The opportunistic pathogen Pseudomonas aeruginosa can establish life-long chronic infections in the airways of cystic fibrosis (CF) patients. Persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. Several reports indicated that P. aeruginosa adapts by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. To validate this model of P. aeruginosa adaptation to CF airways and to identify novel genes involved in this microevolution, we designed a novel approach of positive-selection screening by PCR-based signature-tagged mutagenesis (Pos-STM) in a murine model of chronic airways infection. A systematic positive-selection scheme using sequential rounds of in vivo screenings for bacterial maintenance, as opposed to elimination, generated a list of genes whose inactivation increased the colonization and persistence in chronic airways infection. The phenotypes associated to these Pos-STM mutations reflect alterations in diverse aspects of P. aeruginosa biology which include lack of swimming and twitching motility, lack of production of the virulence factors such as pyocyanin, biofilm formation, and metabolic functions. In addition, Pos-STM mutants showed altered invasion and stimulation of immune response when tested in human respiratory epithelial cells, indicating that P. aeruginosa is prone to revise the interaction with its host during persistent lifestyle. Finally, sequence analysis of Pos-STM genes in longitudinally P. aeruginosa isolates from CF patients identified signs of patho-adaptive mutations within the genome. This novel Pos-STM approach identified bacterial functions that can have important clinical implications for the persistent lifestyle and disease progression of the airway chronic infection.
Vyšlo v časopise: Positive Signature-Tagged Mutagenesis in : Tracking Patho-Adaptive Mutations Promoting Airways Chronic Infection. PLoS Pathog 7(2): e32767. doi:10.1371/journal.ppat.1001270 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1001270
1. BragonziA
ParoniM
NonisA
CramerN
MontanariS
2009 Pseudomonas aeruginosa microevolution during cystic fibrosis lung infection establishes clones with adapted virulence. AJRCCM 180 138 145
2. CiganaC
CurcurùL
LeoneMR
IeranòT
LorèNI
2009 Pseudomonas aeruginosa exploits lipid A and muropeptides modification as a strategy to lower innate immunity during cystic fibrosis lung infection. PLoS One 4 e8439
3. SmithEE
BuckleyDG
WuZ
SaenphimmachakC
HoffmanLR
2006 Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci U S A 30 8487 8492
4. TümmlerB
2006 Clonal variations in Pseudomonas aeruginosa.
RamosJ-L
LevesqueRC
Pseudomonas: molecular biology of emerging issues, vol. 4 New York Springer 35 68
5. BragonziA
WiehlmannL
KlockgetherJ
WorlitzschD
2006 Sequence diversity of the mucABD locus in Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Microbiology 152 3261 3269
6. MahenthiralingamE
CampbellME
SpeertDP
1994 Nonmotility and phagocytic resistance of Pseudomonas aeruginosa isolates from chronically colonized patients with cystic fibrosis. Infect Immun 62 596 605
7. D'ArgenioDA
WuM
KulasekaraHD
DézielE
2007 Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients. Mol Microbiol 64 512 533
8. HoffmanL
EmersonJ
HoustonLS
BurnsJL
2009 Pseudomonas aeruginosa lasR mutants are associated with cystic fibrosis lung disease progression. J Cyst Fibros 8 66 70
9. JainM
RamirezD
SeshadriR
CullinaJF
PowersCA
2004 Type III secretion phenotypes of Pseudomonas aeruginosa strains change during infection of individuals with cystic fibrosis. J Clin Microbiol 42 5229 5237
10. OliverA
CantónR
CampoP
BaqueroF
BlázquezJ
2000 High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 288 1251 1254
11. RakhimovaE
MunderA
BredenbruchF
TümmlerB
2008 Fitness of isogenic colony morphology variants of Pseudomonas aeruginosa in murine airway infection. PLoS One 3 e1685
12. YoungD
HussellT
DouganG
2002 Chronic bacterial infections: living with unwanted guests. Nat Immunol 3 1026 1032
13. NguyenD
SinghPK
2006 Evolving stealth: genetic adaptation of Pseudomonas aeruginosa during cystic fibrosis infections. Proc Natl Acad Sci U S A 30 8305 8306
14. SokurenkoE
ChesnokovaV
DykhuizenDE
OfekI
WuXR
1998 Pathogenic adaptation of Escherichia coli by natural variation of the FimH adhesin. Proc Natl Acad Sci U S A 95 8922 8926
15. MoxonE
MurphyPA
1978 Haemophilus influenzae bacteremia and meningitis resulting from survival of a single organism. Proc Natl Acad Sci U S A 75 1534 1536
16. AkopyantsN
EatonKA
BergDE
1995 Adaptive mutation and cocolonization during Helicobacter pylori infection of gnotobiotic piglets. Infect Immun 63 116 121
17. GalperinM
KooninEV
2010 From complete genome sequence to ‘complete’ understanding? Trends Biotechnol 28 398 406
