The Effect of Cell Growth Phase on the Regulatory Cross-Talk between Flagellar and Spi1 Virulence Gene Expression


Flagellar-mediated motility is fundamental to Salmonella pathogenesis, which takes the lives of hundreds of thousands of people each year. The genes of the Salmonella pathogenicity island 1 and those of the flagellar regulon are part of the same transcriptional hierarchy. We report the novel finding where the key control of this network takes place at the flhDC promoter region. We followed the transcription from the two “live” flhDC promoters as a function of the cell growth phase. P1 comes on early in the cell cycle, while P5 comes on late. Transcription of P5 is HilD dependent, which represents a totally new finding and Salmonella specific: there is no HilD in E. coli flhDC control, no P5 transcription. P1 & P5 can express flhDC to equivalent levels, yet only P1- dependent expression produces motility UNLESS we artificially induce P5 EARLY in the cell cycle. This work is the foundation for the cell cycle stages a Salmonella bacterium experiences during host infection. This is a significant conceptual advance in Salmonella pathogenesis: one can no longer consider gene regulation at 37°C and OD 0.6 as a reflection of the Salmonella infection cycle; the whole cell growth cycle must be considered in understanding this complex biological processes.


Vyšlo v časopise: The Effect of Cell Growth Phase on the Regulatory Cross-Talk between Flagellar and Spi1 Virulence Gene Expression. PLoS Pathog 10(3): e32767. doi:10.1371/journal.ppat.1003987
Kategorie: Research Article
prolekare.web.journal.doi_sk: 10.1371/journal.ppat.1003987

Souhrn

Flagellar-mediated motility is fundamental to Salmonella pathogenesis, which takes the lives of hundreds of thousands of people each year. The genes of the Salmonella pathogenicity island 1 and those of the flagellar regulon are part of the same transcriptional hierarchy. We report the novel finding where the key control of this network takes place at the flhDC promoter region. We followed the transcription from the two “live” flhDC promoters as a function of the cell growth phase. P1 comes on early in the cell cycle, while P5 comes on late. Transcription of P5 is HilD dependent, which represents a totally new finding and Salmonella specific: there is no HilD in E. coli flhDC control, no P5 transcription. P1 & P5 can express flhDC to equivalent levels, yet only P1- dependent expression produces motility UNLESS we artificially induce P5 EARLY in the cell cycle. This work is the foundation for the cell cycle stages a Salmonella bacterium experiences during host infection. This is a significant conceptual advance in Salmonella pathogenesis: one can no longer consider gene regulation at 37°C and OD 0.6 as a reflection of the Salmonella infection cycle; the whole cell growth cycle must be considered in understanding this complex biological processes.


Zdroje

1. KojimaS, BlairDF (2004) The bacterial flagellar motor: structure and function of a complex molecular machine. Int Rev Cytol 233: 93–134.

2. StecherB, HapfelmeierS, MullerC, KremerM, StallmachT, et al. (2004) Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar Typhimurium colitis in streptomycin-pretreated mice. Infect Immun 72: 4138–4150.

3. StecherB, RobbianiR, WalkerAW, WestendorfAM, BarthelM, et al. (2007) Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota. PLoS Biol 5: 2177–2189.

4. BaumlerAJ, KustersJG, StojiljkovicI, HeffronF (1994) Salmonella typhimurium loci involved in survival within macrophages. Infect Immun 62: 1623–1630.

5. YamamotoS, KutsukakeK (2006) FliT acts as an anti-FlhD2C2 factor in the transcriptional control of the flagellar regulon in Salmonella enterica serovar typhimurium. J Bacteriol 188: 6703–6708.

6. HungCC, HainesL, AltierC (2012) The flagellar regulator fliT represses Salmonella pathogenicity island 1 through flhDC and fliZ. PLoS One 7: e34220.

7. ChilcottGS, HughesKT (2000) Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli. Microbiol Mol Biol Rev 64: 694–708.

8. KomedaY, SuzukiH, IshidsuJI, IinoT (1976) The role of cAMP in flagellation of Salmonella typhimurium. Mol Gen Genet 142: 289–298.

9. SoutourinaO, KolbA, KrinE, Laurent-WinterC, RimskyS, et al. (1999) Multiple control of flagellum biosynthesis in Escherichia coli: role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon. J Bacteriol 181: 7500–7508.

