Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Fisheries College, Jimei University, Xiamen 361021, China.
Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Fisheries College, Jimei University, Xiamen 361021, China.
Gene. 2018 Sep 25;672:156-164. doi: 10.1016/j.gene.2018.06.029. Epub 2018 Jun 12.
The success of the pathogenic bacteria is partly attributable to their ability to thwart host innate immune responses, which includes resisting the antimicrobial functions of macrophages. And reactive oxygen species (ROS) is one of the most effective antimicrobial components of macrophages to kill invading bacteria. Our previous studies found that Aeromonas hydrophila can survive in fish macrophages, which suggested that this bacterium might take fish macrophages as their shelters to resist drug killings and other immune damage. But how A. hydrophila survive in host macrophages remains unknown. Since KatG has been reported to have not only catalase activity but also peroxidase and peroxynitritase activity, the amino acid sequence and protein structure of KatG was analyzed in this study, the function of KatG in A. hydrophila survival in and escape from host macrophages was also carried out. The bioinformatics analysis displayed that KatG of A. hydrophila B11 showed >93% homologous to that of KatG in other Aeromonas. KatG of A. hydrophila was stable silenced by shRNA and RT-qPCR confirmed the expression of KatG in KatG-RNAi was significantly reduced. The survival rate of intracellular KatG-RNAi decreased by 80% compared to that of the wild type strain B11, while the intracellular ROS level of the macrophages that phagocytosed KatG-RNAi increased 65.9% when compared to that of the macrophages phagocytosed wild-type strain. The immune escape rate of A. hydrophila decreased by 85% when the expression of KatG was inhibited. These results indicated that (1) The amino acid sequence and protein structure of KatG of A. hydrophila is conserved; (2) KatG helped A. hydrophila to survive in fish macrophages by eliminating the harm of intracellular HO and inhibiting intracellular ROS levels increased; (3) A small portion of intracellular A. hydrophila could escape from host macrophages for further infection, in this process KatG also played important role.
该致病菌的成功部分归因于它们能够挫败宿主固有免疫反应，包括抵抗巨噬细胞的抗菌功能。活性氧（ROS）是巨噬细胞杀死入侵细菌的最有效抗菌成分之一。我们之前的研究发现，嗜水气单胞菌可以在鱼类巨噬细胞中存活，这表明这种细菌可能将鱼类巨噬细胞作为其避难所，以抵抗药物杀伤和其他免疫损伤。但是，嗜水气单胞菌如何在宿主巨噬细胞中存活仍然未知。由于已经报道 KatG 不仅具有过氧化氢酶活性，而且具有过氧化物酶和过氧亚硝酸盐酶活性，因此本研究分析了 KatG 的氨基酸序列和蛋白质结构，并研究了 KatG 在嗜水气单胞菌逃避宿主巨噬细胞杀伤和逃逸中的作用。生物信息学分析显示，嗜水气单胞菌 B11 的 KatG 与其他气单胞菌的 KatG 具有>93%的同源性。通过 shRNA 稳定沉默嗜水气单胞菌的 KatG，并且 RT-qPCR 证实 KatG-RNAi 的表达显著降低。与野生型菌株 B11 相比，细胞内 KatG-RNAi 的存活率降低了 80%，而吞噬 KatG-RNAi 的巨噬细胞的细胞内 ROS 水平增加了 65.9%。当抑制 KatG 的表达时，嗜水气单胞菌的免疫逃避率降低了 85%。这些结果表明：（1）嗜水气单胞菌的 KatG 氨基酸序列和蛋白质结构保守；（2）KatG 通过消除细胞内 HO 的伤害并抑制细胞内 ROS 水平的增加，帮助嗜水气单胞菌在鱼类巨噬细胞中存活；（3）一小部分细胞内嗜水气单胞菌可以逃避宿主巨噬细胞以进行进一步感染，在此过程中 KatG 也发挥了重要作用。
