Azole resistance constitutes a serious clinical problem in the management of infections caused by Candida albicans. This study aimed to explore azole-resistant mechanisms in clinical C. albicans isolates collected in Jinan, Shandong, China. In total, 22 samples were collected and analyzed. Among these, four isolates (28A, 28D, 28I, and 28J) exhibited high level of pan-azole-resistance that was Hsp90 dependent. Gene sequencing revealed that the four Hsp90-dependent strains contained different ERG3 mutations that led to four novel amino acid substitutions (S265Y, N322D, N324S, and E355D) in Erg3. The role of these substitutions in azole resistance development was determined by constructing one copy of the mutated ERG3 from the 28A, 28D, and 28I strains into C. albicans CAI4, respectively. The minimum inhibitory concentration value of fluconazole (FLC) against C. albicans CAI4-ERG328I increased fourfold compared with the wild-type C. albicans strain, suggesting that the novel combination of substitutions S265Y, N322D, and N324S played an important role in mediating azole resistance in 28I. Besides, we identified several different mechanisms in other three isolates. Strains 28A and 28D displayed increased efflux ability and overexpression of MDR1. Strain 28J showed high level of ERG11 expression, but no mutation in its regulator Upc2 was observed. Our study revealed that multiple factors confer azole resistance in clinical C. albicans isolates and combination therapy should be conducted clinically.
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References
1.
DadarM., TiwariR., KarthikK., ChakrabortyS., ShahaliY., and DhamaK.. 2018. Candida albicans—biology, molecular characterization, pathogenicity, and advances in diagnosis and control—an update. Microb. Pathog. 117:128–138.
2.
PfallerM.A., MesserS.A, MoetG.J, JonesR.N, and CastanheiraM.. 2011. Candida bloodstream infections: comparison of species distribution and resistance to echinocandin and azole antifungal agents in Intensive Care Unit (ICU) and non-ICU settings in the SENTRY Antimicrobial Surveillance Program (2008-2009). Int. J. Antimicrob. Agents, 38:65–69.
3.
LiuJ., ShiC., WangY., LiW., ZhaoY., and XiangM.. 2015. Mechanisms of azole resistance in Candida albicans clinical isolates from Shanghai, China. Res. Microbiol. 166:153–161.
4.
MorioF., PagniezF., BesseM., Gay-andrieuF., MiegevilleM., and PapeP.L.. 2013. Deciphering azole resistance mechanisms with a focus on transcription factor-encoding genes TAC1, MRR1 and UPC2 in a set of fluconazole-resistant clinical isolates of Candida albicans. Int. J. Antimicrob. Agents, 42:410–415.
5.
TsaoS., RahkhoodaeeF., and RaymondM.. 2009. Relative contributions of the Candida albicans ABC transporters Cdr1p and Cdr2p to clinical azole resistance. Antimicrob. Agents Chemother. 53:1344–1352.
6.
WuY., GaoN., LiC., GaoJ., and YingC.. 2017. A newly identified amino acid substitution T123I in the 14α-demethylase (Erg11p) of Candida albicans confers azole resistance. FEMS Yeast Res. 17:fox012.
7.
XiangM., LiuJ., NiP., et al.2013. Erg11 mutations associated with azole resistance in clinical isolates of Candida albicans. FEMS Yeast Res. 13:386–393.
8.
MorioF., LogeC., BesseB., HennequinC., and PapeP.L.. 2010. Screening for amino acid substitutions in the Candida albicans Erg11 protein of azole-susceptible and azole-resistant clinical isolates: new substitutions and a review of the literature. Diagn. Microbiol. Infect. Dis. 66:373–384.
9.
MorioF., PagniezF., LacroixC., MiegevilleM., and PapeP.L.. 2012. Amino acid substitutions in the Candida albicans sterol Δ5,6-desaturase (Erg3p) confer azole resistance: characterization of two novel mutants with impaired virulence. J. Antimicrob. Chemother. 67:2131–2138.
