Role of Mycoplasma penetrans endonuclease P40 as a potential pathogenic determinant
Role of Mycoplasma penetrans endonuclease P40 as a potential pathogenic determinant
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DOI：
10.1128/iai.67.9.4456-4462.1999
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发表时间：
1999-09-01
影响因子：
3.1
通讯作者：
Bahraoui, E
中科院分区：
文献类型：
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作者：
Bendjennat, M;Blanchard, A;Bahraoui, E
最近，我们报道了穿透性支原体(M.Bendjennat，A.Blanchard，M.Loutfi，L.Montag nier和E.bahraoui，J.细菌素)产生的依赖于钙和镁的内切酶P40的纯化和性质。179；2210-2220,1997)，首次从人类免疫缺陷病毒感染者的尿液中分离到一种支原体。为了评估这种核酸酶如何与宿主细胞相互作用，我们测试了它对CEM和Molt-4淋巴细胞株以及外周血单核细胞的影响。我们观察到10(-7)~10(-9)M P40能够介导细胞毒作用。结果发现，10(-7)M P40作用24 h，细胞杀伤率为100%，而10(-9)M P40作用72 h，杀伤率仅为40%。相差显微镜下观察到P40处理细胞的形态改变，包括明显的质膜起泡和细胞程序性死亡的细胞质收缩特征，这与琼脂糖凝胶电泳法显示的P40处理的细胞DNA核小体间断裂是一致的。我们发现I-125标记或异硫氰酸荧光素标记的P40能够以剂量依赖的方式与细胞膜特异性结合，这表明P40核酸内切酶的细胞毒性是通过其与细胞表面受体的相互作用而介导的(S)。抑制I-125-P40-CEM复合体形成50%所需的未标记P40浓度约为3×10~(-9)M，显示出高亲和力相互作用。P40的相互作用和细胞毒性都依赖于钙离子。我们的结果表明，体外观察到的穿透支原体的细胞毒性至少部分是由分泌的P40介导的，P40在与宿主细胞相互作用后，可以诱导类凋亡死亡。这些结果强烈表明支原体核酸酶作为潜在的致病决定因素具有重要作用。
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 I-125-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 I-125-P40-CEM complexes was about 3 x 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.