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A Trigger Enzyme in : Impact of the Glycerophosphodiesterase GlpQ on Virulence and Gene Expression


Mycoplasma pneumoniae is a causative agent of atypical pneumonia. The formation of hydrogen peroxide, a product of glycerol metabolism, is essential for host cell cytotoxicity. Phosphatidylcholine is the major carbon source available on lung epithelia, and its utilization requires the cleavage of deacylated phospholipids to glycerol-3-phosphate and choline. M. pneumoniae possesses two potential glycerophosphodiesterases, MPN420 (GlpQ) and MPN566. In this work, the function of these proteins was analyzed by biochemical, genetic, and physiological studies. The results indicate that only GlpQ is an active glycerophosphodiesterase. MPN566 has no enzymatic activity as glycerophosphodiesterase and the inactivation of the gene did not result in any detectable phenotype. Inactivation of the glpQ gene resulted in reduced growth in medium with glucose as the carbon source, in loss of hydrogen peroxide production when phosphatidylcholine was present, and in a complete loss of cytotoxicity towards HeLa cells. All these phenotypes were reverted upon complementation of the mutant. Moreover, the glpQ mutant strain exhibited a reduced gliding velocity. A comparison of the proteomes of the wild type strain and the glpQ mutant revealed that this enzyme is also implicated in the control of gene expression. Several proteins were present in higher or lower amounts in the mutant. This apparent regulation by GlpQ is exerted at the level of transcription as determined by mRNA slot blot analyses. All genes subject to GlpQ-dependent control have a conserved potential cis-acting element upstream of the coding region. This element overlaps the promoter in the case of the genes that are repressed in a GlpQ-dependent manner and it is located upstream of the promoter for GlpQ-activated genes. We may suggest that GlpQ acts as a trigger enzyme that measures the availability of its product glycerol-3-phosphate and uses this information to differentially control gene expression.


Vyšlo v časopise: A Trigger Enzyme in : Impact of the Glycerophosphodiesterase GlpQ on Virulence and Gene Expression. PLoS Pathog 7(9): e32767. doi:10.1371/journal.ppat.1002263
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002263

Souhrn

Mycoplasma pneumoniae is a causative agent of atypical pneumonia. The formation of hydrogen peroxide, a product of glycerol metabolism, is essential for host cell cytotoxicity. Phosphatidylcholine is the major carbon source available on lung epithelia, and its utilization requires the cleavage of deacylated phospholipids to glycerol-3-phosphate and choline. M. pneumoniae possesses two potential glycerophosphodiesterases, MPN420 (GlpQ) and MPN566. In this work, the function of these proteins was analyzed by biochemical, genetic, and physiological studies. The results indicate that only GlpQ is an active glycerophosphodiesterase. MPN566 has no enzymatic activity as glycerophosphodiesterase and the inactivation of the gene did not result in any detectable phenotype. Inactivation of the glpQ gene resulted in reduced growth in medium with glucose as the carbon source, in loss of hydrogen peroxide production when phosphatidylcholine was present, and in a complete loss of cytotoxicity towards HeLa cells. All these phenotypes were reverted upon complementation of the mutant. Moreover, the glpQ mutant strain exhibited a reduced gliding velocity. A comparison of the proteomes of the wild type strain and the glpQ mutant revealed that this enzyme is also implicated in the control of gene expression. Several proteins were present in higher or lower amounts in the mutant. This apparent regulation by GlpQ is exerted at the level of transcription as determined by mRNA slot blot analyses. All genes subject to GlpQ-dependent control have a conserved potential cis-acting element upstream of the coding region. This element overlaps the promoter in the case of the genes that are repressed in a GlpQ-dependent manner and it is located upstream of the promoter for GlpQ-activated genes. We may suggest that GlpQ acts as a trigger enzyme that measures the availability of its product glycerol-3-phosphate and uses this information to differentially control gene expression.


Zdroje

1. SonensheinAL 2007 Control of key metabolic intersections in Bacillus subtilis. Nat Rev Microbiol 5 917 927

2. GörkeBStülkeJ 2008 Carbon catabolite repression in bacteria: Many ways to make the most out of nutrients. Nat Rev Microbiol 6 613 624

3. EisenreichWDandekarTHeesemannJGoebelW 2010 Carbon metabolism of intracellular bacterial pathogens and possible links to virulence. Nat Rev Microbiol 8 40 412

4. WaitesKBTalkingtonDF 2004 Mycoplasma pneumoniae and its role as human pathogen. Clin Microbiol Rev 17 697 728

5. TsiodrasSKelesidisIKelesidisTStamboulisEGiamarellouH 2005 Central nervous system manifestations of Mycoplasma pneumoniae infection. J Infect 51 343 354

