The Role Of Candida Albicans F₁F_o-ATP Synthase I/j Subunit And K Subunit In Mice Lethal Infection Model

Candida albicans is a common conditional-pathogenic fungus.Due to the large amount of antifungal drugs used in the treatment process,the drug resistance of Candida albicans is becoming more and more serious.Therefore,in order to better develop new antifungal drugs,we need to study the pathogenic mechanism of Candida albicans.F₁F_o-ATP synthase is responsible for the synthesis of more than90%of ATP in the cell.Its overall structure is relatively conserved from fungi to mammals,but the i/j subunit and k subunit are unique to fungal F₁F_o-ATP synthase.The i/j subunit and k subunit are very likely to become new targets for antifungal drugs,so we need to understand the role of i/j subunit and k subunit in the lethal infection of Candida albicans on host.ObjectiveIn order to study whether and how the two subunits are involved in lethal infection in mice,Candida albicans F₁F_o-ATP synthase i/j subunit coding gene ATP18 deletion strain and k subunit coding gene ATP19 deletion strain were constructed by using the SAT1-Flipper gene knockout strategy.MethodsPart 1:SAT1-Flipper gene knockout strategy was used to construct ATP18 gene deletion strain and ATP19 gene deletion strain.Part 2:By constructing mice infection models,we compared and analyzed the survival rate,organ bacterial load,serum biochemical detection and histopathology of the mice.Thus,we studied the role of F₁F_o-ATP synthase subunit i/j and k in lethal infection of Candida albicans in mice.Part 3:Studied the effect of F₁F_o-ATP synthase i/j subunit and k subunit on the growth and mitochondrial function of Candida albicans.?1?The growth activity of the ATP18 gene deletion strain and the ATP19 gene deletion strain in vitro was measured by in vitro growth experiments.?2?The mitochondrial function of the ATP18 gene deletion strain and the ATP19gene deletion strain were measured by growth curve experiment,spot assay,intracellular ATP content measurement experiment,mitochondrial membrane potential???m?measurement experiment and intracellular reactive oxygen species?ROS?level measurement experiment.Part 4:To study the effect of F₁F_o-ATP synthase i/j subunit and k subunit on the virulence factors of Candida albicans,the mycelium formation ability,adhesion ability,biofilm formation ability and pathogenicity-related gene expression levels of the ATP18 gene deletion strain and the ATP19 gene deletion strain were measured by the hyphae formation experiment,adhesion experiment,biofilm formation experiment and RT-q PCR.Part 5:Made a primary study on the mechanism of F₁F_o-ATP synthase i/j subunit in the involvement of Candida albicans in lethal infections in mice.?1?The cell wall structure and function of the ATP18 gene deletion strain and the ATP19 gene deletion strain were determined by drug experiment,Alcian blue-binding experiment.?2?The ability of the ATP18 gene deletion strain and the ATP19 gene deletion strain to be engulfed by the mouse macrophage-like cell line RAW264.7 were determined by the interaction experiment with macrophages.?3?The activity of the ATP18 gene deletion strain and the ATP19 gene deletion strain in the model of oxidative stress in simulated macrophages was determined by spot assay and growth curve experiment.ResultsPart 1:PCR results indicated that the ATP18 gene deletion strain and the ATP19gene deletion strain were successfully constructed,and the SAT1 screening marker was removed.RT-q PCR results indicated that the i/j subunit was deleted after the ATP18 gene was deleted,and the k subunit was deleted after the ATP19 gene was deleted.Part 2:?1?After the mice were infected,on the 35th day,67%of the mice infected with the ATP18 gene deletion strain survived,while the mice infected with the parent strain and the ATP19 gene deletion strain all died within 8 days.The ATP18 gene deletion strain was weakly virulent compared with the parent strain?P<0.05?,there was no significant difference between the ATP19 gene deletion strain and the parent strain.?2?Compared with the mice infected with the parent strain,the fungal load of the liver,spleen,and kidney of mice infected with the ATP18 gene deletion strain at 4time points were significantly reduced?P<0.05?,while the fungal load of the liver,spleen,and kidney of mice infected with the ATP19 gene deletion strain at 4 time points had no significant change?P>0.05?.?3?The parent strain and the ATP19 gene deletion strain had same significant effect on liver and kidney metabolism in mice,while the ATP18 gene deletion strain had no significant effect on liver