Abstract：
Outer membrane protein A (OmpA), a major component of outer membrane proteins in gram-negative bacteria, is considered to be an important virulence factor in various pathogenic bacteria, but its underlying mechanisms involved in pathogenic process of Edwardsiella tarda has not yet been fully elucidated. E. tarda is an important facultative intracellular pathogen with a broad host range. This bacterium could survive and replicate in macrophages as an escape mechanism from the host defense. To address the functions of OmpA and its potential roles in the pathogenesis of E. tarda, ΔompA mutant strain and ΔompA-C complementary strain were constructed by the allelic exchange method in this study. Here, we demonstrate that the abilities of motility, biofilm formation and adherence to RAW264.7 cells of ΔompA were significantly impaired, although there was no difference in growth between wild-type (WT) strain and ΔompA. Moreover, inactivation of ompA rendered E. tarda more sensitive to oxidative, heat shock and osmotic stress, which simulate the in vivo conditions that E. tarda encounters within the intramacrophage environment. Consist with this observation, ΔompA was also found to be markedly attenuated for growth within macrophages. In addition, compared with the WT strain, ΔompA activated macrophages to release more inflammatory mediators, including tumor necrosis factor alpha (TNF-α), reactive oxygen species (ROS) and nitric oxide (NO). However, flow cytometry analysis revealed that ΔompA induced less apoptosis of RAW264.7 cells as compared with WT strain, characterized by decreased Annexin V binding and the activation of caspase-3. Overall, our findings suggest an importance of OmpA to E. tarda and provide the first comprehensive insight into its functions and potential roles in the pathogenesis of E. tarda, including its effect on interaction with macrophages.
摘要：
外膜蛋白A(OmpA),革兰氏阴性菌外膜蛋白的主要成分，被认为是各种致病菌的重要毒力因子,但其在塔达爱德华氏菌致病过程中的潜在机制尚未完全阐明。E.tarda是重要的兼性细胞内病原体，具有广泛的宿主范围。这种细菌可以在巨噬细胞中存活和复制，作为宿主防御的逃避机制。为了探讨OmpA的功能及其在E.tarda发病机制中的潜在作用，本研究通过等位基因交换方法构建了ΔompA突变株和ΔompA-C互补株。这里,我们证明了运动能力，ΔompA的生物膜形成和对RAW264.7细胞的粘附力明显受损，尽管野生型（WT）菌株和ΔompA之间的生长没有差异。此外，ompA的失活使E.tarda对氧化更敏感，热休克和渗透胁迫，模拟E.tarda在体内环境中遇到的体内条件。考虑到这一观察，还发现ΔompA在巨噬细胞内的生长明显减弱。此外,与WT菌株相比，ΔompA激活巨噬细胞释放更多的炎症介质，包括肿瘤坏死因子α（TNF-α），活性氧（ROS）和一氧化氮（NO）。然而,流式细胞术分析显示，与WT菌株相比，ΔompA诱导的RAW264.7细胞凋亡较少，以膜联蛋白V结合减少和caspase-3的激活为特征。总的来说,我们的发现表明OmpA对E.tarda的重要性，并首次全面了解其在E.tarda发病机理中的功能和潜在作用，包括它对与巨噬细胞相互作用的影响。
