No abstract

The occurrence, diversity, and pathogenicity of Vibriospp. were investigated in two estuaries along the Italian Adriatic coast. Vibrio alginolyticus was the predominant species, followed by Vibrio parahaemolyticus, non-O1 Vibrio cholerae, and Vibrio vulnificus. By using a biochemical fingerprinting method, all isolates were grouped into nine phenotypes with similarity levels of 75 to 97.5%. The production of toxins capable of causing cytoskeleton-dependent changes was detected in a large number of Vibrio strains. These findings indicate a significant presence of potentially pathogenicVibrio strains along the Adriatic coast.

Toxigenic strains of the anaerobic bacterium Clostridium difficile produce at least two large, single-chain protein exotoxins involved in the pathogenesis of antibiotic-associated diarrhea and colitis. Toxin A (CdA) is a cytotoxic enterotoxin, while toxin B (CdB) is a more potent cytotoxin lacking enterotoxic activity. This study dealt with CdB, providing the first evidence that intestinal cells exposed to this toxin exhibit typical features of apoptosis in that a significant proportion of the treated cells displayed nuclear fragmentation and chromatin condensation. In keeping with ultrastructural data, CdB-treated cells showed the typical flow cytometric hallmark of apoptosis consisting of a distinct sub-G1 peak. The CdB-induced apoptotic response was dose and time dependent and not simply due to the actin-disrupting effect of the toxin or to the subsequent impairment of cell anchorage. Rather, the inhibition of proteins belonging to the Rho family due to CdB seems to play a role in the induction of apoptosis in intestinal cells. The origin of cells and the growth rate may also be cofactors relevant to such a response.

Certain strains of the anaerobic bacterium Bacteroides fragilis are known to produce an enterotoxin of about 20 kDa which is able to induce a fluid response in ligated intestinal loops and a cytotoxic response in HT-29 cells. It presents protease activity, belonging to a family of metalloproteases termed metzincins. In order to investigate the mode of action of the enterotoxin in cultured cells, we performed a study with HT-29 cells, using both fluorescence and electron microscopy. Treated cells underwent morphological changes, mainly consisting of the retraction of the cell body and the formation of numerous blebs on the cell surface. The microfilament system was reorganized, the F-actin being condensed as a ring at the cell periphery, whereas other cell organelles appeared to be unaffected. All these changes, clearly visible after 3 h of exposure to the toxin, were reversed within 24 h of treatment. By inhibiting the protease activity of the toxin with specific metal chelators, the cytoskeletal effects were also prevented. Thus, B. fragilis enterotoxin appears to act on cells by reversibly modifying the actin cytoskeleton, an effect probably dependent on its proteolytic activity. The anaerobic bacterium Bacteroides fragilis constitutes about 1% of the normal bacterial flora in humans (10). However, it has also been isolated from humans with abscesses, soft tissue infections, and bacteriemias (28). Strains of B. fragilis associated with epidemic and endemic diarrheal diseases in lambs and capable of stimulating a secretory response in the ligated intestinal loops of lambs and calves (17) were also associated with endemic diarrheal diseases in humans (18, 25). These enterotoxigenic B. fragilis strains produce a toxin which, in addition to the fluid response in ligated intestinal loops, induces a cytotoxic response in a colon carcinoma cell line, namely, HT-29 cells (30). The enterotoxin (ET) is produced in vitro as an extracellular protein with a molecular mass of about 20,000 Da with no subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (29). Amino acid sequence studies of ET revealed a zinc-binding consensus motif characteristic of metalloproteases termed metzincins. Purified ET contains 1 g-atom of Zn 2ϩ per molecule and is capable of hydrolyzing several proteins such as gelatin, actin, and fibrinogen and also of undergoing autodigestion (16). Although a number of studies dealing with the cytotoxicity of ET have been recently reported (12, 26, 27), the mode of action of the toxin in cultured cells is still unknown. In order to gain more information about the cellular response to ET, we performed a morphological study by using both immunofluorescence and electron microscopy. HT-29 cells, derived from a human colon adenocarcinoma, were used because they are, so far, one of the most sensitive cell lines for ET (29, 30). MATERIALS AND METHODS Cell cultures. HT-29 cells were grown at 37ЊC in RPMI medium, supplemented with 10% fetal calf serum (Flow Laboratories, Irvine, United Kingd...

Experiments done on in vitro-cultured cells exposed to toxin A from C. difficile showed a series of cytopathologic changes leading to cell retraction and rounding accompanied by the marginalization of the nucleus, which localized at one pole of the cell. Cytoskeleton appeared to be strongly involved in such modifications. In particular, the microfflament system seemed to be involved in cell retraction, while microtubule network integrity and function seemed to be necessary for the nuclear displacement. The carboxylic ionophore monensin completely blocked the cytopathic effect when added with the toxin. The serine protease inhibitor chymostatin appeared to be protective also upon addition long after the end of the binding step. The Ca2+-dependent cytosolic protease inhibitors antipain and leupeptin were uneffective in protecting cells. Thus, our results suggest the involvement of an acidic compartment and the action of a serine protease in toxin A-induced cytopathic effect.