YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in
During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.
Vyšlo v časopise:
YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in. PLoS Pathog 6(3): e32767. doi:10.1371/journal.ppat.1000804
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.ppat.1000804
Souhrn
During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.
Zdroje
1. GovanDVJR
1996 Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 60 539 574
2. 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 103 8487 8492
3. HausslerS
ZieglerI
LottelA
von GotzF
RohdeM
2003 Highly adherent small-colony variants of Pseudomonas aeruginosa in cystic fibrosis lung infection. J Med Microbiol 52 295 301
4. KirisitsMJ
ProstL
StarkeyM
ParsekMR
2005 Characterization of colony morphology variants isolated from Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 71 4809 4821
5. HausslerS
TummlerB
WeissbrodtH
RohdeM
SteinmetzI
1999 Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis. Clin Infect Dis 29 621 625
6. ReinhardtA
KohlerT
WoodP
RohnerP
DumasJL
2007 Development and persistence of antimicrobial resistance in Pseudomonas aeruginosa: a longitudinal observation in mechanically ventilated patients. Antimicrob Agents Chemother 51 1341 1350
7. HausslerS
2004 Biofilm formation by the small colony variant phenotype of Pseudomonas aeruginosa. Environ Microbiol 6 546 551
8. HausslerS
LehmannC
BreselgeC
RohdeM
ClassenM
2003 Fatal outcome of lung transplantation in cystic fibrosis patients due to small-colony variants of the Burkholderia cepacia complex. Eur J Clin Microbiol Infect Dis 22 249 253
9. D'ArgenioDA
CalfeeMW
RaineyPB
PesciEC
2002 Autolysis and autoaggregation in Pseudomonas aeruginosa colony morphology mutants. J Bacteriol 184 6481 6489
10. DrenkardE
AusubelFM
2002 Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416 740 743
11. MeissnerA
WildV
SimmR
RohdeM
ErckC
2007 Pseudomonas aeruginosa cupA-encoded fimbriae expression is regulated by a GGDEF and EAL domain-dependent modulation of the intracellular level of cyclic diguanylate. Environ Microbiol 9 2475 2485
12. HickmanJW
TifreaDF
HarwoodCS
2005 A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels. Proc Natl Acad Sci U S A 102 14422 14427
13. StarkeyM
HickmanJH
MaL
ZhangN
De LongS
2009 Pseudomonas aeruginosa rugose small colony variants have adaptations likely to promote persistence in the cystic fibrosis lung. J Bacteriol 191 3492 3503
14. JenalU
2004 Cyclic di-guanosine-monophosphate comes of age: a novel secondary messenger involved in modulating cell surface structures in bacteria? Curr Opin Microbiol 7 185 191
15. GalperinMY
2005 A census of membrane-bound and intracellular signal transduction proteins in bacteria: bacterial IQ, extroverts and introverts. BMC Microbiol 5 35
16. RossP
WeinhouseH
AloniY
MichaeliD
Weinberger-OhanaP
1987 Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325 279 281
17. SchmidtAJ
RyjenkovDA
GomelskyM
2005 The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase: enzymatically active and inactive EAL domains. J Bacteriol 187 4774 4781
18. PaulR
WeiserS
AmiotNC
ChanC
SchirmerT
2004 Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain. Genes Dev 18 715 727
19. ChristenM
ChristenB
FolcherM
SchauerteA
JenalU
2005 Identification and Characterization of a Cyclic di-GMP-specific Phosphodiesterase and Its Allosteric Control by GTP. J Biol Chem 280 30829 30837
20. RyjenkovDA
TarutinaM
MoskvinOV
GomelskyM
2005 Cyclic Diguanylate Is a Ubiquitous Signaling Molecule in Bacteria: Insights into Biochemistry of the GGDEF Protein Domain. J Bacteriol 187 1792 1798
21. ChristenB
ChristenM
PaulR
SchmidF
FolcherM
2006 Allosteric control of cyclic di-GMP signaling. J Biol Chem 281 32015 32024
22. GjermansenM
RagasP
SternbergC
MolinS
Tolker-NielsenT
2005 Characterization of starvation-induced dispersion in Pseudomonas putida biofilms. Environ Microbiol 7 894 906
