Anaerobic fermentation and mitochondrial aerobic respiration of C. albicans compensate and regulate each other during the metabolic process, which makes it difficult to treat fungus infection clinically. In recent years, mitochondrial respiratory chain (MRC) has been considered as a new target for clinical antifungal therapy (Shirazi and Kontoyiannis, 2013; Chamilos et al., 2006).
However, it is not ideal to restrain the growth, reproduction and pathogenicity of C. albicans by inhibiting a complex of mitochondrial ECT (CI, CIII, CIV or CV). In the search for ideal drug targets that can affect multiple energy metabolisms, C. albicans ADHs have earned our particular attention, since this gene family is the key connection to link two major energy metabolisms in non-pathogenic S. cerevisiae. And we found that there was a certain relationship between ADH1 and the azole-resistance genes (Guo et al., 2013). In addition, ADH1 deletion can reduce the virulence of C. albicans, but has no effect on the growth and cell viability (Song et al., 2019).
There are few studies on the combined inhibition of C. albicans fermentation and mitochondrial respiratory pathway. Through comparisons among growth, hyphal formation phenotypes, apoptosis and mitochondrial function between ADH1 null mutant and WT with or without different respiratory inhibitors, we characterize the biological function and pathological roles of ADH1 in this study.
Relevant study showed that the inhibition of CIII or AOX on the growth of yeast cells is obvious, but rotenone (inhibitor of CI) has no obvious effect on proliferation of C. albicans cell (Ruy et al., 2006). We find that,the mitochondrial function of strain WT (SC5314) was slightly damaged by AA (inhibitor of CIII in classical pathway), including ATP and MMP, and AOX was the main respiratory pathway of mitochondria at present. In addition, ROS increased significantly and apoptosis increased slightly. AA also can increase the azole sensitivity and up-regulate the expression of virulence related genes ALS3 and HWP1 in strain WT. It is worth noting that even in the case of a 37% reduction in ATP production, the hyphal formation and cell growth rate did not change significantly. There was a similar situation in WT induced by SHAM (inhibitor of AOX), and CRC was the main respiratory pathway. Only with the combined action of AA and SHAM (PAR is the main respiratory pathway of mitochondria), hyphal formation was obviously limited, and the cell growth was slightly slowed down in strain WT. At this time, the decrease of ATP was similar to that induced by AA.
These results suggest that even if the mitochondrial function of C. albicans is slightly damaged (treated with AA or SHAM), the CRC or AOX pathway can compensate each other, which shows that the cell growth, reproduction and hyphal formation are less affected. Unlike S. cerevisiae, C. albicans is a typical facultative anaerobe that can switch between aerobic respiration and anaerobic pathways depending on the availability of oxygen. The latter allows it to gain more ATP out of glucose when oxygen is available. Also, the multiple-branched metabolic pathways afford C. albicans a more competitive advantage in adapting to different host environments. In spite of several studies focusing on ADH1 contributions to biofilm, virulence and immune response (Klotz et al., 2001; Mukherjee et al., 2006), the roles of C. albicans ADH1 and other ADH isomers in energy metabolism have not yet received much attention, due at least part to the assumption that respiration dominates energy generation in this organism.
The gene encoding ADH1 was disrupted from the C. albicans WT strain SC5314 by means of the SAT1- flipper method, and the ADH1 mutant (EX2) was used in this study. Deletion of ADH1 in this study showed subtle effects on biological phenotypes such as a WT-level azole susceptibility and a slightly delayed germination, perhaps due to a longer generation time observed by us and others (Kwak et al., 2014). We find that, except in cases where growth is truly constrained by ATP availability, all of these phenotypes depend more or less on mitochondrial respiration, since these phenotypes can be remarkably suppressed in both WT and mutant as long as mitochondrial respiration inhibitors are present.
On the other hand, ATP yield is only 35% of WT in ADH1 mutant, due largely to an impaired mitochondrial function. The latter is demonstrated by a 41% reduction of mitochondrial membrane potential, a 2.8-fold higher level of ROS, and more than 10% apoptosis in untreated ADH1 mutant over those of WT (under aerobic conditions). After this impaired respiration was further suppressed by AA, the ATP generating process is nearly abolished in the mutant strain, suggesting that ADH1 is a critical element for ATP generation from the CRC respiration pathway. In an earlier study (Bertram et al., 1996), ADH1 expression reached its maximum at late exponential growth in C. albicans. Under non-glucose media such as glycerol, galactose, lactate, ADH1 mRNA levels were high, but low in glucose or ethanol growth medium. The same authors also speculated that ADH1 is regulated at both the transcriptional and translational levels. We find that a slightly reduced growth of ADH1 mutant on 2% glucose YPD agar is also seen in ethanol, glycerol or oleic acid medium under aerobic conditions in this study. To date we have no answer for carbon dependency of ADH1 activity under aerobic and anaerobic growth conditions. Similar growth behavior on glucose and non-glucose media are perhaps the consequence of inter-substitution by other ADH isomers such as C. albicans Adh2p, which shows a 76 ~ 77% identity to S. cerevisiae Adh1p, Adh2p and Adh3p as well.
The above results suggest that it can’t effectively inhibit C. albicans cells by deleting ADH1 or inhibiting one mitochondrial respiratory pathway. We found that the hyphal formation of ADH1 mutant was obviously limited under the induction of respiratory inhibitor (AA). Even at very low concentrations of AA (0.25 µM), the mutant cells showed no signs of growth (MIC detection and spot assay). And AA could reduce the expression of ALS3 and HWP1 in strain EX2. These results suggest that CIII inhibitors can significantly reduce the pathogenicity of C. albicans ADH1 mutant. And the mutant became hypersensitive to azoles and apoptosis, which may be closely related to the severe damage of respiratory function.
Because ADH1 null mutant strain is highly sensitive to respiratory inhibitors, it is difficult to survive in large numbers in animals. Therefore, the cytotoxicity of both ADH1 and CIII on C. albicans cells is not further studied in rats.
The direct link between ethanol fermentation and mitochondrial metabolism is still something of a mystery in this organism. We have no evidence on which to build an interpretation of this ADH1-dependent respiration event. The divergent metabolic behavior of C. albicans compared to S. cerevisiae suggests that ADH is likely to have a different biological significance in C. albicans. Further characterization of each C. albicans ADH isomer with enzymatic kinetics, location determination and gene regulation in different growth conditions will eventually be needed in order to fully understand how alcohol fermentation links to other energy metabolic pathways in colonization and growth in vivo.
Anaerobic fermentation and mitochondrial aerobic respiration are involved in the energy metabolism of C. albicans. Each pathway contributes to the survival of the fungi and the pathogenicity to the host. Our results indicate that inhibition of anaerobic fermentation (Adh1) and CIII of CRC pathway can significantly attenuate the pathogenicity, thus effectively killing C. albicans cells. This discovery will provide a scientific basis for clinical search for better antifungal drugs and their targets.