分析 PA0262， PA1511 與 PA5266，三個可能與綠膿桿菌PAO1第六型分泌系統(二)相關的 VgrG 蛋白

Characterization of PA0262, PA1511, and PA5266, Three Putative VgrGs Associated with the Type VI Secretion System-II in Pseudomonas aeruginosa PAO1

楊宸鳴(Chen-Ming Yang)

指導教授 ： 張晃猷

國立清華大學/生命科學院/分子醫學研究所/碩士(2013年)

https://doi.org/10.6843/NTHU.2013.00290

摘要

綠膿桿菌為一種常見院內感染病原菌，也常在肺纖維性囊腫病患造成慢性感染。革蘭氏陰性菌為雙層膜結構，因此演化出不同型的分泌系統以將作用蛋白輸送到胞外。綠膿桿菌 PAO1 株，具有三套第六型分泌系統的基因叢集，稱為 T6SS-I, -II, 與 -III。 VgrG 為第六型分泌系統主要的分泌蛋白，其結構與 T4 噬菌體的細胞穿刺構造非常相似。 PAO1 具有十個 vgrG 基因，其中何者與 T6SS-II 相關並不清楚。經由與其他綠膿桿菌基因體的比對，我們認為 PA1511、 PA0262 與 PA5266，很可能是 T6SS-II 的 VgrG 蛋白。利用 InterproScan 預測蛋白保留區域，發現 PA1511 與 PA0262 的羧基端具有一段功能未知的延伸區域，可能用以與真核細胞進行交互作用。本研究首先將帶有 vgrG 的表現載體轉染至真核細胞中，但發現細胞並沒有顯著的死亡。本研究也構築了 PA1511、 PA0262 與 PA5266 等 vgrG 基因剔除株，發現 vgrG 不會影響綠膿桿菌的表型，包括移動能力、酵素活性與菌落形態等。在模式植物之感染試驗中， vgrG 剔除株 ΔPA1511 與 ΔPA5266 的傷口範圍較野生株造成的為小。在免疫轉漬實驗中發現，以 M8 培養的野生株，會分泌蛋白 PA5266 與 Hcp2 至培養液中。本研究雖然未能直接證明這三個 VgrG 與第六型分泌系統相關，但提供了研究這些 VgrG 蛋白的重要基礎。

關鍵字

綠膿桿菌 ； 第六型分泌系統 ； VgrG

並列摘要

Pseudomonas aeruginosa is a common nosocomial pathogen causing acute infections in immunocompromised patients and chronic infections in cystic fibrosis patients. Gram-negative bacteria, especially pathogens, have developed diversified secretion systems to deliver effector molecules through their complex double membrane structure. P. aeruginosa contains three gene clusters named T6SS-I, -II, and -III that can encode a type VI secretion system. The valine-glycine repeat protein G (VgrG), structurally similar with the complex puncturing device of T4 bacteriophage, is one of the proteins secreted by type VI secretion systems P. aeruginosa PAO1 contains 10 vgrG-like genes. While PA0091, PA0095, and PA2685 have been shown to be the VgrGs of T6SS-I, the VgrG associated with T6SS-II is less clear. Based on comparative genomic analysis, either PA1511, PA0262, or PA5266 could be the VgrG associated with T6SS-II. Protein domain analysis revealed that PA1511 and PA0262 contain a functionally unknown extended segment in the C-terminal region. Further cytotoxicity assay of PA1511 and PA0262 by transfecting the genes into cultured HCT-8 cells failed to detect any significant effects. This study then constructed the vgrG deletion strains and examined their biological properties. Among commonly assayed phenotypes, no significant effect could be observed resulting from the vgrG deletions. In contrast, cytotoxicity and virulence to Chinese cabbage leaves are reduced in the ΔPA1511 and ΔPA5266 mutants. Finally, this study demonstrated that PA5266 and Hcp2 are secreted when PAO1 was grown in M8 minimal medium. Although the exact VgrGs secreted by T6SS-II and their functions remain to be determined, this study has established a strong basis for future analysis of the three VgrGs.

並列關鍵字

pseudomonas aeruginosa ； type six secretion system ； VgrG

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