Hasegawa Hiroaki, Häse Claudia C
Department of Biomedical Sciences, Oregon State University, Corvallis, OR 97331, USA.
Microbiology (Reading). 2009 Jul;155(Pt 7):2296-2305. doi: 10.1099/mic.0.028605-0. Epub 2009 Apr 23.
Vibrio tubiashii is a re-emerging pathogen of molluscs that secretes a variety of extracellular products (ECPs), including a metalloprotease and a cytolysin/haemolysin. Previously, we reported that the V. tubiashii haemolysin locus consists of two ORFs (vthB and vthA), similar to that of the homologous haemolysin genes (vvhB and vvhA) found in Vibrio vulnificus. Here, we demonstrate that the concomitant expression of both V. tubiashii genes resulted in significantly higher haemolytic activity than the vthA gene alone. In addition, we created a VthAB- mutant strain of V. tubiashii that was virtually devoid of haemolytic activity in liquid media. Interestingly, significant production of an additional haemolysin(s) was observed on blood plates. Moreover, we have previously reported that in V. tubiashii, proteolytic and haemolytic activities are inversely produced during bacterial growth. Here, we study this correlation in more detail and present evidence that the VtpA metalloprotease inhibits haemolytic activity in culture supernatants, based on the following evidence: (i) loss of metalloprotease activity by either mutation or EDTA inhibition resulted in increased haemolytic activity; (ii) overexpression of the vtpA gene resulted in decreased haemolytic activity; (iii) purified VtpA metalloprotease directly diminished haemolytic activity by purified VthA haemolysin. Importantly, we found not only that vthAB gene expression remained high throughout growth but also that there were no dramatic differences in vthAB gene expression between the parent and VtpA- mutant strains. Thus, our results strongly suggest that the V. tubiashii metalloprotease directly targets its haemolysin.
塔氏弧菌是一种再度出现的软体动物病原体，它能分泌多种胞外产物（ECPs），包括一种金属蛋白酶和一种细胞毒素/溶血素。此前，我们报道过塔氏弧菌溶血素基因座由两个开放阅读框（vthB和vthA）组成，这与创伤弧菌中发现的同源溶血素基因（vvhB和vvhA）类似。在此，我们证明塔氏弧菌这两个基因的协同表达所产生的溶血活性明显高于单独的vthA基因。此外，我们构建了一株塔氏弧菌VthAB -突变株，该菌株在液体培养基中几乎没有溶血活性。有趣的是，在血平板上观察到有额外溶血素的显著产生。而且，我们之前报道过在塔氏弧菌中，蛋白水解活性和溶血活性在细菌生长过程中呈反向产生。在此，我们更详细地研究了这种相关性，并基于以下证据提出VtpA金属蛋白酶抑制培养上清液中溶血活性的证据：（i）通过突变或EDTA抑制使金属蛋白酶活性丧失导致溶血活性增加；（ii）vtpA基因的过表达导致溶血活性降低；（iii）纯化的VtpA金属蛋白酶直接降低了纯化的VthA溶血素的溶血活性。重要的是，我们不仅发现vthAB基因在整个生长过程中表达都很高，而且亲本菌株和VtpA -突变株之间vthAB基因表达没有显著差异。因此，我们的结果有力地表明塔氏弧菌金属蛋白酶直接作用于其溶血素。
