A Bistable Switch and Anatomical Site Control Virulence Gene Expression in the Intestine

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A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients.

Vyšlo v časopise: A Bistable Switch and Anatomical Site Control Virulence Gene Expression in the Intestine. PLoS Pathog 6(9): e32767. doi:10.1371/journal.ppat.1001102
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1001102

Souhrn

Zdroje

1. MoxonR

BaylissC

HoodD

2006 Bacterial contingency loci: the role of simple sequence DNA repeats in bacterial adaptation. Annu Rev Genet 40 307 333

2. DavidsonCJ

SuretteMG

2008 Individuality in bacteria. Annu Rev Genet 42 253 268

3. KaperJB

MorrisJGJr

LevineMM

1995 Cholera. Clin Microbiol Rev 8 48 86

4. KloseKE

2000 The suckling mouse model of cholera. Trends Microbiol 8 189 191

5. LeeSH

HavaDL

WaldorMK

CamilliA

1999 Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection. Cell 99 625 634

6. HouotL

ChangS

AbsalonC

WatnickPI

Vibrio cholerae phosphoenolpyruvate phosphotransferase system control of carbohydrate transport, biofilm formation, and colonization of the germfree mouse intestine. Infect Immun 78 1482 1494

7. DeSN

ChatterjeDN

1953 An experimental study of the mechanism of action of Vibrio cholerae on the intestinal mucous membrane. J Pathol Bacteriol 66 559 562

8. BurrowsW

MusteikisGM

1966 Cholera infection and toxin in the rabbit ileal loop. J Infect Dis 116 183 190

9. RitchieJM

WaldorMK

2009 Vibrio cholerae interactions with the gastrointestinal tract: lessons from animal studies. Curr Top Microbiol Immunol 337 37 59

10. NielsenAT

DolganovNA

OttoG

MillerMC

WuCY

2006 RpoS controls the Vibrio cholerae mucosal escape response. PLoS Pathog 2 e109

11. ColwellRR

1996 Global climate and infectious disease: the cholera paradigm. Science 274 2025 2031

12. NelsonEJ

HarrisJB

MorrisJGJr

CalderwoodSB

CamilliA

2009 Cholera transmission: the host, pathogen and bacteriophage dynamic. Nat Rev Microbiol 7 693 702

13. SchildS

TamayoR

NelsonEJ

QadriF

CalderwoodSB

2007 Genes induced late in infection increase fitness of Vibrio cholerae after release into the environment. Cell Host Microbe 2 264 277

14. HerringtonDA

HallRH

LosonskyG

MekalanosJJ

TaylorRK

1988 Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J Exp Med 168 1487 1492

15. TaylorRK

MillerVL

FurlongDB

MekalanosJJ

1987 Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc Natl Acad Sci U S A 84 2833 2837

16. FinkelsteinRA

LoSpallutoJJ

1969 Pathogenesis of experimental cholera. Preparation and isolation of choleragen and choleragenoid. J Exp Med 130 185 202

17. MillerVL

MekalanosJJ

1984 Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR. Proc Natl Acad Sci U S A 81 3471 3475

18. MatsonJS

WitheyJH

DiRitaVJ

2007 Regulatory networks controlling Vibrio cholerae virulence gene expression. Infect Immun 75 5542 5549

19. XuQ

DziejmanM

MekalanosJJ

2003 Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro. Proc Natl Acad Sci U S A 100 1286 1291

20. LarocqueRC

HarrisJB

DziejmanM

LiX

KhanAI

2005 Transcriptional profiling of Vibrio cholerae recovered directly from patient specimens during early and late stages of human infection. Infect Immun 73 4488 4493

21. BinaJ

ZhuJ

DziejmanM

FaruqueS

CalderwoodS

2003 ToxR regulon of Vibrio cholerae and its expression in vibrios shed by cholera patients. Proc Natl Acad Sci U S A 100 2801 2806

22. MerrellDS

ButlerSM

QadriF

DolganovNA

AlamA

2002 Host-induced epidemic spread of the cholera bacterium. Nature 417 642 645

23. FaruqueSM

BiswasK

UddenSM

AhmadQS

SackDA

2006 Transmissibility of cholera: in vivo-formed biofilms and their relationship to infectivity and persistence in the environment. Proc Natl Acad Sci U S A 103 6350 6355

