A Biofilm-Induced Pathway for Matrix Glucan Delivery: Implications for Drug Resistance

English version České info

Extracellular polysaccharides are key constituents of the biofilm matrix of many microorganisms. One critical carbohydrate component of Candida albicans biofilms, β-1,3 glucan, has been linked to biofilm protection from antifungal agents. In this study, we identify three glucan modification enzymes that function to deliver glucan from the cell to the extracellular matrix. These enzymes include two predicted glucan transferases and an exo-glucanase, encoded by BGL2, PHR1, and XOG1, respectively. We show that the enzymes are crucial for both delivery of β-1,3 glucan to the biofilm matrix and for accumulation of mature matrix biomass. The enzymes do not appear to impact cell wall glucan content of biofilm cells, nor are they necessary for filamentation or biofilm formation. We demonstrate that mutants lacking these genes exhibit enhanced susceptibility to the commonly used antifungal, fluconazole, during biofilm growth only. Transcriptional analysis and biofilm phenotypes of strains with multiple mutations suggest that these enzymes act in a complementary fashion to distribute matrix downstream of the primary β-1,3 glucan synthase encoded by FKS1. Furthermore, our observations suggest that this matrix delivery pathway works independently from the C. albicans ZAP1 matrix formation regulatory pathway. These glucan modification enzymes appear to play a biofilm-specific role in mediating the delivery and organization of mature biofilm matrix. We propose that the discovery of inhibitors for these enzymes would provide promising anti-biofilm therapeutics.

Vyšlo v časopise: A Biofilm-Induced Pathway for Matrix Glucan Delivery: Implications for Drug Resistance. PLoS Pathog 8(8): e32767. doi:10.1371/journal.ppat.1002848
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002848

Souhrn

Zdroje

1. PfallerMA, DiekemaDJ (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20: 133–163.

2. PappasPG, KauffmanCA, AndesD, BenjaminDKJr, CalandraTF, et al. (2009) Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis 48: 503–535.

3. KojicEM, DarouicheRO (2004) Candida infections of medical devices. Clin Microbiol Rev 17: 255–267.

4. DonlanRM, CostertonJW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15: 167–193.

5. DouglasLJ (2003) Candida biofilms and their role in infection. Trends Microbiol 11: 30–36.

6. ChandraJ, KuhnDM, MukherjeePK, HoyerLL, McCormickT, et al. (2001) Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol 183: 5385–5394.

7. RamageG, SavilleSP, ThomasDP, Lopez-RibotJL (2005) Candida biofilms: an update. Eukaryot Cell 4: 633–638.

8. RamageG, BachmannS, PattersonTF, WickesBL, Lopez-RibotJL (2002) Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms. J Antimicrob Chemother 49: 973–980.

9. MukherjeePK, ChandraJ, KuhnDM, GhannoumMA (2003) Mechanism of fluconazole resistance in Candida albicans biofilms: phase-specific role of efflux pumps and membrane sterols. Infect Immun 71: 4333–4340.

10. LaFleurMD, KumamotoCA, LewisK (2006) Candida albicans biofilms produce antifungal-tolerant persister cells. Antimicrob Agents Chemother 50: 3839–3846.

11. BaillieGS, DouglasLJ (2000) Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents. J Antimicrob Chemother 46: 397–403.

12. BegunJ, GaianiJM, RohdeH, MackD, CalderwoodSB, et al. (2007) Staphylococcal biofilm exopolysaccharide protects against Caenorhabditis elegans immune defenses. PLoS Pathog 3: e57.

13. Al-FattaniMA, DouglasLJ (2006) Biofilm matrix of Candida albicans and Candida tropicalis: chemical composition and role in drug resistance. J Med Microbiol 55: 999–1008.

14. NettJE, CrawfordK, MarchilloK, AndesDR (2010) Role of Fks1p and matrix glucan in Candida albicans biofilm resistance to an echinocandin, pyrimidine, and polyene. Antimicrob Agents Chemother 54: 3505–3508.

15. HawserSP, BaillieGS, DouglasLJ (1998) Production of extracellular matrix by Candida albicans biofilms. J Med Microbiol 47: 253–256.

16. NettJ, LincolnL, MarchilloK, MasseyR, HoloydaK, et al. (2007) Putative role of beta-1,3 glucans in Candida albicans biofilm resistance. Antimicrob Agents Chemother 51: 510–520.