23. SpiersAJ
BohannonJ
GehrigSM
RaineyPB
2003 Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. Mol Microbiol 50 15 27
24. SpiersAJ
KahnSG
BohannonJ
TravisanoM
RaineyPB
2002 Adaptive Divergence in Experimental Populations of Pseudomonas fluorescens. I. Genetic and Phenotypic Bases of Wrinkly Spreader Fitness. Genetics 161 33 46
25. LeeVT
MatewishJM
KesslerJL
HyodoM
HayakawaY
LoryS
2007 A cyclic-di-GMP receptor required for bacterial exopolysaccharide production. Mol Microbiol 65 1474 1484
26. KulasekaraHD
VentreI
KulasekaraBR
LazdunskiA
FillouxA
2005 A novel two-component system controls the expression of Pseudomonas aeruginosa fimbrial cup genes. Mol Microbiol 55 368 380
27. KazmierczakBI
LebronMB
MurrayTS
2006 Analysis of FimX, a phosphodiesterase that governs twitching motility in Pseudomonas aeruginosa. Mol Microbiol 60 1026 1043
28. SimmR
MorrM
KaderA
NimtzM
RomlingU
2004 GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol Microbiol 53 1123 1134
29. KulasakaraH
LeeV
BrencicA
LiberatiN
UrbachJ
2006 Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence. Proc Natl Acad Sci U S A 103 2839 2844
30. TischlerAD
CamilliA
2005 Cyclic diguanylate regulates Vibrio cholerae virulence gene expression. Infect Immun 73 5873 5882
31. MerighiM
LeeVT
HyodoM
HayakawaY
LoryS
2007 The second messenger bis-(3′-5′)-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa. Mol Microbiol 65 876 895
32. HickmanJW
HarwoodCS
2008 Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol Microbiol 69 376 389
33. KuchmaSL
ConnollyJP
O'TooleGA
2005 A three-component regulatory system regulates biofilm maturation and type III secretion in Pseudomonas aeruginosa. J Bacteriol 187 1441 1454
34. HuangB
WhitchurchCB
MattickJS
2003 FimX, a multidomain protein connecting environmental signals to twitching motility in Pseudomonas aeruginosa. J Bacteriol 185 7068 7076
35. AlmRA
BoderoAJ
FreePD
MattickJS
1996 Identification of a novel gene, pilZ, essential for type 4 fimbrial biogenesis in Pseudomonas aeruginosa. J Bacteriol 178 46 53
36. MerrittJH
BrothersKM
KuchmaSL
O'TooleGA
2007 SadC Reciprocally Influences Biofilm Formation and Swarming Motility via Modulation of Exopolysaccharide Production and Flagellar Function. J Bacteriol 189 8154 8164
37. BantinakiE
KassenR
KnightCG
RobinsonZ
SpiersAJ
2007 Adaptive divergence in experimental populations of Pseudomonas fluorescens. III. Mutational origins of wrinkly spreader diversity. Genetics 176 441 453
38. GuvenerZT
HarwoodCS
2007 Subcellular location characteristics of the Pseudomonas aeruginosa GGDEF protein, WspR, indicate that it produces cyclic-di-GMP in response to growth on surfaces. Mol Microbiol 66 1459 1473
39. GallagherLA
ManoilC
2001 Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning. J Bacteriol 183 6207 6214
40. CaiazzaNC
O'TooleGA
2004 SadB is required for the transition from reversible to irreversible attachment during biofilm formation by Pseudomonas aeruginosa PA14. J Bacteriol 186 4476 4485
41. CaiazzaNC
MerrittJH
BrothersKM
O'TooleGA
2007 Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14. J Bacteriol 189 3603 3612
42. KuchmaSL
BrothersKM
MerrittJH
LiberatiNT
AusubelFM
2007 BifA, a Cyclic-Di-GMP Phosphodiesterase, Inversely Regulates Biofilm Formation and Swarming Motility by Pseudomonas aeruginosa PA14. J Bacteriol 189 8165 8178
43. HoffmanLR
D'ArgenioDA
MacCossMJ
ZhangZ
JonesRA
2005 Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 436 1171 1175
44. GotohH
ZhangY
DalloSF
HongS
KasaraneniN
2008 Pseudomonas aeruginosa, under DNA replication inhibition, tends to form biofilms via Arr. Res Microbiol 159 294 302
45. KlebensbergerJ
LautenschlagerK
BresslerD
WingenderJ
PhilippB
2007 Detergent-induced cell aggregation in subpopulations of Pseudomonas aeruginosa as a preadaptive survival strategy. Environ Microbiol 9 2247 2259
