Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance

In the paper

“Depletion of HSP90 led to the upregulation of transcripts predicted to be involved in transmembrane transport and downregulation of genes annotated as involved in translation and peptide metabolic processes (Fig. S6 and Table S2).”

In the paper

“In total, 28 transcripts were upregulated and 17 transcripts were downregulated upon Hsp90 perturbation in both organisms (Fig. 6C).”

In the paper

“In C. albicans, filamentation caused by perturbations in Hsp90 was associated with upregulation of filament-associated genes such as HWP1, ECE1, and RBT1, consistent with previous reports (21, 41).”

What the sources say

• Not checked yetcites [21] Shapiro RS, Sellam A, Tebbji F, Whiteway M, Nantel A, Cowen LE 2012 Pho85, Pcl1, and Hms1 signaling governs Candida albicans morphogenesis induced by high temperature or Hsp90 compromise. Curr Biol 22: 461– 470. doi: 10.1016/j.cub.2012.01.062. 22365851 source
• Not checked yetcites [41] Braun BR, Head WS, Wang MX, Johnson AD 2000 Identification and characterization of TUP1-regulated genes in Candida albicans. Genetics 156: 31– 44. 10978273 PMC1461230 source

In the paper

“GO term enrichment analysis of the DEGs in response to Hsp90 inhibition identified upregulation of transcripts involved in ribosomal processes and downregulation of transcripts involved in metabolic and biosynthetic processes (Fig. S6 and Table S2).”

In the paper

“Of these, 107 transcripts showed an overlap under the two conditions, including known regulators of filamentation HWP1, ECE1, RBT1, BRG1, UME6, EFH1, and BCR1, which were upregulated, and YWP1, which was downregulated (Fig. 6A; see also Table S1).”

In the paper

“Although geldanamycin potentiated fluconazole activity against C. albicans, the combination had no effect on azole susceptibility in C. auris (Fig. 1C).”

In the paper

“As CDC-382 and CDC-387 do not contain any known Erg11 substitutions associated with azole resistance and show relatively low levels of CDR1 expression, these two isolates may reflect a C. auris state prior to fluconazole exposure and acquisition of mutations associated with resistance (6).”

In the paper

“GO slim mapping of the DEGs identified 79 and 33 transcripts involved in filamentous growth upon Hsp90 inhibition and depletion, respectively, but none of these showed an overlap with the known regulators of C. albicans filamentation identified above (Fig. 6B; see also Table S3).”

In the paper

“Here, we showed that the impairment of either Hsp90 function or cell cycle progression also leads to filamentous growth in this pathogen.”

In the paper

“Interestingly, all species classified within the “CUG” clade showed filamentous growth in response to geldanamycin to various degrees, while the non-CUG clade species remained exclusively in yeast form (Fig. 5).”

In the paper

“Surprisingly, depletion of HSP90 showed a enrichment profile distinct from that observed upon pharmacological inhibition with Hsp90 in C. albicans, for which transcripts involved in responses to oxidative stress were upregulated and transcripts involved in ribosomal and mitochondrial processes were downregulated (see Fig. S5 and Table S2).”

In the paper

“Notably, geldanamycin did not affect the high-level fluconazole resistance phenotypes of strain Ci6684 or strain CDC-388 or the fungistatic activity of fluconazole against these strains (Fig. 2B).”

In the paper

“Azole resistance mechanisms have been studied most extensively in C. albicans and include alteration or overexpression of the target, activation or increased expression of azole efflux mediators, and induction of cellular stress responses (7).”

What the sources say

• Not checked yetcites [7] Robbins N, Caplan T, Cowen LE 2017 Molecular evolution of antifungal drug resistance. Annu Rev Microbiol 71: 753– 775. doi: 10.1146/annurev-micro-030117-020345. 28886681 source

In the paper

“The most widely deployed class of antifungal is the azoles, which inhibit fungal growth by targeting lanosterol 14-α-demethylase (Erg11), a key component of the pathway for biosynthesis of the membrane sterol ergosterol (7).”