24. KamruzzamanM

UddenSM

CameronDE

CalderwoodSB

NairGB

Quorum-regulated biofilms enhance the development of conditionally viable, environmental Vibrio cholerae. Proc Natl Acad Sci U S A 107 1588 1593

25. NelsonEJ

ChowdhuryA

HarrisJB

BegumYA

ChowdhuryF

2007 Complexity of rice-water stool from patients with Vibrio cholerae plays a role in the transmission of infectious diarrhea. Proc Natl Acad Sci U S A 104 19091 19096

26. ButlerSM

NelsonEJ

ChowdhuryN

FaruqueSM

CalderwoodSB

2006 Cholera stool bacteria repress chemotaxis to increase infectivity. Mol Microbiol 60 417 426

27. NelsonEJ

ChowdhuryA

FlynnJ

SchildS

BourassaL

2008 Transmission of Vibrio cholerae is antagonized by lytic phage and entry into the aquatic environment. PLoS Pathog 4 e1000187

28. AlamA

LarocqueRC

HarrisJB

VanderspurtC

RyanET

2005 Hyperinfectivity of human-passaged Vibrio cholerae can be modeled by growth in the infant mouse. Infect Immun 73 6674 6679

29. KirnTJ

TaylorRK

2005 TcpF is a soluble colonization factor and protective antigen secreted by El Tor and classical O1 and O139 Vibrio cholerae serogroups. Infect Immun 73 4461 4470

30. HigginsDE

NazarenoE

DiRitaVJ

1992 The virulence gene activator ToxT from Vibrio cholerae is a member of the AraC family of transcriptional activators. J Bacteriol 174 6974 6980

31. YuRR

DiRitaVJ

2002 Regulation of gene expression in Vibrio cholerae by ToxT involves both antirepression and RNA polymerase stimulation. Mol Microbiol 43 119 134

32. HulbertRR

TaylorRK

2002 Mechanism of ToxT-dependent transcriptional activation at the Vibrio cholerae tcpA promoter. J Bacteriol 184 5533 5544

33. AndersenJB

SternbergC

PoulsenLK

BjornSP

GivskovM

1998 New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol 64 2240 2246

34. SternbergC

ChristensenBB

JohansenT

Toftgaard NielsenA

AndersenJB

1999 Distribution of bacterial growth activity in flow-chamber biofilms. Appl Environ Microbiol 65 4108 4117

35. NielsenAT

Tolker-NielsenT

BarkenKB

MolinS

2000 Role of commensal relationships on the spatial structure of a surface-attached microbial consortium. Environ Microbiol 2 59 68

36. IwanagaM

YamamotoK

HigaN

IchinoseY

NakasoneN

1986 Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor. Microbiol Immunol 30 1075 1083

37. MedranoAI

DiRitaVJ

CastilloG

SanchezJ

1999 Transient transcriptional activation of the Vibrio cholerae El Tor virulence regulator toxT in response to culture conditions. Infect Immun 67 2178 2183

38. ChiangSL

TaylorRK

KoomeyM

MekalanosJJ

1995 Single amino acid substitutions in the N-terminus of Vibrio cholerae TcpA affect colonization, autoagglutination, and serum resistance. Mol Microbiol 17 1133 1142

39. KirnTJ

LaffertyMJ

SandoeCM

TaylorRK

2000 Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio cholerae. Mol Microbiol 35 896 910

40. SchaechterM

MaaloeO

KjeldgaardNO

1958 Dependency on medium and temperature of cell size and chemical composition during balanced grown of Salmonella typhimurium. J Gen Microbiol 19 592 606

41. DennisPP

BremerH

1974 Differential rate of ribosomal protein synthesis in Escherichia coli B/r. J Mol Biol 84 407 422

42. ZhangX

DennisP

EhrenbergM

BremerH

2002 Kinetic properties of rrn promoters in Escherichia coli. Biochimie 84 981 996

43. BartlettMS

GourseRL

1994 Growth rate-dependent control of the rrnB P1 core promoter in Escherichia coli. J Bacteriol 176 5560 5564

44. AbuaitaBH

WitheyJH

2009 Bicarbonate Induces Vibrio cholerae virulence gene expression by enhancing ToxT activity. Infect Immun 77 4111 4120

45. FordtranJS

LocklearTW

1966 Ionic constituents and osmolality of gastric and small-intestinal fluids after eating. Am J Dig Dis 11 503 521

46. WitheyJH

DiRitaVJ

2006 The toxbox: specific DNA sequence requirements for activation of Vibrio cholerae virulence genes by ToxT. Mol Microbiol 59 1779 1789

