Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Transcriptional and Mycolic Acid Profiling in Mycobacterium bovis BCG In Vitro Show an Effect for c-di-GMP and Overlap between Dormancy and Biofilms

  • Cruz, Miguel A. De la (Unidad de Investigacion Medica en Enfermedades Infecciosas y Parasitarias, Centro Medico Nacional (CMN) Siglo XXI, Instituto Mexicano de Seguro Social (IMSS)) ;
  • Ares, Miguel A. (Unidad de Investigacion Medica en Enfermedades Infecciosas y Parasitarias, Centro Medico Nacional (CMN) Siglo XXI, Instituto Mexicano de Seguro Social (IMSS)) ;
  • Rodriguez-Valverde, Diana (Unidad de Investigacion Medica en Enfermedades Infecciosas y Parasitarias, Centro Medico Nacional (CMN) Siglo XXI, Instituto Mexicano de Seguro Social (IMSS)) ;
  • Vallejo-Cardona, Alba Adriana (Centro de Investigacion y Asistencia en Tecnologia y diseno del Estado de Jalisco (CIATEJ) A.C., Biotecnologia Medica y Farmaceutica) ;
  • Flores-Valdez, Mario Alberto (Centro de Investigacion y Asistencia en Tecnologia y diseno del Estado de Jalisco (CIATEJ) A.C., Biotecnologia Medica y Farmaceutica) ;
  • Nunez, Iris Denisse Cota (Centro de Investigacion y Asistencia en Tecnologia y diseno del Estado de Jalisco (CIATEJ) A.C., Biotecnologia Medica y Farmaceutica) ;
  • Aceves-Sanchez, Michel de Jesus (Centro de Investigacion y Asistencia en Tecnologia y diseno del Estado de Jalisco (CIATEJ) A.C., Biotecnologia Medica y Farmaceutica) ;
  • Lira-Chavez, Jonahtan (Centro de Investigacion y Asistencia en Tecnologia y diseno del Estado de Jalisco (CIATEJ) A.C., Biotecnologia Medica y Farmaceutica) ;
  • Rodriguez-Campos, Jacobo (Centro de Investigacion y Asistencia en Tecnologia y diseno del Estado de Jalisco (CIATEJ) A.C, Unidad de Servicios Analiticos y Metrologicos) ;
  • Bravo-Madrigal, Jorge (Centro de Investigacion y Asistencia en Tecnologia y diseno del Estado de Jalisco (CIATEJ) A.C., Biotecnologia Medica y Farmaceutica)
  • Received : 2019.11.20
  • Accepted : 2020.03.12
  • Published : 2020.06.28
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Abstract

Mycobacterium tuberculosis produces mycolic acids which are relevant for persistence, recalcitrance to antibiotics and defiance to host immunity. c-di-GMP is a second messenger involved in transition from planktonic cells to biofilms, whose levels are controlled by diguanylate cyclases (DGC) and phosphodiesterases (PDE). The transcriptional regulator dosR, is involved in response to low oxygen, a condition likely happening to a subset of cells within biofilms. Here, we found that in M. bovis BCG, expression of both BCG1416c and BCG1419c genes, which code for a DGC and a PDE, respectively, decreased in both stationary phase and during biofilm production. The kasA, kasB, and fas genes, which are involved in mycolic acid biosynthesis, were induced in biofilm cultures, as was dosR, therefore suggesting an inverse correlation in their expression compared with that of genes involved in c-di-GMP metabolism. The relative abundance within trehalose dimycolate (TDM) of α-mycolates decreased during biofilm maturation, with methoxy mycolates increasing over time, and keto species remaining practically stable. Moreover, addition of synthetic c-di-GMP to mid-log phase BCG cultures reduced methoxy mycolates, increased keto species and practically did not affect α-mycolates, showing a differential effect of c-di-GMP on keto- and methoxy-mycolic acid metabolism.

