Adaptation of Mycoplasmas to Fluoroquinolones: Modulation of Proteome and Genotoxicity of Extracellular Vesicles of Acholeplasma laidlawii
Institutes and Faculties
Main page \ Research and Innovations \ Publications \ Publishing activities of KFU \ Uchenye Zapiski Kazanskogo Universiteta \ Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki \ Archive

Adaptation of Mycoplasmas to Fluoroquinolones: Modulation of Proteome and Genotoxicity of Extracellular Vesicles of Acholeplasma laidlawii

E.S. Medvedeva a,b*, T.Y  Malyginaa**, N.B. Baranovaa,b, А.А. Mouzykantova,b, M.N. Davydovab, O.A. Chernova a,b, V.M. Chernov a,b

aKazan Institute of Biochemistry and Biophysics, Kazan Scientific Center,

Russian Academy of Sciences, Kazan, 420111 Russia

bKazan Federal University, Kazan, 420008 Russia

E-mail: *elena-med@list.ru, **redfox-house@mail.ru

Received February 16, 2017

Full text PDF

Abstract

The paper is devoted to the comparative analysis of proteomic profiles and genotoxicity of extracellular vesicles produced by cells that differ in sensitivity to ciprofloxacin of Acholeplasma laidlawii strains – the mycoplasma, being a causative agent of mycoplasmoses of plants and animals, as well as the main contaminant of cell cultures. The relevance of the study is determined by the fact that extracellular vesicles – nanostructures surrounded by a membrane mediating intercellular communication and pathogenesis in bacteria are involved in adaptation of A. laidlawii to antimicrobials and present a new type of infects, the study of which is associated with the prospects of determining the mechanisms of host-parasite systems and solution of problems of pathogen control. The present study has been performed with a view of elucidation of the proteome profile features and assessment of the genotoxicity of extracellular vesicles of A. laidlawii in the development of resistance of the mycoplasma to ciprofloxacin – a drug of fluoroquinolone group which is widely used for inhibiting mycoplasmas. To achieve this goal, we have used the standard microbiological methods, as well as the modern physical and chemical methods, including proteomic profiling with help of 1D-LC-ESI-MS/MS, PCR, automatic scanning, and karyotyping system for assessment of the genotoxicity of vesicles.

It has been shown that a significant part of vesicular proteome of A. laidlawii strains with differential sensitivity to ciprofloxacin represents the bacterial virulence factors, as well as that the vesicles of all strains exhibit genotoxicity to lymphocytes of human peripheral blood in vitro. The most important of these results is the fact that the development of resistance of A. laidlawii to ciprofloxacin is accompanied by a significant modulation of vesicular proteome and the increase of mitotoxicity to eukaryotic cells. The obtained data are essential for fundamental research and applied works in the control system of socially significant infections, contaminations of cell cultures and vaccines. The results of the study provide fundamentally new notions about the processes of adaptation of A. laidlawii to antimicrobial drugs and pathogenicity of extracellular vesicles of mycoplasma, as well as suggest the necessity for correction of the control system of mycoplasmas with a view of bacterial vesicles as a new type of infects.

Keywords: mycoplasmas, Acholeplasma laidlawii, extracellular vesicles, ciprofloxacin, resistance, proteome, virulence factors, genotoxicity, mutagenicity

Acknowledgments. We are grateful to L.R. Samoilova, a genetic doctor at OOO Klinika Nurievykh, for her help in performing the experiments on genotoxicity.

The study was supported by the Russian Foundation for Basic Research (projects no. 15-44-02594, 16-34-00660) and the Grant of the President of the Russian Federation (project no. MK-1099.2017.4).

The investigation was carried out using the equipment of the Interdisciplinary Center of Shared Facilities, Kazan State University with the state support from the Ministry of Education and Science of the Russian Federation (ID RFMEFI59414X0003).

Figure Captions

Fig. 1. Venn's diagram showing “common” and “specific” proteins revealed in vesicles of the following stains: A. laidlawii PG8B, A. laidlawii PG8BCIP+, A. laidlawii PG8R10, A. laidlawii PG8R10CIP–, and A. laidlawii PG8S. The number of identified proteins is given in brackets.

