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

ABSTRACT: 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.

SUBMITTER: Aryamloo P

PROVIDER: S-EPMC6626714 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Cellular apoptosis: An alternative mechanism of action for caspofungin against <i>Candida glabrata</i>.

Aryamloo Parisa P Asgarian-Omran Hossein H Aslani Narges N Hossein-Nataj Hadi H Shokohi Tahereh T Badali Hamid H Nabili Mojtaba M Abdollahi Gohar Atefeh A Moazeni Maryam M

Current medical mycology 20190601 2

<h4>Background and purpose</h4>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 <i>Candida glabrata</i> cell death.<h4>Materials and methods</h4>For the purpose of the study, the minimum inhibitory concentration (MIC) of caspofungin against <i>C.</i> <i>gl ...[more]

PMID: 31321332

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Project description:The direct or indirect interactions that antifungals have with the host immune response may play a significant role in defining their activity in vivo. However, the impact that acquired antifungal resistance has on the immunopharmacologic activity of antifungals is not well described. We assessed the immunopharmacologic activity of caspofungin, micafungin, and voriconazole among isolates of Candida glabrata with or without FKS-mediated echinocandin resistance. Clinical bloodstream isolates of C. glabrata from patients who did (n = 5) or did not (n = 3) develop persistent candidemia and who did (n = 2) or did not (n = 11) harbor FKS gene mutations were included. A cell-based assay was used to compare differences in macrophage activation among isolates when grown in the presence or absence of subinhibitory concentrations of caspofungin, micafungin, or voriconazole. In the absence of antifungals, macrophage activation was significantly lower for index C. glabrata isolates obtained from persistent candidemia patients than for those from nonpersistent patients (33% versus 79% increase over negative controls, respectively; P < 0.01). Growth of isolates possessing wild-type FKS genes in subinhibitory concentrations of micafungin or caspofungin, but not voriconazole, significantly increased macrophage inflammatory responses compared to untreated controls (1.25- to 2.75-fold increase, P < 0.01). For isolates harboring the FKS2 hot spot 1 (HS1) S663P mutation, however, a significant increase was observed only with micafungin treatment (1.75-fold increase versus negative control, P < 0.01). Macrophage activation correlated with the level of unmasking of ?-glucan in the cell wall. The diminished macrophage inflammatory response to isolates that caused persistent candidemia and differential immunopharmacologic activity of echinocandins among FKS mutants suggest that certain strains of C. glabrata may have a higher propensity for immunoevasion and development of antifungal resistance during treatment.

Project description:Microarray was used to analyze azole resistance of Candida glabrata oropharyngeal isolates from 7 hematopoietic stem cell transplant recipients receiving fluconazole prophylaxis. Transcriptional profiling of the sequential-paired clinical isolates by microarray revealed 19 genes upregulated in the majority of resistant isolates compared to their paired-susceptible isolates. All seven resistant isolates had greater than two fold upregulation of CgPDR1, a master transcriptional regulator of PDR network, and all 7 resistant isolates showed upregulation of known CgPDR1-target genes. The altered transcriptome can be explained in part by the observation that all 7 resistant isolates had acquired a single nonsynonymous mutation in their CgPDR1 ORF. Four mutations occurred in the regulatory domain (L280P, L344S, G348A, S391L) and one in the activation domain (G943S) while two mutations (N764I, R772I) occurred in an undefined region. Association of azole resistance and the CgPDR1 mutations was investigated in the same genetic background by introducing the CgPDR1 sequences from one sensitive and five resistant isolates into a laboratory azole-sensitive strain (cgpdr1) via integrative transformation. The cgpdr1 strain was restored to wild-type fluconazole susceptibility when transformed with CgPDR1 from the susceptible isolate but became resistant when transformed with CgPDR1 from the resistant isolates. However, despite the identical genetic background, upregulation of CgPDR1 and CgPDR1-target genes varied between the 5 transformants, independent of the domain locations in which the mutations occurred. In sum, gain-of-function mutations in CgPDR1 not only contributed to the clinical azole resistance but different mutations had varying degrees of impact on the CgPDR1-target genes. Two groups (C1S/84 and C16R/84) consisted of 4 biological replicates in which a complemented strain sample was paired with the laboratory wild type strain 84 sample. Each group included 1 reciprocally labeled sample.

Dataset Information

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

Publications

Cellular apoptosis: An alternative mechanism of action for caspofungin against <i>Candida glabrata</i>.

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