转录因子Gln3、Stp1调控白念珠菌自噬及耐受雷帕霉素的机制研究

发布时间：2018-06-03 05:35

本文选题：白色念珠菌 + 雷帕霉素　；参考：《第二军医大学》2017年硕士论文

【摘要】：白念珠菌是临床免疫力低下患者最容易感染的一种条件致病菌,且致死率极高,这与白念珠菌在宿主内的高适应性有关。而且这种高适应性也使白念珠菌极易产生耐药性,这也是临床上对白念珠菌感染治疗失败的主要原因之一。已有的研究表明,白念珠菌在宿主体内的高适应性与其染色体、基因组的不稳定性,生物被膜的形成,形态的改变等密切相关。但已经发现的机制也难以完全解释白念珠菌的高适应性和高耐药性。近年来,越来越多的研究发现,不同的转录因子参与了对白念珠菌适应性和耐药性的调控。本课题拟利用白念珠菌转录因子缺失菌库,筛选发现调控白念珠菌适应性和耐药性的新的转录因子,并研究其机制。因此,本课题利用48株白念珠菌转录因子缺失菌,筛选其对不同抗真菌药物、化合物、以及酸碱、渗透压等不同刺激的敏感性,发现了一批可能影响白念珠菌不同表型的转录因子。其中Gln3Δ/Δ和Stp1Δ/Δ对雷帕霉素高度耐受,而雷帕霉素结合蛋白TOR是白念珠菌自噬调控通路中的关键蛋白,因此推测,这两个转录因子可能参与了对白念珠菌自噬的调控。细胞自噬是真核细胞在饥饿应答及分化等过程中发生的适应性反应,已成为近年来的研究热点,但目前鲜见对白念珠菌自噬的研究。因此,本课题利用上述基因缺失菌,研究Gln3和Stp1是否参与调控白念珠菌自噬,并考察其可能的调控机制。已有研究表明,在正常培养条件下,细胞内存在胞质定向液泡通路(CVT通路),该通路与自噬发生的过程存在着相似性,并且有研究发现,CVT通路缺陷的菌体,其自噬通路也存在缺陷。在CVT通路和自噬通路缺陷的菌体中,由于自噬小体与液泡融合受阻,细胞表面部分区域会呈现圆形凹陷。因此本实验利用干涉相差显微镜(DIC)观察比较YPD培养基中Gln3Δ/Δ、Stp1Δ/Δ和亲本菌SN250的细胞表面,发现只有少数亲本菌SN250和Gln3Δ/Δ的细胞的表面有圆形凹陷,而Stp1Δ/Δ的大部分细胞表面呈圆形凹陷,表明Stp1Δ/Δ的CVT通路缺陷。而经RAPA诱导后,Gln3Δ/Δ和Stp1Δ/Δ的大部分细胞表面呈圆形凹陷,而SN250表面凹陷的细胞较少。表明Gln3Δ/Δ和Stp1Δ/Δ存在自噬缺陷。自噬的发生通常由自噬相关基因(ATG)的表达增高引起。因此,本课题通过Realtime-PCR检测了不同ATG在m RNA水平上的表达情况,发现RAPA的确能够使菌体TOR1、ATG等自噬相关基因的表达升高,而且大部分基因的升高幅度达10倍以上,Gln3Δ/Δ和Stp1Δ/Δ两株缺失菌在RAPA作用后,上述自噬相关基因虽然也能升高,但升高幅度只在2~3倍左右。上述结果表明,RAPA不能有效诱导Gln3Δ/Δ和Stp1Δ/Δ中的自噬相关基因的表达,所以使上述缺失菌出现自噬缺陷。为了进一步考察验证Gln3和Stp1对白念珠菌自噬的调控作用,本课题构建了Gln3Δ/Δ+AB和Stp1Δ/Δ+AB两株转录因子缺失菌的基因回复菌,并对其基因型进行了鉴定。同时考察回复菌对雷帕霉素(RAPA)的敏感性,发现Gln3Δ/Δ+AB对RAPA的敏感性增加,但Stp1Δ/Δ+AB并对RAPA的敏感性并没有增加,这有可能与异位单拷贝基因回复有关。液泡蛋白氨基肽酶(API)被认为是CVT通路的标志性蛋白。当营养充足时,胞质中的50k D的API前体通过CVT通路运向液泡,在液泡中活化,转变为成熟的API。但处于有限营养或饥饿环境时,API主要通过自噬小体运向液泡。因此,在营养充足时,可以通过定位GFP标记的API是否进入液泡,来考察CVT通路是否缺陷。