0000000000172825
AUTHOR
Trudy M. Wassenaar
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.
Potent membrane-permeabilizing and cytocidal action of Vibrio cholerae cytolysin on human intestinal cells
Many strains of Vibrio cholerae non-O1 and O1 El Tor that cause diarrhea do not harbor genes for a known secretogenic toxin. However, these strains usually elaborate a pore-forming toxin, hitherto characterized as a hemolysin and here designated V. cholerae cytolysin, whose action on intestinal cells has not yet been described. We report that V. cholerae cytolysin binds as a monomer to Intestine 407 cells and then assembles into detergent-stable oligomers that probably represent tetra- or pentamers. Oligomer formation is accompanied by generation of small transmembrane pores that allow rapid flux of K+ but not influx of Ca2+ or propidium iodide. Pore formation is followed by irreversible AT…
Transformation of Campylobacter jejuni
Campylobacter jejuni is a Gram-negative bacterium and is a commensal of many animal species (Skirrow and Blaser, 1992). In humans it is the major cause of human bacterial enteritis both in developed and developing countries (Tauxe, 1992; Taylor, 1992). Many cases of Campylobacter enteritis in humans have been associated with the consumption of or contact with undercooked chicken meat (Deming et al., 1987; Harris et al., 1986).
Binding of Escherichia coli hemolysin and activation of the target cells is not receptor-dependent.
Abstract 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. Competi…
Identification of the membrane penetrating domain of Vibrio cholerae cytolysin as a β-barrel structure
Summary 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…
Mode of primary binding to target membranes and pore formation induced by Vibrio cholerae cytolysin (hemolysin).
Vibrio cholerae cytolysin (VCC) is produced by many non-choleratoxigenic strains of V. cholerae, and possibly represents a relevant pathogenicity determinant of these bacteria. The protein is secreted as a pro-toxin that is proteolytically cleaved to yield the active toxin with a molecular mass of approximately 63 kDa. We here describe a simple procedure for preparative isolation of mature VCC from bacterial culture supernatants, and present information on its mode of binding and pore formation in biological membranes. At low concentrations, toxin monomers interact with a high-affinity binding site on highly susceptible rabbit erythrocytes. This as yet unidentified binding site is absent on…
High-resolution genotyping of Campylobacter strains isolated from poultry and humans with amplified fragment length polymorphism fingerprinting.
ABSTRACT For epidemiological studies of Campylobacter infections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing of Campylobacter jejuni and Campylobacter coli strains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleases Hin dIII and Hha I were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amp…
Toxin production by Campylobacter spp
Of all the virulence factors that were proposed for Campylobacter jejuni and related species to cause disease in humans, the discovery of toxin production was the most promising but led to a rather confusing and even disappointing stream of data. The discussion of whether proteinaceous exotoxins are relevant in disease remains open. One important reason for this lack of consensus is the anecdotal nature of the literature reports. To provide a basis for an unbiased opinion, this review compiles all described exotoxins, compares their reported properties, and provides a summary of animal model studies and clinical data. The toxins are divided into enterotoxins and cytotoxins and are sorted ac…
A cellular metalloproteinase activates Vibrio cholerae pro-cytolysin.
Many strains of Vibrio cholerae produce a cytolysin (VCC) that forms oligomeric transmembrane pores in animal cells. The molecule is secreted as a procytolysin (pro-VCC) of 79 kDa that must be cleaved at the N terminus to generate the active 65-kDa toxin. Processing can occur in solution, and previous studies have described the action of mature VCC thus generated. However, little is known about the properties of pro-VCC itself. In this study, it is shown that pro-VCC exist as a monomer in solution and binds as a monomer to eukaryotic cells. Bound pro-VCC can then be activated either by exogenous, extracellular, or by endogenous, cell-bound proteases. In both cases, cleavage generates the 65…
Evidence of Genomic Instability in Campylobacter jejuni Isolated from Poultry
ABSTRACT Poultry isolates of Campylobacter jejuni derived from a survey of meat processing batches were genotyped by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA to establish the clonal relationships between single-colony isolates. In the majority of batches studied, one or two genotype patterns predominated. However, in one batch (batch A), 21 single-colony isolates gave 14 different PFGE genotypes. The banding patterns obtained with Sma I were sufficiently different to distinguish between genotypes, although the patterns also produced many common bands. The question of whether these isolates represented different clones or had a common clonal ancestry was addressed by additi…
Differential uptake and killing potential of Campylobacter jejuni by human peripheral monocytes/macrophages
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 …
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…
Genotyping of Campylobacter spp.
method. The major disadvantages of both of these techniques are the high number of untypeable strains and the time-consuming and technically demanding requirements of the techniques. Production and quality control of antiserum reagents for serotyping schemes are costly; consequently, these reagents are not widely available. A recently developed scheme (23) based on HS antigens in which modified antibody production and antigen detection techniques are used may be an improvement for routine use, but this scheme does not solve the problem of restricted reagent availability or the problem of the high level of nontypeability. Because of such problems, the value of serotyping techniques for natio…