Fig. 2. Mutagenicity (a) and mitotoxicity (b) of BB strains of A. laidlawii. Control variant – lymphocytes of human peripheral blood without addition of ВВ strains of A. laidlawii. Significant differences from ВВ A. laidlawii PG8B, p < 0.05 are shown with shown with asterisk mark.

Fig. 3. Karyograms (a, c) and metaphase plates (b, d) of lymphocytes in human peripheral blood during cultivation in the presence of ВВ strains of A. laidlawii. a, b – mutagenicity of ВВ A. laidlawii PG8BCIP+; c, d – mutagenicity of ВВ A. laidlawii PG8R10.

References

  1. Borkhsenius S.N., Chernova O.A., Chernov V.M., Vishnyakov I.E. Mycoplasma in Biology and Medicine of the 21st Century. St. Petersburg, Nauka, 2016. 400 p. (In Russian)
  2. Maniloff J. Molecular Biology and Pathogenicity of Mycoplasmas. Phylogeny and Evolution. Razin Sh., Herrman R. (Eds.). New York, Kluwer Academic/Plenum Publ., 2002. 572 p.
  3. Razin Sh. The Prokaryotes. The Genus Mycoplasma and Related Genera (Class Mollicutes). Vol. 4. Bacteria: Firmicutes, Cyanobacteria. 2006, pp. 836–904.
  4. Rottem S., Kosower N.S., Kornspan J.D. Biomedical Tissue Culture. Contamination of Tissue Cultures by Mycoplasmas. Ceccherini-Nelli L., Matteoli B. (Eds.). InTech, 2012, pp. 953–978. doi: 10.5772/51518.
  5. Chernov V.M., Chernova O.A., Sanchez-Vega J.T., Kolpakov A.I., Ilinskaya O.N. Mycoplasma contamination of cell cultures: Vesicular traffic in bacteria and control over infectious agents. Acta Nat., 2014, vol. 6, no. 3, pp. 41–51.
  6. Mariotti E., D'Alessio F., Mirabelli P., Di Noto R., Fortunato G., Del Vecchio L. Mollicutes contamination: A new strategy for an effective rescue of cancer cell lines. Biologicals, 2012, vol. 40, no. 1, pp. 88–91. doi: 10.1016/j.biologicals.2011.10.006.
  7. Uphoff C.C., Drexler H. Elimination of mycoplasmas from infected cell lines using antibiotics. Methods Mol. Biol., 2011, vol. 731, pp. 105–114. doi: 10.1007/978-1-61779-080-5_9.
  8. Bébéar C.M., Pereyre S., Peuchant O. Mycoplasma pneumoniae: Susceptibility and resistance to antibiotics. Future Microbiol., 2011, vol. 6, no. 4. – P. 423–431. doi: 10.2217/fmb.11.18.
  9. Uphoff C.C., Drexler H.G. Detection of mycoplasma contaminations. Methods Mol. Biol., 2013, vol. 946, pp. 1–13. doi: 10.1007/978-1-62703-128-8_1.
  10. Chernov V.M., Chernova O.A., Gorshkov O.V., Baranova N.B., Mouzykantov A.A., Nesterova T.N., Ponomareva A.A. Interaction between mycoplasmas and plants: Extracellular membrane vesicles and phytopathogenicity of Acholeplasma laidlawii PG8. Dokl. Biochem. Biophys., 2013, vol. 450, no. 1, pp. 155–159. doi: 10.1134/S1607672913030083.
  11. Windsor H.M., Windsor G.D., Noordergraaf J.H. The growth and long term survival of Acholeplasma laidlawii in media products used in biopharmaceutical manufacturing. Biologicals, 2010, vol. 38, no. 2, pp. 204–210. doi: 10.1016/j.biologicals.2009.11.009.
  12. Kulp A., Kuehn M.J. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Rev. Microbiol., 2010, vol. 64, pp. 163–184. doi: 10.1146/annurev.micro.091208.073413.
  13. Manning A.J., Kuehn M.J. Contribution of bacterial outer membrane vesicles to innate bacterial defense. BMC Microbiol., 2011, vol. 11, art. 258, pp. 1–14. doi: 10.1186/1471-2180-11-258.