17. NettJE, SanchezH, CainMT, AndesDR Genetic basis of Candida biofilm resistance due to drug-sequestering matrix glucan. J Infect Dis 202: 171–175.

18. NettJE, LepakAJ, MarchilloK, AndesDR (2009) Time course global gene expression analysis of an in vivo Candida biofilm. J Infect Dis 200: 307–313.

19. ChaffinWL (2008) Candida albicans cell wall proteins. Microbiol Mol Biol Rev 72: 495–544.

20. ChaffinWL, Lopez-RibotJL, CasanovaM, GozalboD, MartinezJP (1998) Cell wall and secreted proteins of Candida albicans: identification, function, and expression. Microbiol Mol Biol Rev 62: 130–180.

21. LesageG, BusseyH (2006) Cell wall assembly in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 70: 317–343.

22. GonzalezMM, Diez-OrejasR, MoleroG, AlvarezAM, PlaJ, et al. (1997) Phenotypic characterization of a Candida albicans strain deficient in its major exoglucanase. Microbiology 143 (Pt 9) 3023–3032.

23. ChambersRS, BroughtonMJ, CannonRD, CarneA, EmersonGW, et al. (1993) An exo-beta-(1,3)-glucanase of Candida albicans: purification of the enzyme and molecular cloning of the gene. J Gen Microbiol 139: 325–334.

24. MouynaI, FontaineT, VaiM, MonodM, FonziWA, et al. (2000) Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. J Biol Chem 275: 14882–14889.

25. RagniE, CalderonJ, FascioU, SipiczkiM, FonziWA, et al. (2011) Phr1p, a glycosylphosphatidylinsitol-anchored beta(1,3)-glucanosyltransferase critical for hyphal wall formation, localizes to the apical growth sites and septa in Candida albicans. Fungal Genet Biol 48: 793–805.

26. GoldmanRC, SullivanPA, ZakulaD, CapobiancoJO (1995) Kinetics of beta-1,3 glucan interaction at the donor and acceptor sites of the fungal glucosyltransferase encoded by the BGL2 gene. Eur J Biochem 227: 372–378.

27. SarthyAV, McGonigalT, CoenM, FrostDJ, MeulbroekJA, et al. (1997) Phenotype in Candida albicans of a disruption of the BGL2 gene encoding a 1,3-beta-glucosyltransferase. Microbiology 143 (Pt 2) 367–376.

28. CalderonJ, ZavrelM, RagniE, FonziWA, RuppS, et al. PHR1, a pH-regulated gene of Candida albicans encoding a glucan-remodelling enzyme, is required for adhesion and invasion. Microbiology 156: 2484–2494.

29. FonziWA (1999) PHR1 and PHR2 of Candida albicans encode putative glycosidases required for proper cross-linking of beta-1,3- and beta-1,6-glucans. J Bacteriol 181: 7070–7079.

30. NettJE, SanchezH, CainMT, RossKM, AndesDR (2011) Interface of Candida albicans Biofilm Matrix-Associated Drug Resistance and Cell Wall Integrity Regulation. Eukaryot Cell 10: 1660–1669.

31. NobileCJ, NettJE, HerndayAD, HomannOR, DeneaultJS, et al. (2009) Biofilm matrix regulation by Candida albicans Zap1. PLoS Biol 7: e1000133.

32. NettJE, CrawfordK, MarchilloK, AndesDR (2010) Role of Fks1p and matrix glucan in Candida albicans biofilm resistance to an echinocandin, pyrimidine, and polyene. Antimicrob Agents Chemother 54: 3505–3508.

33. RobbinsN, UppuluriP, NettJ, RajendranR, RamageG, et al. (2011) Hsp90 governs dispersion and drug resistance of fungal biofilms. PLoS Pathog 7: e1002257.

34. AndesD, NettJ, OschelP, AlbrechtR, MarchilloK, et al. (2004) Development and characterization of an in vivo central venous catheter Candida albicans biofilm model. Infect Immun 72: 6023–6031.

35. Saporito-IrwinSM, BirseCE, SypherdPS, FonziWA (1995) PHR1, a pH-regulated gene of Candida albicans, is required for morphogenesis. Mol Cell Biol 15: 601–613.

36. UppuluriP, NettJ, HeitmanJ, AndesD (2008) Synergistic effect of calcineurin inhibitors and fluconazole against Candida albicans biofilms. Antimicrob Agents Chemother 52: 1127–1132.