Keywords

References

  1. Houben RMGJ, Dodd PJ. 2016. The global burden of latent tuberculosis infection: a re-estimation using mathematical modelling. PLoS Med. 13: e1002152. https://doi.org/10.1371/journal.pmed.1002152
  2. Menzies NA, Wolf E, Connors D, Bellerose M, Sbarra AN, Cohen T, et al. 2018. Progression from latent infection to active disease in dynamic tuberculosis transmission models: a systematic review of the validity of modelling assumptions. Lancet Infect. Dis 18: e228-e238. https://doi.org/10.1016/S1473-3099(18)30134-8
  3. Voskuil MI, Schnappinger D, Visconti KC, Harrell MI, Dolganov GM, Sherman DR, et al. 2003. Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J. Exp. Med. 198: 705-713. https://doi.org/10.1084/jem.20030205
  4. Mehra S, Foreman TW, Didier PJ, Ahsan MH, Hudock TA, Kissee R, et al. 2015. The DosR regulon modulates adaptive immunity and is essential for Mycobacterium tuberculosis persistence. Am. J. Rrespir. Crit. Care Med. 191: 1185-1196. https://doi.org/10.1164/rccm.201408-1502OC
  5. Flores-Valdez MA. 2016. Vaccines directed against microorganisms or their products present during biofilm lifestyle: can we make a translation as a broad biological model to Tuberculosis? Front. Microbiol. 7: 14. https://doi.org/10.3389/fmicb.2016.00014
  6. Boyd CD, O'Toole GA. 2012. Second messenger regulation of biofilm formation: breakthroughs in understanding c-di-GMP effector systems. Annu. Rev. Cell Dev. Biol. 28: 439-462. https://doi.org/10.1146/annurev-cellbio-101011-155705
  7. Gupta K, Kumar P, Chatterji D. 2010. Identification, activity and disulfide connectivity of C-di-GMP regulating proteins in Mycobacterium tuberculosis. PLoS One 5: e15072. https://doi.org/10.1371/journal.pone.0015072
  8. Flores-Valdez MA, Aceves-Sanchez Mde J, Pedroza-Roldan C, Vega-Dominguez PJ, Prado-Montes de Oca E, Bravo-Madrigal J, et al. 2015. The cyclic Di-GMP Phosphodiesterase gene Rv1357c/BCG1419c affects BCG Pellicle production and in vivo maintenance. IUBMB Life 67: 129-138. https://doi.org/10.1002/iub.1353
  9. Ojha AK, Baughn AD, Sambandan D, Hsu T, Trivelli X, Guerardel Y, et al. 2008. Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria. Mol. Microbiol. 69: 164-174. https://doi.org/10.1111/j.1365-2958.2008.06274.x
  10. Ojha AK, Jacobs WR Jr, Hatfull GF. 2015. Genetic dissection of mycobacterial biofilms. Methods Mol. Biol. 1285: 215-226. https://doi.org/10.1007/978-1-4939-2450-9_12
  11. Esteban J, Garcia-Coca M. 2017. Mycobacterium Biofilms. Front. Microbiol. 8: 2651. https://doi.org/10.3389/fmicb.2017.02651
  12. Flores-Valdez MA, Segura-Cerda CA, Gaona-Bernal J. 2018. Modulation of autophagy as a strategy for development of new vaccine candidates against tuberculosis. Mol. Immunol. 97: 16-19. https://doi.org/10.1016/j.molimm.2018.03.006
  13. Jahn CE, Charkowski AO, Willis DK. 2008. Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation. J. Microbiol. Methods 75: 318-324. https://doi.org/10.1016/j.mimet.2008.07.004
  14. Aranda PS, LaJoie DM, Jorcyk CL. 2012. Bleach gel: A simple agarose gel for analyzing RNA quality. Electrophoresis 33: 366-369. https://doi.org/10.1002/elps.201100335
  15. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25: 402-408. https://doi.org/10.1006/meth.2001.1262
  16. Arora R, Armitige L, Wanger A, Hunter RL, Hwang SA. 2016. Association of pellicle growth morphological characteristics and clinical presentation of Mycobacterium tuberculosis isolates. Tuberculosis 101: S63-S68. https://doi.org/10.1016/j.tube.2016.09.015
  17. Kremer L, Guerardel Y, Gurcha SS, Locht C, Besra GS. 2002. Temperature-induced changes in the cell-wall components of Mycobacterium thermoresistibile. Microbiology 148: 3145-3154. https://doi.org/10.1099/00221287-148-10-3145