What the sources say

• Not checked yetcites [7] Robbins N, Caplan T, Cowen LE 2017 Molecular evolution of antifungal drug resistance. Annu Rev Microbiol 71: 753– 775. doi: 10.1146/annurev-micro-030117-020345. 28886681 source

In the paper

“Specifically, in C. albicans, the molecular chaperone Hsp90 promotes the evolution of drug resistance by stabilizing regulators of cellular responses to drug-induced stress (8).”

What the sources say

• Not checked yetcites [8] Cowen LE, Lindquist S 2005 Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309: 2185– 2189. doi: 10.1126/science.1118370. 16195452 source

In the paper

“Hsp90 represses filamentous growth in C. albicans such that compromising Hsp90 function induces the yeast-to-filament transition by relieving repression on Ras1-protein kinase A (PKA) signaling (19).”

What the sources say

• Not checked yetcites [19] Shapiro RS, Uppuluri P, Zaas AK, Collins C, Senn H, Perfect JR, Heitman J, Cowen LE 2009 Hsp90 orchestrates temperature-dependent Candida albicans morphogenesis via Ras1-PKA signaling. Curr Biol 19: 621– 629. doi: 10.1016/j.cub.2009.03.017. 19327993 PMC2735497 source

In the paper

“Inhibition of Hsp90 blocks calcineurin-dependent stress responses and cell wall integrity signaling, thereby reducing antifungal tolerance of clinical isolates and transforming azole activity from fungistatic to fungicidal (8–11).”

What the sources say

• Not checked yetcites [8] Cowen LE, Lindquist S 2005 Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309: 2185– 2189. doi: 10.1126/science.1118370. 16195452 source
• Not checked yetcites [9] LaFayette SL, Collins C, Zaas AK, Schell WA, Betancourt-Quiroz M, Gunatilaka AAL, Perfect JR, Cowen LE 2010 PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90. PLoS Pathog 6: e1001069. doi: 10.1371/journal.ppat.1001069. 20865172 PMC2928802 source
• Not checked yetcites [10] Singh SD, Robbins N, Zaas AK, Schell WA, Perfect JR, Cowen LE 2009 Hsp90 governs echinocandin resistance in the pathogenic yeast Candida albicans via calcineurin. PLoS Pathog 5: e1000532. doi: 10.1371/journal.ppat.1000532. 19649312 PMC2712069 source
• Not checked yetcites [11] Caplan T, Polvi EJ, Xie JL, Buckhalter S, Leach MD, Robbins N, Cowen LE 2018 Functional genomic screening reveals core modulators of echinocandin stress responses in Candida albicans. Cell Rep 23: 2292– 2298. doi: 10.1016/j.celrep.2018.04.084. 29791841 source

In the paper

“The ability of C. albicans to transition between yeast and filamentous forms is a key virulence trait that is triggered by a wide variety of environmental cues (20).”

What the sources say

• Not checked yetcites [20] Shapiro RS, Robbins N, Cowen LE 2011 Regulatory circuitry governing fungal development, drug resistance, and disease. Microbiol Mol Biol Rev 75: 213– 267. doi: 10.1128/MMBR.00045-10. 21646428 PMC3122626 source

In the paper

“In C. auris, studies have suggested that point mutations in ERG11 contribute to azole resistance of clinical isolates (15).”

What the sources say

• Not checked yetcites [15] Healey KR, Kordalewska M, Jiménez Ortigosa C, Singh A, Berrío I, Chowdhary A, Perlin DS 2018 Limited ERG11 mutations identified in isolates of Candida auris directly contribute to reduced azole susceptibility. Antimicrob Agents Chemother 62: e01427-18. doi: 10.1128/AAC.01427-18. PMC6153782 30082281 source

In the paper

“Moreover, the impact of Hsp90 on drug resistance has been conserved in a number of other fungal pathogens, including Candida glabrata and Aspergillus fumigatus (12–14).”

