本文選題：白色念珠菌 + 雷帕霉素��； 參考：《第二軍醫(yī)大學》2017年碩士論文

【摘要】：白念珠菌是臨床免疫力低下患者最容易感染的一種條件致病菌,且致死率極高,這與白念珠菌在宿主內(nèi)的高適應性有關。而且這種高適應性也使白念珠菌極易產(chǎn)生耐藥性,這也是臨床上對白念珠菌感染治療失敗的主要原因之一。已有的研究表明,白念珠菌在宿主體內(nèi)的高適應性與其染色體、基因組的不穩(wěn)定性,生物被膜的形成,形態(tài)的改變等密切相關。但已經(jīng)發(fā)現(xiàn)的機制也難以完全解釋白念珠菌的高適應性和高耐藥性。近年來,越來越多的研究發(fā)現(xiàn),不同的轉(zhuǎn)錄因子參與了對白念珠菌適應性和耐藥性的調(diào)控。本課題擬利用白念珠菌轉(zhuǎn)錄因子缺失菌庫,篩選發(fā)現(xiàn)調(diào)控白念珠菌適應性和耐藥性的新的轉(zhuǎn)錄因子,并研究其機制。因此,本課題利用48株白念珠菌轉(zhuǎn)錄因子缺失菌,篩選其對不同抗真菌藥物、化合物、以及酸堿、滲透壓等不同刺激的敏感性,發(fā)現(xiàn)了一批可能影響白念珠菌不同表型的轉(zhuǎn)錄因子。其中Gln3Δ/Δ和Stp1Δ/Δ對雷帕霉素高度耐受,而雷帕霉素結(jié)合蛋白TOR是白念珠菌自噬調(diào)控通路中的關鍵蛋白,因此推測,這兩個轉(zhuǎn)錄因子可能參與了對白念珠菌自噬的調(diào)控。細胞自噬是真核細胞在饑餓應答及分化等過程中發(fā)生的適應性反應,已成為近年來的研究熱點,但目前鮮見對白念珠菌自噬的研究。因此,本課題利用上述基因缺失菌,研究Gln3和Stp1是否參與調(diào)控白念珠菌自噬,并考察其可能的調(diào)控機制。已有研究表明,在正常培養(yǎng)條件下,細胞內(nèi)存在胞質(zhì)定向液泡通路(CVT通路),該通路與自噬發(fā)生的過程存在著相似性,并且有研究發(fā)現(xiàn),CVT通路缺陷的菌體,其自噬通路也存在缺陷。在CVT通路和自噬通路缺陷的菌體中,由于自噬小體與液泡融合受阻,細胞表面部分區(qū)域會呈現(xiàn)圓形凹陷。因此本實驗利用干涉相差顯微鏡(DIC)觀察比較YPD培養(yǎng)基中Gln3Δ/Δ、Stp1Δ/Δ和親本菌SN250的細胞表面,發(fā)現(xiàn)只有少數(shù)親本菌SN250和Gln3Δ/Δ的細胞的表面有圓形凹陷,而Stp1Δ/Δ的大部分細胞表面呈圓形凹陷,表明Stp1Δ/Δ的CVT通路缺陷。而經(jīng)RAPA誘導后,Gln3Δ/Δ和Stp1Δ/Δ的大部分細胞表面呈圓形凹陷,而SN250表面凹陷的細胞較少。表明Gln3Δ/Δ和Stp1Δ/Δ存在自噬缺陷。自噬的發(fā)生通常由自噬相關基因(ATG)的表達增高引起。因此,本課題通過Realtime-PCR檢測了不同ATG在m RNA水平上的表達情況,發(fā)現(xiàn)RAPA的確能夠使菌體TOR1、ATG等自噬相關基因的表達升高,而且大部分基因的升高幅度達10倍以上,Gln3Δ/Δ和Stp1Δ/Δ兩株缺失菌在RAPA作用后,上述自噬相關基因雖然也能升高,但升高幅度只在2~3倍左右。上述結(jié)果表明,RAPA不能有效誘導Gln3Δ/Δ和Stp1Δ/Δ中的自噬相關基因的表達,所以使上述缺失菌出現(xiàn)自噬缺陷。為了進一步考察驗證Gln3和Stp1對白念珠菌自噬的調(diào)控作用,本課題構(gòu)建了Gln3Δ/Δ+AB和Stp1Δ/Δ+AB兩株轉(zhuǎn)錄因子缺失菌的基因回復菌,并對其基因型進行了鑒定。同時考察回復菌對雷帕霉素(RAPA)的敏感性,發(fā)現(xiàn)Gln3Δ/Δ+AB對RAPA的敏感性增加,但Stp1Δ/Δ+AB并對RAPA的敏感性并沒有增加,這有可能與異位單拷貝基因回復有關。液泡蛋白氨基肽酶(API)被認為是CVT通路的標志性蛋白。當營養(yǎng)充足時,胞質(zhì)中的50k D的API前體通過CVT通路運向液泡,在液泡中活化,轉(zhuǎn)變?yōu)槌墒斓腁PI。但處于有限營養(yǎng)或饑餓環(huán)境時,API主要通過自噬小體運向液泡。因此,在營養(yǎng)充足時,可以通過定位GFP標記的API是否進入液泡,來考察CVT通路是否缺陷。經(jīng)過構(gòu)建API和綠色熒光蛋白融合蛋白表達菌體(GFP-API),可以定位觀察API在細胞內(nèi)的定位情況。在RAPA作用后,同時,也觀察到Gln3Δ/Δ和Stp1Δ/Δ胞漿及液泡邊緣明顯存有API前體,并未進入液泡。由于上述結(jié)果表明Stp1Δ/Δ中Cvt通路缺陷,對此結(jié)果用western-blot在蛋白水平進行驗證API-GFP前體蛋白的是否被降解,Western結(jié)果表明,Stp1Δ/Δ中有80k D的未成熟的API-GFP前體蛋白。這說明轉(zhuǎn)錄因子Stp1調(diào)控CVT通路基因表達,轉(zhuǎn)錄因子Stp1缺失,Cvt通路缺陷。并且轉(zhuǎn)錄因子Gln3和Stp1調(diào)控白念珠菌自噬,轉(zhuǎn)錄因子Gln3和Stp1缺失,自噬缺陷。具體的分子調(diào)控機制有待進一步研究。誘導自噬發(fā)生常通過氮饑餓方式,利用缺氮培養(yǎng)基SD-N(含Arg)同時誘導親本菌SN250和基因缺失菌Gln3Δ/Δ和Stp1Δ/Δ,基因回復菌Gln3Δ/Δ+AB和Stp1Δ/Δ+AB自噬。在點板實驗中SD-N培養(yǎng)基上生長并無差異,但是,當在加入RAPA后,增強了其在缺氮培養(yǎng)基上的生存能力。另外,在SD-N(含Arg)誘導的自噬中,發(fā)現(xiàn)以上5株實驗菌株中,液泡內(nèi)均有自噬小體。這說明,Gln3Δ/Δ和Stp1Δ/Δ能夠在氮源誘導的情況下發(fā)生自噬。在SD-N(含Arg)培養(yǎng)基中觀察API定位情況的實驗中發(fā)現(xiàn),與其余4株實驗菌體相比,在Gln3Δ/Δ液泡內(nèi)僅有少部分成熟的API。我們繼續(xù)對5株實驗菌體對SD-N(含Arg)的耐受性進行考察,發(fā)現(xiàn)Gln3Δ/Δ明顯對氮饑餓較為敏感。綜上結(jié)果說明,轉(zhuǎn)錄因子Gln3參與調(diào)控自噬,轉(zhuǎn)錄因子Gln3在參與調(diào)控SD-N誘導的自噬中起著重要的作用,并且可能對增強自噬起到正調(diào)控作用。研究至此,明顯發(fā)現(xiàn),以上實驗菌珠被SD-N(含Arg)誘導的自噬表型,與RAPA誘導的自噬表型并不相同。所以,我們試圖找出菌體耐受RAPA的調(diào)控機制。以上5株實驗菌株經(jīng)RAPA誘導后,胞內(nèi)ATP均增加。所以推測是有氧呼吸產(chǎn)生ATP環(huán)節(jié),在耐受RAPA中起著重要的作用。因此,將全營養(yǎng)培養(yǎng)基YPD中的葡萄糖更換成檸檬酸、甘油等需通過不同呼吸方式產(chǎn)生ATP并加以利用的碳源。結(jié)果顯示,以上5株實驗菌體在檸檬酸替換YPD中的葡萄糖培養(yǎng)基中,對RAPA誘導產(chǎn)生了抗性。在三羧酸循環(huán)(TCA)中,丙酮酸羧化酶將丙酮酸羧化后形成草酰乙酸,草酰乙酸與乙酰輔酶A經(jīng)檸檬酸合酶催化后形成檸檬酸。首先,推測可能是檸檬酸的合成受阻。根據(jù)以上研究結(jié)果,我們推測RAPA誘導的自噬中,Gln3Δ/Δ和Stp1Δ/Δ發(fā)生自噬缺陷,使得三羧酸循環(huán)(TCA)中檸檬酸合酶不能進入液泡內(nèi)進行降解,使得菌體不斷產(chǎn)生ATP加以利用,從而對RAPA產(chǎn)生了耐藥性。經(jīng)過構(gòu)建檸檬酸合酶(CIT1p)綠色熒光融合蛋白GFP-CIT1p菌體,觀察CIT1p定位發(fā)現(xiàn),在RAPA誘導后的以上5株實驗菌株中,Gln3Δ/Δ、Stp1Δ/Δ和Stp1Δ/Δ+AB中的CIT1p位于液泡外部,而液泡內(nèi)并不存在。所以,初步驗證了我們的推測,RAPA誘導的自噬中,Gln3Δ/Δ和Stp1Δ/Δ自噬缺陷,使得三羧酸循環(huán)(TCA)中檸檬酸合酶不能進入液泡內(nèi)進行降解,使得菌體能夠完整的進行TCA產(chǎn)生供給ATP,使得菌體對RAPA產(chǎn)生了抗性。通過RNA-Seq檢測RAPA誘導后的Gln3Δ/Δ和Stp1Δ/Δ的m RNA表達水平,以及考察在有限氮源的環(huán)境中的菌絲生長情況,等生物學表型考察,試圖發(fā)現(xiàn)轉(zhuǎn)錄因子Gln3和Stp1更重要的生物學功能。
