#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Transcriptome Analysis of in
Human Whole Blood and Mutagenesis Studies Identify Virulence Factors Involved in
Blood Survival


During infection Neisseria meningitidis (Nm) encounters multiple

environments within the host, which makes rapid adaptation a crucial factor for

meningococcal survival. Despite the importance of invasion into the bloodstream

in the meningococcal disease process, little is known about how Nm adapts to

permit survival and growth in blood. To address this, we performed a time-course

transcriptome analysis using an ex vivo model of human whole

blood infection. We observed that Nm alters the expression of ≈30% of

ORFs of the genome and major dynamic changes were observed in the expression of

transcriptional regulators, transport and binding proteins, energy metabolism,

and surface-exposed virulence factors. In particular, we found that the gene

encoding the regulator Fur, as well as all genes encoding iron uptake systems,

were significantly up-regulated. Analysis of regulated genes encoding for

surface-exposed proteins involved in Nm pathogenesis allowed us to better

understand mechanisms used to circumvent host defenses. During blood infection,

Nm activates genes encoding for the factor H binding proteins, fHbp and NspA,

genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as

several less characterized surface-exposed proteins that might have a role in

blood survival. Through mutagenesis studies of a subset of up-regulated genes we

were able to identify new proteins important for survival in human blood and

also to identify additional roles of previously known virulence factors in

aiding survival in blood. Nm mutant strains lacking the genes encoding the

hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and

NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate

permease LctP were sensitive to killing by human blood. This increased knowledge

of how Nm responds to adaptation in blood could also be helpful to develop

diagnostic and therapeutic strategies to control the devastating disease cause

by this microorganism.


Vyšlo v časopise: Transcriptome Analysis of in Human Whole Blood and Mutagenesis Studies Identify Virulence Factors Involved in Blood Survival. PLoS Pathog 7(5): e32767. doi:10.1371/journal.ppat.1002027
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002027

Souhrn

During infection Neisseria meningitidis (Nm) encounters multiple

environments within the host, which makes rapid adaptation a crucial factor for

meningococcal survival. Despite the importance of invasion into the bloodstream

in the meningococcal disease process, little is known about how Nm adapts to

permit survival and growth in blood. To address this, we performed a time-course

transcriptome analysis using an ex vivo model of human whole

blood infection. We observed that Nm alters the expression of ≈30% of

ORFs of the genome and major dynamic changes were observed in the expression of

transcriptional regulators, transport and binding proteins, energy metabolism,

and surface-exposed virulence factors. In particular, we found that the gene

encoding the regulator Fur, as well as all genes encoding iron uptake systems,

were significantly up-regulated. Analysis of regulated genes encoding for

surface-exposed proteins involved in Nm pathogenesis allowed us to better

understand mechanisms used to circumvent host defenses. During blood infection,

Nm activates genes encoding for the factor H binding proteins, fHbp and NspA,

genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as

several less characterized surface-exposed proteins that might have a role in

blood survival. Through mutagenesis studies of a subset of up-regulated genes we

were able to identify new proteins important for survival in human blood and

also to identify additional roles of previously known virulence factors in

aiding survival in blood. Nm mutant strains lacking the genes encoding the

hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and

NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate

permease LctP were sensitive to killing by human blood. This increased knowledge

of how Nm responds to adaptation in blood could also be helpful to develop

diagnostic and therapeutic strategies to control the devastating disease cause

by this microorganism.


Zdroje

1. VirjiM

2009

Pathogenic neisseriae: surface modulation, pathogenesis and

infection control.

Nat Rev Microbiol

7

274

286

2. StephensDSGreenwoodBBrandtzaegP

2007

Epidemic meningitis, meningococcaemia, and Neisseria

meningitidis.

Lancet

369

2196

2210

3. BentleySDVernikosGSSnyderLAChurcherCArrowsmithC

2007

Meningococcal genetic variation mechanisms viewed through

comparative analysis of serogroup C strain FAM18.

