Investigation into the mechanism of arachidonic acid increased fluconazole susceptibility in Candida albicans biofilms and application to drug repurposing
Kuloyo, Oluwasegun Olalekan
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A considerable proportion of infections associated with C. albicans are due to its ability to form biofilms. Although there are antifungals available to treat infections caused by C. albicans, the occurrence of antifungal resistance hampers their effectiveness. Polyunsaturated fatty acids (PUFAs) have been reported to have antifungal properties against C. albicans biofilms. Interestingly, the combination of PUFAs, such as arachidonic acid (AA), with existing antifungals of the azole and polyene classes has been shown to increase susceptibility in C. albicans biofilms, but the mechanism involved is unknown. The objective of this study was to investigate the mechanism associated with the increased antifungal susceptibility of C. albicans biofilms in the presence of PUFAs. Our investigation confirmed an increase of C. albicans biofilms susceptibility to azoles (fluconazole and clotrimazole) in the presence of PUFAs (AA and eicosapentaenoic acid). Furthermore, the transcriptional analysis of C. albicans biofilms grown in the presence of AA combined with fluconazole, indicated that the presence of AA might be interfering with ATP generation processes. In addition, in the presence of AA alone or combined with fluconazole, the methionine/cysteine synthesis process, which is essential for biofilm formation, was repressed. However, quantitative analysis of the metabolic products associated with these pathways is required for a comprehensive view of the effects of these compounds on C. albicans biofilms. Several resistance mechanisms are employed by C. albicans biofilms to mitigate the effect of fluconazole treatment. One of the known mechanisms is the overexpression of the CDR1 efflux pump. Although C. albicans upregulates CDR1 in the presence of fluconazole, a significant increase in expression was observed in the presence of AA alone or combined with fluconazole. Nevertheless, this increased mRNA transcription did not necessarily translate into increased efflux when tested with rhodamine 6G. The lack of efflux by Cdr1p, which is a membrane protein, indicates that the presence of AA may have altered the membrane organisation leading to the loss of efflux activity. The ergosterol content in the cell is a factor which influences membrane fluidity. The regulation of ERG11, the fluconazole target, was higher in the treatment containing fluconazole alone compared to those containing AA. It was predicted that the increase in ERG11 expression would enhance the ergosterol content levels. However, the ergosterol content of C. albicans biofilms treated with fluconazole alone did not differ significantly from that treated with fluconazole combined with AA. Fluconazole and PUFAs are known to induce oxidative stress in yeast. In the presence of AA and fluconazole, several oxidative stress-associated genes were downregulated. In addition, the heterozygous mutants of these genes were sensitive to fluconazole treatment, which indicates that they play a role in C. albicans fluconazole resistance. Therefore, the AA may increase drug susceptibility via a number of mechanisms, including inhibition of Cdr1p activity and increased oxidative stress. There is an urgent need for alternative treatment option; hence the use of drug repurposing has progressed because of its reduced cost and time for the discovery of new antifungal drugs. By exploiting drugs against gene families identified in this study, that may play a role in fluconazole resistance, some drugs leads were obtained. However, more research is still required to clarify the associated mechanisms. An understanding of these mechanisms will indicate drug targets which can be explored for the treatment of C. albicans biofilms infections.