46. MillerJH
1972 Experiments in molecular genetics. 352 355 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
47. KovachME
ElzerPH
HillDS
RobertsonGT
FarrisMA
1995 Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166 175 176
48. GuzmanLM
BelinD
CarsonMJ
BeckwithJ
1995 Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177 4121 4130
49. ChoiKH
GaynorJB
WhiteKG
LopezC
BosioCM
2005 A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 2 443 448
50. HoSN
HuntHD
HortonRM
PullenJK
PeaseLR
1989 Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77 51 59
51. HeebS
ItohY
NishijyoT
SchniderU
KeelC
2000 Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria. Mol Plant Microbe Interact 13 232 237
52. AldridgeP
PaulR
GoymerP
RaineyP
JenalU
2003 Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus. Mol Microbiol 47 1695 1708
53. RobertsRC
MohrCD
ShapiroL
1996 Developmental programs in bacteria. Curr Top Dev Biol 34 207 257
54. YuD
EllisHM
LeeEC
JenkinsNA
CopelandNG
2000 An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97 5978 5983
55. UzzauS
Figueroa-BossiN
RubinoS
BossiL
2001 Epitope tagging of chromosomal genes in Salmonella. Proc Natl Acad Sci U S A 98 15264 15269
56. KarimovaG
UllmannA
LadantD
2001 Protein-protein interaction between Bacillus stearothermophilus tyrosyl-tRNA synthetase subdomains revealed by a bacterial two-hybrid system. J Mol Microbiol Biotechnol 3 73 82
57. WinsonMK
SwiftS
HillPJ
SimsCM
GriesmayrG
1998 Engineering the luxCDABE genes from Photorhabdus luminescens to provide a bioluminescent reporter for constitutive and promoter probe plasmids and mini-Tn5 constructs. FEMS Microbiol Lett 163 193 202
58. ValletI
DiggleSP
StaceyRE
CamaraM
VentreI
2004 Biofilm formation in Pseudomonas aeruginosa: fimbrial cup gene clusters are controlled by the transcriptional regulator MvaT. J Bacteriol 186 2880 2890
59. VentreI
GoodmanAL
Vallet-GelyI
VasseurP
SosciaC
2006 Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes. Proc Natl Acad Sci U S A 103 171 176
60. MorganAF
1979 Transduction of Pseudomonas aeruginosa with a mutant of bacteriophage E79. J Bacteriol 139 137 140
61. O'TooleGA
KolterR
1998 Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol 28 449 461
62. ChoiKH
SchweizerHP
2005 An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants. BMC Microbiol 5 30
63. MerrittJH
KadouriDE
O'TooleGA
2005 Growing and analyzing static biofilms. Curr Protoc Microbiol Chapter 1: Unit 1B 1
64. RabinowitzJD
KimballE
2007 Acidic acetonitrile for cellular metabolome extraction from Escherichia coli. Anal Chem 79 6167 6173
65. MichaelisS
InouyeH
OliverD
BeckwithJ
1983 Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli. J Bacteriol 154 366 374
66. KarimovaG
PidouxJ
UllmannA
LadantD
1998 A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A 95 5752 5756
67. WagnerT
SoongG
SokolS
SaimanL
PrinceA
2005 Effects of azithromycin on clinical isolates of Pseudomonas aeruginosa from cystic fibrosis patients. Chest 128 912 919
68. KristianSA
BirkenstockTA
SauderU
MackD
GotzF
2008 Biofilm formation induces C3a release and protects Staphylococcus epidermidis from IgG and complement deposition and from neutrophil-dependent killing. J Infect Dis 197 1028 1035
69. ParsonsLM
LinF
OrbanJ
2006 Peptidoglycan recognition by Pal, an outer membrane lipoprotein. Biochemistry 45 2122 2128
70. BendtsenJD
NielsenH
von HeijneG
BrunakS
2004 Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340 783 795
71. SchultzJ
MilpetzF
BorkP
PontingCP
1998 SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci U S A 95 5857 5864
72. SodingJ
BiegertA
LupasAN
2005 The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res 33 W244 248
73. DuerigA
AbelS
FolcherM
NicollierM
SchwedeT
2009 Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression. Genes Dev 23 93 104
74. PaulR
AbelS
WassmannP
BeckA
HeerklotzH
