Campylobacter jejuni TGH9011 (ATCC43431) secreted through the flagellar apparatus, binds epithelial cells and influences cell invasion.

Type III secretion systems identified in bacterial pathogens of animals and plants transpose effectors and toxins directly into the cytosol of host cells or into the extracellular milieu. Proteins of the type III secretion apparatus are conserved among diverse and distantly related bacteria. Many type III apparatus proteins have homologues in the flagellar export apparatus, supporting the notion that type III secretion systems evolved from the flagellar export apparatus. No type III secretion apparatus genes have been found in the complete genomic sequence of Campylobacter jejuni NCTC11168. In this study, we report the characterization of a protein designated FlaC of C. jejuni TGH9011. FlaC is homologous to the N‐ and C‐terminus of the C. jejuni flagellin proteins, FlaA and FlaB, but lacks the central portion of these proteins. flaC null mutants form a morphologically normal flagellum and are highly motile. In wild‐type C. jejuni cultures, FlaC is found predominantly in the extracellular milieu as a secreted protein. Null mutants of the flagellar basal rod gene (flgF) and hook gene (flgE) do not secrete FlaC, suggesting that a functional flagellar export apparatus is required for FlaC secretion. During C. jejuni infection in vitro, secreted FlaC and purified recombinant FlaC bind to HEp‐2 cells. Invasion of HEp‐2 cells by flaC null mutants was reduced to a level of 14% compared with wild type, suggesting that FlaC plays an important role in cell invasion.

JlpA of Campylobacter jejuni interacts with surface-exposed heat shock protein 90alpha and triggers signalling pathways leading to the activation of NF-kappaB and p38 MAP kinase in epithelial cells.

Campylobacter jejuni is a leading cause of acute bacterial gastroenteritis in humans. The mechanism by which C. jejuni interacts with host cells, however, is still poorly understood. Our previous study has shown that the C. jejuni surface lipoprotein JlpA mediates adherence of the bacterium to epithelial cells. In this report, we demonstrated that JlpA interacts with HEp‐2 cell surface heat shock protein (Hsp) 90α and initiates signalling pathways leading to activation of NF‐κB and p38 MAP kinase. Gel overlay and GST pull down assays showed that JlpA interacts with Hsp90α. Geldanamycin, a specific inhibitor of Hsp90, and anti‐human Hsp90α antibody significantly blocked the interaction between JlpA and Hsp90α, suggesting a direct interaction between JlpA and HEp‐2 cell surface‐exposed Hsp90α. The treatment of HEp‐2 cells with GST‐JlpA initiated two signalling pathways: one leading to the phosphorylation and degradation of IκB and nuclear translocation of NF‐κB; and another one to the phosphorylation of p38 MAP kinase. The activation of NF‐κB and p38 MAP kinase in HEp‐2 cells suggest that JlpA triggers inflammatory/immune responses in host cells following C. jejuni infection.

Campylobacter hyoilei Alderton et al. 1995 and Campylobacter coli Veron and Chatelain 1973 are subjective synonyms.

The taxonomic affiliation of Campylobacter hyoilei was reevaluated by examining a variety of phenotypic and genotypic criteria. Whole-cell protein electrophoresis and a comparison of 66 phenotypic characters revealed that reference strains of C. hyoilei were indistinguishable from Campylobacter coli strains. These data were confirmed by a DNA-DNA hybridization level of 67% between the type strains of the two species. Several species-specific assays based on PCR amplification or probe hybridization further substantiated that C. coli strains and C. hyoilei strains are indistinguishable. It is therefore concluded that C. hyoilei and C. coli represent the same species and that the former name should be regarded as a junior synonym of the latter name.

Campylobacter jejuni Mediated Disruption of Polarized Epithelial Monolayers is Cell-Type Specific, Time Dependent, and Correlates With Bacterial Invasion

The precise mechanism by which the most common cause of bacterial enterocolitis in humans, Campylobacter jejuni, perturbs the intestinal mucosa remains elusive. To define effects of C. jejuni infection on mucosal permeability, Madin-Darby canine kidney (MDCK)-I and T84 cell monolayers were infected with C. jejuni for up to 48 h. All three tested C. jejuni strains caused a 73–78% reduction in transepithelial electrical resistance (TER) in intestinal (T84) cell monolayers, whereas only one strain slightly reduced TER of MDCK-I cells by 25% after 48 h infection. Infection with C. jejuni strains also caused a 2.3–4.5-fold increase in dextran permeability, but only in T84 cells. C. jejuni infection of monolayers also caused morphologic changes in desmosomes, observed by transmission electron microscopy. The cell-type specificity, demonstrated by increased T84 monolayer permeability, correlated with higher bacterial invasion into these cells, relative to MDCK-I cells. In T84 cells, invasion and bacterial translocation preceded barrier disruption and inhibition of C. jejuni invasion using a pharmacological inhibitor of phosphoinositide 3-kinase, reduced the drop in TER. These findings suggest that C. jejuni disruption of monolayers is mediated by invasion, provide new insights into C. jejuni-host epithelial barrier interactions, and offer potential mechanisms of intestinal injury and chronic immune stimulation.

