Skip to main content
SpringerLink
Log in

PknG supports mycobacterial adaptation in acidic environment

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Mycobacterium tuberculosis (Mtb), causative agent of human tuberculosis (TB), has the remarkable ability to adapt to the hostile environment inside host cells. Eleven eukaryotic like serine-threonine protein kinases (STPKs) are present in Mtb. Protein kinase G (PknG) has been shown to promote mycobacterial survival inside host cells. A homolog of PknG is also present in Mycobacterium smegmatis (MS), a fast grower, non-pathogenic mycobacterium. In the present study, we have analyzed the role of PknG in mycobacteria during exposure to acidic environment. Expression of pknG in MS was decreased in acidic medium. Recombinant MS ectopically expressing pknG (MS-G) showed higher growth in acidic medium compared to wild type counterpart. MS-G also showed higher resistance upon exposure to 3.0 pH and better adaptability to acidic pH. Western blot analysis showed differential threonine but not serine phosphorylation of cellular proteins in MS at acidic pH which was restored by ectopic expression of pknG in MS. In Mtb H37Ra (Mtb-Ra), expression of pknG was increased at acidic pH. We also observed decreased expression of pknG in MS during infection in macrophages while the expression of pknG in Mtb-Ra was increased in similar conditions. Taken together, our data strongly suggests that pknG regulates growth of mycobacteria in acidic environment and is differentially transcribed in MS and Mtb under these conditions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. WHO (2016) Global tuberculosis report

  2. Armstrong JA, Hart PD (1971) Response of cultured macrophages to Mycobacterium tuberculosis with observation on fusion of lysosome with phagosome. J Exp Med 134:713–740

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Russell DG, Sturgillkoszycki S, Vanheyningen T, Collins H, Schaible UE (1997) Why intracellular parasitism need not be a degrading experience for Mycobacterium. Philos Trans R Soc Lond B 352:1303–1310. https://doi.org/10.1098/rstb.1997.0114

    Article  CAS  Google Scholar 

  4. Av-gay Y, Everett M (2000) The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis. Trends Microbiol 8:238–244. https://doi.org/10.1016/S0966-842X(00)01734-0

    Article  CAS  PubMed  Google Scholar 

  5. Rieck B, Degiacomi G, Zimmermann M, Cascioferro A, Boldrin F, Lazar-Adler NR, Bottrill AR, le Chevalier F, Frigui W, Bellinzoni M, Lisa MN, Alzari PM, Nguyen L, Brosch R, Sauer U, Manganelli R, O’Hare HM (2017) PknG senses amino acid availability to control metabolism and virulence of Mycobacterium tuberculosis. PLoS Pathog 13:1–31. https://doi.org/10.1371/journal.ppat.1006399

    Article  Google Scholar 

  6. Ventura M, Rieck B, Boldrin F, Degiacomi G, Bellinzoni M, Barilone N, Alzaidi F, Alzari PM, Manganelli R, O’Hare HM (2013) GarA is an essential regulator of metabolism in Mycobacterium tuberculosis. Mol Microbiol 90:356–366. https://doi.org/10.1111/mmi.12368

    CAS  PubMed  Google Scholar 

  7. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, Barrell BG (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544. https://doi.org/10.1038/31159

    Article  CAS  PubMed  Google Scholar 

  8. O’Hare HM, Duran R, Cerveenansky C, Bellinzoni M, Wehenkel AM, Pritsch O, Obal G, Baumgartner J, Vialaret J, Johnsson K, Alzari PM (2008) Regulation of glutamate metabolism by protein kinases in mycobacteria. Mol Microbiol 70:1408–1423. https://doi.org/10.1111/j.1365-2958.2008.06489.x

    Article  PubMed  Google Scholar 

  9. Walburger A, Koul A, Ferrari G, Nguyen L, Prescianotto-Baschong C, Huygen K, Klebl B, Thompson C, Bacher G, Pieters J (2004) Protein kinase G from pathogenic mycobacteria promotes survival within macrophages. Science 304:1–10. https://doi.org/10.1126/science.1099384

