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 LaboratóriumČlánok vyšiel v časopise
PLOS Pathogens
2011 Číslo 5
- Parazitičtí červi v terapii Crohnovy choroby a dalších zánětlivých autoimunitních onemocnění
- Očkování proti virové hemoragické horečce Ebola experimentální vakcínou rVSVDG-ZEBOV-GP
- Koronavirus hýbe světem: Víte jak se chránit a jak postupovat v případě podezření?
Najčítanejšie v tomto čísle
- Crystal Structure and Functional Analysis of the SARS-Coronavirus RNA Cap 2′-O-Methyltransferase nsp10/nsp16 Complex
- The OXI1 Kinase Pathway Mediates -Induced Growth Promotion in Arabidopsis
- The Hexamer Structure of the Rift Valley Fever Virus Nucleoprotein Suggests a Mechanism for its Assembly into Ribonucleoprotein Complexes
- Acquisition of Human-Type Receptor Binding Specificity by New H5N1 Influenza Virus Sublineages during Their Emergence in Birds in Egypt