Kinetic characterisation and inhibitor sensitivity of Candida albicans and Candida auris recombinant AOX expressed in a self-assembled proteoliposome system

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.


Results
To analyse the kinetic parameters of a variety of purified 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). His-tagged NDH-2 was expressed in E. coli C41 and purified to homogeneity as described by Heikal et al. 26 as detailed in Materials and Methods. Figure 1B indicates that NDH-2 purified protein ran as a single band on SDS-PAGE running close to the predicted molecular mass of 44 kDa as described by Heikal et al. 25 and typically possessed a specific activity of 100 µmol NADH oxidised min −1 mg protein −1 (Fig. 1C). 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. Figure 1B indicates the purity of these proteins and, as predicted from their molecular weights, C. albicans AOX2 and C. auris AOX run higher than that of TAO due to the presence of a fungal specific sequence insertion 27 . All membrane-bound AOXs displayed comparable activities (Fig. 1C). However what is particularly noteworthy is the finding that even though only single bands were visualised for both Candida spp. no quinol oxidase activity could be detected for the purified protein with either species in contrast to the high activity observed with TAO.

Measurement of AOX kinetic parameters.
PLs were created by incorporating approximately 50 µg of AOX protein in the presence of Q 10 ranging from 0 to 100 nmol whereas NDH-2 protein was added directly to the activity assay at a concentration which ensured AOX remained rate limiting and this was achieved by creating the PLs in the presence of AOX and titrating AOX activity with exogenous NDH-2 until maximal rates were achieved for a pre-set ubiquinol concentration (see Fig. 2A and Supplementary Fig. 1). As AOX was incorporated during the preparation of the PLs, it is distributed on both sides of the membrane whereas since NDH-2 is added during the assay its location is restricted to the outer surface only.
We confirmed that NDH-2 maintained the Q-pool in a reduced state through the simultaneous measurement of oxygen consumption and the steady-state redox poise of the ubiquinone pool using an Oroboros NextGen-O2k high resolution respirometer fitted with a Q-electrode system 28 the results of which are shown in Fig. 2B. It is apparent from Fig. 2B that in the absence of NDH-2 the small increase in the Q-redox poise on addition of NADH is due to a direct interaction of NADH with the glassy carbon electrode. The subsequent addition of aliquots of NDH-2, however, results in a rapid and complete reduction of the Q-pool to approximately 98% which is associated with a negligible oxygen consumption rate. Following the addition and engagement of 3 aliquots of purified TAO at this NDH-2 concentration, marked and measurable respiratory rates are observed and the Q-pool reduction level has fallen to 93%, 80 and 60% respectively. The subsequent addition of 1 µM ascofuranone (AF) completely inhibits respiration and returns the Q-pool to its fully reduced state confirming that the AOX was fully engaged under these conditions and furthermore that all oxygen consumption was due to AOX activity. www.nature.com/scientificreports/ Of particular importance was the finding that incorporation of the C. albicans and C. auris AOXs into liposomes, following the rationale described for the development of this technique 25,29,30 readily restored enzymatic activity which had been lost after solubilisation and purification. Figure 2C-E shows the Michaelis-Menten curves for AOXs from C. albicans (AOX2), C. auris and TAO respectively 25,29 . Ubiquinone concentrations in the liposome membrane were calculated assuming that 1 mg of phospholipid occupies 1 µL and hence 1 nmol of quinone in 1 mg of phospholipid corresponds to 1 mM 29,30 . In all cases typical Michaelis-Menten Kinetics were observed for all AOXs (Fig. 2C-E), the derived K m and V max values for each of these proteins being summarised in Fig. 2F.
