Pseudomonas syringae pv. tomato DC3000 中 VgrG 和 Hcp 交互作用及 PSPTO_3485、PSPTO_3744和PSPTO_5250 於細菌競爭上所扮演角色之研究

Studies of the VgrG-Hcp interaction, and the roles of PSPTO_ 3485, PSPTO_3744 and PSPTO_5250 involved in interbacterial competition in Pseudomonas syringae pv. tomato DC3000

連彥智(Yen-Chih Lien)

指導教授 ： 林乃君

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201802356

摘要

近年來發現許多革蘭氏陰性菌會利用第六型分泌系統 (Type VI secretion system, T6SS) 作為武器，運輸效應蛋白至真核或原核細胞中對目標細胞造成傷害，以增強自身的致病力或與其他細菌間的競爭能力。Pseudomonas syrnigae pv. tomato (Pst) DC3000為革蘭氏陰性菌，會引起番茄細菌性斑點病，在 Pst DC3000 中有兩組編碼 T6SS 組成分蛋白的基因叢集，分別為 HSI-I 及 HSI-II，其中 HSI-II 對於在細菌間競爭能力上時有較大的貢獻，然而 Pst DC3000 是透過哪些第六型分泌系統效應蛋白的作用達到與其他細菌競爭的目的至今仍不清楚。之前利用生物資訊學方法，在 Pst DC3000 的基因體序列中預測到七個 vgrG 同源基因，其中 vgrG1、vgrG2a 和 vgrG2b 分別位於 HSI-I 和 HSI-II 基因叢集中，而 vgrG2a、vgrG2b 和 vgrG3 可測得基因表現，且以 vgrG3 在所有測試條件下表現量最高。vgrG2a 和 vgrG2b 缺失會影響 Hcp2 的分泌，而透過 pull-down assay 證明 Hcp2 和 VgrG2a 及 VgrG2b 均有交互作用，Hcp2 與 VgrG2b 的結合力較大。有些 vgrG 的下游被預測有可能為第六型分泌系統效應蛋白的基因，然而先前利用突變株的構築與競爭試驗卻未發現這些可能為VgrG-associated 效應蛋白的基因參與於 Pst DC3000 和 E.coli MG1655的競爭上。然而 vgrG3 下游的 PSPTO_3485 在與同屬菌株 Pseudomonas savastanoi pv. phaseolicola (Psph) 1448a 競爭時，扮演重要角色。此外，以 LC-MS/MS 篩選出可能是經由第六型分泌系統分泌之潛力效應蛋白中，PSPTO_3744 和 PSPTO_5250 對於 Pst DC3000 細菌間的競爭力有所貢獻。經序列分析後得知 PSPTO_3485 的 C 端具有脂肪酶活性，而 PSPTO_3744 及 PSPTO_5250 分別為 Peptidyl-prolyl cis-trans isomerase B (PpiB) 及 Polyphosphate kinase (Ppk)。將 PSPTO_3744、PSPTO_5250 和 PSPTO_3485 表現於 Psph 1448a 會造成此菌生長受到抑制，然而此三個蛋白質究竟是否真的是 T6SS 效應蛋白及其是以何種機制造成細菌生長受阻而達到競爭優勢，仍待進一步研究探討。

關鍵字

番茄細菌性斑點病菌 ； 第六型分泌系統 ； 效應蛋白 ； VgrG ； 細菌間競爭作用

並列摘要

Many Gram-negative bacterium can use Type VI secretion system (T6SS) as weapons to translocate effector proteins to eukaryotic or prokaryote cells, for causing damage to the target cells, in order to increase virulence or interbacterial competition. Pseudomonas syrnigae pv. tomato (Pst) DC3000 is a Gram-negative bacterium, which can cause tomato speck disease. Pst DC3000 encodes two T6SS gene clusters, HSI-I and HSI-II, in which HSI-II is the major contribution to its interbacterial competition activity. So far, which T6SS effectors are responsible for interbacterial competition in Pst DC3000 remains unclear. Previous, in silico analysis revealed 7 vgrG genes in the genome of Pst DC3000. vgrG1 is located in HSI-I; vgrG-2a and vgrG-2b are located in HSI-II. In this study, I found the expressions of vgrG2a, vgrG2b, and vgrG3 can be detected, and vgrG3 has the higest expression level under the conditions tested. vgrG2a and vgrG2b have been shown to affect Hcp2 secretion. Here, interaction between Hcp2 and VgrG2a or VgrG2b, particularly stronger interaction between Hcp2 and VgrG2b, was demonstrated by the protein pull-down assay. A putative effector gene was also predicted downstream of each vgrG homolog, but construction of mutants followed by an interbacterial competition assay did not show that these putative VgrG-associated effectors are involved in competition ability of Pst DC3000 toward E.coli MG1655. However, PSPTO_3485, downstream of vgrG3, contributes to its competition with Pseudomonas savastanoi pv. phaseolicola (Psph) 1448a. Using the same strategy, I also found that PSPTO_3744 and PSPTO_5250, originally identified by LC/MS/MS, contribute to interbacterial competition. Sequence analysis indicates that PSPTO_3485 has lipase activity domain at its C-terminus, PSPTO_3744 is homologous to peptidyl-prolyl cis-trans isomerase B (PpiB), and PSPTO_5250 is predicted to be polyphosphate kinase (Ppk). Ectopic expression of PSPTO_3744, PSPTO_5250 or PSPTO_3485 inhibited growth of Psph 1448a. However, whether these proteins are T6SS effectors and how they cause growth retardation of the other bacteria for competitive advantage require further investigation.

並列關鍵字

Pseudomonas syringae pv. tomato ； Type VI secretion system (T6SS) ； Effectors ； Valine-glycine repeat protein G (VgrG) ； interbacterial competition

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