Compensatory response of the cell wall integrity pathway and chitin to caspofungin exposure in Candida glabrata

Abstract

Background: Studies in Saccharomyces cerevisiae and Candida albicans have reported an up-regulation of the cell wall integrity pathway and an increase in cell wall chitin following caspofungin exposure. This study sought to examine the genetic and phenotypic response of the emerging fungal pathogen Candida glabrata to caspofungininduced cell wall damage. Methods: Antifungal susceptibility testing was performed using Saccharomyces cerevisiae deletion strains to identify genes affecting caspofungin activity. Bioinformatics (tBLASTn) searches were used to identify homologs of these sequences in C. glabrata. The XTT colorimetric viability assay and time-kill studies were used to assess the phenotypic response of wild-type C. glabrata ATCC 200989 and the corresponding (Delta symbol)slt2 gene knockout strain to caspofungin. Following caspofungin challenge, relative gene expression of SLT2 (a mitogen-activated protein kinase of the cell wall integrity pathway), CHS3 (chitin synthase III), and SKT5 (activator of chitin synthase III) was determined in triplicate using reverse-transcriptase real-time PCR. Chitin was quantified in triplicate using a colorimetric assay. Results: SLT2, CHS3 and SKT5 deletion in S. cerevisiae resulted in enhanced caspofungin activity. Each of these genes was found to have a homologous region within the C. glabrata genome. Fungal viability of wild-type C. glabrata ATCC 200989 did not fall below 16.3% viability following any caspofungin concentration. In contrast, no viable cells were present in the C. glabrata (Delta symbol)slt2 mutant strain at caspofungin concentrations (Greater than or equal to)0.25 mcg/mL. Similarly, time-kill results demonstrated that caspofungin was only fungicidal against the C. glabrata (Delta symbol)slt2 strain at concentrations of 1 and 16 mcg/mL. Relative gene expression of SLT2, CHS3 and SKT5 increased 2- to 4-fold following caspofungin exposure in wildtype C. glabrata. In addition, chitin content increased 3- to 4.5-fold in wild-type C. glabrata following all caspofungin concentrations tested. In the C. glabrata (Delta symbol)slt2 strain, CHS3 expression and chitin content did not increase following caspofungin challenge. Conclusions: Caspofungin was not fungicidal against the wild-type C. glabrata strain and gene expression of SLT2, CHS3 and SKT5 increased in response to caspofungininduced cell wall damage. Additionally, chitin content was elevated following caspofungin exposure in wild-type C. glabrata. In the corresponding C. glabrata strain with a deletion in SLT2 (a key component of the cell wall integrity pathway) caspofungin activity was significantly enhanced. Also, gene expression of CHS3 and chitin content were not elevated with increasing caspofungin concentrations. This suggests a link between the cell wall integrity pathway, increased chitin concentrations and the decreased caspofungin potency in C. glabrata.