PLoS Genet

3

e23

4. ParkhillJAchtmanMJamesKDBentleySDChurcherC

2000

Complete DNA sequence of a serogroup A strain of Neisseria

meningitidis Z2491.

Nature

404

502

506

5. PengJYangLYangFYangJYanY

2008

Characterization of ST-4821 complex, a unique Neisseria

meningitidis clone.

Genomics

91

78

87

6. SchoenCTettelinHParkhillJFroschM

2009

Genome flexibility in Neisseria meningitidis.

Vaccine

27

Suppl 2

B103

111

7. TettelinHSaundersNJHeidelbergJJeffriesACNelsonKE

2000

Complete genome sequence of Neisseria meningitidis serogroup B

strain MC58.

Science

287

1809

1815

8. ClausHVogelUSwiderekHFroschMSchoenC

2007

Microarray analyses of meningococcal genome composition and gene

regulation: a review of the recent literature.

FEMS Microbiol Rev

31

43

51

9. SunYHBakshiSChalmersRTangCM

2000

Functional genomics of Neisseria meningitidis

pathogenesis.

Nat Med

6

1269

1273

10. HellerudBCStenvikJEspevikTLambrisJDMollnesTE

2008

Stages of meningococcal sepsis simulated in vitro, with emphasis

on complement and Toll-like receptor activation.

Infect Immun

76

4183

4189

11. IsonCAHeydermanRSKleinNJPeakmanMLevinM

1995

Whole blood model of meningococcal bacteraemia–a method for

exploring host-bacterial interactions.

Microb Pathog

18

97

107

12. NolteORickertAEhrhardILedigSSonntagHG

2002

A modified ex vivo human whole blood model of infection for

studying the pathogenesis of Neisseria meningitidis during

septicemia.

FEMS Immunol Med Microbiol

32

91

95

13. SprongTBrandtzaegPFungMPharoAMHoibyEA

2003

Inhibition of C5a-induced inflammation with preserved

C5b-9-mediated bactericidal activity in a human whole blood model of

meningococcal sepsis.

Blood

102

3702

3710

14. WelschJAGranoffD

2007

Immunity to Neisseria meningitidis group B in adults despite lack

of serum bactericidal antibody.

Clin Vaccine Immunol

14

1596

1602

15. FradinCKretschmarMNichterleinTGaillardinCd'EnfertC

2003

Stage-specific gene expression of Candida albicans in human

blood.

Mol Microbiol

47

1523

1543

16. GrahamMRVirtanevaKPorcellaSFBarryWTGowenBB

2005

Group A Streptococcus transcriptome dynamics during growth in

human blood reveals bacterial adaptive and survival

strategies.

Am J Pathol

166

455

465

17. MereghettiLSitkiewiczIGreenNMMusserJM

2008

Extensive adaptive changes occur in the transcriptome of

Streptococcus agalactiae (group B streptococcus) in response to incubation

with human blood.

PLoS ONE

3

e3143

18. Toledo-AranaADussurgetONikitasGSestoNGuet-RevilletH

2009

The Listeria transcriptional landscape from saprophytism to

virulence.

Nature

459

950

956

19. IsonC

2001

Whole-Blood Model.

Pollard AJaMMCJ

Meningococcal Vaccines: Methods and Protocols

Totowa

Human Press Inc

317

329

20. FantappieLMetruccioMMSeibKLOrienteFCartocciE

2009

The RNA chaperone Hfq is involved in stress response and

virulence in Neisseria meningitidis and is a pleiotropic regulator of

protein expression.

Infect Immun

77

1842

1853

21. SeibKLSerrutoDOrienteFDelanyIAdu-BobieJ

2009

Factor H-binding protein is important for meningococcal survival

in human whole blood and serum and in the presence of the antimicrobial

peptide LL-37.

Infect Immun

77

292

299

22. DartonTGuiverMNaylorSJackDLKaczmarskiEB

2009

Severity of meningococcal disease associated with genomic

bacterial load.

Clin Infect Dis

48

587

594

23. HackettSJGuiverMMarshJSillsJAThomsonAP

2002

Meningococcal bacterial DNA load at presentation correlates with

disease severity.

