Transcriptional regulation of a gonococcal gene encoding a virulence factor (L-lactate permease)

English version

Autoři: Julio C. Ayala ^aff001; William M. Shafer ^aff001
Působiště autorů: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America ^aff001; The Emory Antibiotic Resistance Center, Emory University School of Medicine, Atlanta, Georgia, United States of America ^aff002; Laboratories of Bacterial Pathogenesis, Veterans Affairs Medical Center, Decatur, Georgia, United States of America ^aff003
Vyšlo v časopise: Transcriptional regulation of a gonococcal gene encoding a virulence factor (L-lactate permease). PLoS Pathog 15(12): e32767. doi:10.1371/journal.ppat.1008233
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1008233

Souhrn

GdhR is a GntR-type regulator of Neisseria gonorrhoeae encoded by a gene (gdhR) belonging to the MtrR regulon, which comprises multiple genes required for antibiotic resistance such as the mtrCDE efflux pump genes. In previous work we showed that loss of gdhR results in enhanced gonococcal fitness in a female mouse model of lower genital tract infection. Here, we used RNA-Seq to perform a transcriptional profiling study to determine the GdhR regulon. GdhR was found to regulate the expression of 2.3% of all the genes in gonococcal strain FA19, of which 39 were activated and 11 were repressed. Within the GdhR regulon we found that lctP, which encodes a unique L-lactate transporter and has been associated with gonococcal pathogenesis, was the highest of GdhR-repressed genes. By using in vitro transcription and DNase I footpriting assays we mapped the lctP transcriptional start site (TSS) and determined that GdhR directly inhibits transcription by binding to an inverted repeat sequence located 9 bases downstream of the lctP TSS. Epistasis analysis revealed that, while loss of lctP increased susceptibility of gonococci to hydrogen peroxide (H₂O₂) the loss of gdhR enhanced resistance; however, this GdhR-endowed property was reversed in a double gdhR lctP null mutant. We assessed the effect of different carbon sources on lctP expression and found that D-glucose, but not L-lactate or pyruvate, repressed lctP expression within a physiological concentration range but in a GdhR-independent manner. Moreover, we found that adding glucose to the medium enhanced susceptibility of gonococci to hydrogen peroxide. We propose a model for the role of lctP regulation via GdhR and glucose in the pathogenesis of N. gonorrhoeae.

Klíčová slova:

DNA transcription – Gene expression – Gene regulation – Glucose – Hydrogen peroxide – Sequence motif analysis – Electrophoretic mobility shift assay

Zdroje

1. Reported STDs in the United States, 2016 High Burden of STDs Threaten Millions of Americans In: Prevention CfDCa, editor. CDC Fact Sheet: Center for Disease Control and Prevention.

2. Jane Rowley SVH, Eline Korenromp, Nicola Low, Magnus Unemo, Laith J Abu-Raddad, R Matthew Chico, Alex Smolak, Lori Newman, Sami Gottlieb, Soe Thwin, Nathalie Brouteta and Melanie M Taylor (2019) Chlamydia, gonorrhoea, trichomoniasis and syphilis: global prevalence and incidence estimates, 2016. Bulletin of the World Health Organization: 1–43.

3. National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention. Center for Disease Control and Prevention. Atlanta, GA. Press release: New Warning Signs that Gonorrhea Treatment May be Losing Effectiveness. 2016 Sep 21. https://www.cdc.gov/nchhstp/newsroom/2016/2016-std-prevention-conference-press-release.html

4. Zhao S, Duncan M, Tomberg J, Davies C, Unemo M, et al. (2009) Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae. Antimicrob Agents Chemother 53: 3744–3751. doi: 10.1128/AAC.00304-09 19528266

5. Ameyama S, Onodera S, Takahata M, Minami S, Maki N, et al. (2002) Mosaic-like structure of penicillin-binding protein 2 Gene (penA) in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime. Antimicrob Agents Chemother 46: 3744–3749. doi: 10.1128/AAC.46.12.3744-3749.2002 12435671

6. Osaka K, Takakura T, Narukawa K, Takahata M, Endo K, et al. (2008) Analysis of amino acid sequences of penicillin-binding protein 2 in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone. J Infect Chemother 14: 195–203. doi: 10.1007/s10156-008-0610-7 18574654

7. Olesky M, Hobbs M, Nicholas RA (2002) Identification and analysis of amino acid mutations in porin IB that mediate intermediate-level resistance to penicillin and tetracycline in Neisseria gonorrhoeae. Antimicrob Agents Chemother 46: 2811–2820. doi: 10.1128/AAC.46.9.2811-2820.2002 12183233

8. Hagman KE, Pan W, Spratt BG, Balthazar JT, Judd RC, et al. (1995) Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents is modulated by the mtrRCDE efflux system. Microbiology 141 (Pt 3): 611–622.

