Mechanism Of Candida Albicans F₁F_o-ATP Synthase ? Subunit In Mice Lethal Infectious Model

No studies have been reported that F₁F_o-ATP synthase and its subunits are involved in the host infection process of Candida albicans?even other pathogenic fungi?.C.albicans has specific structure of F₁F_o-ATP synthase,most likely to be as a new target of antifungal drugs.Prior to this,it is firstly necessary to clarify the involvement of F₁F_o-ATP synthase in the infection process and its mechanism.ObjectiveMechanism of C.albicans F₁F_o-ATP synthase?subunit in mice lethal infectious model.MethodsPart 1:?1?ATP2 gene deletion?atp2?/??and reconstitution?atp2?/ATP2?mutants were constructed by SAT1-Flipper gene deletion strategy,then PCR and Southern blot were used to conform the results.?2?Spot assay was used to measure the growth of the strains.Part 2:We measured the pathogenicity of these strains in mice disseminated model,include survival rate,organ fungal burden and pathological analysis.Part 3:?1?Growth curves in vitro and cells growth in vivo were used to investigate the growth ability of strains,then colony counting and ATP concentration were used to measure cell vitality.?2?The main virulence factors were measured by hyphae formation,biofilm formation and adhesion experiment.?3?Macrophages?M??co-culture with C.albicans were used to measure the M?killing rate and M?cytotoxicity.?4?Spot assay,alcian blue binding and digital gene expression?DGE?experiments were used to test the sensitivity of cell wall pressure drugs?including Calcium Fluorescence,Congo Red and Caspofungin?,mannan content and transcriptome expression levels of of cell wall related genes.Part 4:?1?Growth curves and spot assay were used to measure growth ability in different carbon source media;colony counting and ATP concentration were used to measure cell vitality in different carbon source media.?2?Hyphae formation and invasion experiment were used to measure virulence of strains in different carbon source media.?3?Spot assay and alcian blue binding were used to test sensitivity of cell wall pressure drugs and mannan content in different carbon source media.Part 5:?1?Colony counting and ATP concentration at different time points were used to analysis the relationship of cell vitality and ATP level.?2?Mitochondrial membrane potential???m?and endogenous reactive oxygen species?ROS?level were used to reflect mitochondrial function.?3?The NAD/NADH ratio was used to determine the equivalent reduction of mutant strains.?4?DGE sequencing and data analysis were used to determine the gene transcriptome expression levels of carbon source metabolism and mitochondrial function.ResultsPart 1:?1?The results of PCR and Southern blot showed that we had constructed atp2?/?and atp2?/ATP2 successfully,and the SAT1 marker had been removed.?2?Spot assay showed that atp2?/?grew well and had no significant difference compared to the wild type?WT?strains.Part 2:In a murine model of disseminated candidiasis,mice infected atp2?/?survived post 30 days while all mice infected with WT were moribund on day 9;mice infected atp2?/?carried a lower fungal burden in tissues;there were no tissue damage and no inflammatory cells infiltration in kidney of mice infected atp2?/?,whereas severe tissue damage,hyphea aggregation and inflammatory cells infiltration happened in mice infected WT and atp2?/ATP2.Part 3:?1?Growth curve and doubling time results displayed that atp2?/?moderately reduced growth rate and the doubling time was prolonged by 0.5 h than WT,there were no significant differences between atp2?/ATP2 and WT?P>0.05?.All of the mice infected intravenously with either 1×10⁵ cells or 1×10⁶ cells of atp2?/?survived post 30 days infection.By contrast,mice infected with WT began to die on day 5 and all mice were moribund on day 14.?2?The ability of hyphae formation,biofilm formation and adhesion were impaired in atp2?/?.?3?M?killing rate was 6 fold higher when atp2?/?co-culture with M?and M?cytotoxicity was 2.5 fold lower than WT group?P<0.05?.?4?The sensitivity of cell wall pressure drugs,mannan content and mRNA expression levels of cell wall related genes had no significant differences between atp2?/?and WT?P>0.05?.Part 4:?1?WT had slower growth and less cellular activity in the non-fermented carbon source than glucose?P<0.05?.The OD₆₀₀00 of the atp2?/?lower than WT significantly in the non-fermented carbon source and did not change substantially,and the cell viability was lower than WT?P<0.05?,there was no significant difference between atp2?/ATP2 and WT.?2?Germination rate,hyphal length,and invasive ability of WT were lower in 2%non-fermentable carbon sources than in 2%glucose?P<0.05?,but higher in 0.2%glucose combined 2%non-fermented carbon source than2%non-fermented carbon source and 0.2%glucose alone,respectively.atp2?/?had lower germination rate,hyphal length,and invasive ability than WT?P<0.05?,and there was no significant difference between atp2?/ATP2 and WT.?3?The growth of WT were inhibited in non-fermented carbon sources media containing CFW or CR.The growth of atp2?/?were promoted CFW medium but inhibited in CR medium.The mannan content in WT grew in non-fermented carbon source was lower than that in glucose.The mannan content in atp2?/?was lower than WT,and there was no significant difference between atp2?/ATP2 and WT.Part 5:?1?The results of colony counting showed that the number of atp2?/?was higher than WT on the first 1-5 days in glucose and began to decrease on day 6,but the number decreased to 0 on day 7 in the non-fermented carbon source.ATP concentration of atp2?/?was higher than WT on the first 1-3 days in glucose and began to decrease on day 4,and it was drastically decreased in the non-fermented carbon source.There was no significant difference between atp2?/ATP2 and WT.?2???m of atp2?/?was significantly lower than that of WT,and ROS level of the atp2?/?had no significant difference from WT.Both??m and ROS had no significant differences between atp2?/ATP2 and WT.?3?The NAD/NADH ratio was higher in atp2?/?than in WT?P<0.05?.?4?The gene transcriptome expression levels of carbon source metabolism and mitochondrial function were up-grade in atp2?/?.ConclusionIn this study,we successfully constructed C.albicans F₁F_o-ATP synthase?subunit ATP2 gene deletion and reconstitution mutants.Then we confirmed that the atp2?/?mutant fails to lead to a fatal infection in mice disseminated model.We identified that the virulence factors and macrophage related factors played key roles in the mechanism.Furthermore,we clarified that the?subunit regulated the metabolic adaptation of carbon sources to maintain the virulence of C.albicans and to prevent phagocytic phagocytosis.The completion of this study will provide a scientific basis for the further screening of antifungal drugs that can act on this target.