Complete sequence of the Campylobacter jejuni glyA gene encoding serine hydroxymethyltransferase

The complete nucleotide sequence of the Campylobacter jejuni glyA gene was determined and the amino acid (aa) sequence of its product, serine hydroxymethyltransferase (SHMT), was deduced. The deduced polypeptide has 414 aa residues (Mr 45,758). The aa sequences of C. jejuni and Escherichia coli show 55.6% identity. Comparative analysis of the aa sequences of the SHMTs of E. coli and C. jejuni identified two new putative functional domains. The translational product of the C. jejuni glyA gene was identified using both minicell and maxicell systems and the transcription start point was mapped. The deduced transcription-regulatory signals, -10 and -35 sequences, show high homology to the corresponding consensus sequences for sigma 70 promoters in E. coli. The C. jejuni glyA promoter may be useful in the construction of shuttle vectors between E. coli and C. jejuni.

Cloning and sequence analysis of the flagellin gene ofCampylobacter jejuni TGH9011

Flagella are essential for motility and have been implicated to be one of the pathogenic determinants. The flagellum ofCampylobacter jejuni is a polymeric structure of a 62-kd protein. Using a high-affinity flagellin antibody to screen a lambda gt 11 phage genomic expression library ofC. jejuni strain TGH9011 (Serotype LIO36), a recombinant phage clone lambda gt 11RK that expresses theC. jejuni flagellin protein was isolated. The recombinant lambda gt 11 RK produced a 56-kd protein upon induction with isopropylthiogalactoside, which reacted specifically with anti-flagellin antibody. The flagellin gene was sequenced, and comparative analysis of the nucleotide and amino acid sequence identified a region of the flagellin that shows hypervariability among differentCampylobacter species and strains.

Cloning and expression of the Campylobacter jejuni glyA gene in Escherichia coli

Genetic studies of Campylobacter jejuni are greatly hampered by the lack of genetic markers and an established classical gene transfer mechanism between strains of this species. To facilitate future genetic studies and to provide a recombinant DNA approach for analyzing genes of C. jejuni, we constructed an extensive genomic library of a pathogenic C. jejuni strain TGH9011 (serotype 0:3) using pBR322. We report the isolation of a number of recombinant plasmids containing the complete structural gene of glyA, that encodes serine hydroxymethyltransferase (SHMT) of C. jejuni. Escherichia coli cells containing this multicopy recombinant plasmid with the glyA gene produce high levels of SHMT. The SHMT-encoding fragment was identified by subcloning and functional complementation. The expression of the C. jejuni glyA gene was probably via transcription initiated from its own promoter.

Mutator genes of baby hamster kidney cells.

Two mutator genes of mammalian cells were demonstrated. One was associated with the ribonucleoside diphosphate reductase, and the other was associated with an extreme adenosine sensitivity.

Isolation and preliminary characterization of 9-β-d-arabinofuranosyladenine-resistant mutants of baby hamster cells

A large number of 9-β-d-arabinofuranosyladenine (araA) -resistant mutants of baby hamster kidney cells (BHK 21/Cl3) were isolated. These mutants can be grouped into three mechanistically distinct classes. All the mutants showed cross-resistance to deoxyadenosine (dAdo). The mechanism of resistance to araA and dAdo in the class I mutants can be attributed to a mutation to adenosine kinase (AK) deficiency. The class II mutants have normal levels of AK, adenosine deaminase, and deoxyadenosine kinase. These mutants also show resistance to 1-β-d-arabinofuranosylcytosine (araC), and the mechanism of resistance is probably due to a mutation in the ribonucleotide reductase gene producing an enzyme that has an increased resistance to the inhibition by 9-β-d-arabinofuranosyladenine 5′-triphosphate (araATP) and 2′-deoxyadenosine 5′-triphosphate (dATP). The class III mutants, unlike those of classes I and II, show extreme adenosine (Ado) sensitivity. The Ados/araAr/dAdor phenotypic properties can be attributed to a single mutation. Classes II and III are novel araA-resistant mutants.