Metabolic fitness of Candida albicans is indispensable for functional drug efflux, ergosterol, and chitin biosynthesis

Background and Purpose: The increment in fungal infections, particularly due to Candida species, is alarming due to the emergence of multidrug resistance (MDR). Hence, the identification of novel drug targets to circumvent the problem of MDR requires immediate attention. The metabolic pathway, such as glyoxylate cycle (GC), which utilizes key enzymes (isocitrate lyase [ICL] and malate synthase [MLS]), enables C. albicans to adapt under glucose-deficient conditions. This study uncovers the effect of GC disruption on the major MDR mechanisms of C. albicans as a human pathogenic fungus. Materials and Methods: For the purpose of the study, efflux pump activity was assessed by phenotypic susceptibilities in the presence of substrates rhodamine 6G (R6G) and Nile red, along with R6G extracellular concentration (527 nm). In addition, ergosterol content was estimated by the alcoholic potassium hydroxide hydrolysis method. The estimation of chitin was also accomplished by the absorbance (520 nm) of glucosamine released by acid hydrolysis. Results: The results revealed that the disruption of ICL enzyme gene (Δicl1) led to the impairment of the efflux activity of multidrug transporters belonging to the ATP - binding cassette superfamily. It was further shown that Δicl1 mutant exhibited diminished ergosterol and chitin contents. In addition, all abrogated phenotypes could be rescued in the reverting strain of Δicl1 mutant. Conclusion: Based on the findings, the disruption of GC affected efflux activity and the synthesis of ergosterol and chitin. The present study for the first time revealed that metabolic fitness was associated with functional drug efflux, ergosterol and chitin biosynthesis and validated GC as an antifungal target. However, further studies are needed to comprehend and exploit this therapeutic opportunity.


Introduction
andida albicans is a prevalent human fungal pathogen and leading cause of nosocomial infections, particularly in individuals with immunocompromised conditions, such as HIV/AIDS, cancer, transplantations, and neonatal infections [1,2]. The impeded progress in the development of new antifungal drugs and concomitant rise in multidrug resistance (MDR) have even worsened the problems associated with such infections [3,4]. Therefore, it is pertinent to identify novel drug targets to circumvent the phenomenon of MDR. The pathogenicity of C. albicans mainly depends upon adaptability to combat diverse stress conditions existing inside the hostile niche by the activation of various stress-induced pathways. Regarding this, the in vitro analysis of these pathways and their roles in stress adaptation have generated considerable interest to identify potential antifungal targets.
Although C. albicans mainly utilizes six-carbon glucose as the key source of metabolism, they frequently encounter sites with low carbon compounds, such as acetate and citrate, which must be utilized to trigger virulence [5,6]. Therefore, the metabolic fitness of C. albicans is a crucial factor, which is required as a strategy to adapt to nutrient-limited conditions and establish a successful infection. Glyoxylate cycle (GC) is one such pathway that operates under metabolic stress and acts as a homeostatic mechanism to adjust various metabolic activities in C. albicans.
In microorganisms, including C. albicans, GC acts as a metabolic shunt for tricarboxylic acid cycle to enable the consumption of the low number of carbon (C2) compounds in a two-step process when glucose is not readily present as a carbon source. Therefore, this cycle not only utilizes C2 compounds but also prevents the loss of the two carbons, bypassing the CO2 generating steps in Krebs cycle [7,8]. The key enzymes of GC include isocitrate lyase (ICL) and malate synthase (MLS) encoded by ICL1 and MLS1, respectively. These enzymatic pathways are absent in humans; therefore, they serve as attractive antifungal targets [9]. It has been already established that C. albicans strains lacking either ICL1 or MLS1 are less virulent [10,11]. However, a comprehensive study deciphering the role of GC in other cellular responses responsible for regulating drug resistance in C. albicans was still elusive. With this background in mind, the present study was conducted to elucidate the effect of GC disruption on drug efflux pump activity and levels of ergosterol and chitin in C. albicans. This is the first study that reports that functional GC is indispensable when one considers dissecting drug resistance mechanisms of C. albicans.

Spot assay
To check phenotypic susceptibility, spot assay was performed as described previously [13]. Briefly, 5 μl of five-fold serially diluted yeast cultures (cells suspended in normal saline to an OD600 of 0.1 nm) was spotted on YNBA plates, supplemented with 2% glycerol in the presence of R6G and NR. Growth difference was measured after incubation for 48 h at 30°C.