Arch Dis Child

86

44

46

24. OvsteboRBrandtzaegPBruslettoBHaugKBLandeK

2004

Use of robotized DNA isolation and real-time PCR to quantify and

identify close correlation between levels of Neisseria meningitidis DNA and

lipopolysaccharides in plasma and cerebrospinal fluid from patients with

systemic meningococcal disease.

J Clin Microbiol

42

2980

2987

25. GarzoniCFrancoisPHuygheACouzinetSTapparelC

2007

A global view of Staphylococcus aureus whole genome expression

upon internalization in human epithelial cells.

BMC Genomics

8

171

26. MaurerAPMehlitzAMollenkopfHJMeyerTF

2007

Gene expression profiles of Chlamydophila pneumoniae during the

developmental cycle and iron depletion-mediated persistence.

PLoS Pathog

3

e83

27. OrihuelaCJRadinJNSublettJEGaoGKaushalD

2004

Microarray analysis of pneumococcal gene expression during

invasive disease.

Infect Immun

72

5582

5596

28. FrancoisPGarzoniCBentoMSchrenzelJ

2007

Comparison of amplification methods for transcriptomic analyses

of low abundance prokaryotic RNA sources.

J Microbiol Methods

68

385

391

29. YeungKYHaynorDRRuzzoWL

2001

Validating clustering for gene expression data.

Bioinformatics

17

309

318

30. HaftDHLoftusBJRichardsonDLYangFEisenJA

2001

TIGRFAMs: a protein family resource for the functional

identification of proteins.

Nucleic Acids Res

29

41

43

31. GrifantiniRBartoliniEMuzziADraghiMFrigimelicaE

2002

Previously unrecognized vaccine candidates against group B

meningococcus identified by DNA microarrays.

Nat Biotechnol

20

914

921

32. FrigimelicaEBartoliniEGalliGGrandiGGrifantiniR

2008

Identification of 2 Hypothetical Genes Involved in Neisseria

meningitidis Cathelicidin Resistance.

J Infect Dis

197

1124

1132

33. DelanyIGrifantiniRBartoliniERappuoliRScarlatoV

2006

Effect of Neisseria meningitidis fur mutations on global control

of gene transcription.

J Bacteriol

188

2483

2492

34. GrifantiniRSebastianSFrigimelicaEDraghiMBartoliniE

2003

Identification of iron-activated and -repressed Fur-dependent

genes by transcriptome analysis of Neisseria meningitidis group

B.

Proc Natl Acad Sci U S A

100

9542

9547

35. PapenfortKVogelJ

2010

Regulatory RNA in bacterial pathogens.

Cell Host Microbe

8

116

127

36. BartoliniEFrigimelicaEGiovinazziSGalliGShaikY

2006

Role of FNR and FNR-regulated, sugar fermentation genes in

Neisseria meningitidis infection.

Mol Microbiol

60

963

972

37. HouseholderTCBelliWALissendenSColeJAClarkVL

1999

cis- and trans-acting elements involved in regulation of aniA,

the gene encoding the major anaerobically induced outer membrane protein in

Neisseria gonorrhoeae.

J Bacteriol

181

541

551

38. NewcombeJEales-ReynoldsLJWoottonLGorringeARFunnellSG

2004

Infection with an avirulent phoP mutant of Neisseria meningitidis

confers broad cross-reactive immunity.

Infect Immun

72

338

344

39. TzengYLZhouXBaoSZhaoSNobleC

2006

Autoregulation of the MisR/MisS two-component signal transduction

system in Neisseria meningitidis.

J Bacteriol

188

5055

5065

40. TzengYLDattaAAmbroseKLoMDaviesJK

2004

The MisR/MisS two-component regulatory system influences inner

core structure and immunotype of lipooligosaccharide in Neisseria

meningitidis.

J Biol Chem

279

35053

35062

41. JametARousseauCMonfortJBFrapyENassifX

2009

A two-component system is required for colonization of host cells

by meningococcus.