It is apparent from Fig. 2F that all AOX proteins possess a similar K m with respect to Q 10 ranging from 4.6 to 7.3 mM whereas V max values varied between 251-578 µmol NADH min −1 mg −1 . Interestingly, the highest V max (or k cat ) was observed with C. albicans AOX2 at 578.0 µmol NADH min −1 mg −1 (404 s −1 ) followed by TAO at 251.6 µmol NADH min −1 mg −1 (146 s −1 ) and C. auris AOX at 166.8 µmol NADH min −1 mg −1 (108 s −1 ). Finally, Fig. 2F shows the catalytic efficiency or pseudo second order rate constant (k cat /K m ), which reflects both substrate binding and catalytic events, for all of the AOXs incorporated into PLs. K m , V max and k cat values are statistically similar between all AOXs, however k cat /K m differed significantly between each one (p < 0.001). These results suggest that C. albicans AOX2 has the best ability to convert substrate to product being more than double that of TAO and 4 times greater than that of C. auris AOX.
AOX inhibitor titrations. The great advantage of the PL system originally described by the Hirst laboratory 25,29 and used in this study is that AOX inhibitors can be titrated against physiologically relevant quinol substrates thereby providing properly defined and robust inhibitor dissociation constants. In this study we titrated all 3 oxidases in self-assembled PL system against Ascofuranone (AF), colletochlorins B and D (CB and CD respectively) and compared the results to that with SHAM (salicylhydroxamic acid), the most widely used AOX inhibitor. AF, CB and CD have previously been shown to behave as mixed type inhibitors against the Trypanosomal protein, with ascofuranone being effective against trypanosomes in vivo 31 (for structures see Supplementary Fig. 2). A Q 10 concentration of 10 mM was selected as being the closest concentration to that within the mitochondrial membrane, thereby approximating the effects of these inhibitors within a mitochondrial envi- PLs suitable for kinetic studies were prepared by inserting Ubiquinone-10 (Q 10 ) and bacterially-expressed AOX proteins into phospholipid vesicles 26 . After PL preparation, NDH-2 was added externally to bind to the membrane of the PLs for oxidation of the electron donor NADH and subsequently Q 10 . Drawn with Microsoft Powerpoint 2016. (B) AOX and NDH-2 proteins were purified to homogeneity from bacterial membranes. Samples of total protein (1), insoluble (2) and soluble membrane fractions (3) and purified protein (4) were resolved by SDS-PAGE and visualised using immunoblotting (1)(2)(3)(4) or coomassie blue staining; NDH-2 (5) TAO (6), C. albicans (7), C. auris (8). (C) Specific activity of membrane-bound AOX was detrmined by measurement of oxygen uptake in a Oroboros Q2k oxygraph. Specific activity of the purified AOX proteins was determined by monitoring the change of absorbance at A 278nm on oxidation of 100 nmol Q 1 H 2 by the oxidase and expressed as µmol. Q 1 H 2 min −1 mg −1 . The specific activity of NDH-2 was determined by monitoring the change of absorbance at A 340nm on oxidation of 1 mM NADH and expressed as µmol. NADH oxidised min −1 mg −1 . Note that zero activity for C. albicans and C. auris AOX was detectable (ND). Original gels and blots are shown in Supplementary Fig. 4 www.nature.com/scientificreports/ ronment. In this case excess NDH-2 was incorporated into the PLs alongside AOX, and not added externally, in order to simplify the experimental setup and minimize handling times during dose-response assays. Literature values for the turnover of NDH-2 and TAO are somewhat comparable, typically within the region of 15-200 s −126,32,33 and therefore to ensure that AOX proteins were rate-limiting during IC 50 measurements, PLs were created with a molar ratio of 400:1 NDH-2:AOX. Such a high ratio ensures that the Q-pool is maintained highly reduced and that the AOX is the rate-limiting step in the system. It is apparent from Fig. 3 that the three inhibitors tested were active against all of the three AOX proteins in the sub-micromolar range-much lower concentrations than SHAM which is effective only at millimolar concentrations. Indeed, SHAM is such a poor inhibitor that the high concentrations required for inhibition interfered with NADH absorbance readings and hence we resorted to oxygen consumption measurements to determine SHAM IC 50 values. For the three AOX proteins, CB displayed the lowest IC 50 , followed by ascofuranone and CD. IC 50 values for TAO were significantly lower than those for C. albicans AOX2 and C. auris AOX (Fig. 3E). Finally, the selectivity of ascofuranone, CB and CD towards all AOXs was evaluated in PLs containing only NDH-2, in which case DCPIP was used as an electron acceptor. Negligible effect of the inhibitors on NDH-2 up to 2.5 µM was detected ( Supplementary Fig. 3).