    Article  Google Scholar 

  10. Chaurasiya SK, Srivastava KK (2008) Differential regulation of protein kinase C isoforms of macrophages by pathogenic and non-pathogenic mycobacteria. Mol Cell Biochem 318:167–174. https://doi.org/10.1007/s11010-008-9866-6

    Article  CAS  PubMed  Google Scholar 

  11. Chaurasiya SK, Srivastava KK (2009) Downregulation of protein kinase C-α enhances intracellular survival of Mycobacteria: role of PknG. BMC Microbiol 9:271. https://doi.org/10.1186/1471-2180-9-271

    Article  PubMed  PubMed Central  Google Scholar 

  12. Webb BL, Hirst SJ, Giembycz MA (2000) Protein kinase C isoenzymes: a review of their structure, regulation and role in regulating airways smooth muscle tone and mitogenesis. Br J Pharmacol 130:1433–1452. https://doi.org/10.1038/sj.bjp.0703452

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. St-Denis A, Caouras V, Gervais F, Descoteaux A (1999) Role of protein kinase C-α in the control of infection by intracellular pathogens in macrophages. J Immunol 163:5505–5511

    CAS  PubMed  Google Scholar 

  14. Zheleznyak A, Brown EJ (1992) Immunoglobulin-mediated phagocytosis by human monocytes requires Protein Kinase C activation. J Biol Chem 267:12042–12048

    CAS  PubMed  Google Scholar 

  15. Holm Å, Tejle K, Gunnarsson T, Magnusson KE, Descoteaux A, Rasmusson B (2003) Role of protein kinase C α for uptake of unopsonized prey and phagosomal maturation in macrophages. Biochem Biophys Res Commun 302:653–658. https://doi.org/10.1016/S0006-291X(03)00231-6

    Article  CAS  PubMed  Google Scholar 

  16. Cowley S, Ko M, Pick N, Chow R, Downing KJ, Gordhan BG, Betts JC, Mizrahi V, Smith DA, Stokes RW, Av-gay Y (2004) The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo. Mol Microbiol 52:1691–1702. https://doi.org/10.1111/j.1365-2958.2004.04085.x

    Article  CAS  PubMed  Google Scholar 

  17. Nguyen L, Walburger A, Houben E, Koul A, Muller S, Morbitzer M, Klebl B, Ferrari G, Pieters J (2005) Role of Protein Kinase G in growth and glutamine metabolism of Mycobacterium bovis BCG. J Bacteriol 187:5852–5856. https://doi.org/10.1128/JB.187.16.5852-5856.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Villarino A, Duran R, Wehenkel A, Fernandez P, England P, Alzari PM (2005) Proteomic Identification of M. tuberculosis Protein Kinase Substrates: PknB Recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions. J Mol Biol 350:953–963. https://doi.org/10.1016/j.jmb.2005.05.049

    Article  CAS  PubMed  Google Scholar 

  19. Su MS, Schlicht S, Gänzle MG (2011) Contribution of glutamate decarboxylase in Lactobacillus reuteri to acid resistance and persistence in sourdough fermentation. Microb Cell Fact 10:S8. https://doi.org/10.1186/1475-2859-10-S1-S8

    Article  PubMed  PubMed Central  Google Scholar 

  20. Damiano MA, Bastianelli D, Dahouk SA, Köhler S, Cloeckaert A, Biase DD, Occhialini A (2014) Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution and contribution to fitness under extremely acidic conditions. Appl Environ Microbiol 81:578–586. https://doi.org/10.1128/AEM.02928-14

    Article  PubMed  Google Scholar 

  21. Biase D, Pennacchietti E (2012) Glutamate decarboxylase-dependent acid resistance in orally acquired bacteria: function, distribution and biomedical implications of the gadBC operon. Mol Microbiol 86:770–786. https://doi.org/10.1111/mmi.12020

    Article  PubMed  Google Scholar 

  22. Biase D, Angela T, Bossa F, Visca P (1999) The response to stationary-phase stress conditions in Escherichia coli: role and regulation of the glutamic acid decarboxylase system. Mol Microbiol 32:1198–1211

