【作者】 王慧；

【导师】 冯露；

【作者基本信息】 南开大学 ， 微生物学， 2013， 博士

【摘要】 沙门氏菌是一类革兰氏阴性致病菌,可以导致小鼠,小牛,鸡和猪的系统性疾病,而且也可以潜伏在家禽和成年牛的体内,在人体内,会导致肠胃炎,包括发烧,腹泻和腹痛。沙门氏菌的致病性主要由鞭毛,菌毛和沙门氏菌致病岛基因决定。沙门氏菌Typhimurium LT2的致病岛主要包括五个致病岛,其中沙门氏菌感染宿主的侵染能力主要是由SPI-1来介导的。SPI-1编码一个三型分泌系统,可以形成一个将细菌效应蛋白注射入宿主细胞质的针状复合体结构。SPI-1分泌效应蛋白可以导致宿主细胞内的许多生理学改变,包括肌动蛋白重排导致的吞噬细菌。沙门氏菌Typhimurium LT2的SPI-2是沙门氏菌在宿主细胞内复制和系统性感染小鼠必须的。SPI-2编码的三型分泌系统运送的效应蛋白,通过改变细胞成分,包括细胞支架结构,信号传导途径,囊泡运输,来促进沙门氏菌在宿主细胞的生存和复制。在细菌中,核酸结合蛋白Fis是一个全局调控因子,可以参与到基因组结构与基因调控等过程。Fis蛋白是同源二聚体结构,在对数期大量表达,在对数早期,Fis在细胞内拷贝数高达50000,然后逐渐减少。之前的研究认为,Fis抑制转录的起始,主要是通过结合在目标启动子上,从而占据RNA聚合酶在目标启动子上的结合位置；或者是通过异构化RNA聚合酶调控的转录复合体影响转录。Fis也是一种传统的转录激活因子,它与RNA聚合酶发生物理接触来激活基因表达；另一个激活机制是通过Fis在负超螺旋区域的结合维持DNA扭曲,使该处转录可以持续进行。有研究通过使用微阵列芯片技术研究Fis在沙门氏菌中的调控基因,报道称Fis蛋白可以调控沙门氏菌SL1344中几百个基因的转录表达,包括致病岛基因。但是受限于微阵列芯片技术的通量,Fis在沙门氏菌中的调控基因报道的并不完全。为了全面细致的研究Fis蛋白在沙门氏菌中的调控网络和调控机制,尤其是对沙门氏菌致病岛基因的调控,我们采用了高通量的测序技术来进行研究。本研究采用了二代测序技术-Solexa测序来研究Fis蛋白在对数中期和对数早期在沙门氏菌中的调控基因,并且通过免疫共沉淀分离Fis的DNA结合位点并测序,得到Fis在沙门氏菌全基因组范围内的结合位点。在对数中期,Fis蛋白在沙门氏菌Typhimurium LT2中上调989个基因,下调657个基因。在同一时期,Fis蛋白的结合位点有895个,分布在943个基因上。综合分析Fis结合位点与调控基因,我们发现Fis调节并结合的基因共有317个,其中207个基因是Fis激活基因,110个基因是Fis抑制基因,其他的1329个Fis调控的基因不包含Fis结合位点。我们推测,这317个Fis调节并结合的基因是受Fis直接调控的,而1329个基因是通过Fis间接调控的。由于Fis可以调控大量的转录调控因子,我们通过实验证明许多Fis调控基因的转录是受上游转录调控因子来控制的。通过转录组数据分析,我们发现更多的未见报道的致病岛基因受Fis的转录调控。其中少数致病岛基因具有Fis结合位点。综合分析Fis结合位点和调控基因,我们推测并实验证明了几种可能的Fis调控致病岛基因的机制,包括：1)Fis直接结合来调控致病岛基因；2)Fis通过调控并结合致病岛调控基因来间接调控致病岛基因；3)Fis通过结合在上游位点,来影响下游一系列基因的转录；4)Fis通过调节OmpR来影响致病岛基因转录。我们在对数早期研究Fis蛋白的调控基因发现,Fis抑制989个基因的表达,激活509个基因的表达,主要起抑制作用,这与对数中期的Fis激活作用相反。通过比较发现,在对数早期和对数中期,受到Fis相同调控趋势的有278个基因,受到Fis相反调控趋势的有个基因398个基因,说明Fis在对数期的调控是瞬间的,且很快发生变化。转录组分析还预测了50个可能的sRNA,且部分受Fis蛋白的调控,这部分还需要进一步研究。更多还原