6. NaritaM 2009 Pathogenesis of neurogenic manifestations of Mycoplasma pneumoniae infection. Pediatr Neurol 41 159 166

7. NaritaM 2010 Pathogenesis of extrapulmonary manifestations of Mycoplasma pneumoniae infection with special reference to pneumonia. J Infect Chemother 16 162 169

8. HalbedelSHamesCStülkeJ 2007 Regulation of carbon metabolism in the mollicutes and its relation to virulence. J Mol Microbiol Biotechnol 12 147 154

9. VeldhuizenRNagKOrgeigSPossmayerF 1998 The role of lipids in pulmonary surfactant. Biochim Biophys Acta 1408 90 108

10. HamesCHalbedelSHoppertMFreyJStülkeJ 2009 Glycerol metabolism is important for cytotoxicity of Mycoplasma pneumoniae. J Bacteriol 191 747 753

11. SomersonNLWallsBEChanockRM 1965 Hemolysin of Mycoplasma pneumoniae: Tentative identification as a peroxide. Science 150 226 228

12. PiloPVileiEMPeterhansEBonvin-KlotzLStoffelMH 2005 A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony. J Bacteriol 187 6824 6831

13. BischofDFVileiEMFreyJ 2009 Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: Characterization of a flavine adenine dinucleotide-binding site deletion mutant. Vet Res 40 35

14. OhshimaNYamashitaSTakahashiNKuroishiCShiroY 2008 Escherichia coli cytosolic glycerophosphodiester phosphodiesterase (UgpQ) requires Mg2+, Co2+, or Mn2+ for its enzyme activity. J Bacteriol 190 1219 1223

15. WongKKKwanHS 1992 Transcription of glpT of Escherichia coli K12 is regulated by anaerobiosis and Fnr. FEMS Microbiol Lett 73 15 18

16. NilssonRPBeijerLRutbergB 1994 The glpT and glpQ genes of the glycerol regulon in Bacillus subtilis. Microbiology 140 723 730

17. AntelmannHScharfCHeckerM 2000 Phosphate starvation-inducible proteins of Bacillus subtilis: Proteomics and transcriptional analysis. J Bacteriol 182 4478 4490

18. BlenckeHMHomuthGLudwigHMäderUHeckerM 2003 Transcriptional profiling of gene expression in response to glucose in Bacillus subtilis: Regulation of the central metabolic pathways. Metab Eng 5 133 149

19. FanXGoldfineHLysenkoEWeiserJN 2001 The transfer of choline from the host to the bacterial cell surface requires glpQ in Haemophilus influenzae. Mol Microbiol 41 1029 1036

20. ForsgrenARiesbeckKJansonH 2008 Protein D of Haemophilus influenzae: A protective nontypeable H. influenzae antigen and a carrier for pneumococcal conjugate vaccines. Clin Infect Dis 46 726 731

21. SchwanTGBattistiJMPorcellaSFRaffelSJSchrumpfME 2003 Glycerol-3-phosphate acquisition in spirochaetes: Distribution and biological activity of glycerophosphodiester phosphodiesterase (GlpQ) among Borrelia species. J Bacteriol 185 1346 1356

22. CommichauFMStülkeJ 2008 Trigger enzymes: Bifunctional proteins active in metabolism and in controlling gene expression. Mol Microbiol 67 692 702

23. BeinertHKennedyMCStoutCD 1996 Aconitase as iron-sulfur protein, enzyme, and iron-regulatory protein. Chem Rev 96 2335 2374

24. ZhuWBeckerDF 2003 Flavin redox state triggers conformational changes in the PutA protein from Escherichia coli. Biochemistry 42 5469 5477

25. SchmalischMHBachemSStülkeJ 2003 Control of the Bacillus subtilis antiterminator protein GlcT by phosphorylation. Elucidation of the phosphorylation chain leading to inactivation of GlcT. J Biol Chem 51108-51115

26. CommichauFMHerzbergCTripalPValeriusOStülkeJ 2007 A regulatory protein-protein interaction governs glutamate biosynthesis in Bacillus subtilis: The glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC. Mol Microbiol 65 642 654

27. HalbedelSStülkeJ 2007 Tools for the genetic analysis of Mycoplasma. Int J Med Microbiol 297 37 44

28. HalbedelSBusseJSchmidlSRStülkeJ 2006 Regulatory protein phosphorylation in Mycoplasma pneumoniae: A PP2C-type phosphatase serves to dephosphorylate HPr(Ser-P). J Biol Chem 281 26253 26259