2007 Activation of the diguanylate cyclase PleD by phosphorylation-mediated dimerization. J Biol Chem 282 29170 29177
75. WassmannP
ChanC
PaulR
BeckA
HeerklotzH
2007 Structure of BeF3- -modified response regulator PleD: implications for diguanylate cyclase activation, catalysis, and feedback inhibition. Structure 15 915 927
76. DeN
PirruccelloM
KrastevaPV
BaeN
RaghavanRV
2008 Phosphorylation-independent regulation of the diguanylate cyclase WspR. PLoS Biol 6 e67 doi:10.1371/journal.pbio.0060067
77. FriedmanL
KolterR
2004 Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix. J Bacteriol 186 4457 4465
78. LeidJG
WillsonCJ
ShirtliffME
HassettDJ
ParsekMR
2005 The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing. J Immunol 175 7512 7518
79. ConwayBA
ChuKK
BylundJ
AltmanE
SpeertDP
2004 Production of exopolysaccharide by Burkholderia cenocepacia results in altered cell-surface interactions and altered bacterial clearance in mice. J Infect Dis 190 957 966
80. GodlewskaR
WisniewskaK
PietrasZ
Jagusztyn-KrynickaEK
2009 Peptidoglycan-associated lipoprotein (Pal) of Gram-negative bacteria: function, structure, role in pathogenesis and potential application in immunoprophylaxis. FEMS Microbiol Lett 298 1 11
81. UedaA
WoodTK
2009 Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885). PLoS Pathog 5 e1000483 doi:10.1371/journal.ppat.1000483
82. BurrowesE
BaysseC
AdamsC
O'GaraF
2006 Influence of the regulatory protein RsmA on cellular functions in Pseudomonas aeruginosa PAO1, as revealed by transcriptome analysis. Microbiology 152 405 418
83. TschowriN
BusseS
HenggeR
2009 The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli. Genes Dev 23 522 534
84. MaL
ConoverM
LuH
ParsekMR
BaylesK
2009 Assembly and development of the Pseudomonas aeruginosa biofilm matrix. PLoS Pathog 5 e1000354 doi:10.1371/journal.ppat.1000354
85. CannonGJ
SwansonJA
1992 The macrophage capacity for phagocytosis. J Cell Sci 101 (Pt 4) 907 913
86. SpeertDP
WrightSD
SilversteinSC
MahB
1988 Functional characterization of macrophage receptors for in vitro phagocytosis of unopsonized Pseudomonas aeruginosa. J Clin Invest 82 872 879
87. GilbertP
DasJ
FoleyI
1997 Biofilm susceptibility to antimicrobials. Adv Dent Res 11 160 167
88. GordonCA
HodgesNA
MarriottC
1988 Antibiotic interaction and diffusion through alginate and exopolysaccharide of cystic fibrosis-derived Pseudomonas aeruginosa. J Antimicrob Chemother 22 667 674
89. SpoeringAL
LewisK
2001 Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J Bacteriol 183 6746 6751
90. GilbertP
CollierPJ
BrownMR
1990 Influence of growth rate on susceptibility to antimicrobial agents: biofilms, cell cycle, dormancy, and stringent response. Antimicrob Agents Chemother 34 1865 1868
91. GirgisHS
LiuY
RyuWS
TavazoieS
2007 A comprehensive genetic characterization of bacterial motility. PLoS Genet 3 e154 doi:10.1371/journal.pgen.0030154
92. GiddensSR
JacksonRW
MoonCD
JacobsMA
ZhangXX
2007 Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment. Proc Natl Acad Sci U S A 104 18247 18252
Štítky
Hygiena a epidemiológia Infekčné lekárstvo LaboratóriumČlánok vyšiel v časopise
PLOS Pathogens
2010 Číslo 3
- Očkování proti virové hemoragické horečce Ebola experimentální vakcínou rVSVDG-ZEBOV-GP
- Parazitičtí červi v terapii Crohnovy choroby a dalších zánětlivých autoimunitních onemocnění
- Koronavirus hýbe světem: Víte jak se chránit a jak postupovat v případě podezření?
Najčítanejšie v tomto čísle
- Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein Binds and Protects a Nuclear Noncoding RNA from Cellular RNA Decay Pathways
- Endocytosis of the Anthrax Toxin Is Mediated by Clathrin, Actin and Unconventional Adaptors
- Perforin and IL-2 Upregulation Define Qualitative Differences among Highly Functional Virus-Specific Human CD8 T Cells
- Inhibition of Macrophage Migration Inhibitory Factor Ameliorates Ocular -Induced Keratitis