  18. Hu Y, Coates AR. 1999. Transcription of two sigma 70 homologue genes, sigA and sigB, in stationary-phase Mycobacterium tuberculosis. J. Bacteriol. 181: 469-476. https://doi.org/10.1128/JB.181.2.469-476.1999
  19. Voskuil MI, Visconti KC, Schoolnik GK. 2004. Mycobacterium tuberculosis gene expression during adaptation to stationary phase and low-oxygen dormancy. Tuberculosis 84: 218-227. https://doi.org/10.1016/j.tube.2004.02.003
  20. Jenal U, Reinders A, Lori C. 2017. Cyclic di-GMP: second messenger extraordinaire. Nat. Rev. Microbiol. 15: 271-284. https://doi.org/10.1038/nrmicro.2016.190
  21. Bhatt A, Molle V, Besra GS, Jacobs WR, Jr., Kremer L. 2007. The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development. Mol. Microbiol. 64: 1442-1454. https://doi.org/10.1111/j.1365-2958.2007.05761.x
  22. Ojha A, Anand M, Bhatt A, Kremer L, Jacobs WR, Jr., Hatfull GF. 2005. GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria. Cell 123: 861-873. https://doi.org/10.1016/j.cell.2005.09.012
  23. Ojha AK, Trivelli X, Guerardel Y, Kremer L, Hatfull GF. 2010. Enzymatic hydrolysis of trehalose dimycolate releases free mycolic acids during mycobacterial growth in biofilms. J. Biol. Chem. 285: 17380-17389. https://doi.org/10.1074/jbc.M110.112813
  24. Chen L, Wen YM. 2011. The role of bacterial biofilm in persistent infections and control strategies. Int. J. Oral Sci. 3: 66-73. https://doi.org/10.4248/IJOS11022
  25. Orme IM. 2013. A new unifying theory of the pathogenesis of tuberculosis. Tuberculosis 94: 8-14. https://doi.org/10.1016/j.tube.2013.07.004
  26. Brosch R, Gordon SV, Garnier T, Eiglmeier K, Frigui W, Valenti P, et al. 2007. Genome plasticity of BCG and impact on vaccine efficacy. Proc. Natl. Acad. Sci. USA 104: 5596-5601. https://doi.org/10.1073/pnas.0700869104
  27. Pedroza-Roldan C, Guapillo C, Barrios-Payan J, Mata-Espinosa D, Aceves-Sanchez MD, Marquina-Castillo B, et al. 2016. The BCG Delta BCG1419c strain, which produces more pellicle in vitro, improves control of chronic tuberculosis in vivo. Vaccine 34: 4763-4770. https://doi.org/10.1016/j.vaccine.2016.08.035
  28. Segura-Cerda CA, Aceves-Sanchez MJ, Marquina-Castillo B, Mata-Espinoza D, Barrios-Payan J, Vega-Dominguez PJ, et al. 2018. Immune response elicited by two rBCG strains devoid of genes involved in c-di-GMP metabolism affect protection versus challenge with M. tuberculosis strains of different virulence. Vaccine 36: 2069-2078. https://doi.org/10.1016/j.vaccine.2018.03.014
  29. Segura-Cerda CA, Aceves-Sanchez MJ, Perez-Koldenkova V, Flores-Valdez MA. 2019. Macrophage infection with combinations of BCG mutants reduces induction of TNF-alpha, IL-6, IL-1beta and increases IL-4. Tuberculosis (Edinb) 115: 42-48. https://doi.org/10.1016/j.tube.2019.01.005
  30. Flores-Valdez MA, Pedroza-Roldan C, Aceves-Sanchez MJ, Peterson EJR, Baliga NS, Hernandez-Pando R, et al. 2018. The BCGDeltaBCG1419c vaccine candidate reduces lung pathology, IL-6, TNF-alpha, and IL-10 during chronic TB infection. Front. Microbiol. 9: 1281. https://doi.org/10.3389/fmicb.2018.01281
  31. Zimhony O, Vilcheze C, Jacobs WR, Jr. 2004. Characterization of Mycobacterium smegmatis expressing the Mycobacterium tuberculosis fatty acid synthase I (fas1) gene. J. Bacteriol. 186: 4051-4055. https://doi.org/10.1128/JB.186.13.4051-4055.2004
  32. Wright CC, Hsu FF, Arnett E, Dunaj JL, Davidson PM, Pacheco SA, et al. 2017. The Mycobacterium tuberculosis MmpL11 cell wall lipid transporter is important for biofilm formation, intracellular growth, and nonreplicating persistence. Infect. Immunity. 85: e00131-17.
