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. 1997 Apr;179(7):2210–2220. doi: 10.1128/jb.179.7.2210-2220.1997

Purification and characterization of Mycoplasma penetrans Ca2+/Mg2+-dependent endonuclease.

M Bendjennat 1, A Blanchard 1, M Loutfi 1, L Montagnier 1, E Bahraoui 1
PMCID: PMC178957  PMID: 9079906

Abstract

The major nuclease from Mycoplasma penetrans has been purified to homogeneity. The enzyme seems to be present as a membrane-associated precursor of 50 kDa and as a peripheral membrane monomeric polypeptide of 40 kDa that is easily removed by washing of cells with isotonic buffers and in the aqueous phase upon Triton partitioning of Triton X-114-solubilized protein. The 40-kDa nuclease was extracted from M. penetrans cells by Triton X-114 and phase fractionation and was further purified by chromatography on Superdex 75 and chelating Sepharose (Zn2+ form) columns. By gel filtration, the apparent molecular mass was 40 kDa. The purified enzyme exhibits both a nicking activity on superhelical and linear double-stranded DNA and a nuclease activity on RNA and single-stranded DNA. No exonuclease activity was found for this enzyme. This nuclease required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity and exhibited a pH optimum between pH 7 and 8 for DNase activity. It was inhibited by Zn2+, Mn2+, heparin, sodium dodecyl sulfate, and chelator agents such EDTA and EGTA, but no effect was observed with ATP, 2-mercaptoethanol, N-ethylmaleimide, dithiothreitol, nonionic detergents, phenylmethylsulfonyl fluoride, and iodoacetamide. Nuclease activity was inhibited by diethylpyrocarbonate at both pH 6 and 8 and by pepstatin, suggesting the involvement of a histidine and an aspartate in the active site. When added to human lymphoblast nuclei, the purified M. penetrans endonuclease induced internucleosomal fragmentation of the chomatin into oligonucleosomal fragments. On the basis of this result, and taking into account the fact that M. penetrans has the capacity to invade eucaryotic cells, one can suggest, but not assert, that produced Ca2+/Mg2+-dependent endonuclease may alter the nucleic acid metabolism of host cells by DNA and/or RNA degradation and may act as a potential pathogenic determinant.