10. CampoyS, JaraM, BusquetsN, de RozasAM, BadiolaI, et al. (2002) Intracellular cyclic AMP concentration is decreased in Salmonella typhimurium fur mutants. Microbiology 148: 1039–1048.

11. KellyA, GoldbergMD, CarrollRK, DaninoV, HintonJC, et al. (2004) A global role for Fis in the transcriptional control of metabolism and type III secretion in Salmonella enterica serovar Typhimurium. Microbiology 150: 2037–2053.

12. StojiljkovicI, BaumlerAJ, HantkeK (1994) Fur regulon in gram-negative bacteria. Identification and characterization of new iron-regulated Escherichia coli genes by a fur titration assay. J Mol Biol 236: 531–545.

13. YanagiharaS, IyodaS, OhnishiK, IinoT, KutsukakeK (1999) Structure and transcriptional control of the flagellar master operon of Salmonella typhimurium. Genes Genet Syst 74: 105–111.

14. SperandioV, TorresAG, KaperJB (2002) Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli. Mol Microbiol 43: 809–821.

15. KoM, ParkC (2000) H-NS-Dependent regulation of flagellar synthesis is mediated by a LysR family protein. J Bacteriol 182: 4670–4672.

16. ErhardtM, HughesKT (2010) C-ring requirement in flagellar type III secretion is bypassed by FlhDC upregulation. Mol Microbiol 75: 376–393.

17. EllermeierCD, SlauchJM (2003) RtsA and RtsB coordinately regulate expression of the invasion and flagellar genes in Salmonella enterica serovar Typhimurium. J Bacteriol 185: 5096–5108.

18. LehnenD, BlumerC, PolenT, WackwitzB, WendischVF, et al. (2002) LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli. Mol Microbiol 45: 521–532.

19. WangQ, ZhaoY, McClellandM, HarsheyRM (2007) The RcsCDB signaling system and swarming motility in Salmonella enterica serovar typhimurium: dual regulation of flagellar and SPI-2 virulence genes. J Bacteriol 189: 8447–8457.

20. MouslimC, GroismanEA (2003) Control of the Salmonella ugd gene by three two-component regulatory systems. Mol Microbiol 47: 335–344.

21. SingerHM, ErhardtM, HughesKT (2013) RflM Functions as a Transcriptional Repressor in the Autogenous Control of the Salmonella Flagellar Master Operon flhDC. J Bacteriol 195: 4274–4282.

22. WeiBL, Brun-ZinkernagelAM, SimeckaJW, PrussBM, BabitzkeP, et al. (2001) Positive regulation of motility and flhDC expression by the RNA-binding protein CsrA of Escherichia coli. Mol Microbiol 40: 245–256.

23. YakhninAV, BakerCS, VakulskasCA, YakhninH, BerezinI, et al. (2013) CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage. Mol Microbiol 87: 851–866.

24. TakayaA, MatsuiM, TomoyasuT, KayaM, YamamotoT (2006) The DnaK chaperone machinery converts the native FlhD2C2 hetero-tetramer into a functional transcriptional regulator of flagellar regulon expression in Salmonella. Mol Microbiol 59: 1327–1340.

25. TomoyasuT, TakayaA, IsogaiE, YamamotoT (2003) Turnover of FlhD and FlhC, master regulator proteins for Salmonella flagellum biogenesis, by the ATP-dependent ClpXP protease. Mol Microbiol 48: 443–452.

26. WadaT, TanabeY, KutsukakeK (2011) FliZ acts as a repressor of the ydiV gene, which encodes an anti-FlhD4C2 factor of the flagellar regulon in Salmonella enterica serovar typhimurium. J Bacteriol 193: 5191–5198.

27. TakayaA, ErhardtM, KarataK, WinterbergK, YamamotoT, et al. (2012) YdiV: a dual function protein that targets FlhDC for ClpXP-dependent degradation by promoting release of DNA-bound FlhDC complex. Mol Microbiol 83: 1268–1284.

28. WadaT, MorizaneT, AboT, TominagaA, Inoue-TanakaK, et al. (2011) EAL domain protein YdiV acts as an anti-FlhD4C2 factor responsible for nutritional control of the flagellar regulon in Salmonella enterica Serovar Typhimurium. J Bacteriol 193: 1600–1611.

29. KrogerC, DillonSC, CameronAD, PapenfortK, SivasankaranSK, et al. (2012) The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium. Proc Natl Acad Sci U S A 109: E1277–1286.

