Pseudomonas syringae pv. tomato DC3000中第六型分泌系統VgrG蛋白之特性分析

Abstract

在大部分的革蘭氏陰性菌中發現具有第六型分泌系統 (Type VI secretion system, T6SS)，為噬菌體尾部同功的微型分子結構，與Hcp蛋白共同堆疊形成類tail-tube結構；而VgrG蛋白作用則為tail-tube端點形成尖刺的結構。植物病原菌Pseudomonas syrigae pv. tomato DC3000 (Pst DC3000) 推測具有兩群的T6SS基因，HIS-I及HIS-II。Hcp2屬於HIS-II基因群，前人研究顯示Hcp2蛋白在被分泌抵來抗腸桿菌科細菌時扮演必須的角色。然而VgrG在細菌中的功能尚未清楚。在本研究中，預測Pst DC3000具有7個類VgrG基因，其中3個屬於HIS-I及HIS-II基因群，另外4個則分散於細菌的基因組中。本研究利用系統性去除 (systematic deletion) 基因的方式證明各個VgrG蛋白在分泌及細菌間競爭中的貢獻程度。在類VgrG基因中，VgrG-2b (PSPTO_5436)為抵抗E. coli主要的貢獻者；VgrG-2a (PSPTO_5415) 在5種不同的植物病害細菌中則有較小的貢獻。上述兩種VgrG蛋白皆屬於HIS-II基因群。此外，VgrG-2b蛋白在離體試驗 (in vitro) 中可直接與Hcp2蛋白作用。綜合本研究結果顯示HIS-II在Pst DC3000與其他細菌競爭作用中扮演主要的角色，進一步證實Pst DC3000具有較佳競爭能力的原因。此外，在T6SS研究中找到11個與VgrG有關聯的基因，顯示其可能具有成潛力為VgrG-2a下游PSPTO_5413的作用蛋白 (effector)。

The type VI secretion system (T6SS), found in most gram-negative bacteria, is a macromolecular structure analogous to bacteriophage tails, with Hcp stacking to form a tail-tube like structure and VgrG, acting as a puncturing device at the tip of this tube. The plant pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is known to have two putative T6SS gene clusters, HSI-I and HSI-II. Hcp2, encoded in the HSI-II gene cluster, previously shown to be secreted and playing an essential role in antimicrobial activity against enterobacteriaceae. However, the role of VgrG in this bacterium is still unknown. In this study, a total of seven vgrG genes were predicted to be present in Pst DC3000, three of which are located within the HSI-I and HSI-II gene clusters with additional four others scattered in the bacterial genome. Here, we demonstrate the secretion of VgrG proteins, and also, the contribution of each VgrG protein in secretion and interbacterial competition by systematic deletion of these genes. Among these VgrG homologs, VgrG-2b (PSPTO_5436) was the major contributor in secretion and interbacterial competition activity against E. coli and five different phytopathogenic bacteria followed by a small contribution of VgrG-2a (PSPTO_5415). Both VgrG proteins are encoded within the HSI-II gene cluster. Furthermore, VgrG-2b was shown to directly interact with Hcp2 in vitro. All these further support the notion that HSI-II plays a major role in Pst DC3000 to compete for niches, thus conferring this bacterium significant fitness advantage against competitive microbes. Additionally, eleven putative VgrG-associated genes were targeted for deletion to observe its role in the T6SS results showed a potential effector activity for PSPTO_5413, located downstream of VgrG-2a.