UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of

English version České info

Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFNγ and TNFα levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens.

Vyšlo v časopise: UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of. PLoS Pathog 7(11): e32767. doi:10.1371/journal.ppat.1002384
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002384

Souhrn

Zdroje

1. BastidasRJHeitmanJ 2009 Trimorphic stepping stones pave the way to fungal virulence. Proc Natl Acad Sci U S A 106 351 352

2. WhitewayMBachewichC 2007 Morphogenesis in Candida albicans. Annu Rev Microbiol 61 529 553

3. ErnstJF 2000 Transcription factors in Candida albicans environmental control of morphogenesis. Microbiol 146 1763 1774

4. DhillonNKSharmaSKhullerGK 2003 Signaling through protein kinases and transcriptional regulators in Candida albicans. Crit Rev Microbiol 29 259 275

5. CarlislePLBanerjeeMLazzellAMonteagudoCLo'pez-RibotJL 2009 Expression levels of a filament-specific transcriptional regulator are sufficient to determine Candida albicans morphology and virulence. Proc Natl Acad Sci U S A 106 599 604

6. LiuH 2002 Co-regulation of pathogenesis with dimorphism and phenotypic switching in Candida albicans, a commensal and a pathogen. Int J Med Microbiol 292 299 311

7. García-SánchezSMavorALRussellCLArgimonSDennisonP 2005 Global roles of Ssn6 in Tup1- and Nrg1-dependent gene regulation in the fungal pathogen, Candida albicans. Mol Biol Cell 16 2913 2925

8. EckertSEShethCCMuhlschlegelFA 2007 Regulation of Morphogenesis in Candida albicans. EnfertCHubeB Candida comparative and functional genomics 263 292

9. ShibataNSuzukiAKobayashHOkawaY 2007 Chemical structure of the cell wall mannan of Candida albicans serotype A and its difference in yeast and hyphal forms. Biochem J 404 365 372

10. RoperJRGütherMLMilneKGFergusonMAJ 2002 Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei. Proc Natl Acad Sci USA 99 5884 5889

11. JinYSamaranayakeLPSamaranayakeYYipHK 2004 Biofilm formation of Candida albicans is variably affected by saliva and dietary sugars. Arch Oral Biol 49 789 798

12. BaillieGSDouglasLJ 2000 Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents. J Antimicrob Chemotherpy 46 397 403

13. HoldenHMRaymentIThodenJB 2003 Structure and function of enzymes of the Leloir pathway for galactose metabolism. J Biol Chem 278 43885 43888

14. BhatPJMurthyTV 2001 Transcriptional control of the GAL/MEL regulon of yeast Saccharomyces cerevisiae: mechanism of galactose mediated signal transduction. Mol Microbiol 40 1059 1066

15. BarberCRostiJRawatAFindlayKRobertsK 2006 Distinct properties of the five UDP-D-glucose/UDP- D -galactose 4-epimerase isoforms of Arabidopsis thaliana. J Biol Chem 281 17276 17285

16. SeibothBKaraVaLSandorEKubicekC 2002 The Hypocrea jecorina gal10 (uridine 50-diphosphate-glucose 4-epimerase-encoding) gene differs from yeast homologs in structure, genomic organization and expression. Gene 295 143 149

17. SinghVSomisettyVMysoreLRSadhalePP 2007 The key enzyme in galactose metabolism, UDPgalactose-4-epimerase, affects cell-wall integrity and morphology in Candida albicans even in the absence of galactose. Fungal Genet Biol 44 563 574

18. WubbenMJ2ndRodermelSRBaumTJ 2004 Mutation of a UDP-glucose-4-epimerase alters nematode susceptibility and ethylene responses in Arabidopsis roots. Plant J 40 712 724

19. QuimbyBBAlanoAAlmashanuSDeSandroAMCowanTM 1997 Characterization of two mutations associated with epimerase- deficiency galactosemia, by use of a yeast expression system for human UDP galactose-4-epimerase. Am J Hum Genet 61 590 598