30. OhlME, MillerSI (2001) Salmonella: a model for bacterial pathogenesis. Annu Rev Med 52: 259–274.

31. GalanJE, CurtissR3rd (1989) Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells. Proc Natl Acad Sci U S A 86: 6383–6387.

32. EichelbergK, GalanJE (1999) Differential regulation of Salmonella typhimurium type III secreted proteins by pathogenicity island 1 (SPI-1)-encoded transcriptional activators InvF and hilA. Infect Immun 67: 4099–4105.

33. HueckCJ (1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62: 379–433.

34. KimbroughTG, MillerSI (2000) Contribution of Salmonella typhimurium type III secretion components to needle complex formation. Proc Natl Acad Sci U S A 97: 11008–11013.

35. LeeCA, JonesBD, FalkowS (1992) Identification of a Salmonella typhimurium invasion locus by selection for hyperinvasive mutants. Proc Natl Acad Sci U S A 89: 1847–1851.

36. MillsDM, BajajV, LeeCA (1995) A 40 kb chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K-12 chromosome. Mol Microbiol 15: 749–759.

37. BajajV, HwangC, LeeCA (1995) hilA is a novel ompR/toxR family member that activates the expression of Salmonella typhimurium invasion genes. Mol Microbiol 18: 715–727.

38. DarwinKH, MillerVL (2001) Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium. EMBO J 20: 1850–1862.

39. EllermeierJR, SlauchJM (2007) Adaptation to the host environment: regulation of the SPI1 type III secretion system in Salmonella enterica serovar Typhimurium. Curr Opin Microbiol 10: 24–29.

40. SchechterLM, LeeCA (2001) AraC/XylS family members, HilC and HilD, directly bind and derepress the Salmonella typhimurium hilA promoter. Mol Microbiol 40: 1289–1299.

41. MacnabRM (2004) Type III flagellar protein export and flagellar assembly. Biochim Biophys Acta 1694: 207–217.

42. CornelisGR (2006) The type III secretion injectisome. Nat Rev Microbiol 4: 811–825.

43. SittkaA, LucchiniS, PapenfortK, SharmaCM, RolleK, et al. (2008) Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq. PLoS Genet 4: e1000163.

44. AltierC, SuyemotoM, LawhonSD (2000) Regulation of Salmonella enterica serovar typhimurium invasion genes by csrA. Infect Immun 68: 6790–6797.

45. WilsonRL, LibbySJ, FreetAM, BoddickerJD, FahlenTF, et al. (2001) Fis, a DNA nucleoid-associated protein, is involved in Salmonella typhimurium SPI-1 invasion gene expression. Mol Microbiol 39: 79–88.

46. TeplitskiM, GoodierRI, AhmerBM (2003) Pathways leading from BarA/SirA to motility and virulence gene expression in Salmonella. J Bacteriol 185: 7257–7265.

47. JonasK, EdwardsAN, AhmadI, RomeoT, RomlingU, et al. (2010) Complex regulatory network encompassing the Csr, c-di-GMP and motility systems of Salmonella Typhimurium. Environ Microbiol 12: 524–540.

48. BaxterMA, JonesBD (2005) The fimYZ genes regulate Salmonella enterica Serovar Typhimurium invasion in addition to type 1 fimbrial expression and bacterial motility. Infect Immun 73: 1377–1385.

49. FortuneDR, SuyemotoM, AltierC (2006) Identification of CsrC and characterization of its role in epithelial cell invasion in Salmonella enterica serovar Typhimurium. Infect Immun 74: 331–339.

50. EllermeierJR, SlauchJM (2008) Fur regulates expression of the Salmonella pathogenicity island 1 type III secretion system through HilD. J Bacteriol 190: 476–486.

51. TeixidoL, CarrascoB, AlonsoJC, BarbeJ, CampoyS (2011) Fur activates the expression of Salmonella enterica pathogenicity island 1 by directly interacting with the hilD operator in vivo and in vitro. PLoS One 6: e19711.

52. TroxellB, SikesML, FinkRC, Vazquez-TorresA, Jones-CarsonJ, et al. (2011) Fur negatively regulates hns and is required for the expression of HilA and virulence in Salmonella enterica serovar Typhimurium. J Bacteriol 193: 497–505.

53. SainiS, SlauchJM, AldridgePD, RaoCV (2010) Role of cross talk in regulating the dynamic expression of the flagellar Salmonella pathogenicity island 1 and type 1 fimbrial genes. J Bacteriol 192: 5767–5777.

