Abstract
Candidemia caused by Candida spp. is a serious threat in hospital settings being a major cause of acquired infection and death and a possible contributor to Covid-19 mortality. Candidemia incidence has been rising worldwide following increases in fungicide-resistant pathogens highlighting the need for more effective antifungal agents with novel modes of action. The membrane-bound enzyme alternative oxidase (AOX) promotes fungicide resistance and is absent in humans making it a desirable therapeutic target. However, the lipophilic nature of the AOX substrate (ubiquinol-10) has hindered its kinetic characterisation in physiologically-relevant conditions. Here, we present the purification and expression of recombinant AOXs from C. albicans and C. auris in a self-assembled proteoliposome (PL) system. Kinetic parameters (Km and Vmax) with respect to ubiquinol-10 have been determined. The PL system has also been employed in dose–response assays with novel AOX inhibitors. Such information is critical for the future development of novel treatments for Candidemia.
Highlights
Candidemia caused by Candida spp. is a serious threat in hospital settings being a major cause of acquired infection and death and a possible contributor to Covid-19 mortality
To analyse the kinetic parameters of a variety of purified alternative oxidase (AOX) proteins, a PL system catalysing NADH:O2 was utilised in which the ubiquinone pool was maintained reduced by an NADH dehydrogenase (NDH-2 from Caldalkalibacillus thermarum) and ubiquinol was reoxidised by AOX (Fig. 1A)
Twin strep-tagged T. brucei, C. albicans and C. auris AOX were purified as outlined in Materials and Methods and visualized through both Western blotting using antistrep antibodies and coomassie staining
Summary
Candidemia caused by Candida spp. is a serious threat in hospital settings being a major cause of acquired infection and death and a possible contributor to Covid-19 mortality. The significant increase in non-albicans infections seen in recent years is largely the result of increasing use of prophylactic antifungal agents such as fluconazole, a drug commonly used to treat c andidaemia[6] One such species of concern is Candida auris which in comparison to C. albicans has shown reduced susceptibility to fluconazole in over 90% of c ases[7]. Given the resistance of C. auris to conventional anti-fungal treatments it is even more imperative that new drugs to treat this fungus are urgently developed Within organisms such as C. auris and albicans, the alternative oxidase (AOX) plays a very key role in facilitating continued respiratory activity and tends to be activated by stress conditions such as the presence of antifungals or oxidative inductors[17,18].
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