Background

Invasive fungal infections are a common cause of morbidity, mortality, and high healthcare costs in hospitals worldwide (Bongomin et al., 2017). Candida species are opportunistic human fungal pathogens, with Candida glabrata being the second to fourth most prevalent etiological agent of Candida bloodstream infections (Chakrabarti et al., 2015; Bongomin et al., 2017; Lamoth et al., 2018; Pfaller et al., 2019). Cells of the innate immune system play an important role in the control of Candida infections through an armamentarium of reactive oxygen and nitrogen species generation, antifungal peptide production, and facilitating fungal antigen presentation to activate the adaptive immune system (Netea et al., 2015; Heung, 2020). C. glabrata encounters reactive oxygen species (ROS)-induced stress upon internalization by host macrophages (Seider et al., 2011; Rai et al., 2012). However, it probably possesses a robust oxidative stress defense system that facilitates its survival and replication in macrophages (Kaur et al., 2007; Seider et al., 2011; Rai et al., 2012). Of note, macrophages are thought to act as Trojan horses for C. glabrata, providing it with a safe niche in the human host (Brunke and Hube, 2013).

Efficient detoxification of ROS is necessary to survive an oxidative environment. C. glabrata shows high levels of resistance towards oxidative stress caused by hydrogen peroxide in vitro (Cuéllar-Cruz et al., 2008). The sole catalase CgCta1 and two superoxide dismutases, CgSod1 and CgSod2, contribute to the effective oxidative stress response system in C. glabrata (Cuéllar-Cruz et al., 2008; Briones-Martin-Del-Campo et al., 2015). Another arm of the oxidative stress defense system is represented by proteins containing flavodoxin-like fold that are implicated in quinone detoxification (Sancho, 2006; Carey et al., 2007; Lodeyro et al., 2012). The flavodoxin-fold is characterized by a 5-stranded parallel β-sheet surrounded by α-helices on both sides, and present in proteins that are involved in electron transfer between varied redox compounds (Sancho, 2006; Carey et al., 2007; Lodeyro et al., 2012; Farías-Rico et al., 2014; Houwman and van Mierlo, 2017). Many proteins with flavodoxin-like fold possess NAD(P)H:quinone oxidoreductase activity and detoxify quinones to hydroquinones by preventing the formation of unstable and highly reactive semiquinone species, and use FMN (flavin mononucleotide) or FAD (flavin adenine dinucleotide) as cofactors (Patridge and Ferry, 2006; Sancho, 2006; Carey et al., 2007; Farías-Rico et al., 2014; Koch et al., 2017). This enzymatic reaction follows a ping-pong bi-bi mechanism, occurs in two half-reactions, reductive and oxidative, and involves two direct hydride transfer steps (Deller, 2008). The reductive half-reaction involves electron donation by the reduced form of nicotinamide adenine dinucleotide, NADH, to cofactors FMN or FAD, with NADH getting oxidized to NAD⁺. In the oxidative half-reaction, the reduced cofactors, FMN(H2) or FAD(H2), transfer the electrons to quinone substrates, which leads to the reduction of quinones to hydroquinones (Deller, 2008).

We have recently identified and characterized four flavodoxin-like proteins (Fld-LPs) in C. glabrata: CgPst2, CgRfs1, CgPst3, and CgYcp4, and shown that CgPst2 is uniquely required to survive menadione and benzoquinone stress (Battu et al., 2021). Since the bacterially purified CgPst2 showed no NAD(P)H:quinone oxidoreductase activity (Battu et al., 2021), we developed a protocol to measure NAD(P)H:quinone oxidoreductase activity in whole cell lysates of wild-type and CgPST2 gene-deleted strains to establish the role of CgPst2 in quinone detoxification. The step-by-step procedure followed to carry out the assay is represented in Figure 1. Our protocol is based on the conserved ping-pong mechanism of flavodoxin-like proteins, wherein we monitored the oxidation of NADH to NAD⁺ at 340 nm as a measure of quinone conversion to quinol/hydroquinone. Since the reduced form of NADH absorbs at 340 nm, while the oxidized form NAD⁺ has no absorbance at 340 nm, NADH oxidation was recorded as a decrease in the absorbance at 340 nm. This reaction mechanism is schematically illustrated in Figure 2.

Using this assay, we were able to show that menadione treatment leads to an increase in cellular NAD(P)H:quinone oxidoreductase activity, which could be partially attributed to elevated CgPst2 levels and enhanced aspartyl protease CgYps1-mediated cleavage of CgPst2, stimulating homo-tetramerization of CgPst2 (Battu et al., 2021). Notably, purified Fld-LPs WrbA and Pst2 exhibited NAD(P)H:quinone oxidoreductase activity in Escherichia coli and Saccharomyces cerevisiae, respectively (Patridge and Ferry, 2006; Koch et al., 2017). However, using the protocol developed, we could show NAD(P)H:quinone oxidoreductase activity in whole cell extracts of C. glabrata leading to NADH oxidation in the presence of menadione in vitro (Battu et al., 2021). Importantly, this activity was absent in strains deleted for the CgPST2 gene, and the activity was regained upon expression of CgPST2 gene in mutant strains (Battu et al., 2021), thereby unequivocally correlating the cellular NAD(P)H:quinone oxidoreductase activity with the presence of CgPst2 in cells.

The advantages of our protocol are many-fold. First, it is an efficient way to measure NAD(P)H:quinone oxidoreductase activity in cellular extracts and does not require cloning, expression, and purification of Fld-LPs in a heterologous expression system. Second, it neither requires expensive chemicals nor sophisticated equipment. Third, our assay is easy-to-use and can readily be applied to study different quinone detoxification activities under varied stress and host-mimicking conditions, as well as in mutants lacking various genes of the oxidative stress response machinery and yeast cells recovered from host immune cells, including macrophages. It can also be adapted to screen inhibitors that are specific for fungal Fld-LPs. Fourth, unlike activity measurement in purified enzymes, no additional supply of FMN/FAD is required as cofactor in our assay. Finally, since this protocol estimates NAD(P)H:quinone oxidoreductase activity in cellular extracts, the loss of enzymatic activity often observed during protein purification is avoided. Thus, this assay can provide key insights into the activity of Fld-LPs present in their native states, including Fld-LPs containing different posttranslational modifications and/or oligomeric Fld-LPs.

Figure 1. Schematic representation of the steps in the protocol

Figure 2. Schematic illustration of the reaction mechanism of flavodoxin-like proteins (Fld-LPs)