Tolerance to Caspofungin in Candida albicans Is Associated with at Least Three Distinctive Mechanisms That Govern Expression of FKS Genes and Cell Wall Remodeling.

MECHANISMS OF RESISTANCE crossm Tolerance to Caspofungin in Candida albicans Is Associated with at Least Three Distinctive Mechanisms That Govern Expression of FKS Genes and Cell Wall Remodeling Feng Yang,a* Lulu Zhang,b Hironao Wakabayashi,a Jason Myers,c Yuanying Jiang,b Yongbing Cao,b Cristina Jimenez-Ortigosa,d David S. Perlin,d Elena Rustchenkoa Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York, USAa; Center for New Drug Research, School of Pharmacy, Second Military Medical University, Shanghai, People's Republic of Chinab; Genomic Research Center, University of Rochester Medical Center, Rochester, New York, USAc; Public Health Research Institute, New Jersey Medical School, Rutgers Biomedical and Health Sciences, Newark, New Jersey, USAd Expanding echinocandin use to prevent or treat invasive fungal infections has led to an increase in the number of breakthrough infections due to resistant Candida species. Although it is uncommon, echinocandin resistance is well documented for Candida albicans, which is among the most prevalent bloodstream organisms. A better understanding is needed to assess the cellular factors that promote tolerance and predispose infecting cells to clinical breakthrough. We previously showed that some mutants that were adapted to growth in the presence of toxic sorbose due to loss of one chromosome 5 (Ch5) also became more tolerant to caspofungin. We found here, following direct selection of mutants on caspofungin, that tolerance can be conferred by at least three mechanisms: (i) monosomy of Ch5, (ii) combined monosomy of the left arm and trisomy of the right arm of Ch5, and (iii) an aneuploidyindependent mechanism. Tolerant mutants possessed cell walls with elevated chitin and showed downregulation of genes involved in cell wall biosynthesis, namely, FKS, located outside Ch5, and CHT2, located on Ch5, irrespective of Ch5 ploidy. Also irrespective of Ch5 ploidy, the CNB1 and MID1 genes on Ch5, which are involved in the calcineurin signaling pathway, were expressed at the diploid level. Thus, multiple mechanisms can affect the relative expression of the aforementioned genes, controlling them in similar ways. Although breakthrough mutations in two specific regions of FKS1 have previously been associated with caspofungin resistance, we found mechanisms of caspofungin tolerance that are independent of FKS1 and thus represent an earlier event in resistance development. ABSTRACT Received 11 January 2017 Returned for modification 29 January 2017 Accepted 13 February 2017 Accepted manuscript posted online 21 February 2017 Citation Yang F, Zhang L, Wakabayashi H, Myers J, Jiang Y, Cao Y, Jimenez-Ortigosa C, Perlin DS, Rustchenko E. 2017. Tolerance to caspofungin in Candida albicans is associated with at least three distinctive mechanisms that govern expression of FKS genes and cell wall remodeling. Antimicrob Agents Chemother 61:e00071-17. https://doi.org/10.1128/ AAC.00071-17. Copyright © 2017 American Society for Microbiology. All Rights Reserved. Address correspondence to Yongbing Cao, , or Elena Rustchenko, . * Present address: Feng Yang, Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel Aviv, Israel. F.Y. and L.Z. contributed equally to this article. KEYWORDS Candida albicans, chromosome 5, molecular mechanisms, caspofungin tolerance T he fungus Candida albicans is part of the normal mycobiota of mucosal surfaces in the human gut and genitals. However, in severely immunocompromised individuals, C. albicans can become an invading pathogen that can cause various degrees of infection, including systemic candidiasis leading to mortality. The echinocandin class drugs are now recommended as first-line therapy for candidiasis because of their low toxicity and high efficacy, especially against Candida isolates resistant to azole drugs (1). They act by interfering with the biosynthesis of the cell wall by inhibiting 1,3-␤-D-glucan synthase. May 2017 Volume 61 Issue 5 e00071-17 Antimicrobial Agents and Chemotherapy aac.asm.org 1 Yang et al. Antimicrobial Agents and Chemotherapy Caspofungin was introduced in 2001 as the first member of the echinocandin drugs. Since that time, clinical resistance of C. albicans to caspofungin has remained low despite increasing reports of cases of resistance. For example, a recent surveillance study revealed an increase in breakthrough infections by resistant strains, from 0.5% in 2001 to 3.1% (6-fold) in 2009 (2), and additional studies also reported low-level resistance among C. albicans strains (3–6). However, C. glabrata showed higher levels of breakthrough infections in some clinical settings (6–9). As echinocandin usage continues to broaden for therapy and prophylaxis, there is a need to better understand the cellular processes that promote resistance. The only known mechanism of C. albicans clinical resistance to caspofungin and other echinocandins is the occurrence of point mutations in the FKS1 (GSC1; orf19.2929) gene on chromosome 1 (Ch1), which encodes a catalytic subunit of the drug target 1,3-␤-D-glucan synthase (10). The FKS1 mutations, clustered in two hot spot regions, HS1 and HS2, reduce glucan synthase sensitivity to echinocandins, elevate MICs, and confer reduced pharmacodynamic responses (reviewed in references 3, 11, and 12). Mutations in FKS1 are also known to cause expression changes of FKS1 relative to that of FKS2 (GSL2; orf19.3269) and FKS3 (GSL1; orf19.2495) (13), as well as enhanced expression of chitin genes, leading to elevated amounts of chitin in the cell wall in some resistant clinical isolates (5, 14). Clinical isolates may show a range of MIC values that are below the clinical breakpoint without the presence of an FKS mutation (15–17). Many of these strains are conditionally drug adapted or tolerant of drug. However, the molecular bases of the tolerance mechanism(s) are not fully resolved. There is a large body of evidence relating caspofungin tolerance in laboratory strains to the cell wall salvage mechanism. This is a reversible mechanism that involves an increase in chitin synthesis that compensates for a diminished amount of cell wall 1,3-␤-D-glucan when glucan synthase is inhibited by caspofungin. The increased chitin level serves to fortify the cell wall, which would otherwise be weakened by the reduced glucan content (14). For example, when C. albicans cells were treated with calcium and calcofluor white to induce elevated cell wall chitin levels and then injected into mice, these cells exhibited reduced susceptibility to echinocandins compared to that of cells with normal levels of chitin (18). In another study involving the paradoxical effect, one C. albicans isolate increased its chitin content ⬃900% and decreased its 1,3-␤-D-glucan and 1,6-␤-D-glucan contents 81% and 73%, respectively (19). In other studies, laboratory mutants were used to demonstrate that susceptibility to caspofungin dec (...truncated)