Influence of polyunsaturated fatty acids on fluconazole susceptibility and drug efflux in Candida krusei

Abstract

Globally, fungal infections affect more than one billion people annually, with an estimated mortality of approximately two million people. The genus Candida is a highly heterogeneous group containing several opportunistic pathogens responsible for the increasing number of life-threatening mycoses, especially in immunocompromised subjects, including cancer patients, human immunodeficiency virus (HIV) positive patients, and organ transplant recipients. Compared to antibacterial counterparts, the number of available antifungals is limited, and the increase in antifungal resistance further complicates this. Resistance or tolerance towards all the available classes of antifungals has been reported. Strikingly, Candida krusei exhibits innate resistance to fluconazole (FLC) and rapid adaptive resistance to other antifungal drugs. Moreover, the role of efflux pumps (e.g. ATP-binding cassette 1, Abc1p) in this inherent FLC resistance remains unclear. This yeast also forms biofilms, which are potentially more resistant to antifungal drugs than planktonic counterparts. Additionally, there is paucity of information on the ability of exogenous polyunsaturated fatty acids (PUFAs) to overcome intrinsic antifungal resistance, such as in the case of C. krusei. In order to address this lack of knowledge, we firstly determined the susceptibility profiles of biofilms of C. krusei strains (and a C. albicans reference strain) towards FLC and five PUFAs [i.e. oleic acid (OA), linoleic (LA), gamma-linolenic acid (GLA), arachidonic acid (AA), and eicosapentaenoic (EPA)]. Our results showed that the antifungal activity of FLC against these strains is concentration-dependent, with C. krusei UFS Y-0277 displaying the least susceptibility. Moreover, the antifungal effect of the PUFAs is dependent on the strain, as well as on the chain length and dose of the fatty acid, with LA and GLA showing the most favourable activity. Upon combination therapy assay, we found that either of the two superior PUFAs (LA or GLA) potentiates the action of FLC towards the biofilms of the most-resistant strain of C. krusei. An initial attempt to examine the mechanism responsible for this revealed that the combination treatments induce the production of extracellular vesicles, cell membrane damage, and cell rupture. A subsequent membrane integrity assay confirmed this deleterious impact on the cell membrane. Our results also showed that antioxidants are capable of protecting C. krusei biofilms from the deleterious effects of the combination treatments. Additionally, these treatments had an inhibitory influence on the activity of efflux pumps, which was directly proportional to the concentration of PUFA used. Furthermore, our in vitro findings were corroborated by in vivo assays in a Caenorhabditis elegans infection model, which demonstrated that the combination treatments promote the overall survival and significantly reduce the intestinal fungal burden of infected nematodes. These observations may reiterate the combination of fatty acids with conventional antifungal drugs as a favourable therapeutic strategy deserving of increased traction and research for resistance reversal and infection control. We also aimed to establish a Clustered Regularly Interspaced Short Palindromic Repeats- Cas associated protein 9 (CRISPR-Cas9)-mediated gene-editing system for C. krusei since the absence of such system has impeded genome engineering, resistance, and virulence studies in this yeast. This was performed through the adaptation of a previously designed C. albicans-specific, CRISPR-Cas9 system (HIS-FLP type). This system's efficacy for geneediting was validated by the successful homozygous deletion of two auxotrophic marker genes, URA3 and ADE2, in this yeast. Using the adapted system, we attempted to construct a Green Fluorescent Fusion (GFP) fusion of Abc1p to assess the influence of AA and FLC on the localisation, expression, and activity of Abc1p – in order to gain better insights into the role of this efflux pump in FLC resistance. However, this was unsuccessful, possibly due to the failure of the yeast to incorporate the supplied ABC1-GFP fusion donor DNA (dDNA). Hence, we resorted to using western blot analysis and efflux pump assay. Results obtained demonstrate that FLC increases the expression and functionality of Abc1p, suggesting that this transporter plays a role in FLC resistance. However, AA reduces the expression of Abc1p, and abrogates its activity in a dose-dependent manner, even in the presence of FLC. These findings highlight AA as a potential inhibitor of Abc1p and lent credence to the role of this transporter in FLC resistance. Taken together, this study demonstrates the FLC-potentiating activity of PUFAs against an intrinsically-resistant C. krusei in vitro and in vivo in a C. elegans infection model – which may pave the way for future studies into novel therapeutic strategies. It also establishes a successful development of a CRISPR-Cas9 system for C. krusei. Although preliminary findings demonstrate the involvement of Abc1p in FLC resistance and show the potential of AA as an inhibitor of this transporter, further studies are necessary for a definitive assertion.