Discussion
AOX is a respiratory protein which is gaining increasing interest in biotechnological applications such as a possible human therapeutic agent and as a molecular target for a novel class of antimicrobial agents. It is therefore imperative to determine relevant biochemical parameters for this enzyme which will guide the future rational design of novel therapies. One such example is the development of antifungal agents effective against drugresistant strains of C. albicans and C. auris which are major sources of hospital infections in the UK and worldwide. With the prevalence of antifungal resistance being both substantial and growing, the development of new bioactive compounds is essential, with drugs which target the switch to more virulent forms of the fungi being a prime candidate. The high resistance of Candida biofilms to anti-fungal compounds, in particular azoles, is one such example with the biofilms being up to 1000 times more resistant than the less virulent yeast forms 34 . Studies aimed at preventing the switch from a yeast to the far more virulent hyphae form associated with invasive candidemia have implicated AOX as a possible therapeutic target. These include both in vitro assays using C. albicans which show that AOX inhibition results in oxidative stress, a reduction in cellular proliferation and an increase in suscepitbilty to fluconazole 35,36 ; and multiple studies suggesting a role for AOX enzymes in activation of the cAMP-PKA signalling pathway, which is one of the signalling pathways controlling hyphae formation [37][38][39][40] . In addition a recent in vivo study using a mouse systemic model of candidiasis caused by C. albicans reinforced the link between AOX expression and hypha formation 41 . This study indicated that AOX enzymes are required for hypha formation upon inhibition of the classical repiratory system.
Considerable complication in the development of antifungal drugs is that the fungi are eukaryotic; this renders them harder to treat due to the obvious need to select pathogen-specific targets, and AOX is of particular interest due to its absence in mammalian electron transport chains. One challenge posed by AOX, however, is www.nature.com/scientificreports/ the fact that it is a monotopic membrane protein and its natural substrate is the highly hydrophobic ubiquinol-10 which is present within the phospholipid bilayer. To the best of our knowledge we present for the first time the characterization of Candida AOX in a PL system which is a truer reflection of the hydrophobic environment of the mitochondrial membrane rather than using detergent-solubilised AOX and the hydrophilic ubiquinol-1 analog. The advantage of the PL system is evident when one compares AOX catalytic rates before and after incorporation of pure protein into PLs. Indeed, the fungal AOXs presented here had no detectable enzymatic activity after removal of the bacterial membrane (Fig. 1C) which was promptly restored following incorporation into PLs (Fig. 2D,E). It is noteworthy that the enzymatic activity of pure AOX in solution is not a good predictor of enzymatic activity in the PL. Based on results obtained with pure (solubilised) protein, TAO has been considered to possess the highest V max to date whereas fungal AOXs have shown notoriously low rates in comparison 42 . However our results demonstrate that C. albicans and C. auris AOX can also possess high V max values and indeed under appropriate conditions surpass that of TAO. Moreover, AOX turnover rates obtained here are comparable to those of other mitochondrial quinol oxidases (the cytochrome bc 1 complex) and a quinone reductase (complex I) in PLs, namely 620 s −1 and 380 s −1 , respectively 29,43 .