and kidney metabolism in mice?P>0.05?.?4?Histopathological results showed that the kidneys of mice infected with the ATP19 gene deletion strain and the parent strain had significant histological changes,while the kidneys of mice infected with the ATP18 gene deletion strain had no significant histological changes.Compared with the parent strain and the ATP19 gene deletion strain,mycelial growth of the ATP18 gene deletion strain was much slower in brain tissue.Part 3:?1?The results of in vitro growth experiments showed that in the medium containing fermentable carbon source glucose,the growth trends of the parent strain,the ATP18 gene deletion strain and the ATP19 gene deletion strain were almost the same.All strains could grow normally,and the difference in doubling time was not statistically significant?P>0.05?.?2?The growth curve experiment results and the spot assay experiment results of Candida albicans in the non-fermented carbon source medium showed that in the non-fermented carbon source medium,the growth trends of the parent strain,the ATP18 gene deletion strain and the ATP19 gene deletion strain were almost the same.?3?The results of intracellular ATP content measurement showed that in the medium containing fermentable carbon source glucose and the medium containing non-fermentable carbon source,compared with the parent strain,the ATP content in the cells of the ATP18 gene deletion strain and the ATP19 gene deletion strain had no significant change?P>0.05?.?4?The results of Candida albicans mitochondrial membrane potential???m?and intracellular reactive oxygen species?ROS?level measurement showed that in culture medium containing fermentable carbon source glucose,compared with the parent strain,the ATP18 gene deletion strain and the ATP19 gene deletion strain had no significant change in the mitochondrial membrane potential and intracellular reactive oxygen levels?P>0.05?.Part 4:?1?The results of mycelium formation experiment,adhesion experiment and biofilm formation experiment showed that compared with the parent strain,there was no significant change in mycelia formation ability,adhesion ability and biofilm activity of the ATP18 gene deletion strain and the ATP19 gene deletion strain?P>0.05?.?2?RT-q PCR results showed that the expression levels of pathogenicity-related genes in the ATP18 gene deletion strain were significantly higher?P<0.05?compared to the parent strain,while the expression levels of pathogenicity-related genes in the ATP19 gene deletion strain had not obvious change?P>0.05?.Part 5:?1?The results of Candida albicans cell wall pressure drug experiments showed that at 37°C,the sensitivity of the ATP18 gene deletion strain to CFW was partially impaired compared with the parent strain and the ATP19 gene deletion strain.Alcian blue-binding experiment results showed that compared with the parent strain,there was no significant change in the content of mannosan in the cell wall of the ATP18gene deletion strain and the ATP19 gene deletion strain?P>0.05?.?2?The results of the interaction reaction with macrophages showed that the survival rate of the parent strain was?28.68±1.27?%,the survival rate of the ATP18gene deletion strain was?20.11±0.94?%,and the survival rate of the ATP18 gene deletion strain was lower than that of the parent strain?P<0.05?.The survival rate of the ATP19 gene deletion strain was?26.96±1.21?%,which was not different from that of the parent strain?P>0.05?.The damage rate of macrophages caused by the ATP18 gene deletion strain was 26%lower compared with the parent strain?P<0.05?.The damage rate of macrophages caused by the ATP19 gene deletion strain was not different from that caused by the parent strain?P>0.05?.?3?Spot assay and growth curve experiment results showed that in the presence of oxidant H₂O₂,the ATP18 gene deletion strain had obvious growth defects?P<0.05?.ConclusionIn this study,the ATP18 gene deletion strain and the ATP19 gene deletion strain were successfully constructed.After deleting the coding gene,the i/j subunit and k subunit of F₁F_o-ATP synthase were deleted,and after the i/j subunit deletion,the pathogenicity of Candida albicans to mice was reduced;it is clear that the i/j subunit and k subunit did not affect the growth activity in vitro,virulence factor and mitochondrial function of Candida albicans,but the impaired cell wall and increased sensitivity to oxidative stress played a key role in pathogenicity reduction of Candida albicans after the i/j subunit deletion.The completion of this study will provide a scientific basis for further screening of targeted antifungal drugs acting on F₁F_o-ATP synthase.