Lethal factor of Clostridium histolyticum kills cells by apoptosis.

In this study, for the first time, Clostridium histolyticum lethal factor was purified by ammonium sulfate precipitation of culture broth, centrifugation through an Amicon filter device and hydrophobic interaction chromatography. The purified lethal factor was devoid of proteolytic activity. At a concentration of 150 ng mL(-1) the lethal factor killed 50% of HeLa cells within 24 h of exposure. Abrogation of actin filaments, activation of caspases, fragmentation of nuclear DNA as well as ultrastructural changes indicated that the cell death occurred by apoptosis. The apoptotic action of the lethal factor is in agreement with changes induced in animal tissues by administration of C. histolyticum culture medium.

Vacuolization of HeLa cells by a partially purified Clostridium histolyticum cytotoxin.

Clostridium histolyticum vacuolating cytotoxin was partially purified from culture broth using ammonium sulfate precipitation, gel filtration and hydrophobic interaction chromatography. The toxin caused vacuolization of HeLa cells visible under a light microscope after 2 h and distinct after 8 h. Transmission electron microscopy revealed the presence of numerous vacuoles, condensation of the mitochondrial matrix, increased cytoplasm density and increased amounts of heterochromatin. Apoptosis was not detected either by electron microscopy or by an apoptosis/necrosis discrimination assay with fluorescein-labeled annexin V and propidium iodide, or DNA fragmentation assay. Calcium ion influx was detected by flow cytometry after labeling cells with Fluo-4 AM. Vacuolation of HeLa cells by C. histolyticum cytotoxin was inhibited by bafilomycin A1, suggesting involvement of H+ -ATPase in the formation of vacuoles.

Determination of the cytotoxic effect of Clostridium histolyticum culture supernatant on HeLa cells in the presence of protease inhibitors.

Clostridium histolyticum culture supernatant contains numerous enzymes, which exert a cytotoxic effect on host cells. This includes lethal toxin, clostripain and high-potassium-sensitive toxin. Since the number of C. histolyticum infections increased during the last several years, it seems worthwhile to evaluate whether protease inhibitors, used for the treatment of many diseases, could influence toxicity, and thus, pathogenicity of C. histolyticum. In this study we evaluated in vitro the influence of four common protease inhibitors: aprotinin, phenylmethylsulphonyl fluoride (PMSF), l-1-chloro-3-[4-tosylamido]-7-amino-2-heptanone-HCl (TLCK) and chymostatin on the toxicity of C. histolyticum supernatant towards human epithelial HeLa cells. We show that aprotinin has no effect, while PMSF, TLCK and chymostatin potentiate the cytotoxic activity of C. histolyticum, probably by hindering natural defence mechanisms of cells. In addition, PMSF and TLCK block clostripain enzymatic activity, while chymostatin leaves it intact. Elevated cytotoxicity of the supernatant is not related to the quantity of high-potassium-sensitive toxin, as was reported previously, since desalted supernatant still exerted its strong toxic effect. Our results show that addition of protease inhibitors for treating diseases complicated by concurrent C. histolyticum infection must require special attention.

Transplants of rat chondrocytes evoke strong humoral response against chondrocyte-associated antigen in rabbits.

Rat chondrocytes transplanted intramuscularly in rabbits produced cartilage. In 1-day-old transplants chondrocytes remained viable. After 1 week peripheral chondrocytes of the transplant were dead and the cartilage was surrounded and resorbed by macrophages. In 2-week-old transplants cartilage deteriorated and was invaded by fibroblast-like cells and macrophages. Sera of rabbits that received two or three consecutive transplants of rat chondrocytes with 2-week intervals contained high titer of antichondrocyte cytotoxic antibodies. A part of the cytotoxic activity could be removed by absorption with rat splenocytes. Western blot analysis of lysates from fresh or 24-h cultured chondrocytes with absorbed sera detected antigen with M(r), of approximately 74 and approximately 23 kDa. Only the latter remained after reduction in 2-mercaptoethanol. In lysates of fibroblasts and endotheliocytes the 23-kDa antigen was not found but the serum reacted with M(r) 39-kDa antigen. In lysates of thymocytes a weak band corresponding to M(r) of 35 kDa was present. Serum from rabbits receiving transplants of living chondrocytes followed by chondrocytes suspended in complete Freund's adjuvant contained antibodies directed against components of crude collagenase used for cell isolation. Such antibodies could not be detected in sera of rabbits receiving transplants of living chondrocytes only. Molecular weight of detected antigen differs from that of collagen type II, core of aggrecan, link proteins, and several other macromolecules of cartilage matrix. It could represent either a component of chondrocyte membrane or a membrane-bound substance resistant to enzymes used for isolation. Availability of antibodies against presumably chondrocyte-specific antigen produced during transplant rejection may help to characterize it more precisely and to ascertain whether its presence may influence results of autogenous chondrocyte transplants in humans.