  33. Schaeffer ML, Agnihotri G, Volker C, Kallender H, Brennan PJ, Lonsdale JT. 2001. Purification and biochemical characterization of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthases KasA and KasB. J. Biol. Chem. 276: 47029-47037. https://doi.org/10.1074/jbc.M108903200
  34. Schaeffer ML, Agnihotri G, Kallender H, Brennan PJ, Lonsdale JT. 2001. Expression, purification, and characterization of the Mycobacterium tuberculosis acyl carrier protein, AcpM. Biochim. Biophys. Acta 1532: 67-78. https://doi.org/10.1016/S1388-1981(01)00116-0
  35. Manganelli R, Dubnau E, Tyagi S, Kramer FR, Smith I. 1999. Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis. Mol. Microbiol. 31: 715-724. https://doi.org/10.1046/j.1365-2958.1999.01212.x
  36. Vilcheze C, Molle V, Carrere-Kremer S, Leiba J, Mourey L, Shenai S, et al. 2014. Phosphorylation of KasB regulates virulence and acid-fastness in Mycobacterium tuberculosis. PLoS Pathog. 10: e1004115. https://doi.org/10.1371/journal.ppat.1004115
  37. Bhatt A, Fujiwara N, Bhatt K, Gurcha SS, Kremer L, Chen B, et al. 2007. Deletion of kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice. Proc. Natl. Acad. Sci. USA 104: 5157-5162. https://doi.org/10.1073/pnas.0608654104
  38. Li W, He ZG. 2012. LtmA, a novel cyclic di-GMP-responsive activator, broadly regulates the expression of lipid transport and metabolism genes in Mycobacterium smegmatis. Nucleic Acids Res. 40: 11292-11307. https://doi.org/10.1093/nar/gks923
  39. Zhang HN, Xu ZW, Jiang HW, Wu FL, He X, Liu Y, et al. 2017. Cyclic di-GMP regulates Mycobacterium tuberculosis resistance to ethionamide. Sci. Rep. 7: 5860. https://doi.org/10.1038/s41598-017-06289-7
  40. Ang ML, Zainul Rahim SZ, Shui G, Dianiskova P, Madacki J, Lin W, et al. 2014. An ethA-ethR-deficient Mycobacterium bovis BCG mutant displays increased adherence to mammalian cells and greater persistence in vivo, which correlate with altered mycolic acid composition. Infect. Immun. 82: 1850-1859. https://doi.org/10.1128/IAI.01332-13
  41. Martinez LC, Vadyvaloo V. 2014. Mechanisms of post-transcriptional gene regulation in bacterial biofilms. Front. Cell Infect. Microbiol. 4: 38. https://doi.org/10.3389/fcimb.2014.00038
  42. Stewart GR, Patel J, Robertson BD, Rae A, Young DB. 2005. Mycobacterial mutants with defective control of phagosomal acidification. PLoS Pathog. 1: 269-278.
  43. Raghunandanan S, Jose L, Gopinath V, Kumar RA. 2019. Comparative label-free lipidomic analysis of Mycobacterium tuberculosis during dormancy and reactivation. Sci. Rep. 9: 3660. https://doi.org/10.1038/s41598-019-40051-5
  44. Shui G, Bendt AK, Pethe K, Dick T, Wenk MR. 2007. Sensitive profiling of chemically diverse bioactive lipids. J. Lipid Res. 48: 1976-1984. https://doi.org/10.1194/jlr.M700060-JLR200
  45. Minnikin DE, Minnikin SM, Dobson G, Goodfellow M, Portaels F, van den Breen L, et al. 1983. Mycolic acid patterns of four vaccine strains of Mycobacterium bovis BCG. J. Gen. Microbiol. 129: 889-891.
  46. Minnikin DE, Parlett JH, Magnusson M, Ridell M, Lind A. 1984. Mycolic acid patterns of representatives of Mycobacterium bovis BCG. J. Gen. Microbiol. 130: 2733-2736.
  47. Belley A, Alexander D, Di Pietrantonio T, Girard M, Jones J, Schurr E, et al. 2004. Impact of methoxymycolic acid production by Mycobacterium bovis BCG vaccines. Infect. Immun. 72: 2803-2809. https://doi.org/10.1128/IAI.72.5.2803-2809.2004
  48. Watanabe M, Aoyagi Y, Ridell M, Minnikin DE. 2001. Separation and characterization of individual mycolic acids in representative mycobacteria. Microbiol-Sgm. 147: 1825-1837. https://doi.org/10.1099/00221287-147-7-1825
  49. Sambandan D, Dao DN, Weinrick BC, Vilcheze C, Gurcha SS, Ojha A, et al. 2013. Keto-mycolic acid-dependent pellicle formation confers tolerance to drug-sensitive Mycobacterium tuberculosis. mBio 4: e00222-00213.
  50. Pang JM, Layre E, Sweet L, Sherrid A, Moody DB, Ojha A, et al. 2012. The polyketide Pks1 contributes to biofilm formation in Mycobacterium tuberculosis. J. Bacteriol. 194: 715-721. https://doi.org/10.1128/JB.06304-11
  51. Ojha A, Hatfull GF. 2007. The role of iron in Mycobacterium smegmatis biofilm formation: the exochelin siderophore is essential in limiting iron conditions for biofilm formation but not for planktonic growth. Mol. Microbiol. 66: 468-483. https://doi.org/10.1111/j.1365-2958.2007.05935.x
  52. Flores-Valdez MA, de Jesus Aceves-Sanchez M, Pedroza-Roldan C, Vega-Dominguez PJ, Prado-Montes de Oca E, Bravo-Madrigal J, et al. 2015. The cyclic di-GMP phosphodiesterase gene Rv1357c/BCG1419c affects BCG Pellicle production and in vivo maintenance. IUBMB Life 67: 129-138. https://doi.org/10.1002/iub.1353
  53. Ares MA, Rios-Sarabia N, De la Cruz MA, Rivera-Gutierrez S, Garcia-Morales L, Leon-Solis L, et al. 2017. The sigma factor SigD of Mycobacterium tuberculosis putatively enhances gene expression of the septum site determining protein under stressful environments. New Microbiol. 40: 199-204.

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