Of particular importance with respect to the use of PLs is that the substrate concentration is not limited by its solubility in aqueous medium which has previously hindered the determination of the true kinetic parameters of AOX 43 . Previous attempts at characterizing AOX have used the hydrophilic ubiquinol-1 analogue which can be used only up to ca. 600 µM before forming micelles. K m values for Q 1 lie in the micromolar range of concentrations 33,42 , however our results using PLs with the natural substrate Q 10 demonstrate that true K m values for AOX are greater by 2 orders of magnitude namely 4-8 mM. The ubiquinol K m value reported for the cytochrome bc 1 complex (~ 10 µM) is, however, much lower than values for AOX, although it is not inconceivable that such values are possibly underestimations since the partitioning of ubiquinol into the lipid phase was not taken into consideration 43 . However, in a biological context this fits well with the AOX's role within the mitochondria-having a significantly higher K m for ubiquinol than that of the cyctochrome bc 1 complex would mean that ubiquinol would preferentially bind to the latter, with the AOX only effectively being engaged as an overflow at time when the ubiquinol pool was highly reduced 21,33 .
The interest in using AOX as a therapeutic target to treat both human and plant pathogens is growing 24,[35][36][37][38][39][40] , and a number of animal studies have implicated AOX for the treatment of human mitochondrial diseases using gene therapy [44][45][46][47][48][49] . The work undertaken here has shown that all 3 ascofuranone derivatives tested are promising candidates with AOX inhibition with IC 50 values in the nM region, with colletochlorin B being the most effective. However, it should be noted that there is a significant decrease in efficacy of the compounds with the fungal AOX's when compared to TAO, requiring a 250 fold increase in concentration. These values are reasonably consistent with those seen against other fungal AOX's 50 , which has largely been attributed to the decrease in size of the hydrophobic cavity in the fungal species when compared to TAO being problematic for inhibitor egress 42,51 .
Novel drug-like molecules are therefore urgently required to translate the recent findings relating to AOX, its role in C. auris and it's potential use as a therapeutic target for treating candidiasis particularly within a Covid-19 setting. We conclude that the incorporation of AOX into the PL system serves as an important tool not only for a more accurate characterisation of AOX catalytic rates but more importantly such a system is required to translate novel drug design currently underway in laboratories across the world into effective therapies.

Materials and methods
Expression and purification of NDH-2. NDH-2 protein expression and purification was based on the method of Heikal et al. 26 . Fresh E. coli C41 transformants carrying the plasmid pTRCndh2 were used to inoculate 10 ml starter cultures of L broth, supplemented with 100 µg ml −1 ampicillin, for growth over-night at 37 °C,180 rpm. Each 10 ml starter culture was used to inoculate 1 L of L-broth supplemented with 100 µg ml −1 carbenicillin for growth at 37 °C, 180 rpm. Once an OD 600 of 0.5 was reached expression of NDH-2 was induced by the addition of 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). Once growth plateaued (typically OD of 1.5 to 1.9) cells were harvested by centrifugation at 7000 × g for 15 min at 4 °C, weighed, and resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 2 mM MgCl 2 , EDTA free protease inhibitor cocktail (Roche)). The cells were lysed using two passes through a pre-cooled cell disruption system (Constant Systems Ltd) set to 30 kPa of pressure. The cell lysate was centrifuged for 15 min at 11,500 × g at 4 °C to collect cells debris and non-lysed cells.
To collect membranes, the supernatant was centrifuged for 60 min at 200,000 × g, 4 °C. Membranes were resuspended in 50 mM Tris HCl pH 8.0 at a concentration of 10 mg ml −1 and solubilised by drop wise addition into an equal volume of solubilisation buffer (50 mM Tris HCl pH 8.0, 2% (w/v) n-octyl-β-D-glucopyranoside (OG), 4 mM imidazole, 150 mM NaCl, EDTA free protease inhibitor cocktail (Roche)) whilst stirring at 4 °C followed by 60 min rotation at 4 °C. Finally, the sample was centrifuged for 30 min at 200,000 × g, 4 °C to collect solubilized membranes.