Microbiology

155

2288

2295

42. OshimaTAibaHMasudaYKanayaSSugiuraM

2002

Transcriptome analysis of all two-component regulatory system

mutants of Escherichia coli K-12.

Mol Microbiol

46

281

291

43. Janiak-SpensFSparlingDPWestAH

2000

Novel role for an HPt domain in stabilizing the phosphorylated

state of a response regulator domain.

J Bacteriol

182

6673

6678

44. LarsonJAHigashiDLStojiljkovicISoM

2002

Replication of Neisseria meningitidis within epithelial cells

requires TonB-dependent acquisition of host cell iron.

Infect Immun

70

1461

1467

45. Perkins-BaldingDRatliff-GriffinMStojiljkovicI

2004

Iron transport systems in Neisseria meningitidis.

Microbiol Mol Biol Rev

68

154

171

46. StojiljkovicIHwaVde Saint MartinLO'GaoraPNassifX

1995

The Neisseria meningitidis haemoglobin receptor: its role in iron

utilization and virulence.

Mol Microbiol

15

531

541

47. LeightonMPKellyDJWilliamsonMPShawJG

2001

An NMR and enzyme study of the carbon metabolism of Neisseria

meningitidis.

Microbiology

147

1473

1482

48. ExleyRMGoodwinLMoweEShawJSmithH

2005

Neisseria meningitidis lactate permease is required for

nasopharyngeal colonization.

Infect Immun

73

5762

5766

49. LeeEHShaferWM

1999

The farAB-encoded efflux pump mediates resistance of gonococci to

long-chained antibacterial fatty acids.

Mol Microbiol

33

839

845

50. HotoppJCGrifantiniRKumarNTzengYLFoutsD

2006

Comparative genomics of Neisseria meningitidis: core genome,

islands of horizontal transfer and pathogen-specific genes.

Microbiology

152

3733

3749

51. ColicchioRRicciSLambertiFPagliaruloCPagliucaC

2009

The meningococcal ABC-Type L-glutamate transporter GltT is

necessary for the development of experimental meningitis in

mice.

Infect Immun

77

3578

3587

52. PagliaruloCSalvatorePDe VitisLRColicchioRMonacoC

2004

Regulation and differential expression of gdhA encoding

NADP-specific glutamate dehydrogenase in Neisseria meningitidis clinical

isolates.

Mol Microbiol

51

1757

1772

53. LoHTangCMExleyRM

2009

Mechanisms of avoidance of host immunity by Neisseria

meningitidis and its effect on vaccine development.

Lancet Infect Dis

9

418

427

54. TurnerDPMarietouAGJohnstonLHoKKRogersAJ

2006

Characterization of MspA, an immunogenic autotransporter protein

that mediates adhesion to epithelial and endothelial cells in Neisseria

meningitidis.

Infect Immun

74

2957

2964

55. van UlsenPAdlerBFasslerPGilbertMvan SchilfgaardeM

2006

A novel phase-variable autotransporter serine protease, AusI, of

Neisseria meningitidis.

Microbes Infect

8

2088

2097

56. ParuchuriDKSeifertHSAjiokaRSKarlssonKASoM

1990

Identification and characterization of a Neisseria gonorrhoeae

gene encoding a glycolipid-binding adhesin.

Proc Natl Acad Sci U S A

87

333

337

57. CapecchiBAdu-BobieJDi MarcelloFCiucchiLMasignaniV

2005

Neisseria meningitidis NadA is a new invasin which promotes

bacterial adhesion to and penetration into human epithelial

cells.

Mol Microbiol

55

687

698

58. ScarselliMSerrutoDMontanariPCapecchiBAdu-BobieJ

2006

Neisseria meningitidis NhhA is a multifunctional trimeric

autotransporter adhesin.

Mol Microbiol

61

631

644

59. SerrutoDAdu-BobieJScarselliMVeggiDPizzaM

2003

Neisseria meningitidis App, a new adhesin with autocatalytic

serine protease activity.