[Abstract]:Candida albicans is one of the most susceptible pathogenic bacteria in clinical immunocompromised patients with high mortality, which is related to the high adaptability of Candida albicans in the host. And this high adaptability also makes Candida albicans highly susceptible to drug resistance, which is one of the main reasons for the clinical failure of Candida albicans infection. Studies have shown that the high adaptability of Candida albicans in the host is closely related to their chromosomes, genomic instability, biofilm formation, and morphological changes. However, the mechanisms that have been found are difficult to fully explain the high adaptability and high resistance of Candida albicans. In recent years, more and more studies have found different transcription factors. In order to regulate the adaptability and resistance of Candida albicans, we use the library of Candida albicans transcriptional factor deletion to screen new transcription factors that regulate the adaptability and resistance of Candida albicans, and study its mechanism. Therefore, this subject uses 48 strains of Candida albicans to screen for different antifungal agents. Gln3 Delta / delta and Stp1 Delta / delta are highly resistant to rapamycin, and rapamycin binding protein TOR is the key protein in the autophagy pathway of Candida albicans, thus speculating that these two transcription factors are available. The autophagy can be involved in the regulation of autophagy of Candida albicans. Autophagy is an adaptive response to the response and differentiation of eukaryotic cells in the process of starvation and differentiation. It has become a research hotspot in recent years. However, there are few studies on autophagy of Candida albicans. Therefore, this subject uses the above gene to study whether Gln3 and Stp1 are involved in the regulation of Candida albicans. The bacteria are autophagy and examine their possible regulatory mechanisms. Studies have shown that cells are in the cytoplasmic orientated vacuole pathway (CVT pathway) under normal culture conditions, and the pathway is similar to the process of autophagy, and studies have found that the bacteria in the CVT pathway are defective in the autophagic pathway. In the CVT pathway and the autophagic pathway, the pathway is deficient. In the trapped mycelium, the surface part of the cell surface will appear circular depression due to the fusion of autophagic bodies and vacuoles. Therefore, this experiment uses interference phase contrast microscope (DIC) to compare the cell surface of Gln3 Delta / Delta, Stp1 Delta / delta and parent strain SN250 in the YPD medium, and found that only a few cells with a few parent bacteria SN250 and Gln3 Delta / delta are round the surface of the cells. The surface of most cells in Stp1 Delta / delta showed a circular depression, indicating that the Stp1 Delta / delta CVT pathway was defective. After RAPA induction, most of the cells on the Gln3 Delta / delta and Stp1 Delta / delta cells showed a circular concave