Allicin modulates expression of cell wall-related genes in Aspergillus fumigatus

Abstract

Background: Aspergillus fumigatus causes life-threatening iinvasive aspergillosis, especially in immunocompromised hosts, and resistance of this filamentous fungus to commonly used antifungal agents has been on the rise. Allicin (diallyl-dithiosulfinate), an allyl-sulfur compound from garlic has been proven to exhibit antimicrobial properties. However, there have been few studies on the antifungal mechanism of allicin against A. fumigatus. Based on our previous studies on the antifungal mechanism of allicin against Candida albicans, an opportunistic yeast pathogen, we hypothesize that allicin would similarly target cell-wall biosynthesis and integrity in A. fumigatus. This study evaluated the expression of cell-wall related genes in A. fumigatus ATCC 36607 upon treatment with allicin. Methods and materials: The minimum inhibitory concentration (MIC) of allicin against A. fumigatus ATCC 36607was determined according to the CLSI M38-A2 guidelines. A. fumigatus was incubated in the presence of allicin at subinhibitory concentrations of 1/4X MIC, 1/2X MIC, and 1X MIC for 48 hours. The cells were harvested post-incubation with allicin and RNA was extracted and reverse-transcribed into cDNA for evaluation of expression of selected genes via real time PCR. Results: The minimum MIC of allicin against A. fumigatus was found to be 3.2 μg/ml. Scanning electron micrographs exhibited significant morphological alteration of A. fumigatus hyphae and conidia after 48 h incubation at 1× MIC, 2× MIC and sub-MIC concentrations of allicin 1/4× MIC and 1/2× MIC in comparison with untreated (growth) control. Upon treatment at 1/4× MIC (0.8 μg/ml), hyphae were seen as wrinkled with patchy and rough surface implying decrease in structural integrity. Further, at 1/2× MIC (1.6 μg/ml), aberrant shape of hyphae with irregular constriction was observed along the length of hypha. At 1X MIC, complete suppression of hyphae growth was observed and only conidia were visible. Real-time PCR findings revealed that significant down-regulation of chsE, chsG, fksA, and gel2 genes were observed at various MICs of allicin tested. On the contrary, rhoD showed an upregulated expression. Conclusion: This study suggests the ability of allicin to affect the expression of cell wall-related genes of A. fumigatus.