Alpha-toxin and gamma-toxin jointly promote Staphylococcus aureus virulence in murine septic arthritis. (1/3113)

Septic arthritis is a common and feared complication of staphylococcal infections. Staphylococcus aureus produces a number of potential virulence factors including certain adhesins and enterotoxins. In this study we have assessed the roles of cytolytic toxins in the development of septic arthritis by inoculating mice with S. aureus wild-type strain 8325-4 or isogenic mutants differing in the expression of alpha-, beta-, and gamma-toxin production patterns. Mice inoculated with either an alpha- or beta-toxin mutant showed degrees of inflammation, joint damage, and weight decrease similar to wild-type-inoculated mice. In contrast, mice inoculated with either double (alpha- and gamma-toxin-deficient)- or triple (alpha-, beta-, and gamma-toxin-deficient)-mutant S. aureus strains showed lower frequency and severity of arthritis, measured both clinically and histologically, than mice inoculated with the wild-type strain. We conclude that simultaneous production of alpha- and gamma-toxin is a virulence factor in S. aureus arthritis.  (+info)

Evolutionary relationships of pathogenic clones of Vibrio cholerae by sequence analysis of four housekeeping genes. (2/3113)

Studies of the Vibrio cholerae population, using molecular typing techniques, have shown the existence of several pathogenic clones, mainly sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones. However, the relationship of the pathogenic clones to environmental V. cholerae isolates remains unclear. A previous study to determine the phylogeny of V. cholerae by sequencing the asd (aspartate semialdehyde dehydrogenase) gene of V. cholerae showed that the sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones had very different asd sequences which fell into separate lineages in the V. cholerae population. As gene trees drawn from a single gene may not reflect the true topology of the population, we sequenced the mdh (malate dehydrogenase) and hlyA (hemolysin A) genes from representatives of environmental and clinical isolates of V. cholerae and found that the mdh and hlyA sequences from the three pathogenic clones were identical, except for the previously reported 11-bp deletion in hlyA in the sixth-pandemic clone. Identical sequences were obtained, despite average nucleotide differences in the mdh and hlyA genes of 1.52 and 3.25%, respectively, among all the isolates, suggesting that the three pathogenic clones are closely related. To extend these observations, segments of the recA and dnaE genes were sequenced from a selection of the pathogenic isolates, where the sequences were either identical or substantially different between the clones. The results show that the three pathogenic clones are very closely related and that there has been a high level of recombination in their evolution.  (+info)

Role of Listeria monocytogenes exotoxins listeriolysin and phosphatidylinositol-specific phospholipase C in activation of human neutrophils. (3/3113)

Polymorphonuclear leukocytes (PMN) are essential for resolution of infections with Listeria monocytogenes. The present study investigated the role of the listerial exotoxins listeriolysin (LLO) and phosphatidylinositol-specific phospholipase C (PlcA) in human neutrophil activation. Different Listeria strains, mutated in individual virulence genes, as well as purified LLO were used. Coincubation of human neutrophils with wild-type L. monocytogenes provoked PMN activation, occurring independently of phagocytosis events, with concomitant elastase secretion, leukotriene generation, platelet-activating factor (PAF) synthesis, respiratory burst, and enhanced phosphoinositide hydrolysis. Degranulation and leukotriene formation were noted to be solely dependent on LLO expression, as these features were absent when the LLO-defective mutant EGD- and the avirulent strain L. innocua were used. These effects were fully reproduced by a recombinant L. innocua strain expressing LLO (INN+) and by the purified LLO molecule. LLO secretion was also required for PAF synthesis. However, wild-type L. monocytogenes was more potent in eliciting PAF formation than mutants expressing LLO, suggesting the involvement of additional virulence factors. This was even more obvious for phosphoinositide hydrolysis and respiratory burst: these events were provoked not only by INN+ but also by the LLO-defective mutant EGD- and by a recombinant L. innocua strain producing listerial PlcA. We conclude that human neutrophils react to extracellularly provided listerial exotoxins by rapid cell activation. Listeriolysin is centrally involved in triggering degranulation and lipid mediator generation, and further virulence factors such as PlcA apparently contribute to trigger neutrophil phosphoinositide hydrolysis and respiratory burst. In this way, listerial exotoxins may influence the host defense against infections with L. monocytogenes.  (+info)

Vibrio parahaemolyticus thermostable direct hemolysin modulates cytoskeletal organization and calcium homeostasis in intestinal cultured cells. (4/3113)