20. MoyrandFLafontaineIFontaineTJanbonG 2008 UGE1 and UGE2 regulate the UDP-Glucose/UDP-Galactose equilibrium in Cryptococcus neoformans. Eukaryotic Cell 7 2069 2077

21. PoulainDJouaultT 2004 Candida albicans cell wall glycans, host receptors and responses: elements for a decisive crosstalk. Curr Opin Microbial 7 342 349

22. NeteaMGGowNAMunroCABatesSCollinsC 2006 Immune sensing of Candida albicans requires cooperative recognition of glucans and mannans by lectin and Toll-like receptors. J Clin Invest 116 1642 1650

23. ThodenJBHoldenHM 2005 The molecular architecture of galactose mutarotase/UDP-galactose-4-epimerase from Saccharomyces cerevisiae. J Biol Chem 280 21900 21907

24. GolaSMartinRWaltherADünklerAWendlandJ 2003 New modules for PCR-based gene targeting in Candida albicans: rapid and efficient gene targeting using 100 bp of flanking homology region. Yeast 20 1339 1347

25. MaYSternRJSchermanMSVissaVDYanW 2001 Drug targeting Mycobacterium tuberculosis cell wall synthesis: genetics of dTDP-Rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dTDP-Glucose to dTDP-Rhamnose. Antimicrob Agents Chemother 45 1407 1416

26. OkaTNemotoTJigamiY 2007 Functional analysis of Arabidopsis thaliana RHM2/MUM4 a multidomain protein involved in UDP-D glucose to UDP-L-rhamnose conversion. J Biol Chem 282 5389 5403

27. MarcilAHarcusDThomasDYWhitewayM 2002 Candida albicans killing by RAW 264.7 mouse macrophage cells: effects of Candida genotype, infection ratios, and gamma interferon treatment. Infect Immun 70 6319 6329

28. BlankenshipJRMitchellAP 2006 How to build a biofilm, a fungal perspective. Curr Opin Microbial 9 588 594

29. SanchezAAJohnstonDAMyersCEdwardsJEMitchellAP 2004 Relationship between Candida albicans virulence during experimental hematogenously disseminated infection and endothelial cell damage In Vitro. Infect Immun 72 598 601

30. ChengSNguyenMHZhangZJiaHHandfieldM 2003 Evaluation of the roles of four Candida albicans genes in virulence by using gene disruption strains that express URA3 from the native locus. Infect Immun 71 6101 6103

31. LeunkRDMoonRJ 1979 Physiological and metabolic alterations accompanying systemic candidiasis in mice. Infect Immun 26 1035 1041

32. KretschmarMHubeBBertschTSanglardDMerkerR 1999 Germ tubes and proteinase activity contribute to virulence of Candida albicans in murine peritonitis. Infect Immun 67 6637 6642

33. DietALinkBSeifertGJSchellenbergBWagnerU 2006 The Arabidopsis root hair cell wall formation mutant lrx1 is suppressed by mutations in the RHM1 gene encoding a UDP-L-rhamnose synthase. Plant Cell 18 1630 1641

34. TorosantucciAChianiPCassoneA 2000 Differential chemokine response of human monocytes to yeast and fungal forms of Candida albicans and its relation to the beta-1,6 glucan of the fungal cell wall. J Leuko Biol 68 923 932

35. BatesSHughesHBMunroCAThomasWPHMacCallumDM 2006 Outer chain N-glycans are required for cell wall integrity and virulence of Candida albicans. J Biol Chem 281 90 98

36. MunroCABatesSBuurmanETHughesHBMacCallumDM 2005 Mnt1p and Mnt2p of Candida albicans are partially redundant alpha-1,2-mannosyltransferases that participate in O-linked mannosylation and are required for adhesion and virulence. J Biol Chem 280 1051 1060

37. BatesSMacCallumDMBertramGMunroCAHughesHB 2005 Candida albicans Pmr1p, a secretory pathway P-type Ca2+/Mn2+-ATPase, is required for glycosylation and virulence. J Biol Chem 280 23408 23415

