創傷弧菌 YJ016中一段與致病相關基因群組的功能探討

Abstract

在創傷弧菌YJ016的小染色體上，我們找到了一個與致病相關的基因群組(VVA0325至VVA0334)，其中有一個RtxL被歸類為“多重覆片段毒素” 家族中一個成員，以及一套第一型的運送系統，和包含一個感應子與四個調控子的訊息傳遞系統。為了要分析rtxL以及包含一個GGDEF功能區域的VVA0326和一個包含EAL功能區域的VVA0328，利用同源互換的原理，我們分別建構了這三個特定基因缺損的突變株。與霍亂弧菌中的細胞毒素RtxA一樣，RtxL也是一個巨大的蛋白質，在長達4655個胺基酸序列中，我們發現了三個長539的重覆片段，然而，在細胞毒殺能力的實驗中卻發現，rtxL缺損株的毒殺能力比較野生株並沒有明顯的差異，也就是說RtxL並不像RTX 家族中的其它成員一樣扮演細胞毒素的角色。缺損了包含一個GGDEF功能區域的突變株 VVA0326-，培養在LB的洋菜膠上，呈現出較小的生長菌落，在測定單位菌落數的結果發現，VVA0326-在生長速度上，相較於野生株並沒有不同，因此我們推論生長速率並不是造成菌落變小的主要因子，除此之外，VVA0326-在游泳的分析培養皿中游的比野生株快，在CAS的分析培養皿上，與野生株比較，VVA0326-有比較好的生長狀態，而缺損了包含一個EAL功能區域的突變株VVA0328-，卻有被抑制生長的現象，可能是因為突變株影響了表面的結構，進而改變了細菌對於HDTMA的敏感層度。此外，我們也發現這兩個蛋白質會有聚集在細菌兩端的行為。 根據pfam資料庫 (Version 17.0)，我們整理同時擁有GGDEF和EAL功能性區域的蛋白質 (GGDEF-EAL)，這些蛋白質可以被區分成兩種型態，在總數621個GGDEF-EAL中有高達606個是A型態，而且它在古老的細菌Aquifex aeolicus VF5的基因體中也能找到，因此我們推測它可能是GGDEF-EAL的袓先，藉由分析創傷弧菌中所包含之GGDEF和EAL的演化關係，大部份的GGDEF-EAL會群聚在樹型相近的位置，進一步比較在EAL樹型上有鄰近關係的蛋白質序列，根據他們的長度、與所包含的功能區域，我們找到了一些重製的演化痕跡，這樣的結果指出，目前看到大多數的GGDEF 和EAL可能都是經過重製產生。

A virulence-associated genomic island (VVA0325 to VVA0334) was annotated on the small chromosome of Vibrio vulnificus YJ016, which contains a gene cluster encoding a large protein RtxL of RTX (repeat in toxin) family, a type I secretion system, and a signal transduction system composed of a sensor and four regulators. In order to analyze the functional roles of rtxL, and VVA0326 and VVA0328, encoding respectively GGDEF- and EAL- proteins of signal transduction system, the gene specific deletion mutants were constructed by gene replacement via homologous recombination. Similar to the large size of V. cholera cytotoxin RtxA, RtxL encodes a protein of 4655 aa containing three repetitive peptides of 539 aa. However, no obvious difference was observed between wild-type and the rtxL- mutant in cytotoxicity assay suggesting that RtxL is not a cytotoxin as the other members of RTX family. The VVA0326- mutant carrying a deletion of the GGDEF domain displayed smaller colony morphology on LB plate while compared to the wild type. No difference in colony forming units was found indicating that growth rate is not likely a major factor to confer the phenotype of small colony. In addition, VVA0326- appeared to swim better than wild type bacteria on the swimming plate. VVA0326- grew better, while VVA0328- mutant, which carried a deletion of the EAL domain, grew worse than wild type when the bacteria were cultured on CAS plate at 30°C. This implied that changes in surface structures of the mutants affected the bacterial susceptibilities to HDTMA contained in CAS plate. Furthermore, subcellular localization analysis revealed that the two GGDEF- and EAL- proteins tended to localize at the cell poles. According to the domain organization, the GGDEF-EAL containing proteins (pfam database, version 17.0) were classified into A and B types. Six hundreds and five of the 621 proteins were in A type, which was also found in the ancient bacteria Aquifex aeolicus VF5 suggesting that this is the ancestor of the GGDEF-EAL proteins. Phylogenetic analysis of GGDEF- and EAL- containing proteins in the genome of V. vulnificus YJ016 revealed that GGDEF-EAL proteins were clustered. Comparative analysis of domain organization of the clads in the EAL tree allowed identification of several duplication events. The results indicated that most of the GGDEF- and EAL- containing proteins arised by gene duplication.