9. Folster JP, Johnson PJ, Jackson L, Dhulipali V, Dyer DW, et al. (2009) MtrR modulates rpoH expression and levels of antimicrobial resistance in Neisseria gonorrhoeae. J Bacteriol 191: 287–297. doi: 10.1128/JB.01165-08 18978065

10. Folster JP, Shafer WM (2005) Regulation of mtrF expression in Neisseria gonorrhoeae and its role in high-level antimicrobial resistance. J Bacteriol 187: 3713–3720. doi: 10.1128/JB.187.11.3713-3720.2005 15901695

11. Rouquette-Loughlin CE, Zalucki YM, Dhulipala VL, Balthazar JT, Doyle RG, et al. (2017) Control of gdhR Expression in Neisseria gonorrhoeae via Autoregulation and a Master Repressor (MtrR) of a Drug Efflux Pump Operon. MBio 8.

12. Claus H, Maiden MC, Wilson DJ, McCarthy ND, Jolley KA, et al. (2005) Genetic analysis of meningococci carried by children and young adults. J Infect Dis 191: 1263–1271. doi: 10.1086/428590 15776372

13. Pagliarulo C, Salvatore P, De Vitis LR, Colicchio R, Monaco C, et al. (2004) Regulation and differential expression of gdhA encoding NADP-specific glutamate dehydrogenase in Neisseria meningitidis clinical isolates. Mol Microbiol 51: 1757–1772. doi: 10.1111/j.1365-2958.2003.03947.x 15009900

14. Monaco C, Tala A, Spinosa MR, Progida C, De Nitto E, et al. (2006) Identification of a meningococcal L-glutamate ABC transporter operon essential for growth in low-sodium environments. Infect Immun 74: 1725–1740. doi: 10.1128/IAI.74.3.1725-1740.2006 16495545

15. Rigali S, Derouaux A, Giannotta F, Dusart J (2002) Subdivision of the helix-turn-helix GntR family of bacterial regulators in the FadR, HutC, MocR, and YtrA subfamilies. J Biol Chem 277: 12507–12515. doi: 10.1074/jbc.M110968200 11756427

16. Aravind L, Anantharaman V (2003) HutC/FarR-like bacterial transcription factors of the GntR family contain a small molecule-binding domain of the chorismate lyase fold. FEMS Microbiol Lett 222: 17–23. doi: 10.1016/S0378-1097(03)00242-8 12757941

17. Zheng M, Cooper DR, Grossoehme NE, Yu M, Hung LW, et al. (2009) Structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains. Acta Crystallogr D Biol Crystallogr 65: 356–365. doi: 10.1107/S0907444909004727 19307717

18. Rigali S, Schlicht M, Hoskisson P, Nothaft H, Merzbacher M, et al. (2004) Extending the classification of bacterial transcription factors beyond the helix-turn-helix motif as an alternative approach to discover new cis/trans relationships. Nucleic Acids Res 32: 3418–3426. doi: 10.1093/nar/gkh673 15247334

19. Exley RM, Wu H, Shaw J, Schneider MC, Smith H, et al. (2007) Lactate acquisition promotes successful colonization of the murine genital tract by Neisseria gonorrhoeae. Infect Immun 75: 1318–1324. doi: 10.1128/IAI.01530-06 17158905

20. Parsons NJ, Boons GJ, Ashton PR, Redfern PD, Quirk P, et al. (1996) Lactic acid is the factor in blood cell extracts which enhances the ability of CMP-NANA to sialylate gonococcal lipopolysaccharide and induce serum resistance. Microb Pathog 20: 87–100. doi: 10.1006/mpat.1996.0008 8722097

