沙門氏菌wzxE與yqiC基因對細菌致病性和人類細胞株宿主反應之效應

The impacts of Sallmonella genes wzxE and yqiC on bacterial virulence and host responses in human cell lines

丁士閔(Shih-Min Ding)

指導教授 ： 陳慶國

共同指導教授 ： 方旭偉 ； 方旭彬(Shiuh-Bin Fang)

國立臺北科技大學/工程學院/生物科技研究所/碩士(2014年)

https://doi.org/10.6841/NTUT.2014.00592

摘要

鼠傷寒沙門氏菌為引起人類腸道疾病之一，並且在世界引起許多疾病和死亡的重要病原菌。過去本實驗室篩選出了鼠傷寒沙門氏菌的ΔwzxE、ΔyqiC基因與該菌感染入侵人類上皮細胞株HeLa的能力有關。在大腸桿菌上的研究發現wzx基因群參與細菌脂多醣(LPS)合成，另外鼠傷寒沙門氏菌的yqiC基因會影響該菌感染小鼠的致病能力，而對於wzxE和yqiC的基因對於沙門氏菌感染人體不同細胞是否會誘導宿主細胞的發炎和免疫的影響所知有限。所以本研究首先利用Gentamicin protection assay將鼠傷寒沙門氏菌野生株SL1344和突變株ΔwzxE、ΔyqiC在感染人類上皮細胞株(HeLa、Caco-2、LS174T)和非上皮細胞株(THP-1)後對於細菌附著和入侵能力的影響，以鼠傷寒沙門氏菌野生株SL1344和兩株突變株ΔwzxE、ΔyqiC感染人類腸道上皮細胞Caco-2、LS174T四小時之後，利用qRT-PCR偵測Interlukin-8(IL-8)及人類β防禦素(human β-defensin, hBD) 1、2、3的mRNA的表現程度；另外在感染七小時之後，利用ELISA偵測腸道上皮細胞的IL-8及hBD1、2、3的蛋白質表現程度。
研究結果顯示，與沙門氏菌野生株SL1344相比，沙門氏菌ΔwzxE與ΔyqiC表示鼠傷寒沙門氏菌的附著和入侵宿主細胞的能力有顯著的降低，而沙門氏菌ΔyqiC在感染宿主細胞後的IL-8 mRNA及蛋白質表現有顯著的降低，另外沙門氏菌ΔwzxE、ΔyqiC、ΔfliC在感染宿主細胞後的hBD3蛋白質的表現有顯著的降低，綜合上述結果指出wzxE與yqiC基因會有顯著的降低鼠傷寒沙門氏菌感染宿主細胞早期的表現，其中yqiC基因會影響沙門氏菌野生株SL1344在感染Caco-2、LS174T細胞後降低IL-8發炎基因的表現，另外wzxE、yqiC、fliC等基因也會影響沙門氏菌野生株SL1344感染LS174T細胞後降低hBD3蛋白質的表現。

關鍵字

鼠傷寒沙門氏菌 ； wzxE ； yqiC ； 鞭毛蛋白(flagellin) ； Interleukin-8 (IL-8) ； human β-defensin (hBD)

並列摘要

Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of non-typhiod Salmonella strains which are the important enteropathogens and cause gastroenteritis in the world. In our preliminary studies, we found two reported Salmonella genes, wzxE and yqiC involved in colonization of S. Typhimurium in human epithelial HeLa cells. In E. coli, the wzx genes mediated lipopolysaccharide (LPS) synthesis. In S.Typhimurium, the yqiC gene was reported account of bacterial virulence in mice. However, it remains little known whether wzxE and yqiC are responsible for colonization (bacterial adherence and invasion) in different human cells and whether these two genes are essential for induction of host cell inflammation and innate immunity. Therefore, we used wild-type SL1344 and wzxE and yqiC mutant strains (ΔwzxE and ΔyqiC) to infect the human epithelial cell lines (Hela, Caco-2 and LS174T) and non-epithelial cell line (THP-1) by using gentamicin protection assay for quantification of colonizing and invading bacteria in these strains. Human intestinal epithelial cells (Caco-2 and LS174T) were infected with wild-type SL1344 and two mutant strains (ΔwzxE and ΔyqiC) for 2 hours, followed by incubation with fresh medium for 1 hour.
Then, we used quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) to detect the mRNA and protein expression of interlukin-8(IL-8), human beta-defensin-1, -2 and -3 (hBD-1, -2 and -3). Finally, we analyzed whether mRNA and protein expression levels of ΔyqiC-infected cells were significantly decreased when compared with wild-type SL1344.
Our data indicated that ΔwzxE and ΔyqiC were significantly attenuated in bacterial colonization and invasion after gentamicin protection assays. The mRNA and protein expression levels of IL-8 were significantly downregulated in Caco-2 and LS174T cells infected by the ΔyqiC. The protein expression of hBD3 was significantly downregulated in LS174T cells by ΔwzxE, ΔyqiC and ΔfliC, when compared with the wild-type SL1344. In conclusion, we found that both wzxE and yqiC genes were required for bacterial colonization and invasion S. Typhimurium during early stage of infectious process. Furthermore the yqiC gene is responsible for inducing IL-8 mRNA expression; wzxE, yqiC and fliC gene are required for hBD3 protein production in Caco-2 and LS174T cells during S. Typhimurium infection in vitro.

並列關鍵字

Salmonella enterica serovar Typhimurium ； ΔwzxE ； ΔyqiC ； Interlukin-8 (IL-8) ； human β-defensin (hBD)

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