Study On Functions Of Drug-resistance-related Gene MXR1in Candida Albicans

More recently, it has been wildly accepted that Candida albicans can exploit severalcellular responses to facilitate tolerance of antifungal agents and can further acquireresistance by multiple mechanisms. Nowadays the adaptation to oxidative stimulimechanism for Candida albicans has been considered to be very important. Manyliteratures have suggested that the oxidative stress may be associated with the azoleresistance of Candida albicans. Therefore, the study of the relationship between oxidativestress mechanism and drug sensitivity is very significant to further explain the drugresistance mechanism of Candida albicans.Goal We successfully built the MXR1gene deletion strains by the URA-Blaster genedeletion strategy. Subsequently, we studied the following phenotypes of the wild strain andthe MXR1mutant strains, including the growth curves, antifungal drug sensitivity,tolerance of H2O2, intracellular reactive oxygen species, glutathione redox system,mitochondrial membrane potential,4important drug-resistance related genes expression,3oxidative stress related genes expression, hyphae growth, Rhodamine efflux and theosmotic sensitivity changes.Method1. We constructed gene deletion plasmid vector by PCR amplification,restrictive enzymes cut and DNA connection method. The plasmid was based on p5921plasmid, including the up and downstream fragments of the MXR1gene ORF and thehisG-Ura3-HisG fragment. We identified the positive plasmids by PCR amplification, andsequencing method.2. Construction of gene mutant strains: We constructed the MXR1gene mutant strains by URA-Blaster strategy. We identified the positive strains by PCRamplification, and Southern-blot method.3. Growth curves: we cultured the wild strainsand MXR1mutant strains by the same concentration, measured the OD600of each strainat different time points, and obtained the growth curves of various strains.4. Drugsensitivity experiments: we studied the drug sensitivity of MXR1mutant strains by theMIC80and Spot-assay experiments.5. H2O2sensitivity experiments: we studied theH2O2sensitivity of MXR1mutant strains by co-culturing with different concentration ofH2O2, Spot-assay experiments.6. Intracellular reactive oxygen species (ROS): we studiedthe ROS content of MXR1mutant strains and wild strains by co-culturing with2,7-dihydro-dichloro-diethyl fluorescein (DCFH-DA). We measured the fluorescencevalues at different time points, so as to determine intracellular reactive oxygen species (ROS) content.7. Oxidized and reduced glutathione content: we studied the oxidized andreduced glutathione contents of MXR1mutant strains and wild strains with the glutathionedetection kit.8. Mitochondrial membrane potential: we studied the mitochondrialmembrane potential of MXR1mutant strains and wild strains with the mitochondrialmembrane potential kit.9. By Real-time RT-PCR method, we studied the expressionchanges of oxidative stress related genes SOD2, TRR1, GLR1between MXR1mutantstrains and wild strains.10. By Real-time RT-PCR method, we studied the expressionchanges of important drug resistance related genes CDR1, CDR2, MDR1and ERG11between MXR1mutant strains and wild strains.11. Hyphae formation: we observed thehyphae formation of strains by culturing on fetal bovine serum (FBS), Lee’s, and Spidermediums.12. The Rhodamine6G efflux experiment: we measured active transport abilityof MXR1mutant strains and wild strains by co-culturing with Rhodamine6G.13. Osmoticsensitivity measurement: we observed the osmotic sensitivity of MXR1mutant strains andwild strains with Spot-assay experiments on SDS, CaCl2, and LiCl mediums.Result1. We successfully constructed MXR1gene mutant strains by Ura-blasterstrategy and identified positive stains by Southern-blot analysis.2. We examined theantifungal drug-resistance of MXR1gene mutant strains and found that the azole drugsensitivity of MXR1mutant strains increased significantly, but at the same time, nosignificant change of the propylene amine and polyene drug sensitivity was observed.3. Inthis study, we successfully investigated the impact of MXR1mutant strains to intracellularoxidative stress mechanism.