surface, while the cells in the SN250 surface were less. It showed that Gln3 Delta / delta and Stp1 Delta / delta existed self ophagy defects. Autophagy was usually caused by autophagy. The expression of ATG was increased. Therefore, we detected the expression of different ATG at the level of M RNA by Realtime-PCR. It was found that RAPA did increase the expression of autophagy related genes such as TOR1 and ATG, and the increase of most genes reached more than 10 times. Gln3 Delta / delta and Stp1 Delta / delta two strains were in RAPA action. Autophagy related genes could also increase, but the increase was only about 2~3 times. The results showed that RAPA could not effectively induce the expression of autophagy related genes in Gln3 Delta / delta and Stp1 Delta / Delta, so that the missing bacteria appeared to have autophagy defects. In order to further examine and verify the regulation of Gln3 and Stp1 on autophagy of Candida albicans The genotypes of Gln3 Delta / delta +AB and Stp1 Delta / delta +AB two strains were identified, and their genotypes were identified. Meanwhile, the sensitivity of restoring bacteria to rapamycin (RAPA) was examined. The sensitivity of Gln3 Delta / delta +AB to RAPA was increased, but Stp1 / delta +AB and RAPA sensitivity did not increase, which might be associated with the heterotopic single copy of Becky. The vacuolar protein aminopeptidase (API) is considered to be a marker protein of the CVT pathway. When sufficient nutrition is sufficient, the API precursor of the 50K D in the cytoplasm is transported to the vacuole through the CVT pathway, and in the vacuole, it is converted into a mature API., but in a limited or starving environment, API is transported to the vacuole mainly through autophagic bodies. Therefore, nutrition is in nutrition. When sufficient, whether or not the GFP labeled API enters the vacuole to investigate whether the CVT pathway is defective. After constructing the API and the green fluorescent protein fusion protein (GFP-API), the localization of API in the cell can be observed. After the action of RAPA, the Gln3 Delta / delta and Stp1 Delta / Delta cytoplasm and the vacuolar margin are also observed. There were API precursors and did not enter the vacuole. Because the above results showed that the Cvt pathway in Stp1 Delta / delta was defective, the results showed that the API-GFP precursor protein was degraded by Western-blot at the protein level. The Western results showed that the Stp1 Delta / Delta had immature API-GFP precursor egg white in 80K D. This indicates that the transcription factor Stp1 regulates the CVT pathway genes. Expression, transcription factor Stp1 deletion, Cvt pathway deficiency, and transcriptional factors Gln3 and Stp1 regulation of Candida albicans autophagy, transcription factor Gln3 and Stp1 deletion, autophagy defects. Specific molecular regulatory mechanisms need further study. The induction of autophagy is often induced by nitrogen starvation, using the nitrogen deficient medium SD-N (Arg) to induce the parent strain SN250 simultaneously. Gene deletion bacteria Gln3 Delta / delta and Stp1 Delta / Delta, gene recovery bacteria Gln3 Delta / delta +AB and Stp1 Delta / delta +AB autophagy. In the point plate experiment, there is no difference in the growth of the SD-N culture medium, but when RAPA is added, its viability on