The Regulating Mechanism Of The Transcription Factors Gln3 And Stp1 Involved In The Autophagy And Rapamycin Resistance Of Candida Albicans

Candida albicans causes the highest mortality rate of fungal infection in patients with impaired immunity.Candida albicans adaptability within the host can enhance its own viability.Autophagy in eukaryotic cells is an adaptive response that occurs during the response and differentiation of cells and the autophagy research has become a hot topic in recent years.Obviously,it is important to study the effects of autophagy on Candida albicans and its adaptability.Rapamycin is a useful tool for inducing autophagy in Candida albicans and we took advantage of rapamycin to induce the transcription factor deletion strains library of Candida albicans autophagy.Using gene knockout techniques,we found the transcription factors involved in the regulation of Candida albicans autophagy and investigated the regulation mechanism of rapamycin-induced autophagy of Candida albicans.By the screening of transcription factors deletion library,Gln3 and Stp1 deletion strains were found to show resistance to rapamycin and probably have an effect on autophagy.Based on this discovery,we thought that it is necessary to explore whether Gln3 and Stp1 participate in the modulation of autophagy.First of all,Gln3 and Stp1 addback strains were constructed.By comparing these two genes deletion strains and addback strains,we could find increased susceptibility to rapamycin of Gln3 addback strain but no increased susceptibility of Stp1 addback.Nitrogen starvation is used to induce the occurrence of autophagy.It was found that Gln3 deletion strain show growth defects in the nitrogen deficiency medium.Since then,we concluded that the deletion of Gln3 may cause autophagy defects in Candida albicans.And a further study of these strains autophagy phenotypes should be conducted.All the parental strains,knockout strains and addbacks show no difference in the nitrogen deficiency medium.However,rapamycin could increase the growth of these strains in nitrogen deficient environment.Therefore,it could be seen that nitrogen deficiency induced autophagy and rapamycin induced autophagy are different in the regulation mechanism.It has been shown in previous research that the transcription factors Gln3 and Stp1 are degraded in the nitrogen deficient environment,which is consistent with the phenotype of Gln3 ?/? and Stp1 ?/? tolerant to rapamycin.This suggests that Gln3 and Stp1 are involved in the regulation of autophagy.The occurrence of autophagy usually leads to an increased expression of autophagy-related genes(ATG).Therefore,real-time PCR is used to detect the expression of ATG in this research.It was found that rapamycin could induce autophagy,and the expression of Gln3 and Stp1 would cause a high level of ATG gene expression.Autophagy is an intuitive indicator of autophagy.Also,in the rapamycin induced Gln3?/? and Stp1?/? strains,autophagic bodies could not bind with vacuoles through DIC observation.However,under the pressure of nitrogen deficiency(with Arg),all SN250,Gln3?/?,and Stp1?/? were able to form autophagosomes.But in the induction of rapamycin,the autophagy of Gln3?/? and Stp1?/? are markedly deficient.Stp1?/? cells were found to be depressed in YPD under the observation of DIC.This result made it necessary to concuct further exploration.It has been found that there exist similarity between cytoplasmic targeted vacuolar passage(CVT pathway)and autophagy process.And CVT defective Candida albicans cells also have defects in autophagy path.The vacuolar protein aminopeptidase(API)is thought to be a marker of the CVT pathway.When nutrition sufficient,the cytoplasmic API precursor was translocated to the vacuole and grew mature.But,when nutrition limited,API mainly was transported to the vacuoles through autophagosome.In the nutrition sufficient environment,the CVT pathway function normally could be tested by API's positioning in vacuoles.In the nutrition deficient environment,API's positioning is a marker to judge if there is deficiency in autophagy.The construction of API and green fluorescent protein fusion protein expression cells(GFP-API)is a way to achieve the positioning of API.It was found that the CVT pathway is not only defective in Stp1?/?,there was a significant presence of API precursors at the margins of Stp1?/? cytoplasm and vacuoles.API precursors did not enter vacuoles.The results were further verified by western-blot at the protein level.The result showed there were indeed immature APIs in and Stp1 ?/? and the immature APIs were degraded upon the effect of rapamycin.The location of API was observed in the nitrogen deficient environment.It is found that API precursors were present at the margins of Stp1?/? vacuoles.This result indicated that Stp1 regulate the expression of the CVT pathway.However,the specific molecular regulatory mechanism still needs to be further explored.In this study,we found that transcription factors Stp1 and Gln3 not only involved in the regulation of autophagy,but also involved in the regulation of autophagy similar CVT pathway gene expression.However,it was found that the autophagic phenotype induced by the nitrogen starvationwas not the same as that induced by RAPA.Since the occurrence of autophagy is a kind of nutritional hunger response,it can be induced by nitrogen starvation and a limited carbon source.Therefore,we replace the whole nutrient medium YPD glucose into citric acid,glycerol,and other carbon sources that need to generate ATP through different breathing modes.The above mentioned five strains of C.albicans in the citrate medium showed resistance to the RAPA.In the tricarboxylic acid cycle,the pyruvate carboxylase carboxylate carboxylation to form oxaloacetic acid,oxaloacetate and acetyl coenzyme A catalyzed by citrate synthase to form citric acid.By detecting the intracellular ATP levels,RAPA could enhance the intracellular ATP levels.Therefore,we speculate that RAPA acts on TCA and affects ATP production,but the formation of oxaloacetic acid is not hindered and may be blocked by the synthesis of citric acid.Based on the above findings,we deduce that during the RAPA induced autophagy,Gln3 ?/? and Stp1 ?/? and Stp1 ?/?+AB deficiencies make the citric acid synthase in the tricarboxylic acid cycle(TCA)disable to enter the vacuole for degradation,so that Candida albicans produce high levels of ATP and resulted in resistance to RAPA.We constructed the GFP-CIT1 p strains and observed its localization in five mentioned strains,CIT1 p is located outside of vacuolar compared with other strains.This phenotype confirmed our deductions mentioned above.In view of the fact that RAPA has been used for clinical antifungal infections for many years and the resistance to RAPA is an important phenomenon,we decided to further explore the RAPA-induced Gln3 ?/? and Stp1 ?/? biological phenotypes.We will use the RNA-Seq to detect the mRNA expression levels of Gln3 ?/? and Stp1 ?/? induced by RAPA and investigate its growth in the environment of limited nitrogen.These results will give us more clues to demonstrate the important biological function of the transcription factors Gln3 and Stp1.