番茄細菌性斑點病菌 Pseudomonas syringae pv. tomato DC3000中第六型分泌系統HSI-II 基因叢集之定性分析

Abstract

Pseudomonas syringae pathovar tomato（Pst）DC3000 是引起番茄細菌型斑點病的主要病原，也被應用在研究細菌與宿主之交互作用而成為常見的模式生物之一。近年來新的第六型分泌系統的發現，帶起科學家一連串的研究，而第六型分泌系統被認為是普遍存在於細菌，如同奈米武器一般可以去傳送作用蛋白到目標細胞中，並提供本身在宿主中的致病力、抗發炎反應或是彼此細菌之間的競爭能力。在 Pst DC3000 中發現，具有稱為 HSI-I 與 HSI-II 的兩組第六型分泌系統基因叢集，而其中位於 HSI-II 基因叢集上的 hcp2 基因，被證實參與在與腸道菌及酵母菌之菌間競爭中；然而，位於此基因叢集之上仍有許多基因的功能還尚未明瞭。在本研究中，我們證實 Pst DC3000 中 HSI-II 基因叢集之活性，對於與不同植物相關的革蘭氏陰性細菌間之競爭是必要的；利用系統性分析所建構各基因的突變株，進一步找出無法表現、分泌此指標蛋白 Hcp2 至細胞外與抑制 Pst DC3000 之菌間競爭能力之相關基因。有趣的是，PSPTO_5413 突變株只影響細菌間之競爭能力，卻不影響其 Hcp2 的分泌，也代表著 PSPTO_5413 蛋白可能是一個毒性效應蛋白。PSPTO_5424 為 Sfa 的同源蛋白，與 sigma 54 同樣參與調控 hcp2 基因的表現，但彼此的關係與如何調控第六型分泌系統相關基因的表現還需要進一步釐清。此外，利用番茄病原性試驗發現，PSPTO_4453 之突變株失去致病能力，但PSPTO_5424 之突變株在番茄中的生長量較野生菌株高更多，且第三型分泌系統之相關基因如：hrpL、avrPto 與 avrPtoB，都有明顯被誘導表現的情形。與第三型分泌系統相似，廣泛性調控子 (global regulator) GacA 與群體感應之調控蛋白 PsyI 與 PsyR，也參與在 hcp2 基因之調控中。綜言之，本篇研究中證實第六型分泌系統在細菌適應力與競爭力上扮演很重要的角色，而 Sfa2、sigma 54 和 GacA 可能組成一調控網絡，負責調節 Pst DC3000 第六型分泌系統相關基因的表現。

Pseudomonas syringae pathovar tomato (Pst) DC3000, the causative agent of tomato speck disease, is well known as a model phytopathogen to study host-pathogen interactions. Recently, a novel secretion system, type six secretion system (T6SS), was discovered and acts like a widespread bacterial nanoweapon for delivery of effector proteins into cell targets, conferring virulence, anti-inflammatory processes, and interbacterial competition. In Pst DC3000, two T6SS gene clusters, namely HSI-I and HSI-II, were identified and hcp2, located in HSI-II, is involved in competition with enterobacteria and yeast. However, the function of the rest genes and how T6SS expression is regulated remain unclear. Here, we demonstrated that interbacterial competition of Pst DC3000 against several Gram-negative plant-associated bacteria requires mainly the activities of HSI-II. With the analysis of each mutant, genes indispensable for Hcp2 expression, secretion and interbacterial competition ability were identified. Interestingly, PSPTO_5413 only affects interbacterial competition ability but not Hcp2 secretion, suggesting that PSPTO_5413 might be a toxic effector. We also demonstrated that PSPTO_5424 (Sfa2) and PSPTO_4453 (sigma 54) are both required for hcp2 expression, but how PSPTO_5424 and PSPTO_4453 regulate hcp2 expression remains to be investigated. Similar to regulation of type III secretion system (T3SS), the global regulator GacA, together with PsyI, contributes to the expression of hcp2. Taken together, T6SS plays an important role in bacterial fitness and competiveness, and GacA, Sfa2 and sigma 54 may construct a regulatory network in Pst DC3000 to modulate the expression of T6SS.