Vibrio parahaemolyticus is a marine bacterium known to be the leading cause of seafood gastroenteritis worldwide. A 46-kDa homodimer protein secreted by this microorganism, the thermostable direct hemolysin (TDH), is considered a major virulence factor involved in bacterial pathogenesis since a high percentage of strains of clinical origin are positive for TDH production. TDH is a pore-forming toxin, and its most extensively studied effect is the ability to cause hemolysis of erythrocytes from different mammalian species. Moreover, TDH induces in a variety of cells cytotoxic effects consisting mainly of cell degeneration which often leads to loss of viability. In this work, we examined the cellular changes induced by TDH in monolayers of IEC-6 cells (derived from the rat crypt small intestine), which represent a useful cell model for studying toxins from enteric bacteria. In experimental conditions allowing cell survival, TDH induces a rapid transient increase in intracellular calcium as well as a significant though reversible decreased rate of progression through the cell cycle. The morphological changes seem to be dependent on the organization of the microtubular network, which appears to be the preferential cytoskeletal element involved in the cellular response to the toxin.  (+info)

Hyperproduction of alpha-hemolysin in a sigB mutant is associated with elevated SarA expression in Staphylococcus aureus. (5/3113)

To evaluate the role of SigB in modulating the expression of virulence determinants in Staphylococcus aureus, we constructed a sigB mutant of RN6390, a prototypic S. aureus strain. The mutation in the sigB gene was confirmed by the absence of the SigB protein in the mutant on an immunoblot as well as the failure of the mutant to activate sigmaB-dependent promoters (e.g., the sarC promoter) of S. aureus. Phenotypic analysis indicated that both alpha-hemolysin level and fibrinogen-binding capacity were up-regulated in the mutant strain compared with the parental strain. The increase in fibrinogen-binding capacity correlated with enhanced expression of clumping factor and coagulase on immunoblots. The effect of the sigB mutation on the enhanced expression of the alpha-hemolysin gene (hla) was primarily transcriptional. Upon complementation with a plasmid containing the sigB gene, hla expression returned to near parental levels in the mutant. Detailed immunoblot analysis as well as a competitive enzyme-linked immunosorbent assay of the cell extract of the sigB mutant with anti-SarA monoclonal antibody 1D1 revealed that the expression of SarA was higher in the mutant than in the parental control. Despite an elevated SarA level, the transcription of RNAII and RNAIII of the agr locus remained unaltered in the sigB mutant. Because of a lack of perturbation in agr, we hypothesize that inactivation of sigB leads to increased expression of SarA which, in turn, modulates target genes via an agr-independent but SarA-dependent pathway.  (+info)

Probing the function of Bordetella bronchiseptica adenylate cyclase toxin by manipulating host immunity. (6/3113)

We have examined the role of adenylate cyclase-hemolysin (CyaA) by constructing an in-frame deletion in the Bordetella bronchiseptica cyaA structural gene and comparing wild-type and cyaA deletion strains in natural host infection models. Both the wild-type strain RB50 and its adenylate cyclase toxin deletion (DeltacyaA) derivative efficiently establish persistent infections in rabbits, rats, and mice following low-dose inoculation. In contrast, an inoculation protocol that seeds the lower respiratory tract revealed significant differences in bacterial numbers and in polymorphonuclear neutrophil recruitment in the lungs from days 5 to 12 postinoculation. We next explored the effects of disarming specific aspects of the immune system on the relative phenotypes of wild-type and DeltacyaA bacteria. SCID, SCID-beige, or RAG-1(-/-) mice succumbed to lethal systemic infection following high- or low-dose intranasal inoculation with the wild-type strain but not the DeltacyaA mutant. Mice rendered neutropenic by treatment with cyclophosphamide or by knockout mutation in the granulocyte colony-stimulating factor locus were highly susceptible to lethal infection by either wild-type or DeltacyaA strains. These results reveal the significant role played by neutrophils early in B. bronchiseptica infection and by acquired immunity at later time points and suggest that phagocytic cells are a primary in vivo target of the Bordetella adenylate cyclase toxin.  (+info)

Resistance of paroxysmal nocturnal hemoglobinuria cells to the glycosylphosphatidylinositol-binding toxin aerolysin. (7/3113)

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder caused by a somatic mutation of the PIGA gene. The product of this gene is required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; therefore, the phenotypic hallmark of PNH cells is an absence or marked deficiency of all GPI-anchored proteins. Aerolysin is a toxin secreted by the bacterial pathogen Aeromonas hydrophila and is capable of killing target cells by forming channels in their membranes after binding to GPI-anchored receptors. We found that PNH blood cells (erythrocytes, lymphocytes, and granulocytes), but not blood cells from normals or other hematologic disorders, are resistant to the cytotoxic effects of aerolysin. The percentage of lysis of PNH cells after aerolysin exposure paralleled the percentage of CD59(+) cells in the samples measured by flow cytometry. The kinetics of red blood cell lysis correlated with the type of PNH erythrocytes. PNH type III cells were completely resistant to aerolysin, whereas PNH type II cells displayed intermediate sensitivity. Importantly, the use of aerolysin allowed us to detect PNH populations that could not be detected by standard flow cytometry. Resistance of PNH cells to aerolysin allows for a simple, inexpensive assay for PNH that is sensitive and specific. Aerolysin should also be useful in studying PNH biology.  (+info)

Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus. (8/3113)

The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membrane-bound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of alpha-toxin.  (+info)