(1) The MXR1mutant strains obtained decreased tolerance toH2O2;(2) The MXR1mutant strains obtained increased intracellular reactive oxygenspecies levels;(3) The MXR1mutant strains obtained increased mitochondrial membranepotential;(4) In the MXR1mutant strains, some important redox-related gene (SOD2,TRR1, GLR1) expression had no significant change;(5) In the MXR1mutant strains, theintracellular GSH/GSSG ratio did not change. These experiment results suggested, MXR1gene of the Candida albicans, possibly played an important role in the oxidative stresssystem.4.(1) In the MXR1mutant strains, CDR1and MDR1expression increased slightly;(2) but CDR2, ERG11expression has no significant change;(3) In the MXR1mutantstrains, the hyphae formation has no significant change; it indicated that MXR1genes maybe not the virulence factor of Candida albicans;(4) MXR1gene deletion did not influenceosmotic sensitivity of Candida albicans.Conclusion In the MXR1mutant strains, the tolerance of H2O2decreased significantly, it indicated that MXR1gene may be involved in the oxidative stress systemof Candida albicans, resulting in the decreased ability of antioxidant. Later, we found thatin the MXR1mutant strains the intracellular reactive oxygen species (ROS) increasedsignificantly. This study demonstrated for the first time that the MXR1gene may reducethe clearance of ROS. Mitochondria are the main site of intracellular ROS production, andmitochondrial membrane potential is an important indicator of mitochondrialfunction. Intracellular reactive oxygen species could be infected by MXR1gene, so wefurther studied the impact of the mitochondrial membrane potential in the MXR1mutantstrains. Results showed that in the MXR1mutant strains, the mitochondrial membranepotential was significantly higher than the wild strains. So we inferred that the decreasedH2O2tolerance of MXR1mutant strains may be due to the increased ROS level and theelevated mitochondrial membrane potential. We further speculated, MXR1gene impactedthe oxidative stress possibly by elevating mitochondrial membrane potential and reducingROS scavenging. In addition, we also found that the MXR1gene deletion did not changeglutathione-reductase system; Some important redox related genes, such as GLR1(glutathione reductase gene), SOD2(manganese-superoxide dismutase gene) and TRR1(thioredoxin protein reductase gene) expression did not change significantly by real-timequantitative RT-PCR. These experiments described, the impact to oxidative stress systemof MXR1gene was not restored by these known oxidation related genes. MXR1wasinvolved in the regulation of oxidative stress in Candida albicans. Real-time RT-PCRresults showed that: in the MXR1gene mutant strains, CDR1and MDR1gene expressionincreased slightly, compared with the wild strain RM1000; but no significant change ofCDR2, and ERG11gene expression was observed. Therefore, we adopted the fluorescentdye rhodamine6G to detect the CDR1efflux ability of Candida albicans. There was nosignificant difference of the efflux of Rhodamine6G fluorescence between the wild strainsand MXR1mutant strains. While this was not consistent with the real-time RT-PCR resultsof CDR1expression rose slightly. We speculated that CDR1gene expression levelincreased only by2.5times, and did not change the encoding and translation process. Inaddition, we found MXR1gene did not change the hyphae formation and osmoticsensitivity. It has been reported that azoles can improve the intracellular reactive oxygenspecies in addition to inhibiting sterol biosynthesis; and the effect of miconazole can besignificantly inhibited by antioxidant; ROS plays an important role in the azole antifungalresistance mechanism; and increased ROS scavenging may be associated with the azole drug resistance. We proved MXR1gene mutant strains increased sensitivity to azoles, andcould lead to elevating the ROS level. So we speculated that MXR1gene deletion resultedin reducing of ROS scavenging, enhancing the killing effect of ROS, and increasingsensitivity to azole drugs such as fluconazole. While propylene amine and polyene had noclear relationship with oxidative stress, resulting that MXR1gene deletion did not changethe sensitivity of these two drugs. We speculated that the gene MXR1was involved in theoxidative stress mechanism of Candida albicans, through clearing intracellular reactiveoxygen (ROS), reducing the mitochondrial membrane potential, which led to resistance toazoles. The MXR1gene was a antifungal drug resistance-related gene of Candida albicans.