NDH-2-His was purified by passage through a 5 ml HiTrap TALON Crude column attached to an ÄKTA FPLC (GE Healthcare Life Sciences). The column was equilibrated at a flow rate of 1 ml per minute in binding buffer (50 mM Tris-HCl pH 8.0, 1% (w/v) OG, 2 mM imidazole, 150 mM NaCl). Protein was bound to the column at a flow rate of 0.5 ml min −1 and washed at a flow rate of 1 ml min −1 until the absorbance at A 280 nm had returned to the baseline. Protein was eluted with elution buffer (50 mM Tris HCl pH 8, 150 mM NaCl, 400 mM imidazole and either 0.05% (w/v) n-Dodecyl β-maltoside (DDM) or 1% (w/v) OG). Peak fractions were collected in a 96 well plate. Fractions containing NDH-2 were pooled, concentrated ~ tenfold via filtration using a 30 kDa weight cut off spin concentrator, supplemented with 10% glycerol and protease inhibitors, aliquoted, flash-frozen in liquid nitrogen and stored at − 80 °C. NDH-2 activity rates were measured pre and post freezing. www.nature.com/scientificreports/ NDH-2 activity assay. The specific activity of the enzyme was measured spectrophotometrically by measuring the oxidation of NADH at A 340 nm. The enzyme was diluted 1 in 50 in 50 mM MOPS pH 7.5 containing 250 µM Q 1 and 300 µM NADH. Typical activity measurements were approximately 100 µmol NADH oxidized min −1 mg −1 NDH-2.
AOX plasmid construction. The AOX from T. brucei, C. albicans and C. auris (GenBank accession numbers: AB070617, AF116872 and XP_018165728 respectively), lacking the mitochondrial targeting sequences (24, 26 and 24 residues respectively), were expressed in a modified version of pET15b, first described by Fedor et. al 29 . in which the 6xHis tag was replaced with a twin-strep tag. Gene constructs were synthesised with codon optimization for expression in E. coli (GenScript, Piscataway, NJ), and contained NdeI and BamHI restriction endonuclease sites at 5' and 3' ends respectively, for insertion into the pET15b.TS expression vector. The resulting plasmids were named pTS.TAO, pTS.CalbAOX2 and pTS.CaAOX.

AOX-strep protein expression and membrane isolation.
Over-expression of AOX was carried out in E. coli strain FN102 32 . There are two AOX genes in C. albicans, AOX1 and AOX2, whilst the former is constitutively expressed, expression of AOX2 is dependent on the growth stage and can be induced by inhibition of complex III or IV of the classical respiratory system. AOX2 was selected for these experiments. AOX-strep was expressed in E. coli as described in Kido et. al. 33 . The protocol described was followed except for the following adaptions: two 50 ml day cultures of K-broth (supplemented with 100 µg ml −1 carbenicillin, 100 µg ml −1 kanamycin, 50 µg ml −1 5-aminolevulinic acid (ALA), 0.2% (w/v) glucose and 50 µl per flask of metal mix (containing 0.5 g of MgSO 4 ·7H 2 O, 0.25 g of FeSO 4 ·7H 2 O, 0.25 g FeCl 3 per 10 ml total volume) were inoculated with 1 ml of over-night culture (Luria broth inoculated with a single bacterial colony, supplemented with 100 µg ml −1 ampicillin, 100 µg ml −1 kanamycin, and 50 µg ml −1 ALA). Cultures were grown at 37 °C in a shaking incubator until OD 600 reached approximately 0.6. Cells were collected by centrifugation, washed to remove the majority of ALA and used to inoculate 4 × 1 L of K-broth (supplemented with 100 µg ml −1 carbenicillin, 100 µg ml −1 kanamycin, 0.2% (w/v) glucose and 1 ml of metal mix) as described in the referenced protocol. AOX activity assay. The specific activity of the purified protein was measured spectrophotometrically by measuring the oxidation of Q 1 H 2 at A 278 nm. The activity was measured in 50 mM MOPS (KOH) pH 7.5 containing 150 µM Q 1 H 2 .
