PKC Signaling Regulates Drug Resistance of the Fungal Pathogen via Circuitry Comprised of Mkc1, Calcineurin, and Hsp90

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Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.

Vyšlo v časopise: PKC Signaling Regulates Drug Resistance of the Fungal Pathogen via Circuitry Comprised of Mkc1, Calcineurin, and Hsp90. PLoS Pathog 6(8): e32767. doi:10.1371/journal.ppat.1001069
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1001069

Souhrn

Zdroje

1. BahnYS

XueC

IdnurmA

RutherfordJC

HeitmanJ

2007 Sensing the environment: lessons from fungi. Nat Rev Microbiol 5 57 69

2. CowenLE

SteinbachWJ

2008 Stress, drugs, and evolution: the role of cellular signaling in fungal drug resistance. Eukaryot Cell 7 747 764

3. CamilliA

BasslerBL

2006 Bacterial small-molecule signaling pathways. Science 311 1113 1116

4. HoganDA

2006 Talking to themselves: autoregulation and quorum sensing in fungi. Eukaryot Cell 5 613 619

5. KellerNP

TurnerG

BennettJW

2005 Fungal secondary metabolism - from biochemistry to genomics. Nat Rev Microbiol 3 937 947

6. RileyMA

WertzJE

2002 Bacteriocins: evolution, ecology, and application. Annu Rev Microbiol 56 117 137

7. YimG

WangHH

DaviesJ

2007 Antibiotics as signalling molecules. Philos Trans R Soc Lond B Biol Sci 362 1195 1200

8. ButlerMS

BussAD

2006 Natural products–the future scaffolds for novel antibiotics? Biochem Pharmacol 71 919 929

9. LamKS

2007 New aspects of natural products in drug discovery. Trends Microbiol 15 279 289

10. EnochDA

LudlamHA

BrownNM

2006 Invasive fungal infections: a review of epidemiology and management options. J Med Microbiol 55 809 818

11. WilsonLS

ReyesCM

StolpmanM

SpeckmanJ

AllenK

2002 The direct cost and incidence of systemic fungal infections. Value Health 5 26 34

12. McNeilMM

NashSL

HajjehRA

PhelanMA

ConnLA

2001 Trends in mortality due to invasive mycotic diseases in the United States, 1980–1997. Clin Infect Dis 33 641 647

13. PfallerMA

DiekemaDJ

2007 Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20 133 163

14. ZaoutisTE

ArgonJ

ChuJ

BerlinJA

WalshTJ

2005 The epidemiology and attributable outcomes of candidemia in adults and children hospitalized in the United States: a propensity analysis. Clin Infect Dis 41 1232 1239

15. BaldaufSL

PalmerJD

1993 Animals and fungi are each other's closest relatives: congruent evidence from multiple proteins. Proc Natl Acad Sci U S A 90 11558 11562

16. WainrightPO

HinkleG

SoginML

StickelSK

1993 Monophyletic origins of the metazoa: an evolutionary link with fungi. Science 260 340 342

17. AndersonJB

SirjusinghC

ParsonsAB

BooneC

WickensC

2003 Mode of selection and experimental evolution of antifungal drug resistance in Saccharomyces cerevisiae. Genetics 163 1287 1298

18. CowenLE

2008 The evolution of fungal drug resistance: modulating the trajectory from genotype to phenotype. Nat Rev Microbiol 6 187 198

19. AndersonJB

2005 Evolution of antifungal-drug resistance: mechanisms and pathogen fitness. Nat Rev Microbiol 3 547 556

20. CowenLE

2009 Hsp90 orchestrates stress response signaling governing fungal drug resistance. PLoS Pathog 5 e1000471

21. PearlLH

ProdromouC

2006 Structure and mechanism of the Hsp90 molecular chaperone machinery. Annu Rev Biochem 75 271 294

22. PrattWB

ToftDO

2003 Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp Biol Med (Maywood) 228 111 133

23. WandingerSK

RichterK

BuchnerJ

2008 The Hsp90 chaperone machinery. J Biol Chem 283 18473 18477

24. CowenLE

CarpenterAE

MatangkasombutO

FinkGR

LindquistS

2006 Genetic architecture of Hsp90-dependent drug resistance. Eukaryot Cell 5 2184 2188

25. CowenLE

LindquistS

2005 Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309 2185 2189

26. ImaiJ

YaharaI

2000 Role of HSP90 in salt stress tolerance via stabilization and regulation of calcineurin. Mol Cell Biol 20 9262 9270

27. SinghSD

RobbinsN

ZaasAK

SchellWA

PerfectJR

2009 Hsp90 governs echinocadin resistance in the pathogenic yeast Candida albicans via calcineurin. PLoS Pathog 5 e1000532

28. FuchsBB

MylonakisE

2009 Our paths might cross: the role of the fungal cell wall integrity pathway in stress response and cross talk with other stress response pathways. Eukaryot Cell 8 1616 1625

29. HeinischJJ

2005 Baker's yeast as a tool for the development of antifungal kinase inhibitors–targeting protein kinase C and the cell integrity pathway. Biochim Biophys Acta 1754 171 182