38. HobsonRPMunroCABatesSMacCallumDMCutlerJE 2004 Loss of cell wall mannosyl phosphate in Candida albicans does not influence macrophage recognition. J Biol Chem 279 39628 39635

39. KruppaMGreeneRRNossILowmanDWWilliamsDL 2011 C. albicans increases cell wall mannoprotein, but not mannan, in response to blood, serum and cultivation at physiological temperature. Glycobiol 21 1173 1180

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

41. LouieABaltchALSmithRPFrankeMARitzWJ 1994 Tumor necrosis factor alpha has a protective role in a murine model of systemic candidiasis. Infect Immun 62 2761 2772

42. KáposztaRTreePMaródiLGordonS 1998 Characteristics of invasive candidiasis in gamma interferon- and interleukin-4-deficient mice: role of macrophages in host defense against Candida albicans. Infect Immun 66 1708 1717

43. LavigneLMSchopfLRChungCLMaylorRSypekJP 1998 The role of recombinant murine IL-12 and IFN-gamma in the pathogenesis of a murine systemic Candida albicans infection. J Immunol 160 284 292

44. d'OstianiCFDel SeroGBacciAMontagnoliCSprecaA 2000 Dendritic cells discriminate between yeasts and hyphae of the fungus Candida albicans. Implications for initiation of T helper cell immunity in vitro and in vivo. J Exp Med 191 1661 1674

45. MencacciADel SeroGCenciEd'Ostiani CFBacciA 1998 Endogenous interleukin 4 is required for development of protective CD4+ T helper type 1 cell responses to Candida albicans. J Exp Med 187 307 317

46. SahaBNandiD 2009 Farnesyl transferase inhibitors reduce ras activation and ameliorate acetaminophen-induced liver injury in mice. Hepatology 50 1547 1557

47. SahaBPrasannaSJChandrasekarBNandiD 2010 Gene modulation and immunoregulatory roles of Interferon gamma. Cytokine 50 1 14

48. AllardSTMBeisKGiraudM-FHegemanADGroMssJW 2002 Toward a structural understanding of the dehydratase mechanism. Structure 10 81 92

49. Moreno-RuizEOrtuGde GrootPWJCottierFLoussertC 2009 The GPI-modified proteins Pga59 and Pga62 of Candida albicans are required for cell wall integrity. Microbiol 155 2004 2020

50. RichardMIbata-OmbettaSDromerFBordon-PallierFJouaultT 2002 Complete glycosylphosphatidylinositol anchors are required in Candida albicans for full morphogenesis, virulence and resistance to macrophages. Mol Microbiol 44 841 853

51. NobleSMFrenchSKohnLAChenVJohnsonAD 2010 Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity. Nat Genetics 42 590 598

52. SlotJCRokasA 2010 Multiple GAL pathway gene clusters evolved independently and by different mechanisms in fungi. Proc Natl Acad Sci USA 107 10136 10141

53. WaltherAWendlandJ 2003 An improved transformation protocol for the human fungal pathogen Candida albicans. Curr Genet 42 339 343

54. ShermanFFinkGRHicksJ 1986 Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor, NY Cold Spring Harbor Laboratory

55. ReuBOVikAKolterRMorschhauserJ 2004 The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. Gene 341 119 127

56. VijayaraghavanUCompanyMAbelsonJ 1989 Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae. Genes Dev 3 1206 1216

57. SamaranayakeYHYeJYauJYCheungBPSamaranayakeLP 2005 In vitro method to study antifungal perfusion in Candida biofilms. J Clin Microbiol 43 818 825

58. GillumAMTsayEYKirschDR 1984 Isolation of the Candida albicans gene for orotidine-5-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet 198 179 182

59. FonziWAIrwinMY 1993 Isogenic Strain Construction and Gene Mapping in Candida albicans. Genetics 134 717 728

60. MehtaDVKabirABhatPJ 1999 Expression of human inositol monophosphatase suppresses galactose toxicity in Saccharomyces cerevisiae: possible implications in galactosemia. Biochim Biophys Acta 1454 217 226

61. MumbergDMullerRFunkM 1995 Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156 119 22