Western blotting. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and visualised with the appropriate antibodies followed by HRP-conjugated secondary antibodies (DAKO, 1:2000). HRP activity was detected using Pierce ECL Western Blotting Substrate followed by exposure to Amersham Hyperfilm. Antibody dilutions used for Western blotting were: anti-AOX (AOA) 52  Preparation of proteoliposomes. PLs were prepared exactly as described in previously published protocols using phospholipids; phosphatidylcholine, phosphatidylethanolamine and cardiolipin, mixed at a ratio of 8:1:1 25,29 . A typical preparation used a total of 10 mg phospholipids and 100 nmol of Q 10 , both of which are essential for PL activity 50 . The only modifications to the protocol were the detergent used to partially solubilise the phospholipid/Q 10 mixture (0.5% DDM in our preparations) and the protein content of the phospholipid vesicles as follows. For IC 50 measurements, 5 µg of AOX protein and 500 µg of NDH-2 were incorporated into the phospholipid vesicles. 100 nmol of Q 10 were added whilst preparing the liposome vesicles for IC 50 measurements.
For measurement of K m and V max 50 µg of AOX protein was incorporated into the phospholipid vesicles. NDH-2 protein was omitted from the PL preparation and instead added directly to the activity assay www.nature.com/scientificreports/ at a concentration to ensure the AOX was rate limiting. A typical preparation resulted in a ratio of 1:100:200 AOX:phospholipid:NDH-2. Between 0 and 160 nmol of Q 10 were added whilst preparing the liposome vesicle for the measurement of K m and V max .
Quantification of protein, Q 10 and lipid incorporation into the phospholipids. Protein and Q 10 quantification were performed as described in previously published protocols 29 . The quantification of both protein (using the Amido black assay) and Q 10 (by HPLC) was performed after the completion of the experiments to ensure any loss of protein or Q 10 during PL preparation did not effect these measurements. Lipid content was determined using the AMES test as described in previously published protocols 25 .
Activity measurements in proteoliposomes. Activity assays were carried out in solutions containing 65 mM MOPS pH 7.5 and 300 µM NADH. AOX was inhibited, when required, by addition of AOX inhibitors at final concentrations ranging from 2.5 pM to 25 µM for all inhibitors except for SHAM where the highest concentration was 500 µM. The selectivity of those inhibitors was evaluated with PLs containing only NDH-2 for which 300 µM 2,6-dichlorophenolindophenol (DCPIP) was used in the reaction medium as an electron acceptor instead of AOX. Rates of NADH oxidation were measured spectrophotometrically at A 340 nm using a Multiscan Sky 96-well plate reader (Thermo Scientific) and baseline rates from the reaction components alone (i.e. in the absence of any enzyme) were subtracted. Additionally, SHAM dose-response assays were performed in the high resolution oxygraph Oroboros Q2k (NextGenO2k, Oroboros Inc.) at concentrations as high as 20 mM by monitoring the PL oxygen consumption rate. The Oroboros was selected for SHAM dose-response assays due to the absorbance spectrum of the compound overlapping with the NADH signal. All other assays were performed using the plate reader for high throughput and to reduce reagent use.
Ubiquinone redox poise measurement. Simultaneous measurement of oxygen consumption and the ubiquinol/ubiquinone ratio was performed in a Oroboros O2k with a glassy carbon and platinum electrodes 28 in 65 mM MOPS (KOH) pH 7.5 and 2 µM Q 2 . 5 µL of a PL prepared without protein (only lipids and 100 nmol Q 10 ) were added in to the 2 ml measurement chamber followed by NADH, NDH-2, AOX and ascofuranone at concentrations described within the results section.
Statistics and reproducibility. Measurements were performed in technical triplicate and each experiment was performed three independent times (e.g. distinct protein purification batches) unless otherwise stated and similar results were obtained each time. No data points were excluded from the analyses. Data fitting and plotting were performed with GraphPad Prism 7.0 (GraphPad Software Inc.). AOX Kinetic parameters and IC 50 values were evaluated with the 2-way ANOVA test followed by Tukey's multiple comparison test.

Data availability
All data used in this study are included within the main text or provided in the supplementary files. Source data for graphs/charts in the main figures is available in Supplementary Data 1. Any additional information and biological material described in the study is available from the corresponding authors upon request.