Protein kinase C activation and vacuolation in HeLa cells invaded by Mycoplasma penetrans.

The AIDS-associated Mycoplasma penetrans is capable of inducing its own uptake by non-phagocytic cells. This study investigated the invasion of HeLa cells and its consequences by confocal laser scanning microscopy. Invasion was dependent on the duration of infection and temperature, diminished by inhibiting microfilament assembly with cytochalasin D and almost completely abolished by disorganising microtubules with vinblastine or taxol. After a short infection period (< 20 min), pronounced activation of protein kinase C was detected in host cells, whereas prolonged infection resulted in intensive vacuolation of the host cells and a pronounced increment in intracellular organic peroxide levels. A marked decrease in the extent of vacuolation was observed when peroxide accumulation was partially prevented by alpha-tocopherol. The possibility that M. penetrans entry into HeLa cells involves the activation of protein kinases and the recruitment of cytoskeleton components is discussed.

The 57-Kilodalton Phosphoprotein of Spiroplasma melliferum Is Autophosphorylated

The partially purified 57-kDa protein of Spiroplasma melliferum was autophosphorylated when incubated with ATP in the presence of ZnCl2. Autophosphorylation was also apparent by showing the in situ phosphorylation of the 57-kDa protein band separated by polyacrylamide gel electrophoresis under nondenaturing conditions. The autophosphorylation was affected neither by the pH of the reaction mixture nor by the presence of NaF. The steady state level of the phosphorylated 57-kDa protein remained constant for up to 15 min, suggesting the absence of a phosphoprotein phosphatase activity in the preparation. As the initial phosphorylation rate did not decrease upon a 100-fold dilution of the 57-kDa protein under constant substrate concentration, it is suggested that the autophosphorylation is an intramolecular process.

Invasion of HeLa cells by Mycoplasma penetrans and the induction of tyrosine phosphorylation of a 145-kDa host cell protein.

The ability of Mycoplasma penetrans to invade eukaryotic cells was studied using a HeLa cell line. The bactericidal antibiotic, gentamicin, in combination with low concentrations of Triton X-100, was utilized to kill mycoplasmas that had not entered the cells, allowing the quantitation of internalized organisms. The intracellular location of the mycoplasma was also documented by transmission electron microscopy. The actin polymerization inhibitor cytochalasin-D markedly inhibited the internalization process, whereas the tyrosine phosphorylation inhibitors, staurosporin and genistein had only a slight effect. As against the invasion of enteropathogenic Escherichia coli which depends on tyrosine phosphorylation of a 90-kDa (Hp90) HeLa cell protein, internalization of M. penetrans by HeLa cells was independent of the phosphorylation of Hp90. Nonetheless, tyrosine phosphorylation of a 145-kDa HeLa cell protein was found to be associated with the interaction of M. penetrans with HeLa cells.