    Article  PubMed  Google Scholar 

  23. Roxas BAP, Li Q (2009) Acid stress response of a mycobacterial proteome: insight from a gene ontology analysis. Int J Clin Exp Med 2:309–328

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Lund P, Tramonti A, De Biase D (2014) Coping with low pH: molecular strategies in neutralophilic bacteria. FEMS Microbiol Rev 38:1091–1125. https://doi.org/10.1111/1574-6976.12076

    Article  CAS  PubMed  Google Scholar 

  25. Li X, Wu J, Han J, Hu Y, Mi K (2015) Distinct responses of Mycobacterium smegmatis to exposure to low and high levels of hydrogen peroxide. PLoS ONE 10:1–15. https://doi.org/10.1371/journal.pone.0134595

    Google Scholar 

  26. Ivy RA, Wiedmann M, Boor KJ (2012) Listeria monocytogenes grown at 7°C shows reduced acid survival and an altered transcriptional response to acid shock compared to L. Monocytogenes grown at 37°C. Appl Environ Microbiol 78:3824–3836. https://doi.org/10.1128/AEM.00051-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Deng J, Bi L, Zhou L, Guo SJ, Fleming J, Jiang HW, Zhou Y, Gu J, Zhong Q, Wang ZX, Liu Z, Deng RP, Gao J, Chen T, Li W, Wang JF, Wang X, Li H, Ge F, Zhu G, Zhang HN, Gu J, Wu FL, Zhang Z, Wang D, Hang H, Li Y, Cheng L, He X, Tao SC, Zhang XE (2014) Mycobacterium tuberculosis proteome microarray for global studies of protein function and immunogenicity. Cell Rep 9:2317–2329. https://doi.org/10.1016/j.celrep.2014.11.023

    Article  CAS  PubMed  Google Scholar 

  28. Cappelli G, Volpe E, Grassi M, Liseo B, Colizzi V, Mariani F (2006) Profiling of Mycobacterium tuberculosis gene expression during human macrophage infection: upregulation of the alternative sigma factor G, a group of transcriptional regulators, and proteins with unknown function. Res Microbiol 157:445–455. https://doi.org/10.1016/j.resmic.2005.10.007

    Article  CAS  PubMed  Google Scholar 

  29. Vandal OH, Nathan CF, Ehrt S (2009) Acid resistance in Mycobacterium tuberculosis. J Bacteriol 191:4714–4721. https://doi.org/10.1128/JB.00305-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Papavinasasundaram KG, Chan B, Chung J, Colston MJ, Davis EO, Av-gay Y (2005) Deletion of the Mycobacterium tuberculosis pknH gene confers a higher bacillary load during the chronic phase of infection in BALB/c mice. J Bacteriol 187:5751–5760. https://doi.org/10.1128/JB.187.16.5751

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Gopalaswamy R, Narayanan S, Chen B, Jacobs WR, Av-gay Y (2009) The serine/threonine protein kinase PknI controls the growth of Mycobacterium tuberculosis upon infection. FEMS Microbiol Lett 295:23–29. https://doi.org/10.1111/j.1574-6968.2009.01570.x

    Article  CAS  PubMed  Google Scholar 

  32. Dannenberg AM (2007) Pathogenesis of human pulmonary tuberculosis: insights from the rabbit model. Clin Infect Dis 44:1257–1258. https://doi.org/10.1086/513587

    Article  Google Scholar 

  33. Schaible UE, Sturgill-koszycki S, Schlesinger PH, Russell DG (1998) Cytokine activation leads to acidification and increases maturation of Mycobacterium avium—containing phagosomes in murine macrophages. J Immunol 160:1290–1296

    CAS  PubMed  Google Scholar 

  34. Schnappinger D, Ehrt S, Voskuil MI, Liu Y, Mangan JA, Monahan IM, Dolganov G, Efron B, Butcher PD, Nathan C, Schoolnik GK (2003) Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment. J Exp Med 198:693–704. https://doi.org/10.1084/jem.20030846