Rhodamine 6G Efflux
The efflux of R6G was determined as described previously [13]. Briefly, 1×10 6 yeast cells from the overnight-grown cultures of WT, Δicl1, and Δicl1+ ICL1 were transferred to YNB medium, supplemented with 2% glycerol and allowed to grow for 5 h. The cells were then pelleted, washed twice with phosphatebuffered saline (PBS; without glucose), and resuspended as a 2% cell suspension, corresponding to 10 8 cells in PBS without glucose.
The cells were then de-energized to deplete any ATP for 45 min in 2-DOG (5 mM) and 2,4 DNP (5 mM) in PBS (without glucose). The de-energized cells were pelleted, washed, resuspended as a 2% cell suspension (wt/vol) in PBS without glucose, and then added with R6G at a final concentration of 10 µM and incubated for 40 min at 30°C. Subsequently, the equilibrated cells with R6G were washed and resuspended as a 2% cell suspension (wt/vol) in PBS without glucose. The samples with a volume of 1 ml were withdrawn at the indicated time and centrifuged at 9,000 g for 2 min. The supernatant was then collected, and absorption was measured at 527 nm. Energy-dependent efflux (at the indicated time) was measured after the addition of glucose (2%) to the cells resuspended in PBS (without glucose). Glucose-free negative controls were also included in all experiments.

Quantitation of ergosterol
Sterols were extracted by the alcoholic KOH method; additionally, the percentage of ergosterol was calculated as described previously [14]. Briefly, a single C. albicans colony from the overnight cultures of WT, Δicl1, and Δicl1+ICL1 was used to inoculate 50 mL YNB medium, supplemented with 2% glycerol. Both ergosterol and 24(28)-DHE were absorbed at 281.5 nm, whereas only 24(28)-DHE was absorbed at 230 nm. Ergosterol content was determined by subtracting the amount of 24(28)-DHE (calculated from OD230) from the total ergosterol plus 24(28)-DHE content (calculated from OD281.5). Ergosterol content was measured as the percentage of the wet weight of the cells using the following equations:

Chitin estimation
The chitin content was estimated by measuring the absorbance of glucosamine released by acid hydrolysis as described previously with some modifications [15]. Briefly, the overnight-grown cultures of WT, Δicl1, and Δicl1+ICL1 were inoculated in YNB medium supplemented with 2% glycerol, and 1×10 6 yeast cells were collected by centrifugation. The cells were then washed, suspended in sterile distilled water, and disrupted with 0.5-mm glass beads in a homogenizer. mL of 6M HCl at 100°C for 17 h. After evaporation at 65°C, 1 mL of sterile distilled water was added. A volume of 0.1 mL of this sample was mixed with 0.1 mL of 1.5 M Na2CO3 in 4% acetyl acetone. The mixture was incubated at 100°C for 20 min, and then added with 0.7 mL of 96% ethanol and 0.1 mL of 1.6 g of p-dimethylaminobenzaldehyde in 30 mL of concentrated HCl and 30 mL of ethanol. After 1 h, the absorbance was measured at 520 nm using a spectrophotometer. The glucosamine concentration in each sample was determined from a standard curve of 0-0.3 mg/ml of glucosamine. Subsequently, chitin content was calculated as the percentage of cell wall dry weight.

Statistical analysis
All experiments were performed in triplicates (n=3). The results were reported as mean±standard deviation and analyzed using Student's t-test in which only a p-value of < 0.05 for WT vs. Δicl1 was considered statistically significant.

Ethical considerations
Not applicable in this study.

Inhibitory effect of glyoxylate cycle disruption against ATP-binding cassette superfamily efflux pumps activity
Initially, the phenotypic susceptibility of WT, Δicl1, and Δicl1+ICL1 was monitored in the presence of R6G and NR. The results were indicative of the susceptibility of Δicl1 to R6G with no effect on WT and Δicl1+ICL1 strains. This suggested that the disruption of ICL1 led to the induction of some inhibitory effects against ABC superfamily transporters functioning as R6G in their specific substrate ( Figure  1). Additionally, in order to investigate the probable effects of ICL1 disruption on major facilitator superfamily (MFS) transporters, the susceptibility of substrate NR was evaluated. Based on the results, Δicl1 was not susceptible to NR, confirming that ICL1 deletion has no effect on the activity of MFS transporters (Figure 1).
To further confirm this hypothesis, the functionality of efflux pumps in WT, Δicl1, and Δicl1+ ICL1 was checked by monitoring R6G efflux, which is a widely used substrate for ABC transporters. To our expectations, the extracellular concentration of R6G was decreased in Δicl1 mutant in comparison to those in WT and Δicl1+ICL1 strains, confirming the impaired activity of ABC superfamily drug transporters (Figure 2).

