Trudy M. Wassenaar

Inactivation of Campylobacter jejuni flagellin genes by homologous recombination demonstrates that flaA but not flaB is required for invasion.

The role of the Campylobacter jejuni flagella in adhesion to, and penetration into, eukaryotic cells was investigated. We used homologous recombination to inactivate the two flagellin genes flaA and flaB of C. jejuni, respectively. Mutants in which flaB but not flaA is inactivated remain motile. In contrast a defective flaA gene leads to immotile bacteria. Invasion studies showed that mutants without motile flagella have lost their potential to adhere to, and penetrate into, human intestinal cells in vitro. Invasive properties could be partially restored by centrifugation of the mutants onto the tissue culture cells, indicating that motility is a major, but not the only, factor involved in invasion.

Colonization of chicks by motility mutants of Campylobacter jejuni demonstrates the importance of flagellin A expression.

Campylobacter jejuni strain 81116 contains two flagellin genes, flaA and flaB. Wild-type (WT) bacteria express flaA only, but flaB can be expressed under certain conditions. We have determined the importance of flagella for colonization of the avian caecum, which appears to be the natural environment for these bacteria. Mutants in which flaA or flaB, or both had been inactivated, and motility variants, were investigated. Flagella are not a requisite for colonization, but mutants lacking both flagellin genes colonized less efficiently than WT. Inactivation of the flaB gene, which had no effect on bacterial motility, enhanced chicken caecal colonization 1000-fold compared to WT. A variant (SF-1) with flagella composed of flagellin A, but with poor motility, also colonized better than WT. Conversely, mutants with an inactivated flaA gene colonized 100- to 1000-fold less efficiently than WT, regardless of their motility conferred by truncated or full-length flagellin B flagella. These results suggest that the presence of flagellin A, rather than motility, is essential for optimal bacterial colonization of chicken caeca.

Pathophysiology of Campylobacter jejuni infections of humans

Campylobacter jejuni and closely related organisms are major causes of human bacterial enteritis. These infections can lead to extraintestinal disease and severe long-term complications. Of these, neurological damage, apparently due to the immune response of the host, is the most striking. This review examines current knowledge of the pathophysiology of the organism. Diversity of C. jejuni isolates in genotypic and phenotypic characteristics now is recognized and clinically relevant examples are presented. Expected future directions are outlined.

Variation of the flagellin gene locus of Campylobacter jejuni by recombination and horizontal gene transfer

The capacity of Campylobacter jejuni to generate genetic diversity was determined for its flagellar region. Recombination within a genome, as well as recombination after the uptake of exogenous DNA, could be demonstrated. The subunit of the flagellar filament of C. jejuni is encoded by two tandem genes, flaA and flaB, which are highly similar and therefore subject to recombination. A spontaneous recombination within this locus was demonstrated in a bacterial clone containing an antibiotic-resistance gene inserted in flaA. A recombinant was isolated in which the antibiotic-resistance gene had been repositioned into flaB, indicating that genetic information can be exchanged between the two flagellin genes of C. jejuni. The occurrence of recombinational events after the uptake of exogenous DNA by naturally competent bacteria was demonstrated with two mutants containing different antibiotic-resistance markers in their flagellin genes. Double-resistant transformants were formed when purified chromosomal donor DNA was added to a recipient strain, when the two bacterial cultures were mixed under conditions that induce natural competence, or when the two strains were cocultured. Both mechanisms of recombination may be used by the pathogenic organism to escape the immunological responses of the host or otherwise adapt to the environment.

Differential uptake and killing potential of Campylobacter jejuni by human peripheral monocytes/macrophages

Abstract The ability of Campylobacter jejuni to survive in monocytes after phagocytic uptake was tested in a new in vitro model using adherent macrophages derived from human peripheral monocytes. The cells were stimulated with cytokines before use to ensure full phagocytic and killing activity. The kinetics of uptake and killing of bacteria was followed for 72 h with 16 strains, including stool and blood isolates and laboratory adapted strains. Significant bacterial strain differences were not observed, but the viability of phagocytosed bacteria was dependent on the individual donating the macrophages. The majority of blood donors carried macrophages that killed phagocytosed Campylobacter within 24 or 48 h. There was no correlation between the source of isolation of the strains and relative intracellular survival. Bacterial mutants of superoxide dismutase, catalase or polyphosphate kinase were all as sensitive to macrophage killing as their isogenic wild-type strain. In contrast, about 10% of the voluntary blood donors carried monocytes which were incapable of killing phagocytosed bacteria. Such macrophages displayed normal uptake, but killing was insufficient and bacterial growth was observed with all strains and mutants tested. We conclude that (1) since in most cases activated human macrophages kill C. jejuni efficiently after phagocytosis, intra-phagocytic survival is not a common phenomenon during Campylobacter infection; and (2) those individuals carrying macrophages that are unable to destroy phagocytosed bacteria are at risk to develop a bacteremia during Campylobacter infection.

Binding of Escherichia coli Hemolysin and Activation of the Target Cells Is Not Receptor-dependent

Production of a single cysteine substitution mutant, S177C, allowed Escherichia coli hemolysin (HlyA) to be radioactively labeled with tritiated N-ethylmaleimide without affecting biological activity. It thus became possible to study the binding characteristics of HlyA as well as of toxin mutants in which one or both acylation sites were deleted. All toxins bound to erythrocytes and granulocytes in a nonsaturable manner. Only wild-type toxin and the lytic monoacylated mutant stimulated production of superoxide anions in granulocytes. An oxidative burst coincided with elevation of intracellular Ca2+, which was likely because of passive influx of Ca2+ through the toxin pores. Competition experiments showed that binding to the cells was receptor-independent, and preloading of cells with a nonlytic HlyA mutant did not abrogate the respiratory burst provoked by a subsequent application of wild-type HlyA. In contrast to a previous report, expression or activation of the β2 integrin lymphocyte function-associated antigen-1 did not affect binding of HlyA. We conclude that HlyA binds nonspecifically to target cells and a receptor is involved neither in causing hemolysis nor in triggering cellular reactions.

