【摘要】：豬鏈球菌(Streptococcus suis)是一種人獸共患的傳染病病原,在全球有16個(gè)國(guó)家和地區(qū)都有豬鏈球菌病流行的報(bào)道,不僅對(duì)養(yǎng)豬業(yè)造成巨大的經(jīng)濟(jì)損失,而且嚴(yán)重威脅人類健康。豬鏈球菌根據(jù)莢膜抗原成分的不同分成33個(gè)血清型,其中豬鏈球菌2型(SS2)能夠感染人,是流行最廣、毒力最強(qiáng)的優(yōu)勢(shì)血清型。1998年和2005年中國(guó)爆發(fā)了兩次由SS2感染人引起的疫情,總共造成229人發(fā)病,52人死亡的嚴(yán)重后果。隨著抗生素的廣泛使用,豬鏈球菌的耐藥問(wèn)題日益嚴(yán)重。病原細(xì)菌的細(xì)胞分裂蛋白質(zhì)機(jī)器(protein machinary)是新的抗菌藥物創(chuàng)制的重要靶標(biāo)。與桿菌不同,鏈球菌具有特殊的細(xì)胞分裂機(jī)制。然而,鏈球菌細(xì)胞分裂機(jī)制缺乏深入研究。隔膜的形成及正確定位是細(xì)菌細(xì)胞分裂的關(guān)鍵環(huán)節(jié)。細(xì)菌細(xì)胞分裂骨架蛋白FtsZ與多種細(xì)胞分裂相關(guān)蛋白質(zhì)相互作用形成Z環(huán)和隔膜。最近的研究發(fā)現(xiàn),在枯草芽孢桿菌中存在一種膜結(jié)合蛋白SepF,可作為Z環(huán)與細(xì)胞膜之間的“橋梁”蛋白,將Z環(huán)精確的錨定到細(xì)胞中部的細(xì)胞膜上。SepF在鏈球菌細(xì)胞分裂過(guò)程中的功能是否與桿菌一樣尚不清楚。本研究以豬鏈球菌為研究材料,研究SepF在鏈球菌生長(zhǎng)和細(xì)胞分裂中的功能,取得的主要研究結(jié)果如下:1.SepF對(duì)SS2生長(zhǎng)和細(xì)胞形態(tài)的影響:以SS2野生菌株SC-19為親本菌株,構(gòu)建了sepF基因缺失菌株和互補(bǔ)菌株。生長(zhǎng)曲線、革蘭氏染色和掃描電鏡觀察發(fā)現(xiàn),sepF基因缺失后,SS細(xì)胞生長(zhǎng)速度減慢,細(xì)菌鏈變長(zhǎng),透射電鏡和超分辨顯微鏡觀察發(fā)現(xiàn),sepF基因缺失后SS細(xì)胞呈兩頭稍尖的紡錘狀,細(xì)胞分裂位點(diǎn)的隔膜連接線與細(xì)胞縱軸不垂直、隔膜向胞內(nèi)凹陷錯(cuò)位�？梢�(jiàn),SepF蛋白的確影響豬鏈球菌的生長(zhǎng)和細(xì)胞分裂。2.SepF蛋白與FtsZ蛋白的相互作用:細(xì)菌雙雜交實(shí)驗(yàn)發(fā)現(xiàn)SepF蛋白與FtsZ蛋白能夠互作。分別克隆、表達(dá)和純化SepF和FtsZ蛋白,體外實(shí)驗(yàn)也證實(shí)了二者的相互作用,并且SepF能夠促進(jìn)FtsZ蛋白的聚合形成多聚體。此外還發(fā)現(xiàn)SepF蛋白能與肽聚糖合成酶MurG互作。這些結(jié)果預(yù)示與桿菌一樣,SepF蛋白可能在鏈球菌中也協(xié)助正確選擇細(xì)胞分裂位點(diǎn),影響細(xì)胞分裂中隔膜形態(tài),在維持細(xì)胞形態(tài)具有重要功能。
[Abstract]:Streptococcus suis (Streptococcus suis) is a zoonotic infectious disease, which is reported in 16 countries and regions in the world, which not only causes huge economic loss to pig industry, but also seriously threatens human health. Streptococcus suis is divided into 33 serotypes according to the different components of capsule antigen. Among them, Streptococcus suis type 2 (SS2) can infect people and is the most prevalent. The most virulent predominant serotype. In 1998 and 2005, there were two outbreaks in China caused by SS2 infection, resulting in 229 cases and 52 deaths. With the widespread use of antibiotics, the drug resistance of Streptococcus suis is becoming more and more serious. (protein machinary), the cell division protein machine of pathogenic bacteria, is an important target for the creation of new antimicrobial agents. Unlike Bacillus, Streptococcus has a special cell division mechanism. However, the mechanism of streptococcus cell division is not well understood. The formation and correct location of diaphragm is the key link of bacterial cell division. Bacterial mitotic cytoskeleton protein (FtsZ) interacts with a variety of cell division related proteins to form Z rings and membranes. Recent studies have found that there is a membrane binding protein SepF, in Bacillus subtilis that acts as a "bridge" protein between Z ring and cell membrane. The Z loop was precisely anchored to the cell membrane in the middle of the cell. It was not clear whether SepF played the same role in the cell division of streptococcus as that of the bacillus. The function of SepF in the growth and cell division of Streptococcus suis was studied in this study. The main results were as follows: the effect of 1.SepF on the growth and cell morphology of SS2: the wild SS2 strain SC-19 was used as parent strain. SepF gene deletion strain and complementary strain were constructed. Growth curve, Gram staining and scanning electron microscopy showed that after sepF gene deletion, the growth rate of SS cells slowed down and the bacterial chain became longer. After the deletion of sepF gene, the SS cells were spindle-like at both ends, the connective line of the cell division site was not perpendicular to the longitudinal axis of the cell, and the diaphragm was dislocated into the cell. The interaction between 2.SepF protein and FtsZ protein was observed. The results showed that SepF protein and FtsZ protein could interact with each other. SepF and FtsZ proteins were cloned, expressed and purified, and their interaction was confirmed by experiments in vitro, and SepF promoted the polymerization of FtsZ proteins to form polymers. It was also found that SepF protein could interact with peptidoglycan synthase MurG. These results suggest that, like bacillus, SepF protein may also assist in the correct selection of cell division sites in streptococcus, affect the membrane morphology in cell division, and play an important role in maintaining cell morphology.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.611

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