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Via LE, Fratti RA, McFalone M, Pagan-Ramos E, Deretic D, Deretic V (1998) Effects of cytokines on mycobacterial phagosome maturation. J Cell Sci 111:897–905

    CAS  PubMed  Google Scholar 

  36. MacMicking JD, Taylor GA, McKinney JD (2003) Immune control of tuberculosis by IFN-gamma-inducible LRG-47. Science 302:654–659. https://doi.org/10.1126/science.1088063

    Article  CAS  PubMed  Google Scholar 

  37. Fisher MA, Plikaytis BB, Shinnick TM (2002) Microarray analysis of the Mycobacterium tuberculosis transcriptional response to the acidic conditions found in phagosomes. J Bacteriol 184:4025–4032. https://doi.org/10.1128/JB.184.14.4025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Rohde K, Yates RM, Purdy GE, Russell DG (2007) Mycobacterium tuberculosis and the environment within the phagosome. Immunol Rev 219:37–54. https://doi.org/10.1111/j.1600-065X.2007.00547.x

    Article  CAS  PubMed  Google Scholar 

  39. Saviola B (2012) Response of mycobacterial species to an acidic environment, Understanding Tuberculosis- Deciphering the secret life of the bacilli: Dr. Pere-Joan Cardona (Ed.), ISBN: 978-953-307- 946-2, InTech, Europe

  40. O’brien LM, Gordon SV, Roberts LS, Andrew PW (1996) Response of Mycobacterium smegmatis to acid stress. FEMS Microbiol Lett 139:11–17. https://doi.org/10.1111/j.1574-6968.1996.tb08173.x

    Article  PubMed  Google Scholar 

  41. Wolff KA, Nguyen HT, Cartabuke RH, Singh A, Ogwang S, Nguyen L (2009) Protein kinase G is required for intrinsic antibiotic resistance in mycobacteria. Antimicrob Agents Chemother 53:3515–3519. https://doi.org/10.1128/AAC.00012-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Wu F, Liu Y, Jiang H, Luan Y, Zhang H, He X, Xu Z, Hou J, Ji L, Xie Z, Czajkowsky D, Yan W, Deng J, Bi L, Zhang X, Tao S (2017) The ser/thr protein kinase protein-protein interaction map of M. tuberculosis. Mol Cell Proteom. https://doi.org/10.1074/mcp.M116.065771

    Google Scholar 

  43. Wolff KA, de la Peña AH, Nguyen HT, Pham TH, Amzel LM, Gabelli SB, Nguyen L (2015) A redox regulatory system critical for Mycobacterial survival in macrophages and biofilm development. PLoS Pathog 11:1–20. https://doi.org/10.1371/journal.ppat.1004839

    Article  Google Scholar 

  44. Houben ENG, Walburger A, Ferrari G, Nguyen L, Thompson CJ, Miess C, Vogel G, Mueller B, Pieters J (2009) Differential expression of a virulence factor in pathogenic and non-pathogenic mycobacteria. Mol Microbiol 72:41–52. https://doi.org/10.1111/j.1365-2958.2009.06612.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Chapman JS, Bernard JS (1962) The tolerance of unclassified mycobacteria. Am Rev Respir Dis 86:582–583. https://doi.org/10.1164/arrd.1962.86.4.582

    CAS  PubMed  Google Scholar 

  46. Cosma CL, Sherman DR, Ramakrishnan L (2003) The secret lives of the pathogenic mycobacteria. Annu Rev Microbiol 57:641–676. https://doi.org/10.1146/annurev.micro.57.030502.091033

    Article  CAS  PubMed  Google Scholar 

  47. Sundaramurthy V, Korf H, Singla A, Scherr N, Nguyen L, Ferrari G, Landmann R, Huygen K, Pieters J (2017) Survival of Mycobacterium tuberculosis and Mycobacterium bovis BCG in lysosomes in vivo. Microbes Infect. https://doi.org/10.1016/j.micinf.2017.06.008