10.
SunJ., QiC., LafleurM.D, and QiQ.G.. 2009. Fluconazole susceptibility and genotypic heterogeneity of oral Candida albicans colonies from the patients with cancer receiving chemotherapy in China. Int. J. Oral Sci. 1:56–162.
11.
YangC., GongW., LuJ., ZhuX., and QiQ.. 2010. Antifungal drug susceptibility of oral Candida albicans isolates may be associated with apoptotic responses to Amphotericin B. J. Oral Pathol. Med. 39:182–187.
12.
SikorskiR., and HieterP.. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics, 122:19–27.
13.
FonziW.A., and IrwinM.Y.. 1993. Isogenic strain construction and gene mapping in Candida albicans. Genetics, 134:717–728.
14.
Clinical and Laboratory Standards Institute. 2008. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard. 3rd ed. CLSI Document M27-A3. CLSI, Wayne, PA.
15.
Clinical and Laboratory Standards Institute. 2017. Performance Standards for Antifungal Susceptibility Testing of Yeasts. 1st ed. CLSI Supplement M60. CLSI, Wayne, PA.
16.
ZhangJ., LiuW., TanJ., SunY., WanZ., and LiR.. 2013. Antifungal activity of geldanamycin alone or in combination with fluconazole against Candida species. Mycopathologia. 175:273–279.
17.
LaFayetteS.L., CollinsC., ZaasA.K, et al.2010. PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90. PLoS Pathog. 6:e1001069.
18.
XieF., ChangW., ZhangM., et al.2016. Quinone derivatives isolated from the endolichenic fungus Phialocephala fortinii are Mdr1 modulators that combat azole resistance in Candida albicans. Sci. Rep. 6:33687.
19.
CaoY.Y., CaoY.B, XuZ., et al.2005. cDNA microarray analysis of differential gene expression in Candida. albicans biofilm exposed to farnesol. Antimicrob. Agents Chemother. 49:584–589.
20.
ChauA.S., MendrickC.A, SabatelliF.J, LoebenbergD., and McNicholasP.M.. 2004. Application of real-time quantitative PCR to molecular analysis of Candida albicans strains exhibiting reduced susceptibility to azoles. Antimicrob. Agents Chemother. 48:2124–2131.
21.
VersalovicJ., KoeuthT., and LupskiJ.R.. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19:6823–6831.
22.
ShahanaS., ChildersD.S, BallouE.R, et al.2014. New Clox Systems for rapid and efficient gene disruption in Candida albicans. PLoS One. 9:e100390.
23.
AntoniA.D., and GallwitzD.. 2000. A novel multi-purpose cassette for repeated integrative epitope tagging of genes in Saccharomyces cerevisiae. Gene. 246:179–185.
24.
YangJ., and ZhangY.. 2015. I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res. 43:W174–W181.
25.
MonkB.C., TomasiakT.M, KeniyaM.V, et al. 2014. Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer. Proc. Natl. Acad. Sci. U. S. A. 111:3865–3870.
26.
GarnaudC., García-OliverE., WangY., et al.2018. The Rim pathway mediates antifungal tolerance in Candida albicans through newly identified Rim101 transcriptional targets, including Hsp90 and Ipt1. Antimicrob. Agents Chemother. 62:e01785-17.
27.
CruzM.C., GoldsteinA.L, BlankenshipJ.R, et al.2002. Calcineurin is essential for survival during membrane stress in Candida albicans. EMBO J. 21:546–559.
28.
ZhangM.R., ZhaoF., WangS., et al.2020. Molecular mechanism of azoles resistant Candida albicans in a patient with chronic mucocutaneous candidiasis. BMC Infect. Dis. 20:126.
29.
SchubertS., BarkerK.S, ZnaidiS., et al.2011. Regulation of efflux pump expression and drug resistance by the transcription factors Mrr1, Upc2, and Cap1 in Candida albicans. Antimicrob. Agents Chemother. 55:2212–2223.