18. HenselM
SheaJE
GleesonC
JonesMD
DaltonE
1995 Simultaneous identification of bacterial virulence genes by negative selection. Science 269 400 403
19. CoulterSN
SchwanWR
NgEY
LanghorneMH
RitchieHD
1998 Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments. Mol Microbiol 30 393 404
20. HerbertMA
HayesS
DeadmanME
TangCM
HoodDW
2002 Signature Tagged Mutagenesis of Haemophilus influenzae identifies genes required for in vivo survival. Microb Pathog 33 211 23
21. BragonziA
2010 Murine models of acute and chronic lung infection with cystic fibrosis pathogens. IJMM 300 584 93
22. PotvinE
LehouxDE
Kukavica-IbruljI
RichardKL
SanschagrinF
2003 In vivo functional genomics of Pseudomonas aeruginosa for high-throughput screening of new virulence factors and antibacterial targets. Environ Microbiol 5 1294 1308
23. BragonziA
PierGB
TimpertP
UlrichM
2005 Nonmucoid Pseudomonas aeruginosa expresses alginate in the lungs of patients with cystic fibrosis and in a mouse model. J Infect Dis 192 410 419
24. ReadAF
TaylorLH
2001 The ecology of genetically diverse infections. Science 292 1099 1102
25. FeldmanM
BryanR
RajanS
SchefflerL
BrunnertS
1998 Role of flagella in pathogenesis of Pseudomonas aeruginosa pulmonary infection. Infect Immun 66 43 51
26. TangHB
DiMangoE
GambelloM
IglewskiBH
1996 Contribution of specific Pseudomonas aeruginosa virulence factors to pathogenesis of pneumonia in a neonatal mouse model of infection. Infect Immun 64 37 43
27. BohnYS
BrandesG
RakhimovaE
HoratzekS
SalunkheP
2009 Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection. Mol Microbiol 71 730 747
28. O'TooleGA
KolterR
1998 Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30 295 304
29. OchsnerUA
FiechterA
ReiserJ
1994 Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis. J Biol Chem 269 19787 19795
30. Abdel-MawgoudAM
LépineF
2010 Rhamnolipids: diversity of structures, microbial origins and roles. Appl Microbiol Biotechnol 86 1323 1336
31. LuCD
YangZ
LiW
2004 Transcriptome analysis of the ArgR regulon in Pseudomonas aeruginosa. J Bacteriol 186 3855 3861
32. HaasD
GalimandsM
GamperM
ZimmermannA
1990 Arginine network of Pseudomonas aeruginosa: specific and global controls.
SilverS
ChakrabartyA-M
IglewskiB
KaplanS
Pseudomonas: biotransformations, pathogenesis, and evolving biotechnology Washington, DC American Society for Microbiology 303 316
33. ItohY
1997 Cloning and characterization of the aru genes encoding enzymes of the catabolic arginine succinyltransferase pathway in Pseudomonas aeruginosa. J Bacteriol 179 7280 7729
34. WorlitzschD
TarranR
SchwabU
CekiciA
2002 Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J Clin Invest 109 317 325
35. Alvarez-OrtegaC
HarwoodCS
2007 Responses of Pseudomonas aeruginosa to low oxygen indicate that growth in the cystic fibrosis lung is by aerobic respiration. Mol Microbiol 65 153 165
36. MartinDW
MohrCD
2000 Invasion and intracellular survival of Burkholderia cepacia. Infection and Immunity 68 24 29
37. PhilpottDJ
BelaidD
TroubadourP
ThibergeJM
TankovicJ
2002 Reduced activation of inflammatory responses in host cells by mouse-adapted Helicobacter pylory isolates. Cell Microbiol 4 285 296
38. MonackD
MuellerA
FalkowS
2004 Persistent bacterial infections: the interface of the pathogen and the host immune system. Nat Rev Microbiol 2 747 765
39. FinlayB
1997 Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 61 136 169
40. KaufmannSHE
1993 Immunity to intracellular bacteria. Annu Rev Immunol 11 129 163
41. LamotheJ
HuynhKK
GrinsteinS
ValvanoMA
2007 Intracellular survival of Burkholderia cenocepacia in macrophages is associated with a delay in the maturation of bacter. Cell Microbiol 9 40 53
42. PierG
GroutM
ZaidiTS
OlsenJC
JohnsonLG
1996 Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections. Science 271 64 67
43. PierG
1997 Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung. Proc Natl Acad Sci U S A 94 12088 12093
44. KumarP
HenikoffS
NgPC
2009 Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 4 1073 1081
45. HollowayB
KrishnapillaiV
MorganAF
1979 Chromosomal genetics of Pseudomonas. Microbiol Rev 43 73 102
46. KwonY
RickeSC
2000 Efficient amplification of multiple transposon-flanking sequences. J Microbiol Methods 41 195 199
47. MontanariS
OliverA
SalernoP
MenaA
BertoniG
2007 Biological cost of hypermutation in Pseudomonas aeruginosa strains from patients with cystic fibrosis. Microbiology 153 1445 1454
48. PironeL
BragonziA
FarcomeniA
AuricheC
2008 Burkholderia cenocepacia strains isolated from cystic fibrosis patients are apparently more invasive and more virulent than rhizosphere strains. Environ Microbiol 10 2773 2784
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