Rock Jessica L, Nelson David R
Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA.
Infect Immun. 2006 May;74(5):2777-86. doi: 10.1128/IAI.74.5.2777-2786.2006.
Vibrio anguillarum is a causative agent of vibriosis in fish. Hemolytic activity has been suggested as a virulence factor by contributing to hemorrhagic septicemia and diarrhea. In order to identify and characterize the hemolysin genes and examine the role of hemolytic activity in virulence, a mini-Tn10Kan mutagenesis clone bank of V. anguillarum was screened. While no hemolysin-negative strains were observed, several mutants with two- to threefold-increased hemolytic activity were found. The region containing the insertion mutation was cloned, sequenced, and found to contain the V. anguillarum hemolysin (vah1) and two other open reading frames, coding for a putative lactonizing lipase (llpA) and a putative phospholipase (plp). The mini-Tn10Kan was inserted into plp. Site-directed mutagenesis of each gene revealed that mutations in vah1 and llpA did not affect hemolytic activity, but insertions into plp caused a two- to threefold increase in hemolysis. Double mutations in plp and either vah1 or llpA resulted in wild-type hemolytic activity. Complementation of plp restored hemolytic activity to wild-type levels. Spectrophotometric determination of hemolysin specific activity revealed that activity on a per cell basis peaked during the first 2 h of growth in LB20. Real-time quantitative reverse transcriptase PCR used to quantitate transcription of the hemolysin genes plp and vah1 in V. anguillarum wild-type strains M93Sm and NB10 revealed that transcription of plp and vah1 peaked at 2 h of growth in LB20. Additionally, expression of vah1 measured in the plp mutant strain, JL01, during the first 2 h of growth was >8 times higher than that in M93Sm. Mutations in plp and llpA did not affect virulence of V. anguillarum. The mutation in vah1 attenuated V. anguillarum virulence in fish. These data show that several genes are responsible for hemolytic activity in V. anguillarum. At least three genes (plp, llpA, and vah1) are responsible for one hemolytic activity. The data also suggest that plp acts as a negative regulator of vah1 and llpA.
鳗弧菌是鱼类弧菌病的病原体。溶血活性被认为是一种毒力因子，可导致出血性败血症和腹泻。为了鉴定和表征溶血素基因，并研究溶血活性在毒力中的作用，对鳗弧菌的一个mini-Tn10Kan诱变克隆文库进行了筛选。虽然未观察到溶血素阴性菌株，但发现了几株溶血活性增加两到三倍的突变体。对包含插入突变的区域进行了克隆、测序，发现其含有鳗弧菌溶血素（vah1）和另外两个开放阅读框，分别编码一种假定的内酯化脂肪酶（llpA）和一种假定的磷脂酶（plp）。mini-Tn10Kan插入到了plp中。对每个基因进行定点诱变表明，vah1和llpA中的突变不影响溶血活性，但插入plp会导致溶血增加两到三倍。plp与vah1或llpA的双突变导致野生型溶血活性。plp的互补使溶血活性恢复到野生型水平。分光光度法测定溶血素比活性表明，在LB20中生长的前2小时内，每细胞的活性达到峰值。用于定量鳗弧菌野生型菌株M93Sm和NBvah1转录的实时定量逆转录PCR显示，plp和vah1的转录在LB20中生长2小时时达到峰值。此外，在plp突变体菌株JL01生长的前2小时内测定的vah1表达比M93Sm中的高 >8倍。plp和llpA中的突变不影响鳗弧菌的毒力。vah1中的突变减弱了鳗弧菌在鱼类中的毒力。这些数据表明，几个基因负责鳗弧菌的溶血活性。至少三个基因（plp、llpA和vah1）负责一种溶血活性。数据还表明，plp作为vah1和llpA的负调节因子。