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Selected References

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  1. Andreev J., Borovsky Z., Rosenshine I., Rottem S. Invasion of HeLa cells by Mycoplasma penetrans and the induction of tyrosine phosphorylation of a 145-kDa host cell protein. FEMS Microbiol Lett. 1995 Oct 15;132(3):189–194. doi: 10.1111/j.1574-6968.1995.tb07832.x. [DOI] [PubMed] [Google Scholar]
  2. Blanchard A., Montagnier L. AIDS-associated mycoplasmas. Annu Rev Microbiol. 1994;48:687–712. doi: 10.1146/annurev.mi.48.100194.003351. [DOI] [PubMed] [Google Scholar]
  3. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  4. Chowdhury I. H., Munakata T., Koyanagi Y., Kobayashi S., Arai S., Yamamoto N. Mycoplasma can enhance HIV replication in vitro: a possible cofactor responsible for the progression of AIDS. Biochem Biophys Res Commun. 1990 Aug 16;170(3):1365–1370. doi: 10.1016/0006-291x(90)90545-x. [DOI] [PubMed] [Google Scholar]
  5. Dahl C. E., Sacktor N. C., Dahl J. S. Acylated proteins in Acholeplasma laidlawii. J Bacteriol. 1985 Apr;162(1):445–447. doi: 10.1128/jb.162.1.445-447.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fraser C. M., Gocayne J. D., White O., Adams M. D., Clayton R. A., Fleischmann R. D., Bult C. J., Kerlavage A. R., Sutton G., Kelley J. M. The minimal gene complement of Mycoplasma genitalium. Science. 1995 Oct 20;270(5235):397–403. doi: 10.1126/science.270.5235.397. [DOI] [PubMed] [Google Scholar]
  7. Gabridge M. G., Johnson C. K., Cameron A. M. Cytotoxicity of Mycoplasma pneumoniae Membranes. Infect Immun. 1974 Nov;10(5):1127–1134. doi: 10.1128/iai.10.5.1127-1134.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Girón J. A., Lange M., Baseman J. B. Adherence, fibronectin binding, and induction of cytoskeleton reorganization in cultured human cells by Mycoplasma penetrans. Infect Immun. 1996 Jan;64(1):197–208. doi: 10.1128/iai.64.1.197-208.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grau O., Slizewicz B., Tuppin P., Launay V., Bourgeois E., Sagot N., Moynier M., Lafeuillade A., Bachelez H., Clauvel J. P. Association of Mycoplasma penetrans with human immunodeficiency virus infection. J Infect Dis. 1995 Sep;172(3):672–681. doi: 10.1093/infdis/172.3.672. [DOI] [PubMed] [Google Scholar]
  10. Halden N. F., Wolf J. B., Leonard W. J. Identification of a novel site specific endonuclease produced by Mycoplasma fermentans: discovery while characterizing DNA binding proteins in T lymphocyte cell lines. Nucleic Acids Res. 1989 May 11;17(9):3491–3499. doi: 10.1093/nar/17.9.3491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Lemaître M., Henin Y., Destouesse F., Ferrieux C., Montagnier L., Blanchard A. Role of mycoplasma infection in the cytopathic effect induced by human immunodeficiency virus type 1 in infected cell lines. Infect Immun. 1992 Mar;60(3):742–748. doi: 10.1128/iai.60.3.742-748.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lo S. C., Hayes M. M., Kotani H., Pierce P. F., Wear D. J., Newton P. B., 3rd, Tully J. G., Shih J. W. Adhesion onto and invasion into mammalian cells by mycoplasma penetrans: a newly isolated mycoplasma from patients with AIDS. Mod Pathol. 1993 May;6(3):276–280. [PubMed] [Google Scholar]
  14. Lo S. C., Hayes M. M., Tully J. G., Wang R. Y., Kotani H., Pierce P. F., Rose D. L., Shih J. W. Mycoplasma penetrans sp. nov., from the urogenital tract of patients with AIDS. Int J Syst Bacteriol. 1992 Jul;42(3):357–364. doi: 10.1099/00207713-42-3-357. [DOI] [PubMed] [Google Scholar]
  15. Lo S. C., Tsai S., Benish J. R., Shih J. W., Wear D. J., Wong D. M. Enhancement of HIV-1 cytocidal effects in CD4+ lymphocytes by the AIDS-associated mycoplasma. Science. 1991 Mar 1;251(4997):1074–1076. doi: 10.1126/science.1705362. [DOI] [PubMed] [Google Scholar]
  16. McConkey D. J., Hartzell P., Orrenius S. Rapid turnover of endogenous endonuclease activity in thymocytes: effects of inhibitors of macromolecular synthesis. Arch Biochem Biophys. 1990 Apr;278(1):284–287. doi: 10.1016/0003-9861(90)90261-v. [DOI] [PubMed] [Google Scholar]