21. Yates E, Gao L, Woodcock N, Parsons N, Cole J, et al. (2000) In a medium containing glucose, lactate carbon is incorporated by gonococci predominantly into fatty acids and glucose carbon incorporation is increased: implications regarding lactate stimulation of metabolism. Int J Med Microbiol 290: 627–639. doi: 10.1016/S1438-4221(00)80012-0 11200544

22. Atack JM, Ibranovic I, Ong CL, Djoko KY, Chen NH, et al. (2014) A role for lactate dehydrogenases in the survival of Neisseria gonorrhoeae in human polymorphonuclear leukocytes and cervical epithelial cells. J Infect Dis 210: 1311–1318. doi: 10.1093/infdis/jiu230 24737798

23. Blake MS, Wetzler LM, Gotschlich EC, Rice PA (1989) Protein III: structure, function, and genetics. Clin Microbiol Rev 2 Suppl: S60–63.

24. Smith H, Yates EA, Cole JA, Parsons NJ (2001) Lactate stimulation of gonococcal metabolism in media containing glucose: mechanism, impact on pathogenicity, and wider implications for other pathogens. Infect Immun 69: 6565–6572. doi: 10.1128/IAI.69.11.6565-6572.2001 11598023

25. Smith H, Tang CM, Exley RM (2007) Effect of host lactate on gonococci and meningococci: new concepts on the role of metabolites in pathogenicity. Infect Immun 75: 4190–4198. doi: 10.1128/IAI.00117-07 17562766

26. Haydon DJ, Guest JR (1991) A new family of bacterial regulatory proteins. FEMS Microbiol Lett 63: 291–295. doi: 10.1016/0378-1097(91)90101-f 2060763

27. Grant CE, Bailey TL, Noble WS (2011) FIMO: scanning for occurrences of a given motif. Bioinformatics 27: 1017–1018. doi: 10.1093/bioinformatics/btr064 21330290

28. Suvorova IA, Korostelev YD, Gelfand MS (2015) GntR Family of Bacterial Transcription Factors and Their DNA Binding Motifs: Structure, Positioning and Co-Evolution. PLoS One 10: e0132618. doi: 10.1371/journal.pone.0132618 26151451

29. Aguilera L, Campos E, Gimenez R, Badia J, Aguilar J, et al. (2008) Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. J Bacteriol 190: 2997–3005. doi: 10.1128/JB.02013-07 18263722

30. Georgi T, Engels V, Wendisch VF (2008) Regulation of L-lactate utilization by the FadR-type regulator LldR of Corynebacterium glutamicum. J Bacteriol 190: 963–971. doi: 10.1128/JB.01147-07 18039772

31. Gao C, Hu C, Zheng Z, Ma C, Jiang T, et al. (2012) Lactate utilization is regulated by the FadR-type regulator LldR in Pseudomonas aeruginosa. J Bacteriol 194: 2687–2692. doi: 10.1128/JB.06579-11 22408166

32. Britigan BE, Klapper D, Svendsen T, Cohen MS (1988) Phagocyte-derived lactate stimulates oxygen consumption by Neisseria gonorrhoeae. An unrecognized aspect of the oxygen metabolism of phagocytosis. J Clin Invest 81: 318–324. doi: 10.1172/JCI113323 3123517

33. Fu HS, Hassett DJ, Cohen MS (1989) Oxidant stress in Neisseria gonorrhoeae: adaptation and effects on L-(+)-lactate dehydrogenase activity. Infect Immun 57: 2173–2178. 2543633

34. Quillin SJ, Hockenberry AJ, Jewett MC, Seifert HS (2018) Neisseria gonorrhoeae Exposed to Sublethal Levels of Hydrogen Peroxide Mounts a Complex Transcriptional Response. mSystems 3.