54. LinD, RaoCV, SlauchJM (2008) The Salmonella SPI1 type three secretion system responds to periplasmic disulfide bond status via the flagellar apparatus and the RcsCDB system. J Bacteriol 190: 87–97.

55. KageH, TakayaA, OhyaM, YamamotoT (2008) Coordinated regulation of expression of Salmonella pathogenicity island 1 and flagellar type III secretion systems by ATP-dependent ClpXP protease. J Bacteriol 190: 2470–2478.

56. IyodaS, KamidoiT, HiroseK, KutsukakeK, WatanabeH (2001) A flagellar gene fliZ regulates the expression of invasion genes and virulence phenotype in Salmonella enterica serovar Typhimurium. Microb Pathog 30: 81–90.

57. ChubizJE, GolubevaYA, LinD, MillerLD, SlauchJM (2010) FliZ regulates expression of the Salmonella pathogenicity island 1 invasion locus by controlling HilD protein activity in Salmonella enterica serovar typhimurium. J Bacteriol 192: 6261–6270.

58. WangQ, FryeJG, McClellandM, HarsheyRM (2004) Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes. Mol Microbiol 52: 169–187.

59. EllermeierCD, EllermeierJR, SlauchJM (2005) HilD, HilC and RtsA constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator hilA in Salmonella enterica serovar Typhimurium. Mol Microbiol 57: 691–705.

60. LaishramRS, GowrishankarJ (2007) Environmental regulation operating at the promoter clearance step of bacterial transcription. Genes Dev 21: 1258–1272.

61. MouslimC, DelgadoM, GroismanEA (2004) Activation of the RcsC/YojN/RcsB phosphorelay system attenuates Salmonella virulence. Mol Microbiol 54: 386–395.

62. WozniakCE, LeeC, HughesKT (2009) T-POP array identifies EcnR and PefI-SrgD as novel regulators of flagellar gene expression. J Bacteriol 191: 1498–1508.

63. FontaineF, StewartEJ, LindnerAB, TaddeiF (2008) Mutations in two global regulators lower individual mortality in Escherichia coli. Mol Microbiol 67: 2–14.

64. MacnabRM (2003) How bacteria assemble flagella. Annu Rev Microbiol 57: 77–100.

65. LestasI, VinnicombeG, PaulssonJ (2010) Fundamental limits on the suppression of molecular fluctuations. Nature 467: 174–178.

66. AlonU (2007) Network motifs: theory and experimental approaches. Nat Rev Genet 8: 450–461.

67. CummingsLA, WilkersonWD, BergsbakenT, CooksonBT (2006) In vivo, fliC expression by Salmonella enterica serovar Typhimurium is heterogeneous, regulated by ClpX, and anatomically restricted. Mol Microbiol 61: 795–809.

68. StewartMK, CummingsLA, JohnsonML, BerezowAB, CooksonBT (2011) Regulation of phenotypic heterogeneity permits Salmonella evasion of the host caspase-1 inflammatory response. Proc Natl Acad Sci U S A 108: 20742–20747.

69. SainiS, EllermeierJR, SlauchJM, RaoCV (2010) The role of coupled positive feedback in the expression of the SPI1 type three secretion system in Salmonella. PLoS Pathog 6: e1001025.

70. CleggS, HughesKT (2002) FimZ is a molecular link between sticking and swimming in Salmonella enterica serovar Typhimurium. J Bacteriol 184: 1209–1213.

71. GolubevaYA, SadikAY, EllermeierJR, SlauchJM (2012) Integrating global regulatory input into the Salmonella pathogenicity island 1 type III secretion system. Genetics 190: 79–90.

72. PesaventoC, BeckerG, SommerfeldtN, PosslingA, TschowriN, et al. (2008) Inverse regulatory coordination of motility and curli-mediated adhesion in Escherichia coli. Genes Dev 22: 2434–2446.

73. YangX, ThornburgT, SuoZ, JunS, RobisonA, et al. (2012) Flagella overexpression attenuates Salmonella pathogenesis. PLoS One 7: e46828.

74. ShinD, LeeEJ, HuangH, GroismanEA (2006) A positive feedback loop promotes transcription surge that jump-starts Salmonella virulence circuit. Science 314: 1607–1609.

75. ScarlatoV, PrugnolaA, AricoB, RappuoliR (1990) Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis. Proc Natl Acad Sci U S A 87: 10067.

