Deletion of the <i>Riemerella anatipestifer</i> type IX secretion system gene <i>sprA</i> results in differential expression of outer membrane proteins and virulence

<i>Riemerella anatipestifer</i> (RA), the causative agent of infectious serositis that targets ducklings and other poultry, secretes protein via the type IX secretion system (T9SS). The proteins transported by T9SS are located on the bacterial cell surface or secreted into the extracellular milieu. In this study, a <i>sprA</i> deletion mutant was constructed encoding a core protein of T9SS to investigate its influence on outer membrane protein expression and its role in virulence. Compared with the wild-type RA-YM strain, the deletion mutant Δ<i>sprA</i> failed to digest gelatin, showed the same growth rate in the logarithmic phase and exhibited greater sensitivity to the bactericidal activity of duck sera, whereas the complemented strain restored these phenotypes. The outer membrane proteome of RA-YM and the Δ<i>sprA</i> mutant were analyzed by Tandem Mass Tags, which revealed 198 proteins with predicted localization to the cell envelope. Sixty-three of these proteins were differentially expressed in the outer membrane, with 43 up-regulated and 20 down-regulated. Among the twelve outer membrane proteins which were secreted by T9SS, four proteins were up-regulated and one protein was down-regulated. Animal experiments demonstrated that the median lethal dose of the mutant strain Δ<i>sprA</i> was about 500 times higher than that of the wild-type RA-YM strain, and bacterial loads in blood, brain, heart, liver and spleen of the Δ<i>sprA</i>-infected ducks were significantly reduced. Our results indicate that the SprA is a virulence-associated factor of RA, and its absence results in altered abundance of outer membrane proteins, and secretion disorders associated with some of the T9SS effector proteins.鸭疫里默氏杆菌（Riemerella anatipestifer，以下简称RA）是引发雏鸭及其他禽类感染性浆膜炎的病原菌，可通过IX型分泌系统（Type IX Secretion System，T9SS）分泌蛋白质。经T9SS转运的蛋白质定位于细菌细胞表面，或被分泌至胞外环境中。本研究构建了一株编码T9SS核心蛋白的sprA基因缺失突变株，以探究其对细菌外膜蛋白表达的影响，以及其在病原菌毒力中的作用。与野生型RA-YM菌株相比，ΔsprA缺失突变株无法降解明胶，在对数生长期的生长速率与野生型一致，但对鸭血清的杀菌活性表现出更高的敏感性，而互补菌株可恢复上述表型。本研究采用串联质量标签（Tandem Mass Tags，TMT）技术分析RA-YM菌株与ΔsprA突变株的外膜蛋白质组，共鉴定得到198个预测定位于细胞被膜的蛋白质。其中63个蛋白质在外膜中呈现差异表达，43个表达上调，20个表达下调。在12种经T9SS分泌的外膜蛋白质中，4种表达上调，1种表达下调。动物实验结果显示，ΔsprA突变株的半数致死量约为野生型RA-YM菌株的500倍，且感染ΔsprA的雏鸭其血液、脑、心脏、肝脏及脾脏中的细菌载量显著降低。本研究结果表明，SprA是鸭疫里默氏杆菌的毒力相关因子，其缺失会导致外膜蛋白质丰度发生改变，并引发部分T9SS效应蛋白的分泌紊乱。