47. KovacikovaG

SkorupskiK

1999 A Vibrio cholerae LysR homolog, AphB, cooperates with AphA at the tcpPH promoter to activate expression of the ToxR virulence cascade. J Bacteriol 181 4250 4256

48. DiRitaVJ

ParsotC

JanderG

MekalanosJJ

1991 Regulatory cascade controls virulence in Vibrio cholerae. Proc Natl Acad Sci U S A 88 5403 5407

49. HaseCC

MekalanosJJ

1998 TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci U S A 95 730 734

50. KovacikovaG

SkorupskiK

2002 Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter. Mol Microbiol 46 1135 1147

51. MillerVL

TaylorRK

MekalanosJJ

1987 Cholera toxin transcriptional activator toxR is a transmembrane DNA binding protein. Cell 48 271 279

52. FerrellJEJr

2002 Self-perpetuating states in signal transduction: positive feedback, double-negative feedback and bistability. Curr Opin Cell Biol 14 140 148

53. TotsikaM

BeatsonSA

HoldenN

GallyDL

2008 Regulatory interplay between pap operons in uropathogenic Escherichia coli. Mol Microbiol 67 996 1011

54. VeeningJW

SmitsWK

KuipersOP

2008 Bistability, epigenetics, and bet-hedging in bacteria. Annu Rev Microbiol 62 193 210

55. DubnauD

LosickR

2006 Bistability in bacteria. Mol Microbiol 61 564 572

56. YuRR

DiRitaVJ

1999 Analysis of an autoregulatory loop controlling ToxT, cholera toxin, and toxin-coregulated pilus production in Vibrio cholerae. J Bacteriol 181 2584 2592

57. ProutyMG

OsorioCR

KloseKE

2005 Characterization of functional domains of the Vibrio cholerae virulence regulator ToxT. Mol Microbiol 58 1143 1156

58. HungDT

ShakhnovichEA

PiersonE

MekalanosJJ

2005 Small-molecule inhibitor of Vibrio cholerae virulence and intestinal colonization. Science 310 670 674

59. ShakhnovichEA

HungDT

PiersonE

LeeK

MekalanosJJ

2007 Virstatin inhibits dimerization of the transcriptional activator ToxT. Proc Natl Acad Sci U S A 104 2372 2377

60. GorkeB

StulkeJ

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

61. SkorupskiK

TaylorRK

1997 Cyclic AMP and its receptor protein negatively regulate the coordinate expression of cholera toxin and toxin-coregulated pilus in Vibrio cholerae. Proc Natl Acad Sci U S A 94 265 270

62. SilvaAJ

BenitezJA

2004 Transcriptional regulation of Vibrio cholerae hemagglutinin/protease by the cyclic AMP receptor protein and RpoS. J Bacteriol 186 6374 6382

63. LiangW

Pascual-MontanoA

SilvaAJ

BenitezJA

2007 The cyclic AMP receptor protein modulates quorum sensing, motility and multiple genes that affect intestinal colonization in Vibrio cholerae. Microbiology 153 2964 2975

64. KovacikovaG

SkorupskiK

2001 Overlapping binding sites for the virulence gene regulators AphA, AphB and cAMP-CRP at the Vibrio cholerae tcpPH promoter. Mol Microbiol 41 393 407

65. KrukonisES

YuRR

DiritaVJ

2000 The Vibrio cholerae ToxR/TcpP/ToxT virulence cascade: distinct roles for two membrane-localized transcriptional activators on a single promoter. Mol Microbiol 38 67 84

66. OgiermanMA

VossE

MeaneyC

FaastR

AttridgeSR

1996 Comparison of the promoter proximal regions of the toxin-co-regulated tcp gene cluster in classical and El Tor strains of Vibrio cholerae O1. Gene 170 9 16

67. ThomasS

WilliamsSG

ManningPA

1995 Regulation of tcp genes in classical and El Tor strains of Vibrio cholerae O1. Gene 166 43 48

68. PostmaPW

LengelerJW

JacobsonGR

1993 Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. Microbiol Rev 57 543 594