Mol Microbiol

48

323

334

60. McNeilGVirjiMMoxonER

1994

Interactions of Neisseria meningitidis with human

monocytes.

Microb Pathog

16

153

163

61. AnjumMFStevaninTMReadRCMoirJW

2002

Nitric oxide metabolism in Neisseria

meningitidis.

J Bacteriol

184

2987

2993

62. SeibKLTsengHJMcEwanAGApicellaMAJenningsMP

2004

Defenses against oxidative stress in Neisseria gonorrhoeae and

Neisseria meningitidis: distinctive systems for different

lifestyles.

J Infect Dis

190

136

147

63. DunnKLFarrantJLLangfordPRKrollJS

2003

Bacterial [Cu,Zn]-cofactored superoxide dismutase

protects opsonized, encapsulated Neisseria meningitidis from phagocytosis by

human monocytes/macrophages.

Infect Immun

71

1604

1607

64. CardinaleJAClarkVL

2000

Expression of AniA, the major anaerobically induced outer

membrane protein of Neisseria gonorrhoeae, provides protection against

killing by normal human sera.

Infect Immun

68

4368

4369

65. LeuzziRSerinoLScarselliMSavinoSFontanaMR

2005

Ng-MIP, a surface-exposed lipoprotein of Neisseria gonorrhoeae,

has a peptidyl-prolyl cis/trans isomerase (PPIase) activity and is involved

in persistence in macrophages.

Mol Microbiol

58

669

681

66. SchneiderMCExleyRMRamSSimRBTangCM

2007

Interactions between Neisseria meningitidis and the complement

system.

Trends Microbiol

15

233

240

67. JarvaHRamSVogelUBlomAMMeriS

2005

Binding of the complement inhibitor C4bp to serogroup B Neisseria

meningitidis.

J Immunol

174

6299

6307

68. MadicoGNgampasutadolJGulatiSVogelURicePA

2007

Factor H binding and function in sialylated pathogenic neisseriae

is influenced by gonococcal, but not meningococcal, porin.

J Immunol

178

4489

4497

69. OrienteFScarlatoVDelanyI

2010

Expression of factor H binding protein of meningococcus responds

to oxygen limitation through a dedicated FNR-regulated

promoter.

J Bacteriol

192

691

701

70. LewisLANgampasutadolJWallaceRReidJEVogelU

2010

The meningococcal vaccine candidate neisserial surface protein A

(NspA) binds to factor H and enhances meningococcal resistance to

complement.

PLoS Pathog

6

e1001027

71. van UlsenPvan AlphenLten HoveJFransenFvan der LeyP

2003

A Neisserial autotransporter NalP modulating the processing of

other autotransporters.

Mol Microbiol

50

1017

1030

72. Ait-TaharKWooldridgeKGTurnerDPAttaMToddI

2000

Auto-transporter A protein of Neisseria meningitidis: a potent

CD4+ T-cell and B-cell stimulating antigen detected by expression

cloning.

Mol Microbiol

37

1094

1105

73. PeiserLMakepeaceKPluddemannASavinoSWrightJC

2006

Identification of Neisseria meningitidis nonlipopolysaccharide

ligands for class A macrophage scavenger receptor by using a novel

assay.

Infect Immun

74

5191

5199

74. PluddemannAHoeJCMakepeaceKMoxonERGordonS

2009

The macrophage scavenger receptor A is host-protective in

experimental meningococcal septicaemia.

PLoS Pathog

5

e1000297

75. StohlEACrissAKSeifertHS

2005

The transcriptome response of Neisseria gonorrhoeae to hydrogen

peroxide reveals genes with previously uncharacterized roles in oxidative

damage protection.

Mol Microbiol

58

520

532

76. TidharAFlashnerYCohenSLeviYZaubermanA

2009

The NlpD lipoprotein is a novel Yersinia pestis virulence factor

essential for the development of plague.

PLoS ONE

4

e7023

77. FeaversIMPizzaM

2009

Meningococcal protein antigens and vaccines.