What the sources say

• Not checked yetcites [12] Cowen LE, Singh SD, Köhler JR, Collins C, Zaas AK, Schell WA, Aziz H, Mylonakis E, Perfect JR, Whitesell L, Lindquist S 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. doi: 10.1073/pnas.0813394106. 19196973 PMC2650349 source
• Not checked yetcites [13] Singh-Babak SD, Babak T, Diezmann S, Hill JA, Xie JL, Chen Y-LL, Poutanen SM, Rennie RP, Heitman J, Cowen LE 2012 Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata. PLoS Pathog 8: e1002718. doi: 10.1371/journal.ppat.1002718. 22615574 PMC3355103 source
• Not checked yetcites [14] Lamoth F, Juvvadi PR, Gehrke C, Steinbach WJ 2013 In vitro activity of calcineurin and heat shock protein 90 inhibitors against Aspergillus fumigatus azole- and echinocandin-resistant strains. Antimicrob Agents Chemother 57: 1035– 1039. doi: 10.1128/AAC.01857-12. 23165466 PMC3553695 source

In the paper

“In addition to its role in enabling the emergence and maintenance of azole resistance, Hsp90 governs temperature-dependent morphogenesis in C. albicans (19).”

What the sources say

• Not checked yetcites [19] Shapiro RS, Uppuluri P, Zaas AK, Collins C, Senn H, Perfect JR, Heitman J, Cowen LE 2009 Hsp90 orchestrates temperature-dependent Candida albicans morphogenesis via Ras1-PKA signaling. Curr Biol 19: 621– 629. doi: 10.1016/j.cub.2009.03.017. 19327993 PMC2735497 source

In the paper

“However, exposure to various cues that induce C. albicans filamentation does not induce morphogenesis in C. auris, suggesting divergence in the circuitry governing the yeast-to-filament transition between the two Candida species (23).”

What the sources say

• Not checked yetcites [23] Larkin E, Hager C, Chandra J, Mukherjee PK, Retuerto M, Salem I, Long L, Isham N, Kovanda L, Borroto-Esoda K, Wring S, Angulo D, Ghannoum M 2017 The emerging pathogen Candida auris: growth phenotype, virulence factors, activity of antifungals, and effect of SCY-078, a novel glucan synthesis inhibitor, on growth morphology and biofilm formation. Antimicrob Agents Chemother 61: e02396-16. doi: 10.1128/AAC.02396-16. 28223375 PMC5404565 source

In the paper

“The fluconazole-potentiating effect of Hsp90 inhibition is reminiscent of the results of a study of a series of C. albicans clinical isolates from an AIDS patient, with the early clinical isolates showing an Hsp90-dependent fluconazole tolerance phenotype (8, 27, 28).”

What the sources say

• Not checked yetcites [8] Cowen LE, Lindquist S 2005 Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309: 2185– 2189. doi: 10.1126/science.1118370. 16195452 source
• Not checked yetcites [27] White TC 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. 9210670 PMC163944 source
• Not checked yetcites [28] Hill JA, O’Meara TR, Cowen LE 2015 Fitness trade-offs associated with the evolution of resistance to antifungal drug combinations. Cell Rep 10: 809– 819. doi: 10.1016/j.celrep.2015.01.009. 25660029 source

In the paper

“Only one study has reported filamentous growth in C. auris to date, and that growth occurred under conditions of high salt concentrations (31).”

What the sources say

• Not checked yetcites [31] Wang X, Bing J, Zheng Q, Zhang F, Liu J, Yue H, Tao L, Du H, Wang Y, Wang H, Huang G 2018 The first isolate of Candida auris in China: clinical and biological aspects. Emerg Microbes Infect 7: 93. doi: 10.1038/s41426-018-0095-0. 29777096 PMC5959928 source

In the paper

“Over time, these isolates showed stepwise increases in fluconazole resistance that became independent of Hsp90 as the isolates acquired mutations in ERG11 and in the transcriptional activator of CDR1, TAC1 (8, 27, 28).”

