Pseudomonas syringae pv. tomato DC3000 中第六型分泌系統之分析

Abstract

革蘭氏陰性菌中，具有五種不同的分泌系統負責將蛋白質運送至胞外。然而最新的研究顯示，有一群基因產物能夠調控及分泌特定的蛋白質，但結構似乎與其他系統不同，故將其命名為第六型分泌系統 ( type VI secretion system, T6SS)。現階段研究結果指出，此分泌系統包括第四型分泌系統重要成員之一的 IcmF 同源物、具 ATPase 功能的 ClpV 、位於膜上的脂蛋白、含有FHA domain 的蛋白質及可調節其作用的蛋白質激酶和去磷酸酶，以及能藉此系統分泌出去的 VgrG 和 Hcp 蛋白質等。在某些重要的致病菌，如霍亂弧菌 ( Vibrio cholerae )中，第六型分泌系統在致病力上扮演關鍵的角色；如同第三型分泌系統一樣，致病菌可能會利用第六型分泌系統將其作用蛋白 (effector proteins) 傳送到真核細胞中，以影響宿主細胞的正常代謝途徑或抑制宿主的免疫反應。此外，第六型分泌系統需在特定情況下才能被誘導；且菌體可藉由轉錄和轉譯後修飾，來達到調節第六型分泌系統的基因表現和系統的組裝。Pseudomonas syringae 為常見的植物病原菌，其宿主範圍相當廣泛；經由生物資訊學的分析發現，其中一模式菌株 Pseudomonas syringae pv. tomato (Pst) DC3000 具有兩組可能的第六型分泌系統，因此我們希望藉由突變 icmFs (PSPTO_2554 和PSPTO_5418)、整個 T6SS 基因叢集 (PSPTO_2543-2554 和 PSPTO_5414-5427) 和 vgrGs (PSPTO_2538 和 PSPTO_5415) 來探討第六型分泌系統對 Pst DC3000 在蕃茄、圓葉菸草及模式植物阿拉伯芥上致病力的影響。結果發現，同時將 icmF1 及 icmF2 突變後，此突變株在圓葉菸草中的菌數會比野生型少，且在蕃茄上的病徵較為緩和；將整個基因叢聚突變後，不論是 T6SS1 或是 T6SS2 的突變株，在蕃茄上造成的病斑數及第七天時在葉片中的細菌數量，都比野生型少。此外，所有的 vgrG 突變株在致病力上均與野生型無顯著差異。由此病原性分析得知，第六型分泌系統對於 Pst DC3000 在不同宿主的致病力上，會有不同程度的影響。此外，VgrG2a 在 hrp minimal medium 或是有植物萃取物的誘導下會表現；同樣培養條件下，當持續表現 VgrG2a 時，VgrG2a 可被分泌到胞外。

Gram-negative proteobacteria possess five secretion systems (T1SS-T5SS) to secrete effector proteins into the external medium. Recently, a new secretion system, the type VI secretion system (T6SS), was described and characterized in several pathogens. The core components of T6SS include the T4SS IcmF-like proteins, a putative lipoprotein, the ClpV AAA+ ATPase, FHA domain-containing protein, STPK and STPP, and the secreted Hcp (haemolysin co-regulated protein) and VgrG (valine–glycine repeats) proteins. In some important pathogenic bacteria, like Vibrio cholerae, T6SS clearly plays a key role in virulence, and it also has been implicated in the translocation of a potential effector protein into the eukaryotic cells to disturb metabolisms or suppress the immune system in the hosts. Moreover, T6SS only expresses under certain conditions, and the regulatory mechanisms of T6SS involve transcriptional activators and the translational modification. Pseudomonas syringae is a widespread plant pathogen, which has broad host range. By using bioinformatic methods, we indentified two putative T6SS gene clusters in Pseudomonas syringae pv. tomato (Pst) DC3000. Here, we characterized the T6SS systems in Pst DC3000 by deletion of the icmFs (PSPTO_2554 and PSPTO_5418), the whole T6SS gene clusters (PSPTO_2543-2554 and PSPTO_5414-5427), and vgrGs (PSPTO_2538 and PSPTO_5415). We found that deletion of both icmF1 and icmF2 caused reduction of bacterial population in Nicotiana benthaminana and milder symptoms on tomato leaves. When T6SS1 or T6SS2 cluster was deleted, the symptoms and bacterial populations were both reduced. However, there was no effect on disease development on either tomato or N. benthamiana when vgrG1 or vgrG2a was deleted. The data from the pathogenicity assay indicate that the T6SS gene clusters has different degree of impacts on the virulence of Pst DC3000 on different hosts. Preliminary data showed that VgrG2a was expressed when Pst DC3000 was grown in hrp-minimal medium or minimal medium supplemented with 10% tomato extract. Furthermore, VgrG2a can be secreted into the media when overexpression on a plasmid in wild type Pst DC3000.