Mathew J A, Tan Y P, Srinivasa Rao P S, Lim T M, Leung K Y
Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Kent Ridge Crescent, Singapore1192601.
Microbiology (Reading). 2001 Feb;147(Pt 2):449-457. doi: 10.1099/00221287-147-2-449.
Edwardsiella tarda is a Gram-negative bacterium that causes a systemic infection, edwardsiellosis, in fish. The virulence factors of this pathogen and its genetic determinants have not been systematically examined. In this study, TnphoA transposon mutagenesis was used to construct a library of 440 alkaline phosphatase (PhoA(+)) fusion mutants from a total of 400000 transconjugants derived from Ed. tarda PPD130/91. This library included genes for secreted and membrane-associated proteins normally involved in virulence. The library was screened for four virulence factors: siderophore production, motility, serum resistance and catalase production. Eight mutants deficient in one or more of these phenotypes were grouped into four classes. They were further characterized for their stimulation of reactive oxygen intermediate production by fish phagocytes, for their adhesion to and internalization into EPC (epithelioma papillosum of carp) cells, and for attenuation of virulence in blue gourami. Mutants 2A and 34 were highly attenuated in fish, with LD(50) values about 10 times higher than for the wild-type. These strains had mutations in the genes encoding arylsulfate sulfotransferase (mutant 2A) and a catalase precursor protein (mutant 34). One hyperinvasive/adhesive mutant and four pst mutants that were pleiotropic and slightly attenuated in fish were also isolated.
迟缓爱德华氏菌是一种革兰氏阴性菌，可在鱼类中引起全身性感染，即爱德华氏菌病。该病原体的毒力因子及其遗传决定因素尚未得到系统研究。在本研究中，利用TnphoA转座子诱变技术，从迟缓爱德华氏菌PPD130/91产生的总共400000个接合子中构建了一个包含440个碱性磷酸酶（PhoA(+)）融合突变体的文库。该文库包括通常参与毒力的分泌蛋白和膜相关蛋白的基因。对该文库筛选了四种毒力因子：铁载体产生、运动性、血清抗性和过氧化氢酶产生。八个在一种或多种这些表型上有缺陷的突变体被分为四类。进一步对它们进行了表征，包括它们对鱼类吞噬细胞产生活性氧中间体的刺激作用、它们对EPC（鲤鱼乳头状瘤上皮细胞）细胞的粘附和内化作用，以及它们在蓝曼龙中的毒力减弱情况。突变体2A和34在鱼类中的毒力高度减弱，其半数致死剂量（LD(50)）值比野生型高约10倍。这些菌株在编码芳基硫酸酯磺基转移酶（突变体2A）和过氧化氢酶前体蛋白（突变体34）的基因中发生了突变。还分离出了一个高侵袭性/粘附性突变体和四个在鱼类中具有多效性且毒力略有减弱的pst突变体。