Structure and genotypic plasticity of the Campylobacter fetus sap locus

The Campylobacter fetus surface layer proteins (SLPs), encoded by five to nine sapA homologues, are major virulence factors. To characterize the sapA homologues further, a 65.9 kb C. fetus genomic region encompassing the sap locus from wild‐type strain 23D was completely sequenced and analysed; 44 predicted open reading frames (ORFs) were recognized. The 53.8 kb sap locus contained eight complete and one partial sapA homologues, varying from 2769 to 3879 bp, sharing conserved 553–2622 bp 5′ regions, with partial sharing of 5′ and 3′ non‐coding regions. All eight sapA homologues were expressed in Escherichia coli as antigenic proteins and reattached to the surface of SLP– strain 23B, indicating their conserved function. Analysis of the sap homologues indicated three phylogenetic groups. Promoter‐specific polymerase chain reactions (PCRs) and sapA homologue‐specific reverse transcription (RT)‐PCRs showed that the unique sapA promoter can potentially express all eight sapA homologues. Reciprocal DNA recombination based on the 5′ conserved regions can involve each of the eight sapA homologues, with frequencies from 10−1 to 10−3. Intragenic recombination between sapA7 and sapAp8, mediated by their conserved regions with a 10−1−10−2 frequency, allows the formation of new sap homologues. As divergent SLP C‐termini possess multiple antigenic sites, their reciprocal recombination behind the unique sap promoter leads to continuing antigenic variation.

Pro-inflammatory feedback activation cycle evoked by attack of Vibrio cholerae cytolysin on human neutrophil granulocytes

Vibrio cholerae cytolysin (VCC) is a pore-forming toxin that is secreted in precursor form (pro-VCC) and requires proteolytic cleavage in order to attain membrane-permeabilizing properties. Pro-VCC can be activated both in solution and membrane-bound state. Processing of membrane-bound pro-VCC can in turn be achieved through the action of both cell-associated and soluble proteases. The current investigation describes the interaction of VCC with human neutrophil granulocytes. It is shown that pro-VCC binds to these cells and is cleaved by cell-bound serine proteases. Membrane permeabilization leads to granulocyte activation, as witnessed by the generation of reactive oxygen metabolites and liberation of granule constituents. A mutant toxin with unaltered binding properties but devoid of pore-forming activity did not elicit these effects. The secreted proteases cleave and activate further bound- and non-bound pro-VCC. A positive feedback loop is thus created that results in enhanced cytotoxicity towards both the targeted granulocytes and towards bystander cells that are not primarily killed by the protoxin. Thus, activation of neutrophil granulocytes by VCC fuels a positive feedback cycle that will cripple immune defence, augment inflammation, and enhance the cytotoxic action of the toxin on neighbouring tissue cells.

Putative identification of an amphipathic alpha-helical sequence in hemolysin of Escherichia coli (HlyA) involved in transmembrane pore formation.

Abstract Escherichia coli hemolysin is a pore-forming protein belonging to the RTX toxin family. Cysteine scanning mutagenesis was performed to characterize the putative pore-forming domain of the molecule. A single cysteine residue was introduced at 48 positions within the sequence spanning residues 170–400 and labeled with the polarity-sensitive dye badan. Spectrofluorimetric analyses indicated that several amino acids in this domain are inserted into the lipid bilayer during pore formation. An amphipathic α-helix spanning residues 272–298 was identified that may line the aqueous pore. The importance of this sequence was highlighted by the introduction of two prolines at positions 284 and 287. Disruption of the helix structure did not affect binding properties, but totally abolished the hemolytic activity of the molecule.

Identification of the membrane penetrating domain of Vibrio cholerae cytolysin as a β‐barrel structure

Vibrio cholerae cytolysin (VCC) is an oligomerizing pore‐forming toxin that is related to cytolysins of many other Gram‐negative organisms. VCC contains six cysteine residues, of which two were found to be present in free sulphydryl form. The positions of two intramolecular disulphide bonds were mapped, and one was shown to be essential for correct folding of protoxin. Mutations were created in which the two free cysteines were deleted, so that single cysteine substitution mutants could be generated for site‐specific labelling. Employment of polarity‐sensitive fluorophores identified amino acid side‐chains that formed part of the pore‐forming domain of VCC. The sequence commenced at residue 311, and was deduced to form a β‐barrel in the assembled oligomer with the subsequent odd‐numbered residues facing the lipid bilayer and even‐numbered residues facing the lumen. Pro328/Lys329 were tentatively identified as the position at which the sequence turns back into the membrane and where the antiparallel β‐strand commences. This was deduced from fluorimetric analyses combined with experiments in which the pore was reversibly occluded by derivatization of sulphydryl groups with a bulky moiety. Our data support computer‐based predictions that the membrane‐permeabilizing amino acid sequence of VCC is homologous to the β‐barrel‐forming sequence of staphylococcal cytolysins and identify the β‐barrel as a membrane‐perforating structure that is highly conserved in evolution.