35. Stohl EA, Seifert HS (2006) Neisseria gonorrhoeae DNA recombination and repair enzymes protect against oxidative damage caused by hydrogen peroxide. J Bacteriol 188: 7645–7651. doi: 10.1128/JB.00801-06 16936020

36. Stohl EA, Chan YA, Hackett KT, Kohler PL, Dillard JP, et al. (2012) Neisseria gonorrhoeae virulence factor NG1686 is a bifunctional M23B family metallopeptidase that influences resistance to hydrogen peroxide and colony morphology. J Biol Chem 287: 11222–11233. doi: 10.1074/jbc.M111.338830 22334697

37. Zheng HY, Hassett DJ, Bean K, Cohen MS (1992) Regulation of catalase in Neisseria gonorrhoeae. Effects of oxidant stress and exposure to human neutrophils. J Clin Invest 90: 1000–1006. doi: 10.1172/JCI115912 1522209

38. Turner S, Reid E, Smith H, Cole J (2003) A novel cytochrome c peroxidase from Neisseria gonorrhoeae: a lipoprotein from a Gram-negative bacterium. Biochem J 373: 865–873. doi: 10.1042/BJ20030088 12720546

39. Tseng HJ, Srikhanta Y, McEwan AG, Jennings MP (2001) Accumulation of manganese in Neisseria gonorrhoeae correlates with resistance to oxidative killing by superoxide anion and is independent of superoxide dismutase activity. Mol Microbiol 40: 1175–1186. doi: 10.1046/j.1365-2958.2001.02460.x 11401721

40. Skaar EP, Tobiason DM, Quick J, Judd RC, Weissbach H, et al. (2002) The outer membrane localization of the Neisseria gonorrhoeae MsrA/B is involved in survival against reactive oxygen species. Proc Natl Acad Sci U S A 99: 10108–10113. doi: 10.1073/pnas.152334799 12096194

41. Hofer B, Muller D, Koster H (1985) The pathway of E. coli RNA polymerase-promoter complex formation as visualized by footprinting. Nucleic Acids Res 13: 5995–6013. doi: 10.1093/nar/13.16.5995 3898021

42. Carpousis AJ, Gralla JD (1985) Interaction of RNA polymerase with lacUV5 promoter DNA during mRNA initiation and elongation. Footprinting, methylation, and rifampicin-sensitivity changes accompanying transcription initiation. J Mol Biol 183: 165–177. doi: 10.1016/0022-2836(85)90210-4 2409292

43. Antunes A, Golfieri G, Ferlicca F, Giuliani MM, Scarlato V, et al. (2015) HexR Controls Glucose-Responsive Genes and Central Carbon Metabolism in Neisseria meningitidis. J Bacteriol 198: 644–654. doi: 10.1128/JB.00659-15 26644430

44. Leyn SA, Li X, Zheng Q, Novichkov PS, Reed S, et al. (2011) Control of proteobacterial central carbon metabolism by the HexR transcriptional regulator: a case study in Shewanella oneidensis. J Biol Chem 286: 35782–35794. doi: 10.1074/jbc.M111.267963 21849503

45. Novichkov PS, Kazakov AE, Ravcheev DA, Leyn SA, Kovaleva GY, et al. (2013) RegPrecise 3.0—a resource for genome-scale exploration of transcriptional regulation in bacteria. BMC Genomics 14: 745. doi: 10.1186/1471-2164-14-745 24175918

46. Reizer J, Hoischen C, Titgemeyer F, Rivolta C, Rabus R, et al. (1998) A novel protein kinase that controls carbon catabolite repression in bacteria. Mol Microbiol 27: 1157–1169. doi: 10.1046/j.1365-2958.1998.00747.x 9570401

47. Gao L, Parsons NJ, Curry A, Cole JA, Smith H (1998) Lactate causes changes in gonococci including increased lipopolysaccharide synthesis during short-term incubation in media containing glucose. FEMS Microbiol Lett 169: 309–316. doi: 10.1111/j.1574-6968.1998.tb13334.x 9868775

48. Britigan BE, Cohen MS (1986) Effects of human serum on bacterial competition with neutrophils for molecular oxygen. Infect Immun 52: 657–663. 3086230

49. Podinovskaia M, Lee W, Caldwell S, Russell DG (2013) Infection of macrophages with Mycobacterium tuberculosis induces global modifications to phagosomal function. Cell Microbiol 15: 843–859. doi: 10.1111/cmi.12092 23253353

50. van Zwieten R, Wever R, Hamers MN, Weening RS, Roos D (1981) Extracellular proton release by stimulated neutrophils. J Clin Invest 68: 310–313. doi: 10.1172/JCI110250 6265500

51. Hurst JK, Barrette WC Jr. (1989) Leukocytic oxygen activation and microbicidal oxidative toxins. Crit Rev Biochem Mol Biol 24: 271–328. doi: 10.3109/10409238909082555 2548810