  17. McGarrity G. J., Vanaman V., Sarama J. Cytogenetic effects of mycoplasmal infection of cell cultures: a review. In Vitro. 1984 Jan;20(1):1–18. doi: 10.1007/BF02633326. [DOI] [PubMed] [Google Scholar]
  18. Minion F. C., Goguen J. D. Identification and preliminary characterization of external membrane-bound nuclease activities in Mycoplasma pulmonis. Infect Immun. 1986 Jan;51(1):352–354. doi: 10.1128/iai.51.1.352-354.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Minion F. C., Jarvill-Taylor K. J., Billings D. E., Tigges E. Membrane-associated nuclease activities in mycoplasmas. J Bacteriol. 1993 Dec;175(24):7842–7847. doi: 10.1128/jb.175.24.7842-7847.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Morowitz H. J., Bode H. R., Kirk R. G. The nucleic acids of mycoplasma. Ann N Y Acad Sci. 1967 Jul 28;143(1):110–114. doi: 10.1111/j.1749-6632.1967.tb27650.x. [DOI] [PubMed] [Google Scholar]
  21. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  22. Neyrolles O., Ferris S., Behbahani N., Montagnier L., Blanchard A. Organization of Ureaplasma urealyticum urease gene cluster and expression in a suppressor strain of Escherichia coli. J Bacteriol. 1996 Feb;178(3):647–655. doi: 10.1128/jb.178.3.647-655.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nozaki-Renard J., Iino T., Sato Y., Marumoto Y., Ohta G., Furusawa M. A fluoroquinolone (DR-3355) protects human lymphocyte cell lines from HIV-1-induced cytotoxicity. AIDS. 1990 Dec;4(12):1283–1286. doi: 10.1097/00002030-199012000-00018. [DOI] [PubMed] [Google Scholar]
  24. Nyström S., Wallbrandt P., Wieslander A. Membrane protein acylation. Preference for exogenous myristic acid or endogenous saturated chains in Acholeplasma laidlawii. Eur J Biochem. 1992 Feb 15;204(1):231–240. doi: 10.1111/j.1432-1033.1992.tb16629.x. [DOI] [PubMed] [Google Scholar]
  25. Paton G. R., Jacobs J. P., Perkins F. T. Chromosome changes in human diploid-cell cultures infected with Mycoplasma. Nature. 1965 Jul 3;207(992):43–45. doi: 10.1038/207043a0. [DOI] [PubMed] [Google Scholar]
  26. Phillips D. M., Pearce-Pratt R., Tan X., Zacharopoulos V. R. Association of mycoplasma with HIV-1 and HTLV-I in human T lymphocytes. AIDS Res Hum Retroviruses. 1992 Nov;8(11):1863–1868. doi: 10.1089/aid.1992.8.1863. [DOI] [PubMed] [Google Scholar]
  27. Pollack J. D., Hoffmann P. J. Properties of the nucleases of mollicutes. J Bacteriol. 1982 Oct;152(1):538–541. doi: 10.1128/jb.152.1.538-541.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Quillent C., Grau O., Clavel F., Montagnier L., Blanchard A. Inhibition of HIV type 1 reverse transcriptase assay by nucleases produced by contaminating mycoplasmas. AIDS Res Hum Retroviruses. 1994 Oct;10(10):1251–1257. doi: 10.1089/aid.1994.10.1251. [DOI] [PubMed] [Google Scholar]
  29. RAZIN S., KNYSZYNSKI A., LIFSHITZ Y. NUCLEASES OF MYCOPLASMA. J Gen Microbiol. 1964 Aug;36:323–332. doi: 10.1099/00221287-36-2-323. [DOI] [PubMed] [Google Scholar]
  30. Razin S. Peculiar properties of mycoplasmas: the smallest self-replicating prokaryotes. FEMS Microbiol Lett. 1992 Dec 15;100(1-3):423–431. doi: 10.1111/j.1574-6968.1992.tb14072.x. [DOI] [PubMed] [Google Scholar]
  31. Razin S. The mycoplasmas. Microbiol Rev. 1978 Jun;42(2):414–470. doi: 10.1128/mr.42.2.414-470.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Robertson J. A., Stemler M. E., Stemke G. W. Immunoglobulin A protease activity of Ureaplasma urealyticum. J Clin Microbiol. 1984 Feb;19(2):255–258. doi: 10.1128/jcm.19.2.255-258.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Roganti F. S., Rosenthal A. L. DNases of Acholeplasma spp. J Bacteriol. 1983 Aug;155(2):802–805. doi: 10.1128/jb.155.2.802-805.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rottem S., Hasin M., Razin S. Differences in susceptibility to phospholipase C of free and membrane-bound phospholipids of Mycoplasma hominis. Biochim Biophys Acta. 1973 Nov 16;323(4):520–531. doi: 10.1016/0005-2736(73)90160-0. [DOI] [PubMed] [Google Scholar]
  35. Salman M., Rottem S. The cell membrane of Mycoplasma penetrans: lipid composition and phospholipase A1 activity. Biochim Biophys Acta. 1995 May 4;1235(2):369–377. doi: 10.1016/0005-2736(95)80026-c. [DOI] [PubMed] [Google Scholar]
