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Infection and Immunity, September 1999, p. 4456-4462, Vol. 67, No. 9
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Role of Mycoplasma penetrans Endonuclease P40 as a Potential Pathogenic Determinant

Mourad Bendjennat,1,2 Alain Blanchard,3 Mohammed Loutfi,2 Luc Montagnier,3 and Elmostafa Bahraoui1,*

Laboratory of Immunovirology UFR SVT, University of Paul Sabatier, 31062 Toulouse,1 and Department of AIDS and Retroviruses, Viral Oncology Unit, CNRS URA 1157, Institut Pasteur, 75724 Paris,3 France, and Laboratory of Biochemistry, University Hassan II, Casablanca, Morocco2

Received 25 March 1999/Returned for modification 28 April 1999/Accepted 28 May 1999

Recently, we reported the purification to homogeneity and characterization of Ca2+- and Mg2+-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 125I-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 125I-P40-CEM complexes was about 3 × 10-9 M, indicating a high-affinity interaction. Both P40 interaction and cytotoxicity are Ca2+ 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.

* Corresponding author. Mailing address: Laboratory of Immunovirology UFR SVT, University of Paul Sabatier, Bât. 4, R. 3, 118 route de Narbonne, 31062 Toulouse, France. Phone: 33 5 61 55 86 67. Fax: 33 5 61 55 86 06. E-mail: bahraoui{at}cict.fr.

Infection and Immunity, September 1999, p. 4456-4462, Vol. 67, No. 9
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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