蛋白酶ClpQY影響沙門氏菌的致病性

The Two Component Protease ClpQY Play Roles in Salmonella Pathogenicity

郭珏伶(Chueh-Ling Kuo)

指導教授 ： 吳蕙芬

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10164

摘要

沙門氏菌透過毒性因子的作用，改變腸道上皮細胞骨架，而使細菌入侵細胞。當沙門氏菌被巨噬細胞吞噬後，沙門氏菌啟動一連串毒性因子的表現，最終巨噬細胞失去殺菌的能力，沙門氏菌得以在其內存活並且增生而造成感染。細菌在惡劣的環境下，一連串的機制會被啟動，使細菌能夠克服所遭遇的困境。其中，蛋白酶扮演著重要的角色。Lon﹝Takaya et al., 2002﹞與ClpXP﹝Yamamoto et al., 2001﹞分別已被證明與沙門氏菌毒性有關。此外，在更早之前，ClpQY 已被證實了與Lon弁鄐W的重疊﹝Wu et al., 1999﹞，然而卻沒有文獻指出ClpQY與沙門氏菌致病力的相關性，因此，本實驗室透過突變株的建構，探討蛋白酶ClpQY與沙門氏菌毒性的關係。在細菌入侵細胞能力的測試中，我們發現各突變株在不同的培養條件下﹝震盪或靜置﹞，對細菌入侵上皮細胞的影響有顯著的差異。當細菌震盪培養，我們得知clpQ的突變並不會造成細菌入侵力顯著的影響，然而，不同於clpQ，clpY或clpQY的缺失，造成細菌入侵力只剩一半。在Lon蛋白酶缺失的背景下，clpQ與clpY任一的缺失都會造成細菌入侵上皮細胞的能力顯著的下降。然而，當細菌為靜置培養，我們發現，clpQ基因的缺失反而使細菌入侵力較野生株稍微提升，而整個clpQY操作子的缺失，細菌對上皮細胞的入侵力則沒有改變。另外，與野生株比較，clpY-或lon-突變株的入侵力僅為野生株的百分之七十五，其中，lon-突變株的入侵效能與 Takaya (2002)等人的文獻有很大的出入，然而，在lon-的背景下，clpQ基因的缺失則使細菌入侵力提升為野生株的3-4倍。另在巨噬細胞的細菌存活率分析中，我們發現，在感染細胞20小時後，相較於野生株，各突變株的存活率只剩不到三分之一。此外，在細菌細胞毒性分析中，我們發現Lon蛋白酶的缺失，使沙門氏菌的細胞毒性大幅提升，然而，在Lon蛋白酶缺失的背景下，clpQ與clpY任一的缺失都會造成細菌細胞毒性的減低，與震盪培養之細菌入侵細胞能力測試的結果相似。因此，蛋白酶ClpQY與沙門氏菌的致病力有顯著的相關。為進一步了解ClpQY在沙門氏菌感染時扮演的角色，我們分析各突變株的胞外蛋白質表現，發現確實有明顯差異之處，這些差異可以幫助我們了解各突變基因與細菌致病力的關係。此外，在細菌群泳能力分析中，我們發現一般情況下，clpQ或clpY基因與clpQ-clpY-雙重變株的缺失，不影響沙門氏菌的移動性，而所有lon-突變株的移動能力下降，尤其以clpY- lon-的雙重突變株最為顯著。然而，在不同的培養狀態下，各突變株移動的能力有些釭漣幭隉A細菌移動的能力可能影響其入侵效能。

關鍵字

蛋白酶沙門氏菌 ； 細菌致病力 ； 熱休克蛋白ClpQY

並列摘要

Salmonellae are pathogenic bacteria to both human and animals. They infect millions of people annually and cause a health hazard world-wide. The disease process is initiated by bacterial interaction with and invasion of the intestinal epithelium. Salmonella invades intestinal epithelial cells by using a type III secretion system (TTSS) encoded by Salmonella pathogenicity island 1 (SPI1). The SPI1 TTSS forms a needle-like structure that injects effector proteins directly into the cytosol of host cells (Kimbrough et al., 2000). When bacteria encounter hostile situation, several mechanisms are switched on to counteract the stress. And the proteases play important roles to make bacteria survive. There are four kinds of ATP dependent proteases in bacteria, including Lon, the Clp family (ClpAP and ClpXP), HslUV, and FtsH. Evidences were found that several heat shock proteins, such as ClpX and ClpP are associated with bacterial pathogenicity and were identified to be an essential factor in the systemic infection of mice with Salmonella typhimurium (Tomoko et al., 2001). Disruption of the lon gene in S. enterica serovar Typhimurium markedly stimulates invasion of epithelial cells. It inferred that Lon may regulate SPI genes expression by proteolysis of putative regulators that activate invasive genes expression (Akiko et al., 2002). Besides, our favorite gene ClpQY was demonstrated to share redundant activities with Lon (Wu et al., 1999). However, there is no experiment to point out the relationship of ClpQY with Salmonella virulence. Therefore, we would like to know whether ClpQ and/or ClpY contribute to the pathogenicity of Salmonella typhimurium. In the invasion assay, we found that the cell growth condition, with or without agitation, is critical to evaluate the potential roles of the ClpQ and ClpY involved in bacterial pathogenicity. When bacteria were grown with agitation, we found that the deletion of clpY gene results in deficient invasiveness. The deletion of clpQ or clpY genes in lon mutants made the bacterial invasion efficiency decrease to 7 or 2 fold further, respectively. Alternatively, when bacterial cells were grown without agitation, the deletion of clpQ genes increased the bacterial invasiveness and the loss of clpQY operon had no influences on its invasion efficiency. Interestingly, in our test the lon- strain only retained 75% invasiveness as the wild-type did. This data is different from the result reported by Takaya in 2002. To reason this, the bacterial cell grown under different conditions may lead to observe dissimilar results. In macrophage survival test, lon-/lon- double mutants and clpQ-clpY- strain are fail in surviving in macrophage 20 hrs postinfection. Besides, we tested the bacterial cytotoxicity, lon- strain showed profound cytotoxicity in cultured macrophages. However, further deletion of clpQ or clpY in lon- strain background leads to loss of strong bacterial cytotoxicity. Therefore, the protease/chaperone ClpQY has an effect on Salmonella virulence. We also analyzed the secretion protein pattern of clpQ, clpY or/and lon single or double mutants. Our data showed that each mutant strain has its own secretion protein pattern and it reveals the correlation between the ClpQY and the bacterial virulent protein secretions. Furthermore, in the bacterial motility assay, among above mentioned mutants, we again found that the growth conditions (with/without agitation) and also the cells incubated temperature had influences on their motility. Our data indicated that the bacterial motility probably have effects on S. typhimurium invasiveness

並列關鍵字

Bacterial pathogenicity ； protease ； Salmonella typhimurium ； clpQY

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