52. McRipley RJ, Sbarra AJ (1967) Role of the phagocyte in host-parasite interactions. XI. Relationship between stimulated oxidative metabolism and hydrogen peroxide formation, and intracellular killing. J Bacteriol 94: 1417–1424. 4383408

53. Isabella VM, Clark VL (2011) Deep sequencing-based analysis of the anaerobic stimulon in Neisseria gonorrhoeae. BMC Genomics 12: 51. doi: 10.1186/1471-2164-12-51 21251255

54. Falsetta ML, Steichen CT, McEwan AG, Cho C, Ketterer M, et al. (2011) The Composition and Metabolic Phenotype of Neisseria gonorrhoeae Biofilms. Front Microbiol 2: 75. doi: 10.3389/fmicb.2011.00075 21833322

55. Phillips NJ, Steichen CT, Schilling B, Post DM, Niles RK, et al. (2012) Proteomic analysis of Neisseria gonorrhoeae biofilms shows shift to anaerobic respiration and changes in nutrient transport and outermembrane proteins. PLoS One 7: e38303. doi: 10.1371/journal.pone.0038303 22701624

56. Falsetta ML, McEwan AG, Jennings MP, Apicella MA (2010) Anaerobic metabolism occurs in the substratum of gonococcal biofilms and may be sustained in part by nitric oxide. Infect Immun 78: 2320–2328. doi: 10.1128/IAI.01312-09 20231417

57. Kellogg DS Jr., Peacock WL Jr., Deacon WE, Brown L, Pirkle DI (1963) Neisseria gonorrhoeae. I. Virulence Genetically Linked to Clonal Variation. J Bacteriol 85: 1274–1279. 14047217

58. Dillard JP (2011) Genetic Manipulation of Neisseria gonorrhoeae. Curr Protoc Microbiol Chapter 4: Unit4A 2.

59. Silver LE, Clark VL (1995) Construction of a translational lacZ fusion system to study gene regulation in Neisseria gonorrhoeae. Gene 166: 101–104. doi: 10.1016/0378-1119(95)00605-6 8529870

60. Rio DC (2015) Denaturation and electrophoresis of RNA with formaldehyde. Cold Spring Harb Protoc 2015: 219–222. doi: 10.1101/pdb.prot080994 25646498

61. Edgar R, Domrachev M, Lash AE (2002) Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30: 207–210. doi: 10.1093/nar/30.1.207 11752295

62. Lee EH, Rouquette-Loughlin C, Folster JP, Shafer WM (2003) FarR regulates the farAB-encoded efflux pump of Neisseria gonorrhoeae via an MtrR regulatory mechanism. J Bacteriol 185: 7145–7152. doi: 10.1128/JB.185.24.7145-7152.2003 14645274

63. Wang H, Ayala JC, Benitez JA, Silva AJ (2012) Interaction of the histone-like nucleoid structuring protein and the general stress response regulator RpoS at Vibrio cholerae promoters that regulate motility and hemagglutinin/protease expression. J Bacteriol 194: 1205–1215. doi: 10.1128/JB.05900-11 22194453

64. Ayala JC, Wang H, Benitez JA, Silva AJ (2018) Molecular basis for the differential expression of the global regulator VieA in Vibrio cholerae biotypes directed by H-NS, LeuO and quorum sensing. Mol Microbiol 107: 330–343. doi: 10.1111/mmi.13884 29152799

65. Wang H, Ayala JC, Benitez JA, Silva AJ (2014) The LuxR-type regulator VpsT negatively controls the transcription of rpoS, encoding the general stress response regulator, in Vibrio cholerae biofilms. J Bacteriol 196: 1020–1030. doi: 10.1128/JB.00993-13 24363348

66. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory.

67. Grant JR, Stothard P (2008) The CGView Server: a comparative genomics tool for circular genomes. Nucleic Acids Res 36: W181–184. doi: 10.1093/nar/gkn179 18411202

68. Thorvaldsdottir H, Robinson JT, Mesirov JP (2013) Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14: 178–192. doi: 10.1093/bib/bbs017 22517427

69. Hill AV (1910) The possible effects of the aggregation of the molecules of hæmoglobin on its dissociation curves. Journal of Physiology 40 Suppl: iv–vii.