VHH/TLH溶血素基因在海洋弧菌中分布的研究

Genes encoding the Vibrio harveyi haemolysin (VHH)/thermolabile haemolysin (TLH) are widespread in Vibrios

摘要:

哈维氏弧菌（V. harveyi）的VHH溶血素是对海水养殖鱼类的潜在致病因子。哈维氏弧菌的VHH溶血素基因与副溶血弧菌（V. parahaemolyticus）的TLH热不稳定性溶血素基因具有高度相似性，其氨基酸序列的相似性达到85.6%。根据哈维氏弧菌vhhA溶血素基因序列，合成一个地高辛标记的VHH基因探针，利用其进行Southern Blot，检测VHH溶血素基因在57株弧菌（包括26株国际标准菌株，20株哈维氏弧菌，11株副溶血弧菌）中的分布情况。结果显示，VHH基因探针与13株弧菌标准菌株有强杂交信号，包括2株溶藻胶弧菌（V. alginolyticus），2株哈维氏弧菌以及1株霍氏格里蒙菌（Grimontia hollisae），坎贝氏弧菌（V. campbellii），辛辛那提弧菌（V. cincinatiensis），费氏弧菌（V. fischeri），拟态弧菌（V. mimicus），飘浮弧菌（V. natriegens），副溶血弧菌，解蛋白弧菌（V. proteolyticus）和火神弧菌（V. logei）。与6株弧菌标准菌株有弱杂交信号，包括鳗弧菌（V. anguillarum），河口弧菌（V. aestuarianus），美人鱼发光杆菌（Photobacterium damselae subsp. damselae），河弧菌（V. fluvialis），弗尼斯弧菌（V. furnissii）和创伤弧菌（V. vulnificus），而另外7株弧菌标准菌株中无杂交信号。所有的哈维氏弧菌菌株至少含有一条杂交带，其中菌株VIB 645, VIB 648 和 SF-1分别含有2条杂交带。11株副溶血弧菌中均含有一条杂交带。上述数据表明，vhh/tlh溶血素基因广泛分布于弧菌中，尤其是哈维氏弧菌相关菌株和费氏弧菌相关菌株中。另外对鳗弧菌VIB 72, 坎贝氏弧菌VIB 285, 飘浮弧菌VIB 299和哈维氏弧菌VIB 647的vhh/tlh溶血素基因进行克隆并测序，其氨基酸序列与VHH溶血素和TLH溶血素氨基酸序列的同源性分别为67%～99%和69%～91%。对vhh/tlh溶血素基因在弧菌中的分布研究，将有助于进一步确定这类溶血素基因在病原弧菌致病性中的作用。

Abstract:

V. harveyi VHH haemolysin, which shows high homology to the TLH haemolysin (the identities of their deduced amino acid sequences are up to 85.6%), is a putative virulence factor to marine cultured fish. A VHH probe, which is specific to V. harveyi vhhA haemolysin gene, was used to screen EcoRⅠ digests of total DNA from 57 vibrio strains，including 26 vibrio type strains, 20 V. harveyi isolates and 11 V. parahaemolyticus isolates. As a result, 1 strong hybridisation band was detected in 13 type strains, including 2 of Vibrio alginolyticus, 2 of V. harveyi, and 1 strain each of Grimontia hollisae, V. campbellii, V. cincinnatiensis, V. fischeri, V. mimicus, V. natriegens, V. parahaemolyticus, V. proteolyticus and V. logei. Also, 1 weak band was detected in 6 type strains, including V. anguillarum, V. aestuarianus, Photobacterium damselae subsp. damselae, V. fluvialis, V. furnissii and V. vulnificus. There was not any hybridization signal in other type strains. Also, vhh/tlh was present in all isolates of V. harveyi and V. parahaemolyticus. Moreover, 3 isolates of V. harveyi，i.e. VIB 645, VIB 648 and SF1, had duplicated vhh genes. The data indicates that vhh/tlh is widespread in vibrios, especially in V. harveyi related species and V. fischeri related species. To support this conclusion, the vhh/tlh homologue genes in V. anguillarum VIB 72, V. campbellii VIB 285, V. natriegens VIB 299 and V. harveyi VIB 647 were cloned and sequenced, and the deduced amino acid sequences showed high degree of identities to VHH (67%～99%) and TLH haemolysin (69％～91%). This study will help us to identify the role of vhh/tlh haemolysin gene in the pathogenicity of vibrios.