76. MurrayHD, ApplemanJA, GourseRL (2003) Regulation of the Escherichia coli rrnB P2 promoter. J Bacteriol 185: 28–34.

77. WozniakCE, ChevanceFF, HughesKT (2010) Multiple promoters contribute to swarming and the coordination of transcription with flagellar assembly in Salmonella. J Bacteriol 192: 4752–4762.

78. TanabeY, WadaT, OnoK, AboT, KutsukakeK (2011) The transcript from the sigma(28)-dependent promoter is translationally inert in the expression of the sigma(28)-encoding gene fliA in the fliAZ operon of Salmonella enterica serovar Typhimurium. J Bacteriol 193: 6132–6141.

79. KarlinseyJE, TanakaS, BettenworthV, YamaguchiS, BoosW, et al. (2000) Completion of the hook-basal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription. Mol Microbiol 37: 1220–1231.

80. FryeJ, KarlinseyJE, FeliseHR, MarzolfB, DowidarN, et al. (2006) Identification of new flagellar genes of Salmonella enterica serovar Typhimurium. J Bacteriol 188: 2233–2243.

81. MisselwitzB, BarrettN, KreibichS, VonaeschP, AndritschkeD, et al. (2012) Near surface swimming of Salmonella Typhimurium explains target-site selection and cooperative invasion. PLoS Pathog 8: e1002810.

82. VegotskyA, LimF, FosterJF, KofflerH (1965) Disintegration of flagella by acid. Arch Biochem Biophys 111: 296–307.

83. SantosRL, ZhangS, TsolisRM, BaumlerAJ, AdamsLG (2002) Morphologic and molecular characterization of Salmonella typhimurium infection in neonatal calves. Vet Pathol 39: 200–215.

84. ReisBP, ZhangS, TsolisRM, BaumlerAJ, AdamsLG, et al. (2003) The attenuated sopB mutant of Salmonella enterica serovar Typhimurium has the same tissue distribution and host chemokine response as the wild type in bovine Peyer's patches. Vet Microbiol 97: 269–277.

85. KnodlerLA, VallanceBA, CelliJ, WinfreeS, HansenB, et al. (2010) Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia. Proc Natl Acad Sci U S A 107: 17733–17738.

86. Rivera-ChavezF, WinterSE, LopezCA, XavierMN, WinterMG, et al. (2013) Salmonella uses energy taxis to benefit from intestinal inflammation. PLoS Pathog 9: e1003267.

87. PrussBM, CampbellJW, Van DykTK, ZhuC, KoganY, et al. (2003) FlhD/FlhC is a regulator of anaerobic respiration and the Entner-Doudoroff pathway through induction of the methyl-accepting chemotaxis protein Aer. J Bacteriol 185: 534–543.

88. Davis RW, DBotstein, J. RRoth and Cold Spring Harbor N.Y. Laboratory of Quantitative Biology. (1980) Advanced bacterial genetics: a manual for genetic engineering. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

89. GoodierRI, AhmerBM (2001) SirA orthologs affect both motility and virulence. J Bacteriol 183: 2249–2258.

90. KarlinseyJE, HughesKT (2006) Genetic transplantation: Salmonella enterica serovar Typhimurium as a host to study sigma factor and anti-sigma factor interactions in genetically intractable systems. J Bacteriol 188: 103–114.

91. UzzauS, Figueroa-BossiN, RubinoS, BossiL (2001) Epitope tagging of chromosomal genes in Salmonella. Proc Natl Acad Sci U S A 98: 15264–15269.

92. SchaggerH (2006) Tricine-SDS-PAGE. Nat Protoc 1: 16–22.

93. GraingerDC, OvertonTW, ReppasN, WadeJT, TamaiE, et al. (2004) Genomic studies with Escherichia coli MelR protein: applications of chromatin immunoprecipitation and microarrays. J Bacteriol 186: 6938–6943.

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2014 Číslo 3

Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Eozinofilní granulomatóza s polyangiitidou
nový kurz

Betablokátory a Ca antagonisté z jiného úhlu
Autori: prof. MUDr. Michal Vrablík, Ph.D., MUDr. Petr Janský

Autori: doc. MUDr. Petr Čáp, Ph.D.

Farmakoterapie akutní a chronické bolesti

Získaná hemofilie - Povědomí o nemoci a její diagnostika

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Nemáte účet?  Registrujte sa

Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa