Abstract
BackgroundIn the fungal pathogen Candida albicans, amino acid substitutions of 14alpha-demethylase (CaErg11p, CaCYP51) are associated with azole antifungals resistance. This is an area of research which is very dynamic, since the stakes concern the screening of new antifungals which circumvent resistance. The impact of amino acid substitutions on azole interaction has been postulated by homology modeling in comparison to the crystal structure of Mycobacterium tuberculosis (MT-CYP51). Modeling of amino acid residues situated between positions 428 to 459 remains difficult to explain to date, because they are in a major insertion loop specifically present in fungal species.Methodology/Principal FindingFluconazole resistance of clinical isolates displaying Y447H and V456I novel CaErg11p substitutions confirmed in vivo in a murine model of disseminated candidiasis. Y447H and V456I implication into fluconazole resistance was then studied by site-directed mutagenesis of wild-type CaErg11p and by heterogeneously expression into the Pichia pastoris model. CLSI modified tests showed that V447H and V456I are responsible for an 8-fold increase in fluconazole MICs of P. pastoris mutants compared to the wild-type controls. Moreover, mutants showed a sustained capacity for producing ergosterol, even in the presence of fluconazole. Based on these biological results, we are the first to propose a hybrid homology structure-function model of Ca-CYP51 using 3 different homology modeling programs. The variable position of the protein insertion loop, using different liganded or non-liganded templates of recently solved CYP51 structures, suggests its inherent flexibility. Mapping of recognized azole-resistant substitutions indicated that the flexibility of this region is probably enhanced by the relatively high glycine content of the consensus.Conclusions/SignificanceThe results highlight the potential role of the insertion loop in azole resistance in the human pathogen C. albicans. This new data should be taken into consideration for future studies aimed at designing new antifungal agents, which circumvent azole resistance.
Highlights
Candida albicans is an opportunistic fungal pathogen that causes severe blood and disseminated infections (BSIs)
Whereas 5 mg/kg of FLC had a significant survival effect on mice infected by the susceptible CAAL97 isolate, both CAAL37 and CAAL61 clinical isolates showed a drastically reduced FLC susceptibility in vivo
The understanding of the molecular mechanisms underlying the resistance to azole in C. albicans is essential, both in providing guidance in the selection of the appropriate antifungal agent at the onset of infection, and in the discovery of new antifungals which circumvent azole resistance
Summary
Candida albicans is an opportunistic fungal pathogen that causes severe blood and disseminated infections (BSIs) The incidence of these infections has markedly increased over the past decade, due to the increase of immunocompromised and neutropenic patients after organ transplantation, cancer therapy or AIDS [1]. In the fungal pathogen Candida albicans, amino acid substitutions of 14alpha-demethylase (CaErg11p, CaCYP51) are associated with azole antifungals resistance. This is an area of research which is very dynamic, since the stakes concern the screening of new antifungals which circumvent resistance. Modeling of amino acid residues situated between positions 428 to 459 remains difficult to explain to date, because they are in a major insertion loop present in fungal species
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