37. NoriceCT, SmithFJJr, SolisN, FillerSG, MitchellAP (2007) Requirement for C. albicans Sun41 in Biofilm Formation and Virulence. Eukaryot Cell 6: 2046–55.

38. PopoloL, VaiM (1998) Defects in assembly of the extracellular matrix are responsible for altered morphogenesis of a Candida albicans phr1 mutant. J Bacteriol 180: 163–166.

39. DouglasCM, D'IppolitoJA, SheiGJ, MeinzM, OnishiJ, et al. (1997) Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors. Antimicrob Agents Chemother 41: 2471–2479.

40. NobileCJ, AndesDR, NettJE, SmithFJ, YueF, et al. (2006) Critical role of Bcr1-dependent adhesins in C. albicans biofilm formation in vitro and in vivo. PLoS Pathog 2: e63.

41. NobileCJ, SchneiderHA, NettJE, SheppardDC, FillerSG, et al. (2008) Complementary adhesin function in C. albicans biofilm formation. Curr Biol 18: 1017–1024.

42. FlemmingHC, WingenderJ (2010) The biofilm matrix. Nat Rev Microbiol 8: 623–633.

43. SadovskayaI, VinogradovE, LiJ, HachaniA, KowalskaK, et al. (2010) High-level antibiotic resistance in Pseudomonas aeruginosa biofilm: the ndvB gene is involved in the production of highly glycerol-phosphorylated beta-(1->3)-glucans, which bind aminoglycosides. Glycobiology 20: 895–904.

44. ColvinKM, GordonVD, MurakamiK, BorleeBR, WozniakDJ, et al. The pel polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. PLoS Pathog 7: e1001264.

45. DouglasCM (2001) Fungal beta(1,3)-D-glucan synthesis. Med Mycol 39 Suppl 1: 55–66.

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

47. BowenWH, KooH Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Res 45: 69–86.

48. EnloeB, DiamondA, MitchellAP (2000) A single-transformation gene function test in diploid Candida albicans. J Bacteriol 182: 5730–5736.

49. WilsonRB, DavisD, MitchellAP (1999) Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol 181: 1868–1874.

50. NobleSM, JohnsonAD (2005) Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans. Eukaryot Cell 4: 298–309.

51. NettJE, SanchezH, CainMT, AndesDR (2010) Genetic basis of Candida biofilm resistance due to drug-sequestering matrix glucan. J Infect Dis 202: 171–175.

52. NobileCJ, SolisN, MyersCL, FayAJ, DeneaultJS, et al. (2008) Candida albicans transcription factor Rim101 mediates pathogenic interactions through cell wall functions. Cell Microbiol 10: 2180–2196.

53. BassoLRJr, BartissA, MaoY, GastCE, CoelhoPS, et al. (2010) Transformation of Candida albicans with a synthetic hygromycin B resistance gene. Yeast 27: 1039–1048.

54. LivakKJ, SchmittgenTD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408.

55. TaffHT, NettJE, AndesDR (2011) Comparative analysis of Candida biofilm quantitation assays. Med Mycol 50: 214–8.

56. NettJE, CainMT, CrawfordK, AndesDR (2011) Optimizing a Candida biofilm microtiter plate model for measurement of antifungal susceptibility by tetrazolium salt assay. J Clin Microbiol 49: 1426–1433.

57. RamageG, Lopez-RibotJL (2005) Techniques for antifungal susceptibility testing of Candida albicans biofilms. Methods Mol Med 118: 71–79.

58. CLSI (2008) Clinical Laboratory Standards Institute. Reference method for broth microdilution antifungal susceptibility testing Document M27-A3. Wayne, PA: Clinical Laboratory Standards Institute.

59. BrunoVM, KalachikovS, SubaranR, NobileCJ, KyratsousC, et al. (2006) Control of the C. albicans cell wall damage response by transcriptional regulator Cas5. PLoS Pathog 2: e21.

60. DijkgraafGJ, BrownJL, BusseyH (1996) The KNH1 gene of Saccharomyces cerevisiae is a functional homolog of KRE9. Yeast 12: 683–692.

61. MansfieldBE, OlteanHN, OliverBG, HootSJ, LeydeSE, et al. (2010) Azole drugs are imported by facilitated diffusion in Candida albicans and other pathogenic fungi. PLoS Pathog 6: e1001126.