Effects Of ADH3 On Candida Albicans Virulence And Resistance To Macrophage Killing

Objective:ADH3 encodes alcohol dehydrogenase III?Adh3p?,which is mainly involved in ethanol degradation,but its effects on pathogenicity of pathogenic microorganisms have not been reported.We knocked out ADH3 from Candida albicans in order to explore its effects on Candida albicans virulence and resistance to macrophage killing,and to provide support for clarifying the pathogenicity of ADH3 to the host and its mechanism.Method:Part I:The wild type?WT?strain SC5314 of Candida albicans was used as the parent strain to construct ADH3 double mutant strains?adh3?/??by SAT1-Flipper gene knockout strategy.The success of ADH3 knockout was verified by PCR and sequencing.Part II:?1?The OD₆₀₀00 of WT and adh3?/?at 0-96 h through in vitro growth kinetics experiments and growth curves were plotted to determine the effects of ADH3 deletion on the growth of Candida albicans.?2?To study the effects of ADH3 gene on the ability of hyphae formation,we observed the hyphae formation of strains by culturing on solid and liquid hyphae induction medium.?3?To study the effects of ADH3 gene on the ability of surface hydrophobicity,we observed hydrocarbon adsorption of strains by microbial hydrocarbon adsorption method.?4?To study the effects of ADH3 gene on the biofilm formation,we observed biofilm activity of strains by XTT method.?5?To study the effects of ADH3 gene on Candida albicans adhesion,we observed strains adhereed the 12-well plate by adhesion test.Part III:?1?To explore the effects of ADH3 gene on the killing effect of immune cells,the strains were co-cultured with THP-1 macrophage in vitro.?2?To explore the effects of ADH3 gene on the endogenousr reactive oxygen species?ROS?,we observed the ROS of strains by DCFH-CA probe.?3?To explore the effects of ADH3 gene on redox balance,we observed the NAD⁺/NADH of strains by using NAD⁺/NADH-GloTM Assay kit.Result:Part I:The results of PCR showed that the band size of parents was 1224 bp,and that of ADH3 knockout strains was 555 bp.There was only a single target band,indicating that the double allele of ADH3 had been knocked out.The sequencing results of PCR further showed that the parent strain contained complete ADH3 sequence,while the knockout strain did not contain ADH3sequence,suggesting that the ADH3 double mutant strains were successfully constructed.Part II:?1?In vitro growth kinetics showed that there was no difference in growth curve between strains,and the doubling time of adh3?/?and WT were 2.46 h and 2.39 h,respectively,with no significant difference?P>0.05?,indicating that the deletion of ADH3 did not affect the growth and reproduction of Candida albicans.?2?The results of liquid mycelium formation experiment showed that adh3?/?and WT could form normal slender mycelium.There was no significant difference in mycelium morphology,length and germination rate.Solid mycelium formation experiment showed that strains could form larger colonies,which were close to the surface of culture medium,with obvious wrinkles on the surface of colonies,dense interweaving of mycelia on the edge and growth infiltration.There was no significant difference in hyphal morphology between the two strains,suggesting that the absence of ADH3 did not affect the mycelial formation ability of Candida albicans.?3?The results of cell surface hydrophobicity showed that the average relative hydrophobicity of adh3?/?was 34.5%,and that of WT was47.6%.The difference was statistically significant?P<0.05?,indicating that the absence of ADH3reduced the cell surface hydrophobicity of Candida albicans.?4?XTT results showed that the average absorbance of adh3?/?was 1.81,and that of WT was 1.84.There was no significant difference?P>0.05?,indicating that the absence of ADH3 did not affect the activity of Candida albicans biofilm.?5?Adhesion test showed that there was no significant difference between adh3?/?and WT in adhesion to 12-well plate,indicating that the absence of ADH3 did not affect the adhesion ability of Candida albicans.Part III:The results of macrophage killing experiment showed that the average survival rate of adh3?/?and WT were 52.0%and 66.6%,respectively.The difference was statistically significant?P<0.05?,indicating that the deletion of ADH3 was not conducive to the resistance of Candida albicans to macrophage killing.?2?The results of intracellular ROS assay showed that the average ROS content of adh3?/?was 5689,and WT was 3564.The adh3?/?was 1.6 times higher than the WT.The difference was statistically significant?P<0.05?,indicating that ADH3deletion could increase the ROS.?3?The results of NAD⁺/NADH ratios showed that the average NAD⁺/NADH ratios of adh3?/?and WT were 20.2 and 15.1,respectively.The adh3?/?was 1.3times higher than WT,and the difference was statistically significant?P<0.05?,indicating that the reduction equivalent of Candida albicans decreased after the deletion of ADH3.Conclusion:The Candida albicans ADH3 double mutant strains are successfully constructed bySAT1-Flipper gene knockout strategy.It is found that ADH3 cannot affect the virulence factors including growth,mycelial morphological transformation,biofilm and adhesion,but reduce the hydrophobicity of cell surface.The deletion of ADH3 reduces the resistance of Candida albicans to macrophage killing,which are probably due to the increase of ROS in cells and the decrease of hydrophobicity on cell surface.The study can provide basis for the further study on pathogenicity of Candida albicans to host and its mechanism.