Role ofMycoplasma penetransEndonuclease P40 as a Potential Pathogenic Determinant

Abstract

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 10−7to 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 of125I-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. penetransobserved 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.

Mycoplasma pneumoniae Mpn133 is a cytotoxic nuclease with a glutamic acid-, lysine- and serine-rich region essential for binding and internalization but not enzymatic activity

“…6C). However, the reduction of viability was not as dramatic as observed with M. penetrans endonuclease P40, which caused 100% cell death (Bendjennat et al ., 1999). Also, unlike previous studies that reported induction of internucleosomal fragmentation with certain pathogenic mycoplasma nucleases (Paddenberg et al ., 1996; Paddenberg et al ., 1998) we failed to detect such cell damage.…”

“…Since evidence exists that several mycoplasma nucleases can initiate apoptotic events (Paddenberg et al ., 1998; Bendjennat et al ., 1999), we investigated the effect of rMpn133 on A549 cells. Airway cell viability was assessed after 24 h exposure to rMpn133 using 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT) as indicator of viability.…”

“…The prevalence of nucleases in Mycoplasma species suggests not only their important functional role in mycoplasma biological integrity but also their contribution to pathogenicity (Paddenberg et al ., 1998; Sokolova et al ., 1998; Bendjennat et al ., 1999). Still, little information is available concerning M. pneumoniae nucleases, and nothing has been reported about their cytotoxic potential.…”

“…Similarly, the 40 kDaCa 2+ and Mg 2+ ion‐dependent nuclease of M . penetrans triggered apoptosis in cultured lymphocytes (Bendjennat et al ., 1999). However, nuclease activity in M. pneumoniae and its possible effect on mammalian cells have not yet been investigated.…”

Mycoplasma arthritidis-derived superantigen (MAM) displays DNase activity

“…Chromosomal mutations of the host have also been detected (Paton et al , 1965). Another striking phenomenon is that DNA degradation is accompanied by apoptotic effects to the cells (Paddenberg et al , 1998; Bendjennat et al , 1999).…”