Srinivasa Rao P S, Lim T M, Leung K Y
Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 119260.
Infect Immun. 2001 Sep;69(9):5689-97. doi: 10.1128/IAI.69.9.5689-5697.2001.
Edwardsiella tarda is responsible for hemorrhagic septicemia (edwardsiellosis) in fish and also causes diseases in higher vertebrates such as birds, reptiles, and mammals, including humans. Interactions of E. tarda with blue gourami phagocytes were studied by light microscopy as well as by adherence, intracellular replication, and superoxide anion assays. Both nonopsonized virulent (PPD130/91 and AL9379) and avirulent (PPD125/87 and PPD76/87) bacteria could adhere to and survive and replicate within phagocytes, while only opsonized virulent strains replicated within the phagocytes. Furthermore, only avirulent E. tarda elicited a higher rate of production of reactive oxygen intermediates (ROIs) by phagocytes, indicating that they were unable to avoid and/or resist reactive oxygen radical-based killing by the fish phagocytes. TnphoA transposon mutagenesis was used to construct a library of 200 alkaline phosphatase (PhoA+) fusion mutants from a total of 182,000 transconjugants derived from E. tarda PPD130/91. Five of these mutants induced more ROI production in phagocytes than the wild-type strain. Two mutants had lower replication ability inside phagocytes and moderately higher 50% lethal dose values than the wild-type strain. Sequence analysis revealed that three of these mutants had insertions at sequences having homology to PhoS, dipeptidase, and a surface polymer ligase of lipid A core proteins of other pathogens. These three independent mutations might have changed the cell surface characteristics of the bacteria, which in turn induced phagocytes to produce increased ROIs. Sequences from two other mutants had no homology to known genes, indicating that they may be novel genes for antiphagocytic killing. The present study showed that there are differences in the interactions of virulent and avirulent E. tarda organisms with fish phagocytes and PhoA+ fusion mutants that could be used successfully to identify virulence genes. The information elucidated here would help in the development of suitable strategies to combat the disease caused by E. tarda.
迟缓爱德华氏菌可引发鱼类的出血性败血症（爱德华氏菌病），还会在包括人类在内的鸟类、爬行动物和哺乳动物等高等脊椎动物中引发疾病。通过光学显微镜以及黏附、细胞内复制和超氧阴离子检测等方法，研究了迟缓爱德华氏菌与蓝曼龙吞噬细胞的相互作用。未调理的强毒株（PPD130/91和AL9379）和无毒株（PPD125/87和PPD76/87）均可黏附于吞噬细胞并在其中存活和复制，而只有调理后的强毒株能在吞噬细胞内复制。此外，只有无毒的迟缓爱德华氏菌能促使吞噬细胞产生更高比例的活性氧中间体（ROIs），这表明它们无法避免和/或抵抗鱼类吞噬细胞基于活性氧自由基的杀伤作用。利用TnphoA转座子诱变技术，从迟缓爱德华氏菌PPD130/91的182,000个接合子中构建了一个包含200个碱性磷酸酶（PhoA+）融合突变体的文库。其中五个突变体在吞噬细胞中诱导产生的ROIs比野生型菌株更多。两个突变体在吞噬细胞内的复制能力较低，50%致死剂量值比野生型菌株略高。序列分析表明，其中三个突变体在与其他病原体脂多糖核心蛋白的PhoS、二肽酶和表面聚合物连接酶具有同源性的序列处发生了插入。这三个独立的突变可能改变了细菌的细胞表面特征，进而诱导吞噬细胞产生更多的ROIs。另外两个突变体的序列与已知基因无同源性，表明它们可能是抗吞噬杀伤的新基因。本研究表明，强毒和无毒的迟缓爱德华氏菌与鱼类吞噬细胞的相互作用存在差异，PhoA+融合突变体可成功用于鉴定毒力基因。此处阐明的信息将有助于制定合适的策略来对抗由迟缓爱德华氏菌引起的疾病。