Effect of glyoxylate cycle disruption on reducing ergosterol content
The perturbation of membrane structure and function in response to GC disruption was also evident from the fact that ergosterol levels, which are targetsfor azole and polyenes, were considerably reduced. Accordingly, C. albicans ergosterol biosynthesis was investigated based on the total ergosterol isolated by alcoholic KOH as described previously. The results revealed a significant decline (P<0.05) in ergosterol levels in Δicl1 mutant that could be rescued in the revertant Δicl1+ ICL1 strain (Figure 3).

Effect of glyoxylate cycle disruption on reducing chitin content
This study also involved the examination of the effect of GC disruption on chitin biosynthesis, which is a target for echinocandins. The cell wall is a characteristic structure of fungi; moreover, components of the fungal cell wall are not present in humans; accordingly, this structure is an excellent target for antifungal therapy. To this end, the chitin content in Δicl1 mutant was estimated, and the results revealed the reduction of chitin content as a result of ICL1 deletion in comparison to those subjected to WT and Δicl1+ICL1 (revertant) strains (Figure 4). These observations suggested that the disruption of GC also affected cell wall integrity.

Discussion
Candidiasis is one of the major causes of nosocomial infections with a high mortality rate across the world. Among the various mechanisms, which drive Candida to escape from the action of antifungal drugs, the overexpression of drug efflux pumps, as well as alteration in cell membrane and cell wall, are the most dominant strategies for drug resistance. The GC is one of the key cycles, which enables C. albicans to survive in a wide variety of glucose-depleted niches to establish candidiasis. Recently, GC cycle has been found to be essential for the virulence of several other pathogens and explored as a potential drug target since it is absent in humans.
In C. albicans, MDR is mediated by drug efflux pumps, which belong to either ABC superfamily or MFS [16,17]. The major transporters involved are Candida drug resistance proteins, namely CaCdr1p and CaCdr2p, from ABC superfamily, along with CaMdr1p belonging to MFS [18,19]. These results established that blockage in GC would lead to impaired efflux pump activity. In addition, the enhanced susceptibility in the presence of R6G contrary to NR reflected that GC disruption would result in the inhibition of the activity of drug efflux pumps belonging to ABC superfamily ( Figure 1). Moreover, the reduced exracellular concentration of R6G in Δicl1 further confirmed the specific inhibition of ABC transporter efflux (Figure 2).
The cell membrane of C. albicans is a critical interface that mediates a variety of functions, including sensing and signaling to the external environment [20]. In this study, the inhibition of efflux pump activity encouraged us to examine the composition of the cell membrane more closely. Hence, we quantified the level of ergosterol, which is one of the main components of the cell membrane and known to regulate drug resistance in C. albicans [21]. The result confirmed that GC disruption leads to perturbation in the cell membrane homeostasis. Moreover, the reduction of ergosterol level in Δicl1 could be one of the reasons for the impaired efflux pump activity observed while considering the fact that ABC transporters display different lipid specificities and are targeted to membrane rafts rich in ergosterol [21,22], which gets depleted in Δicl1 mutant.
The disruption of membrane homeostasis further prompted us to study cell wall, which is another crucial target for drugs belonging to the class of echinocandins, administered against C. albicans [23,24]. The cell wall is an essential component in fungal homeostasis. The lack of a covering wall in human cells makes this component an attractive target for antifungal development. Furthermore, elevated chitin content is associated with reduced susceptibility of Candida species to caspofungin. Given the observation of a decrease in chitin content in Δicl1 mutant in the present study, it was hypothesized that the disruption of GC also affected chitin biosynthesis.

Conclusion
Considering the fact that drug efflux is the major MDR mechanism and that ergosterol and chitin biosynthesis are the targets for antifungal azoles and echinocandins, the crosstalks exposed in this study were only fitting. Taken together, the data presented in the current study revealed the indispensability of GC for functional drug efflux, as well as membrane and cell wall integrity in C. albicans. However, the mechanisms that underlie this crosstalk remain to be elucidated. Based on these findings, it is required to design the pharmacological inhibitors of GC to aid in combating MDR. The identification of these mechanisms may hold promise for revealing new therapeutic targets for the treatment of life-threatening fungal diseases.