Department of Marine Biology, Ocean University of China, Qingdao, China.
Fish Shellfish Immunol. 2012 Sep;33(3):514-21. doi: 10.1016/j.fsi.2012.06.002. Epub 2012 Jun 13.
Bacterial sialidases are a group of glycohydrolases that are known to play an important role in invasion of host cells and tissues. In this study, we examined in a model of Japanese flounder (Paralichthys olivaceus) the potential function of NanA, a sialidase from the fish pathogen Edwardsiella tarda. NanA is composed of 670 residues and shares low sequence identities with known bacterial sialidases. In silico analysis indicated that NanA possesses a sialidase domain and an autotransporter domain, the former containing five Asp-boxes, a RIP motif, and the conserved catalytic site of bacterial sialidases. Purified recombinant NanA (rNanA) corresponding to the sialidase domain exhibited glycohydrolase activity against sialic acid substrate in a manner that is pH and temperature dependent. Immunofluorescence microscopy showed binding of anti-rNanA antibodies to E. tarda, suggesting that NanA was localized on cell surface. Mutation of nanA caused drastic attenuation in the ability of E. tarda to disseminate into and colonize fish tissues and to induce mortality in infected fish. Likewise, cellular study showed that the nanA mutant was significantly impaired in the infectivity against cultured flounder cells. Immunoprotective analysis showed that rNanA in the form of a subunit vaccine conferred effective protection upon flounder against lethal E. tarda challenge. rNanA vaccination induced the production of specific serum antibodies, which enhanced complement-mediated bactericidal activity and reduced infection of E. tarda into flounder cells. Together these results indicate that NanA plays an important role in the pathogenesis of E. tarda and may be exploited for the control of E. tarda infection in aquaculture.
细菌唾液酸酶是一类糖水解酶，已知其在宿主细胞和组织的侵袭中发挥重要作用。在本研究中，我们在牙鲆（Paralichthys olivaceus）模型中研究了鱼类病原体迟缓爱德华氏菌（Edwardsiella tarda）的唾液酸酶 NanA 的潜在功能。NanA 由 670 个残基组成，与已知的细菌唾液酸酶具有较低的序列同一性。计算机分析表明，NanA 具有唾液酸酶结构域和自转运结构域，前者包含五个 Asp-box、RIP 基序和细菌唾液酸酶的保守催化位点。与唾液酸酶结构域相对应的纯化重组 NanA（rNanA）表现出对唾液酸底物的糖水解酶活性，这种活性依赖于 pH 值和温度。免疫荧光显微镜显示抗 rNanA 抗体与 E. tarda 结合，表明 NanA 定位于细胞表面。nanA 突变导致 E. tarda 在鱼组织中传播和定植的能力以及感染鱼的死亡率急剧降低。同样，细胞研究表明，nanA 突变体在感染牙鲆细胞的感染力方面显著受损。免疫保护分析表明，rNanA 作为亚单位疫苗在牙鲆中赋予了针对致命性 E. tarda 挑战的有效保护。rNanA 疫苗接种诱导产生了特异性血清抗体，增强了补体介导的杀菌活性，并减少了 E. tarda 感染牙鲆细胞。这些结果表明，NanA 在 E. tarda 的发病机制中发挥重要作用，可能被用于水产养殖中控制 E. tarda 感染。