What the sources say

• Not checked yetcites [8] Cowen LE, Lindquist S 2005 Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309: 2185– 2189. doi: 10.1126/science.1118370. 16195452 source
• Not checked yetcites [27] White TC 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. 9210670 PMC163944 source
• Not checked yetcites [28] Hill JA, O’Meara TR, Cowen LE 2015 Fitness trade-offs associated with the evolution of resistance to antifungal drug combinations. Cell Rep 10: 809– 819. doi: 10.1016/j.celrep.2015.01.009. 25660029 source

In the paper

“Although universal changes in cell surface composition upon morphogenesis have not been identified in fungi, cell surface proteins are often important regulators of morphogenesis and many are required for flocculation and adhesion, which are phenotypes associated with specific morphogenetic states (43).”

What the sources say

• Not checked yetcites [43] Gow NAR, Latge J-P, Munro CA 2017 The fungal cell wall: structure, biosynthesis, and function. Microbiol Spectr 5. doi: 10.1128/microbiolspec.FUNK-0035-2016. PMC11687499 28513415 source

In the paper

“Our use of the tetracycline-repressible promoter system to regulate the expression of HSP90 in C. auris establishes a precedent for genetic analysis of essential genes in this pathogen.”

In the paper

“Although morphogenesis is an important virulence trait in C. albicans and other fungal pathogens (40), the role of filamentous growth in C. auris is unclear.”

What the sources say

• Not checked yetcites [40] Gow NAR, Brown AJP, Odds FC 2002 Fungal morphogenesis and host invasion. Curr Opin Microbiol 5: 366– 371. 12160854 source

In the paper

“This analysis validates the idea that a core transcriptional response accompanies C. albicans morphogenesis in response to perturbation of Hsp90 function and provides a powerful platform for comparative analyses with the transcriptional response in C. auris.”

In the paper

“In addition, antifungal exposure has been shown to elicit distinct transcriptional responses between C. albicans and C. auris, suggesting that distinct genes may contribute to antifungal resistance in C. auris (18).”

What the sources say

• Not checked yetcites [18] Muñoz JF, Gade L, Chow NA, Loparev VN, Juieng P, Berkow EL, Farrer RA, Litvintseva AP, Cuomo CA 2018 Genomic insights into multidrug-resistance, mating and virulence in Candida auris and related emerging species. Nat Commun 9: 5346. 30559369 PMC6297351 source

In the paper

“In C. albicans, filamentous growth is also associated with extensive cell surface remodeling as indicated by changes in cell surface components at the transcriptomic and proteomic levels (32).”

What the sources say

• Not checked yetcites [32] Nantel A, Dignard D, Bachewich C, Harcus D, Marcil A, Bouin A-PP, Sensen CW, Hogues H, van Het Hoog M, Gordon P, Rigby T, Benoit F, Tessier DC, Thomas DY, Whiteway M, Van Het HM, Gordon P, Rigby T, Benoit F, Tessier DC, Thomas DY, Whiteway M 2002 Transcription profiling of Candida albicans cells undergoing the yeast-to-hyphal transition. Mol Biol Cell 13: 3452– 3465. doi: 10.1091/mbc.e02-05-0272. 12388749 PMC129958 source

In the paper

“Although the specific genes that are regulated during the morphogenetic transition are largely distinct between the two species, the regulation of cell surface-associated genes during filamentous growth appears to be conserved between C. auris and C. albicans.”

In the paper

“This highlights that azole resistance in these strains is likely due to the poor engagement of fluconazole with its cellular target Erg11 and/or to the upregulation of azole efflux pumps, thereby minimizing drug-induced cellular stress.”

In the paper

“The absence of nonsynonymous mutations in plasmids was verified by sequencing.”

In the paper

“RNA was prepared in quadruplicate as described above.”