Pseudomonas syringae pv. tomato DC3000 中嵌合型第六型分泌系統效應蛋白 PSPTO_5413L 之特性分析

Abstract

第六型分泌系統 (type VI secretion system, T6SS) 為革蘭氏陰性菌所使用之分子武器，其功能之一為分泌具有毒性的效應蛋白 (effector) 以介導細菌間的競爭。在目前已知的第六型分泌系統效應蛋白中，大多僅具有單一催化結構域，以展現特定之毒素活性；相較之下，具有多重催化結構域並發揮功能多樣性的第六型分泌系統效應蛋白之研究仍相當有限。本研究自植物病原菌 Pseudomonas syringae pv. tomato (Pst) DC3000 中鑑定出一個嵌合型 (chimeric) 第六型分泌系統效應蛋白 PSPTO_5413L，並透過結構預測與分析揭示此效應蛋白包含三個分別與 Autolysin、Lysostaphin 和 Lysozyme 相似的催化結構域以及三個 SH3/SH3b 細胞壁結合結構域，顯示其為一潛在具三重催化活性之效應蛋白。細菌間競爭試驗結果證實 PSPTO_5413L 的 AL 與 LZ 催化結構域對 E. coli MG1655 之抗菌活性具重要功能。此外，生物資訊分析進一步鑑定出分別與 AL 與 LZ 這兩個催化結構域對應之免疫蛋白 (immunity protein) PSPTO_5414 與 PSPTO_5412-2，並以在 E. coli MG1655 中表現免疫蛋白後進行之細菌間競爭試驗，支持此兩個免疫蛋白之專一性以及保護能力的推論。後續以胺基酸序列進行同源蛋白分佈分析顯示，PSPTO_5413L 之同源效應蛋白同時存在於植物病原性與促進植物生長之 Pseudomonas spp. 中。本研究亦以植物環境生態角色與 Pst DC3000 不同之促進植物生長根棲細菌 (plant growth-promoting rhizobacterium, PGPR) P. kribbensis (Pk) XP1-6 為例，證實 PSPTO_5413L 的同源效應蛋白 PK_5712 同樣具備相同之抗菌能力，並進一步預測出其對應之免疫蛋白 PK_5711 與 PK_5713。最後，核酸序列 GC 含量分析以及在其他菌種中發現 PSPTO_5413L 各結構區域分別獨立存在之同源蛋白結果，支持 PSPTO_5413L 之不同結構區域可能經由水平基因轉移 (horizontal gene transfer)，並於後續演化過程中透過基因重組 (recombination) 整合成此特殊的單一嵌合型效應蛋白。總結來說，本研究鑑定出一類新型、具三重催化結構域之嵌合型第六型分泌系統效應蛋白，其可能透過擴增單一效應蛋白之潛在作用目標範圍，提供細菌在葉表與土壤根圈等高度競爭之生態棲位中，更為顯著的競爭優勢。

The type VI secretion system (T6SS) is a molecular weapon employed by Gram-negative bacteria. One of its key functions is to deliver toxic effector proteins that mediate interbacterial competition. Most characterized T6SS effectors (T6Es) known thus far contain only a single catalytic domain that exerts specific antibacterial activity; in contrast, effectors containing multi-catalytic domains and exhibiting functional versatility remain largely unexplored. In this study, a chimeric T6E, PSPTO_5413L, from the phytopathogenic bacterium Pseudomonas syringae pv. tomato (Pst) DC3000 was identified, and structural prediction and analysis revealed that PSPTO_5413L contains three distinct catalytic domains, including Autolysin-, Lysostaphin-, and Lysozyme-like peptidoglycan hydrolases, together with three SH3/SH3b cell wall-binding domains, indicating its potential as a multi-functional effector. Interbacterial competition assays demonstrated that the AL and LZ catalytic domains contribute to its antibacterial activity against E. coli MG1655. Furthermore, bioinformatic analyses identified two corresponding immunity proteins, PSPTO_5414 and PSPTO_5412-2. Their specificity and protective functions were supported by heterologously expressing either of these two proteins in E. coli MG1655 followed by interbacterial competition assays. Sequence-based homolog distribution analysis revealed that orthologs of PSPTO_5413L are distributed among both phytopathogenic and plant growth-promoting Pseudomonas spp. Further analyses using the plant growth-promoting rhizobacterium (PGPR) P. kribbensis (Pk) XP1-6 as a representative ecologically distinct species confirmed that the homologous effector PK_5712 in Pk XP1-6 exhibits comparable antibacterial activity. Its corresponding immunity proteins PK_5711 and PK_5713 were also predicted. Finally, GC content analysis together with the identification of homologs corresponding to individual structural regions of PSPTO_5413L in other bacterial species supports the hypothesis that these domains were acquired through horizontal gene transfer and subsequently integrated into a single chimeric effector via recombination during evolutionary processes. In conclusion, this study identifies a novel class of chimeric T6Es with three peptidoglycan-targeting catalytic domains. Such multi-catalytic effectors likely expand the target range of a single effector and provide bacteria with a competitive advantage in highly contested ecological niches, such as the phyllosphere or rhizosphere.