  14. Medvedeva E.S., Baranova N.B., Mouzykantov A.A., Grigorieva (Malygina) T.Y., Davydova M.N., Trushin M.V., Chernova O.A., Chernov V.M. Adaptation of mycoplasmas to antimicrobial agents: Acholeplasma laidlawii extracellular vesicles mediate the export of ciprofloxacin and a mutant gene related to the antibiotic target. Sci. World J., 2014, vol. 2014, art. 150615, pp. 1–7. doi: 10.1155/2014/150615.
  15. Chernov V.M., Chernova O.A., Medvedeva E.S., Sorvina A.I., Davydova M.N., Rogova M.A., Serebryakova M.V. Responses of Acholeplasma laidlawii PG8 cells to cold shock and oxidative stress: Proteomic analysis and stress-reactive mycoplasma proteins. Dokl. Biochem. Biophys., 2010, vol. 432, pp. 126–130. doi: 10.1134/S1607672910030099.
  16. Chernov V.M., Chernova O.A., Medvedeva E.S., Davydova M.N. Adaptation of mycoplasmas to environmental conditions: Features of the proteome shift in Acholeplasma laidlawii PG8 at persistent exposure to stressors. Dokl. Biochem. Biophys., 2011, vol. 438, no. 1, pp. 134–137. doi: 10.1134/S1607672911030057.
  17. Medvedeva E.S., Baranova N.B., Mouzykantov A.A., Grigoreva T.Y., Davydova M.N., Chernova O.A., Chernov V.M. Extracellular vesicles of mycoplasmas and development of resistance to quinolones in bacteria. Dokl. Biochem. Biophys., 2014, vol. 454, no. 1, pp. 34–37. doi: 10.1134/S1607672914010104.
  18. Chernov V.M., Chernova O.A., Mouzykantov A.A., Efimova I.R., Shaymardanova G.F., Medvedeva E.S., Trushin M.V. Extracellular vesicles derived from Acholeplasma laidlawii PG8. Sci. World J., 2011, vol. 11, pp. 1120–1130. doi: 10.1100/tsw.2011.109.
  19. Chernov V.M., Chernova O.A., Medvedeva E.S., Mouzykantov A.A., Ponomareva A.A., Shaymardanova G.F., Gorshkov O.V., Trushin M.V. Unadapted and adapted to starvation Acholeplasma laidlawii cells induce different responses of Oryza sativa, as determined by proteome analysis. J. Proteomics, 2011, vol. 74, no. 12, pp. 2920–2936. doi: 10.1016/j.jprot.2011.07.016.
  20. Mouzykantov A.A., Baranova N.B., Medvedeva E.S., Grigor'eva T.Y., Chernova O.A., Chernov V.M. Exported mycoplasmal proteins: Proteome of extracellular membrane vesicles of Acholeplasma laidlawii PG8. Dokl. Biochem. Biophys., 2014, vol. 455, no. 1, pp. 43–48. doi: 10.1134/S160767291402001X.
  21. Medvedeva E.S., Davydova M.N., Mouzykantov A.A., Baranova N.B., Grigoreva T.Y., Siniagina M.N., Boulygina E.A., Chernova O.A., Chernov V.M. Genomic and proteomic profiles of Acholeplasma laidlawii strains differing in sensitivity to ciprofloxacin. Dokl. Biochem. Biophys., 2016, vol. 466, no. 1, pp. 23–27. doi: 10.1134/S1607672916010075.
  22. Chernov V.M., Mouzykantov A.A,, Baranova N.B., Medvedeva E.S., Grygorieva T.Y., Trushin M.V., Vishnyakov I.E., Sabantsev A.V., Borchsenius S.N., Chernova O.A. Extracellular membrane vesicles secreted by mycoplasma Acholeplasma laidlawii PG8 are enriched in virulence proteins. J. Proteomics, 2014, vol. 110, pp. 117–128. doi: 10.1016/j.jprot.2014.07.020.