the nitrogen deficient medium is enhanced. In addition, among the 5 experimental strains, in the SD-N (Arg) induced autophagy, the vacuoles are found in the vacuoles. There were autophagic bodies. This shows that Gln3 Delta / delta and Stp1 Delta / delta can occur autophagy in the case of nitrogen source induced. In the experiment of observing API localization in the SD-N (Arg) medium, we found that compared with the other 4 experimental strains, only a few mature API. in the Gln3 Delta / delta vacuoles continue to tolerate the tolerance of 5 experimental strains to SD-N (Arg). The results show that Gln3 Delta / delta is more sensitive to nitrogen hunger. The results show that the transcription factor Gln3 participates in the regulation of autophagy, and the transcription factor Gln3 plays an important role in regulating autophagy induced by SD-N, and may play a positive role in enhancing autophagy. The induction of autophagic phenotype is not the same as the autophagic phenotype induced by RAPA. Therefore, we try to find the regulatory mechanism of the bacterial tolerance to RAPA. The intracellular ATP increases after RAPA induction. Therefore, it is speculated that aerobic respiration produces ATP links and plays an important role in the tolerance of RAPA. Therefore, the staphylococcus is in the full nutrient medium YPD. When glucose is replaced with citric acid, glycerol, etc., need to produce a carbon source of ATP and used in different breathing modes. The results show that the 5 experimental strains are resistant to RAPA induced in the glucose medium of citric acid replacing YPD. In the three carboxylic acid cycle (TCA), acetonate carboxylase is carboxylation of pyruvate to the formation of acetoacetic acid and acyl Acetic acid and acetyl coenzyme A are catalyzed by citrate synthase to form citric acid. First, it is presumed that the synthesis of citric acid may be hindered. According to the above results, we speculate that the autophagy in the RAPA induced autophagy, Gln3 Delta / delta and Stp1 Delta / Delta, makes the citric acid synthase in the three carboxylic acid cycle (TCA) unable to enter the vacuole to degrade and make the bacteria ATP was used to produce resistance to RAPA. After constructing the citric acid synthase (CIT1p) green fluorescent fusion protein GFP-CIT1p strain, the CIT1p localization found that among the 5 experimental strains of RAPA induced, Gln3 Delta / Delta, Stp1 Delta / delta and Stp1 Delta / delta +AB CIT1p were located outside the vacuole, but the vacuoles did not exist. We have preliminarily verified our speculations that the autophagy induced by RAPA, Gln3 Delta / delta and Stp1 Delta / delta autophagy, makes the citric acid synthase in the three carboxylic acid cycle (TCA) can not be degraded into the vacuole, so that the bacteria can complete TCA production and supply ATP, causing the strain to produce resistance to RAPA. The Gln3 delta of RAPA induced by RNA-Seq is detected by RNA-Seq. The level of M RNA expression of delta and Stp1 Delta / Delta, as well as the growth of mycelium in the environment of limited nitrogen sources, and other biological phenotypes, try to find the more important biological functions of the transcription factor Gln3 and Stp1.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96

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本文編號：1971662