30.
DunkelN., LiuT.T, BarkerK.S, HomayouniR., MorschhäuserJ., and RogersP.D.. 2008. A Gain-of-Function mutation in the transcription factor Upc2p causes upregulation of ergosterol biosynthesis genes and increased fluconazole resistance in a clinical Candida albicans isolate. Eukaryot. Cell, 7:1180–1190.
31.
Alvarez-RuedaN., FleuryA., MorioF., PagniezF., GastinelL., and PapeP.L.. 2011. Amino acid substitutions at the major insertion loop of Candida albicans sterol 14α-demethylase are involved in fluconazole resistance. PLoS One, 6:e21239.
32.
MarichalP., KoymansL., WillemsensS., et al.1999. Contribution of mutations in the cytochrome P450 14α-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans. Microbiology, 145:2701–2713.
33.
RosanaY., YasmonA., and LestariD.C.. 2015. Overexpression and mutation as genetic mechanism of fluconazole resistance in Candida albicans isolated from human immunodeficiency virus patients in Indonesia. J. Med. Microbiol. 64:1046–1052.
34.
LiX., BrownN., ChauA.S, et al.2004. Changes in susceptibility to posaconazole in clinical isolates of Candida albicans. J. Antimicrob. Chemother. 53:74–80.
35.
GuoL.N., YuS.Y, XiaoM., et al. 2020. Species distribution and antifungal susceptibility of invasive Candidiasis: a 2016–2017 multicenter surveillance study in Beijing, China. Infect. Drug Resist. 13:2443–2452.
36.
CowenL.E., SinghS.D, KöhlerJ.R, et al. 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.
37.
LiL., AnM., ShenH., et al.2015. The non-Geldanamycin Hsp90 inhibitors enhanced the antifungal activity of fluconazole. Am. J. Transl. Res. 7:2589–2602.
38.
O'MearaT.R., RobbinsN., and CowenL.E.. 2017. The Hsp90 chaperone network modulates Candida virulence traits. Trends Microbiol. 25:809–819.
39.
Cowen, L.E. 2008. The evolution of fungal drug resistance: modulating the trajectory from genotype to phenotype. Nat. Rev. Microbiol. 6:187–198.
40.
CowenL.E., and LindquistS.. 2005. Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science, 309:2185–2189.
41.
MartelC.M., ParkerJ.E, BaderO., et al.2010. Identification and characterization of four azole-resistant erg3 mutants of Candida albicans. Antimicrob. Agents Chemother. 54:4527–4533.
42.
McTaggartL.R., CabreraA., CroninK., and KusJ.V.. 2020. Antifungal susceptibility of clinical yeast isolates from a large Canadian Reference Laboratory and application of whole-genome sequence analysis to elucidate mechanisms of acquired resistance. Antimicrob. Agents Chemother. 64:e00402-20.
43.
Luna-TapiaA., ButtsA., and PalmerG.E.. 2018. Loss of C-5 sterol desaturase activity in Candida albicans: azole resistance or merely trailing growth? Antimicrob. Agents Chemother. 63:e01337-18.
44.
Ramírez-ZavalaB., MogaveroS., SchöllerE., SasseC., RogersP.D, and MorschhäuserJ.. 2014. SAGA/ADA complex subunit Ada2 is required for Cap1- but not Mrr1-mediated upregulation of the Candida albicans multidrug efflux pump MDR1. Antimicrob. Agents Chemother. 58:5102–5110.
45.
FengW., YangJ., YangL., et al.2018. Research of Mrr1, Cap1 and MDR1 in Candida albicans resistant to azole medications. Exp. Ther. Med. 15:1217–1224.
46.
TapiaC.V., HermosillaG., FortesP., et al.2017. Genotyping and persistence of Candida albicans from pregnant women with vulvovaginal candidiasis. Mycopathologia, 182:339–347.
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