  23. Lee E.Y., Choi D.Y., Kim D.K. Gram–positive bacteria produce membrane vesicles: Proteomics-based characterization of Staphylococcus aureus-derived membrane vesicles. Proteomics, 2009, vol. 9, no. 24, pp. 5425–5436. doi: 10.1002/pmic.200900338.
  24. Prokhorova I.M., Fomicheva P.N., Kovaleva M.I. Estimation of Mitotoxic and Mutagenic Effects of Environmental Factors. Yaroslavl, Yarosl. Gos. Univ., 2003. 32 p. (In Russian)
  25. Lazarev V.N., Levitskii S.A., Basovskii Y.I., Chukin M.M., Akopian T.A., Vereshchagin V.V., Kostrjukova E.S., Kovaleva G.Y., Kazanov M.D., Malko D.B., Vitreschak A.G., Sernova N.V., Gelfand M.S., Demina I.A., Serebryakova M.V., Galyamina M.A., Vtyurin N.N., Rogov S.I., Alexeev D.G., Ladygina V.G., Govorun V.M. Complete genome and proteome of Acholeplasma laidlawii. J. Bacteriol., 2011, vol. 193, no. 18, pp. 4943–4953. doi: 10.1128/JB.05059-11.
  26. Clements M.O., Eriksson S., Thompson A., Lucchini S., Hinton J.C., Normark S., Rhen M. Polynucleotide phosphorylase is a global regulator of virulence and persistency in Salmonella enterica. Proc. Natl. Acad. Sci. U. S. A., 2002, vol. 99, no. 13, pp. 8784–8789.
  27. Henderson B., Martin A. Bacterial moonlighting proteins and bacterial virulence. Curr. Top. Microbiol. Immunol., 2013, vol. 358, pp. 155–213. doi: 10.1007/82_2011_188.
  28. Thomas C.M., Jacobs E., Dumke R. Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumoniae. Microbiology, 2013, vol. 159, pt. 2, pp. 352–365. doi: 10.1099/mic.0.061184-0.
  29. Goulhen F., Hafezi A., Uitto V.J., Hinode D., Nakamura R., Grenier D., Mayrand D. Subcellular localization and cytotoxic activity of the GroEL-like protein isolated from Actinobacillus actinomycetemcomitans. Infect. Immun., 1998, vol. 66, no. 11, pp. 5307–5313.
  30. De Rycke J., Ducommun B. Bacterial cyclostatin, or how do bacteria manipulate the eukaryotic cell cycle. Med. Sci. (Paris), 2003, vol. 19, no. 11, pp. 1128–1136. (in French)
  31. Smitherman L.S., Minnick M.F. Bartonella bacilliformis GroEL: Effect on growth of human vascular endothelial cells in infected cocultures. Ann. N. Y. Acad. Sci., 2005, vol. 1063, pp. 286–298.
  32. Li Z.T., Zhang R.L., Bi X.G., Xu L., Fan M., Xie D., Xian Y., Wang Y., Li X.J., Wu Z.D., Zhang K.X. Outer membrane vesicles isolated from two clinical Acinetobacter baumannii strains exhibit different toxicity and proteome characteristics. Microb. Pathog., 2015, vol. 81, pp. 46–52. doi: 10.1016/j.micpath.2015.03.009.
  33. Jeon H., Oh M.H., Jun S.H., Kim S.I., Choi C.W., Kwon H.I., Na S.H., Kim Y.J., Nicholas A., Selasi G.N., Lee J.C. Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells. Microb. Pathog., 2016, vol. 93, pp. 185–193. doi: 10.1016/j.micpath.2016.02.014.

For citation: Medvedeva E.S., Malygina T.Y., Baranova N.B., Mouzykantov А.А., Davy­dova M.N., Chernova O.A., Chernov V.M. Adaptation of mycoplasmas to fluoroquinolones: Modulation of proteome and genotoxicity of extracellular vesicles of Acholeplasma laidlawii. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2017, vol. 159, no. 2, pp. 248–261. (In Russian)


The content is available under the license Creative Commons Attribution 4.0 License.

Вход в личный кабинет
Ваш логин
Ваш пароль
Забыли пароль? Регистрация