Functional analysis of a novel cell envelope integrity protein in Brucella ovis

摘要 这项研究的长期目标是确定细菌如何保持细胞包膜的完整性， 面对恶劣和波动的环境条件。革兰氏阴性菌的细胞被膜 由细胞质膜、肽聚糖细胞壁和脂多糖组成 （LPS）的外膜。在胞内病原体中，布鲁氏菌属，的组合物 LPS对宿主的毒力和持久性至关重要。我们小组最近发现了一种周质 功能未知的蛋白质，命名为EipA，在α-变形菌纲中是保守的， 包括布鲁氏菌流产布鲁氏菌eipA基因缺失导致对包膜应激物敏感 以及在小鼠感染模型中的毒力减弱。在羊种布鲁氏菌中， 平滑LPS、eipA是必需的。B中eipA表达的条件性耗竭。ovis使细胞 形成链;这为EipA在细胞分裂中的作用提供了证据。初级低温电子 显微镜结果表明，这些连锁细胞无法控制之间的距离， 细胞质膜和外膜，导致eipA缺失的细胞中周质延长。 细胞在目的1中，我将研究eipA缺失对细胞包膜结构的影响。 具体来说，我将测试eipA在a）肽聚糖合成和隔部重塑中的作用， B）EipA与磷脂的结合。在目标2中，我将描述抑制基因突变， 生长至eipA耗竭菌株，并进行无偏下拉测定，目的是 鉴定EipA的分子相互作用伴侣。本提案中描述的实验将 阐明了必需基因eipA在B中的作用。ovis并将增加我们对 α-变形菌细胞被膜

Abstract The long-term goal of this research is to determine how bacteria maintain cell envelope integrity in the face of harsh and fluctuating environmental conditions. The cell envelope of Gram-negative bacteria is composed of the cytoplasmic membrane, peptidoglycan cell wall, and the lipopolysaccharide (LPS)-containing outer membrane. In the intracellular pathogens, Brucella spp., the composition of the LPS is critical for virulence and persistence in the host. Our group recently identified a periplasmic protein of unknown function, named EipA, that is conserved across the class Alphaproteobacteria, including Brucella. Deletion of eipA in Brucella abortus results in a sensitivity to envelope stressors and an attenuation of virulence in a mouse model of infection. In Brucella ovis, a species that lacks smooth LPS, eipA is essential. Conditional depletion of eipA expression in B. ovis causes cells to form chains; this provides evidence for a role for EipA in cell division. Preliminary cryo-electron microscopy results indicate that these chained cells fail to control the distance between the cytoplasmic membrane and outer membrane, resulting in an extended periplasm in eipA-depleted cells. In aim 1, I will investigate the effect of eipA depletion on the cell envelope architecture. Specifically, I will test the role of eipA in a) peptidoglycan synthesis and remodeling at the septum and b) the binding of EipA to phospholipids. In aim 2, I will characterize suppressor mutations that restore growth to the eipA depletion strain and perform an unbiased pull-down assay with the goal of identifying the molecular interaction partners of EipA. The experiments described in this proposal will clarify the role of the essential gene eipA in B. ovis and will add to our understanding of the alphaproteobacterial cell envelope.