29. HalbedelSHamesCStülkeJ 2004 In vivo activity of enzymatic and regulatory components of the phosphoenolpyruvate:sugar phosphotransferase system in Mycoplasma pneumoniae. J Bacteriol 186 7936 7943

30. HalbedelSEilersHJonasBBusseJHeckerM 2007 Transcription in Mycoplasma pneumoniae: Analysis of the promoters of the ackA and ldh genes. J Mol Biol 371 596 607

31. SchmidlSRGronauKHamesCBusseJBecherD 2010 The stability of cytadherence proteins in Mycoplasma pneumoniae requires activity of the protein kinase PrkC. Infect Immun 78 184 192

32. FrithMCSaundersNFKobeBBaileyTL 2008 Discovering sequence motifs with arbitrary insertions and deletions. PLoS Comput Biol 4 e1000071

33. KannanTRBasemanJB 2006 ADP-ribosylating and vacuolating cytotoxin of Mycoplasma pneumoniae represents unique virulence determinant among bacterial pathogens. Proc Natl Acad Sci U S A 103 6724 6729

34. VileiEMFreyJ 2001 Genetic and biochemical characterization of glycerol uptake in Mycoplasma mycoides subsp. mycoides SC: Its impact on H2O2 production and virulence. Clin Diagn Lab Immunol 8 85 92

35. YusEMaierTMichalodimitrakisKvan NoortVYamadaT 2009 Impact of genome reduction on bacterial metabolism and its regulation. Science 326 1263 1268

36. BrownDRFarleyJMZacherLACarltonJMClippingerTL 2001 Mycoplasma alligatoris sp. nov., from American alligators. Int J Syst Evol Microbiol 51 419 424

37. ShevchenkoDVAkinsDRRobinsonEJLiMShevchenkoOV 1997 Identification of homologs for thioredoxin, peptidyl prolyl cis-trans isomerase, glcerophosphodiester phosphodiesterase in outer membrane fractions of Treponema pallidum, the syphilis spirochaete. Infect Immun 65 4179 4189

38. StebeckCEShafferJMArrollTWLukehartSAvan VoohisWC 1997 Identification of the Treponema pallidum subsp. pallidum glcerophosphodiester phosphodiesterase homologue. FEMS Microbiol Lett 154 303 310

39. JefferyCJ 1999 Moonlighting proteins. Trends Biochem Sci 24 8 11

40. OkazakiYOhshimaNYoshizawaIKameiYMarrigioS 2010 A novel glycerophosphodiester phosphodiesterase, GDE5, controls skeletal muscle development via a non-enzymatic mechanism. J Biol Chem 285 27652 27663

41. GüellMvan NoortVYusEChenWHLeigh-BellJ 2009 Transcriptome complexity in a genome-reduced bacterium. Science 328 1268 1271

42. SchmidlSRGronauKPietackNHeckerMBecherD 2010 The phosphoproteome of the minimal bacterium Mycoplasma pneumoniae: Analysis of the complete known Ser/Thr kinome suggests the existence of novel kinases. Mol Cell Proteomics 9 1228 1242

43. Waldo RH3rdPophamPLRomero-ArroyoCEMothershedEALeeKK 1998 Transcriptional analysis of the hmw gene cluster of Mycoplasma pneumonia. J Bacteriol 181 4978 4985

44. SambrookJFritschEFManiatisT 1989 Molecular cloning: A laboratory manual, 2nd ed. N.Y Cold Spring Harbor Laboratory, Cold Spring Harbor

45. PichOQBurgosRPlanellRQuerolEPiñolJ 2006 Comparative analysis of antibiotic resistance gene markers in Mycoplasma genitalium: Application to studies of the minimal gene complement. Microbiology 152 519 527

46. MerzbacherMDetschCHillenWStülkeJ 2004 Mycoplasma pneumoniae HPr kinase/phosphorylase: Assigning functional roles to the P-loop and the HPrK/P signature sequence motif. Eur J Biochem 271 367 374

47. HamesCHalbedelSStülkeJ 2005 Multiple-mutation reaction: A method for simultaneous introduction of multiple mutations into the glpK gene of Mycoplasma pneumoniae. Appl Environ Microbiol 71 4097 4100

48. LarsonTJEhrmannMBoosW 1983 Periplasmic glycerophosphodiester phosphodiesterase of Escherichia coli, a new enzyme of the glp regulon. J Biol Chem 258 5428 5432

49. AbramoffMDMagelhaesPJRamSJ 2004 Image processing with ImageJ. Biophotonics Int 11 36 42

50. CrooksGEHonGChandoniaJMBrennerSE 2004 WebLogo: A sequence logo generator. Genome Res 14 1188 1190

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