30. LevinDE

2005 Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 69 262 291

31. ZhaoX

MehrabiR

XuJR

2007 Mitogen-activated protein kinase pathways and fungal pathogenesis. Eukaryot Cell 6 1701 1714

32. ParaviciniG

MendozaA

AntonssonB

CooperM

LosbergerC

1996 The Candida albicans PKC1 gene encodes a protein kinase C homolog necessary for cellular integrity but not dimorphism. Yeast 12 741 756

33. MongeRA

RomanE

NombelaC

PlaJ

2006 The MAP kinase signal transduction network in Candida albicans. Microbiology 152 905 912

34. BlankenshipJR

FanningS

HamakerJJ

MitchellAP

2010 An extensive circuitry for cell wall regulation in Candida albicans. PLoS Pathog 6 e1000752

35. MarkovichS

YekutielA

ShalitI

ShadkchanY

OsherovN

2004 Genomic approach to identification of mutations affecting caspofungin susceptibility in Saccharomyces cerevisiae. Antimicrob Agents Chemother 48 3871 3876

36. Reinoso-MartinC

SchullerC

Schuetzer-MuehlbauerM

KuchlerK

2003 The yeast protein kinase C cell integrity pathway mediates tolerance to the antifungal drug caspofungin through activation of Slt2p mitogen-activated protein kinase signaling. Eukaryot Cell 2 1200 1210

37. WalkerLA

MunroCA

de BruijnI

LenardonMD

McKinnonA

2008 Stimulation of chitin synthesis rescues Candida albicans from echinocandins. PLoS Pathog 4 e1000040

38. RogersB

DecottigniesA

KolaczkowskiM

CarvajalE

BalziE

2001 The pleitropic drug ABC transporters from Saccharomyces cerevisiae. J Mol Microbiol Biotechnol 3 207 214

39. WhiteTC

1997 Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 41 1482 1487

40. WhiteTC

1997 The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14alpha demethylase in Candida albicans. Antimicrob Agents Chemother 41 1488 1494

41. WhiteTC

PfallerMA

RinaldiMG

SmithJ

ReddingSW

1997 Stable azole drug resistance associated with a substrain of Candida albicans from an HIV-infected patient. Oral Dis 3 Suppl 1 S102 109

42. EppE

VanierG

HarcusD

LeeAY

JansenG

Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence. PLoS Pathog 6 e1000753

43. HerbertJM

AugereauJM

GleyeJ

MaffrandJP

1990 Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 172 993 999

44. MarxMH

PiantadosiC

NosedaA

DanielLW

ModestEJ

1988 Synthesis and evaluation of neoplastic cell growth inhibition of 1-N-alkylamide analogues of glycero-3-phosphocholine. J Med Chem 31 858 863

45. SussmanA

HussK

ChioLC

HeidlerS

ShawM

2004 Discovery of cercosporamide, a known antifungal natural product, as a selective Pkc1 kinase inhibitor through high-throughput screening. Eukaryot Cell 3 932 943

46. SugawaraF

StrobelS

StrobelG

LarsenRD

BerglundDL

1991 The structure and biological activity of cercosporamide from Cercosporidium henningsii. J Org Chem 56 909 910

47. OmuraS

IwaiY

HiranoA

NakagawaA

AwayaJ

1977 A new alkaloid AM-2282 OF Streptomyces origin. Taxonomy, fermentation, isolation and preliminary characterization. J Antibiot (Tokyo) 30 275 282

48. WatanabeM

ChenCY

LevinDE

1994 Saccharomyces cerevisiae PKC1 encodes a protein kinase C (PKC) homolog with a substrate specificity similar to that of mammalian PKC. J Biol Chem 269 16829 16836

49. WhiteTC

MarrKA

BowdenRA

1998 Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 11 382 402

50. LevinDE

FieldsFO

KunisawaR

BishopJM

ThornerJ

1990 A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle. Cell 62 213 224

51. YoshidaS

IkedaE

UnoI

MitsuzawaH

1992 Characterization of a staurosporine- and temperature-sensitive mutant, stt1, of Saccharomyces cerevisiae: STT1 is allelic to PKC1. Mol Gen Genet 231 337 344

52. Navarro-GarciaF

EismanB

FiuzaSM

NombelaC

PlaJ

2005 The MAP kinase Mkc1p is activated under different stress conditions in Candida albicans. Microbiology 151 2737 2749

53. JungUS

LevinDE

1999 Genome-wide analysis of gene expression regulated by the yeast cell wall integrity signalling pathway. Mol Microbiol 34 1049 1057

54. BaetzK

MoffatJ

HaynesJ

ChangM

AndrewsB

2001 Transcriptional coregulation by the cell integrity mitogen-activated protein kinase Slt2 and the cell cycle regulator Swi4. Mol Cell Biol 21 6515 6528

55. KamadaY

JungUS

PiotrowskiJ

LevinDE

1995 The protein kinase C-activated MAP kinase pathway of Saccharomyces cerevisiae mediates a novel aspect of the heat shock response. Genes Dev 9 1559 1571