  36. Sasaki Y., Blanchard A., Watson H. L., Garcia S., Dulioust A., Montagnier L., Gougeon M. L. In vitro influence of Mycoplasma penetrans on activation of peripheral T lymphocytes from healthy donors or human immunodeficiency virus-infected individuals. Infect Immun. 1995 Nov;63(11):4277–4283. doi: 10.1128/iai.63.11.4277-4283.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shang H., Miyakawa Y., Sasaki T., Nakashima H., Ito M. Suppression of HIV-1 reverse transcriptase activity by culture supernatants of mycoplasmas. Microbiol Immunol. 1995;39(12):987–993. doi: 10.1111/j.1348-0421.1995.tb03302.x. [DOI] [PubMed] [Google Scholar]
  38. Shibata K., Sasaki T., Watanabe T. AIDS-associated mycoplasmas possess phospholipases C in the membrane. Infect Immun. 1995 Oct;63(10):4174–4177. doi: 10.1128/iai.63.10.4174-4177.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shibata K., Watanabe T. Purification and characterization of an aminopeptidase from Mycoplasma salivarium. J Bacteriol. 1987 Aug;169(8):3409–3413. doi: 10.1128/jb.169.8.3409-3413.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sladek T. L., Nowak J. A., Maniloff J. Mycoplasma restriction: identification of a new type of restriction specificity for DNA containing 5-methylcytosine. J Bacteriol. 1986 Jan;165(1):219–225. doi: 10.1128/jb.165.1.219-225.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Somerson N. L., Walls B. E., Chanock R. M. Hemolysin of Mycoplasma pneumoniae: tentative identification as a peroxide. Science. 1965 Oct 8;150(3693):226–228. doi: 10.1126/science.150.3693.226. [DOI] [PubMed] [Google Scholar]
  42. Stephens M. A. Partial purification and cleavage specificity of a site-specific endonuclease, SciNI, isolated from Spiroplasma citri. J Bacteriol. 1982 Feb;149(2):508–514. doi: 10.1128/jb.149.2.508-514.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Takahashi K., Chang W. J. The structure and function of acid proteases. V. Comparative studies on the specific inhibition of acid proteases by diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(p-nitrophenoxy) propane and pepstatin. J Biochem. 1976 Sep;80(3):497–506. doi: 10.1093/oxfordjournals.jbchem.a131304. [DOI] [PubMed] [Google Scholar]
  44. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Valerie K., Delers A., Bruck C., Thiriart C., Rosenberg H., Debouck C., Rosenberg M. Activation of human immunodeficiency virus type 1 by DNA damage in human cells. Nature. 1988 May 5;333(6168):78–81. doi: 10.1038/333078a0. [DOI] [PubMed] [Google Scholar]
  46. Vasudevachari M. B., Mast T. C., Salzman N. P. Suppression of HIV-1 reverse transcriptase activity by mycoplasma contamination of cell cultures. AIDS Res Hum Retroviruses. 1990 Mar;6(3):411–416. doi: 10.1089/aid.1990.6.411. [DOI] [PubMed] [Google Scholar]
  47. Wang R. Y., Shih J. W., Grandinetti T., Pierce P. F., Hayes M. M., Wear D. J., Alter H. J., Lo S. C. High frequency of antibodies to Mycoplasma penetrans in HIV-infected patients. Lancet. 1992 Nov 28;340(8831):1312–1316. doi: 10.1016/0140-6736(92)92493-y. [DOI] [PubMed] [Google Scholar]
  48. Wang R. Y., Shih J. W., Weiss S. H., Grandinetti T., Pierce P. F., Lange M., Alter H. J., Wear D. J., Davies C. L., Mayur R. K. Mycoplasma penetrans infection in male homosexuals with AIDS: high seroprevalence and association with Kaposi's sarcoma. Clin Infect Dis. 1993 Oct;17(4):724–729. doi: 10.1093/clinids/17.4.724. [DOI] [PubMed] [Google Scholar]
  49. Wira C. R., Munck A. Glucocorticoid-receptor complexes in rat thymus cells. "Cytoplasmic"-nuclear transformations. J Biol Chem. 1974 Aug 25;249(16):5328–5336. [PubMed] [Google Scholar]
  50. el-Farrash M. A., Kannagi M., Kuroda M. J., Yoshida T., Harada S. The mycoplasma-related inhibitor of HIV-1 reverse transcriptase has a DNase activity and is present in the particle-free supernatants of contaminated cultures. J Virol Methods. 1994 Apr;47(1-2):73–82. doi: 10.1016/0166-0934(94)90067-1. [DOI] [PubMed] [Google Scholar]

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