【Abstract】 Salmonella are Gram-negative bacterial pathogens that a capable of infecting a wide range of animals including mice, young cows, chicken and pig, however they are also able to colonize poultry and adult cattle without symptoms. In humans, infection with Salmonella results in a self-limiting gastroenteritis involving fever, diarrhea, and abdominal pain. The virulence of Salmonella is determined by flagella, fimbriae, and Salmonella pathogenicity islands (SPI) genes. There have been five SPIs identified in S. enterica serovar Typhimurium LT2, and invasion is mediated by a type III secretion system encoded on SPI-1. The SPI-1type III secretion system forms a needle-like complex that is responsible for the injection of bacterial effector proteins into the host cell cytosol. SPI-1effector proteins elicit several physiological changes in the host cell, including actin rearrangement leading to engulfment of the bacterium. After invasion of intestinal epithelial cells, Salmonella are able to disseminate to any tissue in the body, where the bacteria propagate inside macrophages, a process that requires a second type III secretion system encoded on SPI-2. The effectors facilitate Salmonella survival and replication in host cells by altering a variety of cell properties including its cytoskeletal structure, signal transduction pathways, and vesicular trafficking.The nucleoid-associated proteins Fis is a global regulator in bacteria, it could involve in maintaining genomic structure and gene regulation. Fis is a homodimer protein, and the intracellular level of Fis protein is growth-dependent and changes from less than100copies in stationary phase to more than50,000copies per cell in log phase. Fis represses transcription initiation either by imposing a blockade at the target promoter and excluding RNA polymerase or by a more subtle mechanism, in which it modulates the RNA polymerase-mediated isomerization of the closed transcription complex to an open complex. Fis can also be a conventional transcription activator, making physical contact with RNA polymerase. In addition, another mechanism involves the displacement of DNA twisting from an upstream site to the target promoter by Fis-mediated DNA bending.In the previously report, by using microarray technic, Fis was detected to regulate hundreds of genes transcription in S. enterica Typhimurium SL1344, including SPI genes. In order to obtain the regulation of global gene transcription by Fis and the regulatory mechanism, especially the regulatory mechanism on SPI genes, we used the high-throughput sequencing technic to perform ChIP-seq and RNA-seq in this study. We clarified the genome-wide distribution of binding regions and global regulation pattern of Fis in S. enterica Typhimurium LT2.In mid-exponential phase, Fis could up-regulate989genes and down-regulated657genes in LT2. And in the same time, Fis has895binding sites on genome-wide, spreading on943genes. Combined the two datasets, we find Fis regulates and binds on317genes, including207Fis up-regulated genes, and110Fis down-regulated genes. It shows that317Fis-regulated and Fis-binding genes are probably regulated by Fis directly, and1329Fis-regulated genes without Fis-binding are probably regulated by Fis indirectly. Since Fis regulates the transcriptions of several transcriptional factors genes, by using gene knockout and RT-PCR, we proved Fis controls the transcription of many genes by regulating corresponding transcriptional factors.We detect that several SPI genes are Fis-regulated genes and several SPI-genes are also Fis-binding genes. In this study, we firstly conclude the existence of several modes of Fis regulatory mechanism on SPI genes, and provide a regulatory network of Fis on SPI genes, including:1) Fis binds and regulates SPI genes directly;2) Fis binds to and activates the SPI positive regulator genes;3) Fis binds to the ORF region of SPI genes to mediate transcriptional activation of these genes and those downstream genes;4) Fis positively influences the SPI regulators by binding to and activating ompR.We also performed RNA-seq in early-exponential phase, and found that Fis could up-regulate509genes and down-regulated989genes in LT2. Fis is a repressor in early-exponential phase which is opposite with as an activator in mid-exponential phase. Compared the Fis-regulated genes in both phases, it shows that278genes have same Fis-regulatory tendency, whereas398genes have opposite Fis-regulatory tendency.We forecasted50sRNA from RNA-seq, and many of which are Fis-regulated.更多还原