56. BonillaM

CunninghamKW

2003 Mitogen-activated protein kinase stimulation of Ca(2+) signaling is required for survival of endoplasmic reticulum stress in yeast. Mol Biol Cell 14 4296 4305

57. BonillaM

NastaseKK

CunninghamKW

2002 Essential role of calcineurin in response to endoplasmic reticulum stress. Embo J 21 2343 2353

58. HomannOR

DeaJ

NobleSM

JohnsonAD

2009 A phenotypic profile of the Candida albicans regulatory network. PLoS Genet 5 e1000783

59. ReedyJL

FillerSG

HeitmanJ

2010 Elucidating the Candida albicans calcineurin signaling cascade controlling stress response and virulence. Fungal Genet Biol 47 107 116

60. RoyJ

LiH

HoganPG

CyertMS

2007 A conserved docking site modulates substrate affinity for calcineurin, signaling output, and in vivo function. Mol Cell 25 889 901

61. Stathopoulos-GerontidesA

GuoJJ

CyertMS

1999 Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation. Genes Dev 13 798 803

62. RobbinsN

CollinsC

MorhayimJ

CowenLE

2010 Metabolic control of antifungal drug resistance. Fungal Genet Biol 47 81 93

63. KarababaM

ValentinoE

PardiniG

CosteAT

BilleJ

2006 CRZ1, a target of the calcineurin pathway in Candida albicans. Mol Microbiol 59 1429 1451

64. MillsonSH

TrumanAW

KingV

ProdromouC

PearlLH

2005 A two-hybrid screen of the yeast proteome for Hsp90 interactors uncovers a novel Hsp90 chaperone requirement in the activity of a stress-activated mitogen-activated protein kinase, Slt2p (Mpk1p). Eukaryot Cell 4 849 860

65. ShapiroRS

UppuluriP

ZaasAK

CollinsC

SennH

2009 Hsp90 orchestrates temperature-dependent Candida albicans morphogenesis via Ras1-PKA signaling. Curr Biol 19 621 629

66. CowenLE

SinghSD

KohlerJR

CollinsC

ZaasAK

2009 Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease. Proc Natl Acad Sci U S A 106 2818 2823

67. BaderT

BodendorferB

SchroppelK

MorschhauserJ

2003 Calcineurin is essential for virulence in Candida albicans. Infect Immun 71 5344 5354

68. BlankenshipJR

HeitmanJ

2005 Calcineurin is required for Candida albicans to survive calcium stress in serum. Infect Immun 73 5767 5774

69. SanglardD

IscherF

MarchettiO

EntenzaJ

BilleJ

2003 Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence. Mol Microbiol 48 959 976

70. MunroCA

SelvagginiS

de BruijnI

WalkerL

LenardonMD

2007 The PKC, HOG and Ca2+ signalling pathways co-ordinately regulate chitin synthesis in Candida albicans. Mol Microbiol 63 1399 1413

71. KumamotoCA

2008 Molecular mechanisms of mechanosensing and their roles in fungal contact sensing. Nat Rev Microbiol 6 667 673

72. GouldCM

KannanN

TaylorSS

NewtonAC

2009 The chaperones Hsp90 and Cdc37 mediate the maturation and stabilization of protein kinase C through a conserved PXXP motif in the C-terminal tail. J Biol Chem 284 4921 4935

73. McClellanAJ

XiaY

DeutschbauerAM

DavisRW

GersteinM

2007 Diverse cellular functions of the Hsp90 molecular chaperone uncovered using systems approaches. Cell 131 121 135

74. ZhaoR

DaveyM

HsuYC

KaplanekP

TongA

2005 Navigating the chaperone network: An integrative map of physical and genetic interactions mediated by the Hsp90 chaperone. Cell 120 715 727

75. Diez-OrejasR

MoleroG

Navarro-GarciaF

PlaJ

NombelaC

1997 Reduced virulence of Candida albicans MKC1 mutants: a role for mitogen-activated protein kinase in pathogenesis. Infect Immun 65 833 837

76. AranaDM

Alonso-MongeR

DuC

CalderoneR

PlaJ

2007 Differential susceptibility of mitogen-activated protein kinase pathway mutants to oxidative-mediated killing by phagocytes in the fungal pathogen Candida albicans. Cell Microbiol 9 1647 1659

77. KumamotoCA

2005 A contact-activated kinase signals Candida albicans invasive growth and biofilm development. Proc Natl Acad Sci U S A 102 5576 5581

78. GerikKJ

BhimireddySR

RyerseJS

SpechtCA

LodgeJK

2008 PKC1 is essential for protection against both oxidative and nitrosative stresses, cell integrity, and normal manifestation of virulence factors in the pathogenic fungus Cryptococcus neoformans. Eukaryot Cell 7 1685 1698

79. GonelliA

MischiatiC

GuerriniR

VoltanR

SalvadoriS

2009 Perspectives of protein kinase C (PKC) inhibitors as anti-cancer agents. Mini Rev Med Chem 9 498 509

80. MackayHJ

TwelvesCJ

2007 Targeting the protein kinase C family: are we there yet? Nat Rev Cancer 7 554 562

81. BlankenshipJR

SteinbachWJ

PerfectJR