69. LowdenMJ

SkorupskiK

PellegriniM

ChiorazzoMG

TaylorRK

2010 Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes. Proc Natl Acad Sci U S A 107 2860 2865

70. LiuZ

MiyashiroT

TsouA

HsiaoA

GoulianM

2008 Mucosal penetration primes Vibrio cholerae for host colonization by repressing quorum sensing. Proc Natl Acad Sci U S A 105 9769 9774

71. DietzJ

FieldM

1973 Ion transport in rabbit ileal mucosa. IV. Bicarbonate secretion. Am J Physiol 225 858 861

72. ForstnerJF

RoomiNW

FahimRE

ForstnerGG

1981 Cholera toxin stimulates secretion of immunoreactive intestinal mucin. Am J Physiol 240 G10 16

73. DiRitaVJ

1992 Co-ordinate expression of virulence genes by ToxR in Vibrio cholerae. Mol Microbiol 6 451 458

74. CaiL

FriedmanN

XieXS

2006 Stochastic protein expression in individual cells at the single molecule level. Nature 440 358 362

75. BalabanNQ

MerrinJ

ChaitR

KowalikL

LeiblerS

2004 Bacterial persistence as a phenotypic switch. Science 305 1622 1625

76. KerenI

ShahD

SpoeringA

KaldaluN

LewisK

2004 Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J Bacteriol 186 8172 8180

77. van SinderenD

LuttingerA

KongL

DubnauD

VenemaG

1995 comK encodes the competence transcription factor, the key regulatory protein for competence development in Bacillus subtilis. Mol Microbiol 15 455 462

78. MaamarH

DubnauD

2005 Bistability in the Bacillus subtilis K-state (competence) system requires a positive feedback loop. Mol Microbiol 56 615 624

79. ChungJD

StephanopoulosG

IretonK

GrossmanAD

1994 Gene expression in single cells of Bacillus subtilis: evidence that a threshold mechanism controls the initiation of sporulation. J Bacteriol 176 1977 1984

80. Gonzalez-PastorJE

HobbsEC

LosickR

2003 Cannibalism by sporulating bacteria. Science 301 510 513

81. VeeningJW

HamoenLW

KuipersOP

2005 Phosphatases modulate the bistable sporulation gene expression pattern in Bacillus subtilis. Mol Microbiol 56 1481 1494

82. ChaiY

ChuF

KolterR

LosickR

2008 Bistability and biofilm formation in Bacillus subtilis. Mol Microbiol 67 254 263

83. MantleM

HusarSD

1994 Binding of Yersinia enterocolitica to purified, native small intestinal mucins from rabbits and humans involves interactions with the mucin carbohydrate moiety. Infect Immun 62 1219 1227

84. IwanagaM

YamamotoK

1985 New medium for the production of cholera toxin by Vibrio cholerae O1 biotype El Tor. J Clin Microbiol 22 405 408

85. BradfordMM

1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 248 254

86. FullnerKJ

MekalanosJJ

1999 Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae. Infect Immun 67 1393 1404

87. LambertsenL

SternbergC

MolinS

2004 Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins. Environ Microbiol 6 726 732

88. KochB

JensenLE

NybroeO

2001 A panel of Tn7-based vectors for insertion of the gfp marker gene or for delivery of cloned DNA into Gram-negative bacteria at a neutral chromosomal site. J Microbiol Methods 45 187 195

89. MeibomKL

LiXB

NielsenAT

WuCY

RosemanS

2004 The Vibrio cholerae chitin utilization program. Proc Natl Acad Sci U S A 101 2524 2529

90. TusherVG

TibshiraniR

ChuG

2001 Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98 5116 5121

91. SchoolnikGK

VoskuilMI

SchnappingerD

YildizFH

MeibomK

2001 Whole genome DNA microarray expression analysis of biofilm development by Vibrio cholerae O1 E1 Tor. Methods Enzymol 336 3 18

92. KovacikovaG

LinW

SkorupskiK

2003 The virulence activator AphA links quorum sensing to pathogenesis and physiology in Vibrio cholerae by repressing the expression of a penicillin amidase gene on the small chromosome. J Bacteriol 185 4825 4836