Cellular apoptosis: An alternative mechanism of action for caspofungin against Candida glabrata.

BACKGROUND AND PURPOSE: Although the mechanism of action for echinocandins is known, the physiological mechanisms by which these antifungal agents cause cell death via the classical apoptotic pathways are not well-defined yet. Regarding this, the present study aimed to evaluate the mechanisms of caspofungin-induced Candida glabrata cell death. MATERIALS AND METHODS: For the purpose of the study, the minimum inhibitory concentration (MIC) of caspofungin against C. glabrata (ATCC 90030) was determined using the broth microdilution reference method (CLSI M27-A2 and M27-S4). The annexin V and propidium iodide staining was performed to determine the way through which caspofungin acts against C. glabrata (i.e., through the induction of apoptosis and/or necrosis). Additionally, the possible effect of caspofungin on inducing the expression of two apoptotic genes, namely MCA1 and NUC, was studied using the real-time polymerase chain reaction assay. RESULTS: According to the obtained MIC value (0.5 µg/mL), C. glabrata, exposed to 0.25, 0.5, and 1 µg/mL of caspofungin, exhibited the features of late apoptosis/necrosis after 18 h of incubation. Furthermore, the use of 0.25, 0.5, and 1 µg/ml caspofungin induced apoptosis (early/late) in 14.67%, 17.04%, and 15.89% of the cells, respectively. The results showed a significant difference between the percentages of early-apoptotic cells at the three concentrations (P<0.05). In addition, the rate of necrosis was significantly greater than that of apoptosis in response to caspofungin. Accordingly, necrosis occurred in 71.26%, 71.26%, and 61.26% of the cells at the caspofungin concentrations of 0.25, 0.5, and 1 µg/mL, respectively (P<0.05). The analysis of the data in the REST software demonstrated a significant increase in the expression of MCA1 and NUC1 genes (P<0.05). CONCLUSION: As the findings of the present study indicated, caspofungin promoted both necrosis and apoptosis of C. glabrata cells at concentrations higher than or equal to the MIC value.

Glabridin induces overexpression of two major apoptotic genes, MCA1 and NUC1, in Candida albicans.

OBJECTIVES: The growing trend in emergence of antifungal-resistant Candida strains has recently inspired researchers to design new antifungal agents with novel mechanisms of action. Glabridin is a natural substrate with multiple biological activities. In this study, the antifungal effects and possible mechanism of action of glabridin were investigated. METHODS: Minimum inhibitory concentrations (MICs) of glabridin against fluconazole (FLU)-resistant and FLU-susceptible Candida albicans strains were investigated according to Clinical and Laboratory Standards Institute (CLSI) guidelines. To investigate the possible mechanism of action, expression of two critical genes involved in yeast apoptosis (MCA1 and NUC1) was assayed by real-time PCR. RESULTS: FLU-susceptible and FLU-resistant C. albicans strains showed the same glabridin MICs (MIC50, 8µg/mL). Therefore, a distinct azole-independent mechanism might be responsible for the inhibitory activity of glabridin. Overexpression of MCA1 and NUC1 was observed in C. albicans cells treated with glabridin, suggesting the involvement of apoptosis signalling in C. albicans strains exposed to glabridin. CONCLUSION: This study suggests that glabridin might be considered a safe agent to fight against C. albicans strains.

Amino acid substitutions in Erg11p of azole-resistant Candida glabrata: Possible effective substitutions and homology modelling.

Understanding the mechanisms responsible for fluconazole resistance in Candida glabrata is not only crucial for the development of new antifungals but is also important in choosing appropriate antifungals for patients at the earliest stages. The aim of this study was to determine the Erg11p amino acid substitutions in fluconazole-resistant C. glabrata isolates. Sixty clinical isolates of C. glabrata were investigated. In vitro antifungal activities of fluconazole, itraconazole and voriconazole were determined using the broth microdilution reference method. The ERG11 gene for resistant (n=4) and susceptible (n=1) isolates were sequenced and multi-aligned using MEGA6 software. A homology model of the C. glabrata ERG11 gene was created by SWISS-MODEL software using the crystal structure of Saccharomyces cerevisiae Erg11p as a template, and the predicted binding sites to fluconazole were investigated. Fluconazole and multi-azole resistance were observed in 6.7% and 3.3% of the isolates, respectively. Several amino acid substitutions were identified, among which some were also identified in susceptible isolates. The amino acid substitution G236V was at the binding site, and substitutions H146Q and D234E were near to the binding site of triazoles according to the SWISS-MODEL. According to the homology modelling results, the amino acid substitution G236V is highly likely to play a key role in azole resistance development.