经过构建API和绿色荧光蛋白融合蛋白表达菌体(GFP-API),可以定位观察API在细胞内的定位情况。在RAPA作用后,同时,也观察到Gln3Δ/Δ和Stp1Δ/Δ胞浆及液泡边缘明显存有API前体,并未进入液泡。由于上述结果表明Stp1Δ/Δ中Cvt通路缺陷,对此结果用western-blot在蛋白水平进行验证API-GFP前体蛋白的是否被降解,Western结果表明,Stp1Δ/Δ中有80k D的未成熟的API-GFP前体蛋白。这说明转录因子Stp1调控CVT通路基因表达,转录因子Stp1缺失,Cvt通路缺陷。并且转录因子Gln3和Stp1调控白念珠菌自噬,转录因子Gln3和Stp1缺失,自噬缺陷。具体的分子调控机制有待进一步研究。诱导自噬发生常通过氮饥饿方式,利用缺氮培养基SD-N(含Arg)同时诱导亲本菌SN250和基因缺失菌Gln3Δ/Δ和Stp1Δ/Δ,基因回复菌Gln3Δ/Δ+AB和Stp1Δ/Δ+AB自噬。在点板实验中SD-N培养基上生长并无差异,但是,当在加入RAPA后,增强了其在缺氮培养基上的生存能力。另外,在SD-N(含Arg)诱导的自噬中,发现以上5株实验菌株中,液泡内均有自噬小体。这说明,Gln3Δ/Δ和Stp1Δ/Δ能够在氮源诱导的情况下发生自噬。在SD-N(含Arg)培养基中观察API定位情况的实验中发现,与其余4株实验菌体相比,在Gln3Δ/Δ液泡内仅有少部分成熟的API。我们继续对5株实验菌体对SD-N(含Arg)的耐受性进行考察,发现Gln3Δ/Δ明显对氮饥饿较为敏感。综上结果说明,转录因子Gln3参与调控自噬,转录因子Gln3在参与调控SD-N诱导的自噬中起着重要的作用,并且可能对增强自噬起到正调控作用。研究至此,明显发现,以上实验菌珠被SD-N(含Arg)诱导的自噬表型,与RAPA诱导的自噬表型并不相同。所以,我们试图找出菌体耐受RAPA的调控机制。以上5株实验菌株经RAPA诱导后,胞内ATP均增加。所以推测是有氧呼吸产生ATP环节,在耐受RAPA中起着重要的作用。因此,将全营养培养基YPD中的葡萄糖更换成柠檬酸、甘油等需通过不同呼吸方式产生ATP并加以利用的碳源。结果显示,以上5株实验菌体在柠檬酸替换YPD中的葡萄糖培养基中,对RAPA诱导产生了抗性。在三羧酸循环(TCA)中,丙酮酸羧化酶将丙酮酸羧化后形成草酰乙酸,草酰乙酸与乙酰辅酶A经柠檬酸合酶催化后形成柠檬酸。首先,推测可能是柠檬酸的合成受阻。根据以上研究结果,我们推测RAPA诱导的自噬中,Gln3Δ/Δ和Stp1Δ/Δ发生自噬缺陷,使得三羧酸循环(TCA)中柠檬酸合酶不能进入液泡内进行降解,使得菌体不断产生ATP加以利用,从而对RAPA产生了耐药性。经过构建柠檬酸合酶(CIT1p)绿色荧光融合蛋白GFP-CIT1p菌体,观察CIT1p定位发现,在RAPA诱导后的以上5株实验菌株中,Gln3Δ/Δ、Stp1Δ/Δ和Stp1Δ/Δ+AB中的CIT1p位于液泡外部,而液泡内并不存在。所以,初步验证了我们的推测,RAPA诱导的自噬中,Gln3Δ/Δ和Stp1Δ/Δ自噬缺陷,使得三羧酸循环(TCA)中柠檬酸合酶不能进入液泡内进行降解,使得菌体能够完整的进行TCA产生供给ATP,使得菌体对RAPA产生了抗性。通过RNA-Seq检测RAPA诱导后的Gln3Δ/Δ和Stp1Δ/Δ的m RNA表达水平,以及考察在有限氮源的环境中的菌丝生长情况,等生物学表型考察,试图发现转录因子Gln3和Stp1更重要的生物学功能。
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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