    PubMed  Google Scholar 

  48. Crowle AJ, Dahl R, Ross E, May MH (1991) Evidence that vesicles containing living, virulent Mycobacterium tuberculosis or Mycobacterium avium in cultured human macrophages are not acidic. Infect Immun 59:1823–1831.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Wu QL, Kong D, Lam K, Husson RN (1997) Sigma factor involved in survival following stress. J Bacteriol 179:2922–2929.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Scherr N, Müller P, Perisa D, Combaluzier B, Jenö P, Pieters J (2009) Survival of pathogenic mycobacteria in macrophages is mediated through autophosphorylation of protein kinase G. J Bacteriol 191:4546–4554. https://doi.org/10.1128/JB.00245-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Jayakumar D, Jacobs WR, Narayanan S (2008) Protein kinase E of Mycobacterium tuberculosis has a role in the nitric oxide stress response and apoptosis in a human macrophage model of infection. Cell Microbiol 10:365–374. https://doi.org/10.1111/j.1462-5822.2007.01049.x

    CAS  PubMed  Google Scholar 

  52. Kruh NA, Troudt J, Izzo A, Prenni J, Dobos KM (2010) Portrait of a pathogen: the Mycobacterium tuberculosis proteome in vivo. PLoS ONE 5:e13938. https://doi.org/10.1371/journal.pone.0013938

    Article  PubMed  PubMed Central  Google Scholar 

  53. Hatzios SK, Baer CE, Rustad TR, Siegrist MS, Pang JM, Ortega C, Alber T, Grundner C, Sherman DR, Bertozzi CR (2013) Osmosensory signaling in Mycobacterium tuberculosis mediated by a eukaryotic-like ser/thr protein kinase. Proc Natl Acad Sci 110:E5069–E5077. https://doi.org/10.1073/pnas.1321205110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Park ST, Kang C-M, Husson RN (2008) Regulation of the SigH stress response regulon by an essential protein kinase in Mycobacterium tuberculosis. Proc Natl Acad Sci 105:13105–13110. https://doi.org/10.1073/pnas.0801143105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Anandan T, Han J, Baun H, Nyayapathy S, Brown JT, Dial RL, Moltalvo JA, Kim M, Yang SH, Ronning DR, Husson RN, Suh J, Kang C (2014) Phosphorylation regulates mycobacterial proteasome. J Microbiol 52:743–754. https://doi.org/10.1007/s12275-014-4416-2

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgment

We are grateful to Prof. Kishore K Srivastava, CSIR-Central Drug Research Institute, Lucknow for providing mycobacterial cultures, and THP-1 cell lines. RP is recipient of DST-INSPIRE Junior Research Fellowship. Real time PCR facility at SIC, Dr HS Gour University, Sagar was used on payment basis.

Funding

This study was funded by the SERB, Department of Science and Technology, India (Grant # SB/Y/LS-143/2013), Department of Biotechnology, India (Grant # BT/PR8640/AGR/36/785/2013), and University Grants Commission, India (Grant # 30-12/2014(BSR) grants sanctioned to Dr Shivendra K Chaurasiya.

Author information

Authors and Affiliations

  1. Host–Pathogen Interaction and Signal Transduction Laboratory, Department of Microbiology, School of Biological Sciences, Dr. Hari Singh Gour University, Sagar, MP, 470003, India

    Ruchi Paroha, Rajesh Mondal & Shivendra K. Chaurasiya

  2. Department of Chemistry, School of Chemical Sciences, Dr. Hari Singh Gour University, Sagar, MP, 470003, India

    Rashmi Chourasia

Authors
  1. Ruchi Paroha
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rashmi Chourasia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rajesh Mondal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shivendra K. Chaurasiya
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SKC conceived the idea, accumulated grants, and resources, designed study, supervised the research, interpreted data, and wrote manuscript. RP performed and recorded experiments, interpreted observations and prepared figures. RC helped in manuscript writing.

Corresponding author

Correspondence to Shivendra K. Chaurasiya.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 1449 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Paroha, R., Chourasia, R., Mondal, R. et al. PknG supports mycobacterial adaptation in acidic environment. Mol Cell Biochem 443, 69–80 (2018). https://doi.org/10.1007/s11010-017-3211-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-017-3211-x

Keywords

Search

Navigation