Vaccine

27

Suppl 2

B42

50

78. GiulianiMMAdu-BobieJComanducciMAricoBSavinoS

2006

A universal vaccine for serogroup B

meningococcus.

Proc Natl Acad Sci U S A

103

10834

10839

79. PizzaMScarlatoVMasignaniVGiulianiMMAricoB

2000

Identification of vaccine candidates against serogroup B

meningococcus by whole-genome sequencing.

Science

287

1816

1820

80. MetruccioMMPigozziERoncaratiDBerlanda ScorzaFNoraisN

2009

A novel phase variation mechanism in the meningococcus driven by

a ligand-responsive repressor and differential spacing of distal promoter

elements.

PLoS Pathog

5

e1000710

81. SeibKLOrienteFAdu-BobieJMontanariPFerliccaF

2010

Influence of serogroup B meningococcal vaccine antigens on growth

and survival of the meningococcus in vitro and in ex vivo and in vivo models

of infection.

Vaccine

28

2416

2427

82. MuzziAMasignaniVRappuoliR

2007

The pan-genome: towards a knowledge-based discovery of novel

targets for vaccines and antibacterials.

Drug Discov Today

12

429

439

83. YuNYWagnerJRLairdMRMelliGReyS

2010

PSORTb 3.0: improved protein subcellular localization prediction

with refined localization subcategories and predictive capabilities for all

prokaryotes.

Bioinformatics

26

1608

1615

84. ReySAcabMGardyJLLairdMRdeFaysK

2005

PSORTdb: a protein subcellular localization database for

bacteria.

Nucleic Acids Res

33

D164

168

85. ExleyRMShawJMoweESunYHWestNP

2005

Available carbon source influences the resistance of Neisseria

meningitidis against complement.

J Exp Med

201

1637

1645

86. CarpenterBMWhitmireJMMerrellDS

2009

This is not your mother's repressor: the complex role of fur

in pathogenesis.

Infect Immun

77

2590

2601

87. JosephBSchneiker-BekelSSchramm-GluckABlomJClausH

2010

Comparative genome biology of a serogroup B carriage and disease

strain supports a polygenic nature of meningococcal

virulence.

J Bacteriol

192

5363

5377

88. HughesTRMaoMJonesARBurchardJMartonMJ

2001

Expression profiling using microarrays fabricated by an ink-jet

oligonucleotide synthesizer.

Nat Biotechnol

19

342

347

89. CharbonnierYGettlerBFrancoisPBentoMRenzoniA

2005

A generic approach for the design of whole-genome oligoarrays,

validated for genomotyping, deletion mapping and gene expression analysis on

Staphylococcus aureus.

BMC Genomics

6

95

90. SaalLHTroeinCVallon-ChristerssonJGruvbergerSBorgA

2002

BioArray Software Environment (BASE): a platform for

comprehensive management and analysis of microarray data.

Genome Biol

3

SOFTWARE0003

91. StoreyJDTibshiraniR

2003

Statistical significance for genomewide studies.

Proc Natl Acad Sci U S A

100

9440

9445

92. AryeeMJGutierrez-PabelloJAKramnikIMaitiTQuackenbushJ

2009

An improved empirical bayes approach to estimating differential

gene expression in microarray time-course data: BETR (Bayesian Estimation of

Temporal Regulation).

BMC Bioinformatics

10

409

93. GiancarloRScaturroDUtroF

2008

Computational cluster validation for microarray data analysis:

experimental assessment of Clest, Consensus Clustering, Figure of Merit, Gap

Statistics and Model Explorer.

BMC Bioinformatics

9

462

94. SaeedAISharovVWhiteJLiJLiangW

2003

TM4: a free, open-source system for microarray data management

and analysis.

Biotechniques

34

374

378

95. IevaRAlaimoCDelanyISpohnGRappuoliR

2005

CrgA is an inducible LysR-type regulator of Neisseria

meningitidis, acting both as a repressor and as an activator of gene

transcription.

J Bacteriol

187

3421

3430

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2011 Číslo 5
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
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

#ADS_BOTTOM_SCRIPTS#