Related Experiment Videos
Role of Mycoplasma penetrans endonuclease P40 as a potential pathogenic determinant
M Bendjennat1, A Blanchard, M Loutfi
1Laboratory of Immunovirology UFR SVT, University of Paul Sabatier, 31062 Toulouse, France.
Abstract:
Recently, we reported the purification to homogeneity and characterization of Ca(2+)- and Mg(2+)-dependent endonuclease P40 produced by Mycoplasma penetrans (M. Bendjennat, A. Blanchard, M. Loutfi, L. Montagnier, and E. Bahraoui, J. Bacteriol. 179; 2210-2220, 1997), a mycoplasma which was isolated for the first time from the urine of human immunodeficiency virus-infected patients. To evaluate how this nuclease could interact with host cells, we tested its effect on CEM and Molt-4 lymphocytic cell lines and on peripheral blood mononuclear cells. We observed that 10(-7) to 10(-9) M P40 is able to mediate a cytotoxic effect. We found that 100% of cells were killed after 24 h of incubation with 10(-7) M P40 while only 40% cytotoxicity was obtained after 72 h of incubation with 10(-9) M P40. Phase-contrast microscopy observations of P40-treated cells revealed morphological changes, including pronounced blebbing of the plasma membrane and cytoplasmic shrinkage characteristic of programmed cell death, which is in agreement with the internucleosomal fragmentation of P40-treated cell DNA as shown by agarose gel electrophoresis. We showed that (125)I-radiolabeled or fluorescein isothiocyanate-labeled P40 was able to bind specifically in a dose-dependent manner to the cell membrane of CEM cells, which suggested that the cytotoxicity of P40 endonuclease was mediated by its interaction with the cell surface receptor(s). The concentration of unlabeled P40 required to inhibit by 50% the formation of (125)I-P40-CEM complexes was about 3 x 10(-9) M, indicating a high-affinity interaction. Both P40 interaction and cytotoxicity are Ca(2+) dependent. Our results suggest that the cytotoxicity of M. penetrans observed in vitro is mediated at least partially by secreted P40, which, after interaction with host cells, can induce an apoptosis-like death. These results strongly suggest a major role of mycoplasmal nucleases as potential pathogenic determinants.
Insights
Mycoplasma penetrans endonuclease P40 induces apoptosis-like cell death in human lymphocytes. This secreted nuclease binds to cell surfaces, suggesting a role in mycoplasma pathogenicity and potential therapeutic targets for infections.
Area of Science:
- Molecular Biology
- Cell Biology
- Microbiology
- Immunology
Background:
- Mycoplasma penetrans is a bacterium isolated from HIV-infected patients.
- The Ca(2+)- and Mg(2+)-dependent endonuclease P40 is secreted by M. penetrans.
- The pathogenic role of M. penetrans and its secreted factors requires further investigation.
Purpose of the Study:
- To investigate the cytotoxic effects of M. penetrans endonuclease P40 on human lymphocytic cell lines and peripheral blood mononuclear cells.
- To elucidate the mechanism of P40-mediated cytotoxicity, including cell surface interactions and induction of programmed cell death.
- To assess the potential role of P40 as a pathogenic determinant in M. penetrans infections.
Main Methods:
- Treatment of CEM, Molt-4, and peripheral blood mononuclear cells with varying concentrations of purified P40.
- Assessment of cell viability and morphological changes using phase-contrast microscopy.
- Analysis of DNA fragmentation via agarose gel electrophoresis.
- Binding assays using radiolabeled and fluorescently labeled P40 to determine cell surface interaction and affinity.
Main Results:
- P40 exhibited dose-dependent cytotoxicity, killing 100% of cells at 10(-7) M within 24 hours and 40% at 10(-9) M within 72 hours.
- Morphological changes and DNA fragmentation indicated P40 induces apoptosis-like programmed cell death.
- P40 specifically bound to the cell membrane of CEM cells with high affinity, dependent on Ca(2+).
- Both P40 interaction with cells and its cytotoxicity were calcium-dependent.
Conclusions:
- Mycoplasma penetrans endonuclease P40 mediates cytotoxicity against human host cells through an apoptosis-like pathway.
- The interaction of P40 with host cell surface receptors is crucial for its cytotoxic effect.
- Secreted mycoplasma nucleases, like P40, are likely significant contributors to the pathogenicity of Mycoplasma infections.