Pathogenic strategies of the swine pathogen Streptococcus suis

猪链球菌（血清型2）是一种导致全球养猪业重大经济损失的猪病原体。由这种病原体引起的主要感染包括关节炎、败血症、脑膜炎和心内膜炎。本实验室的研究项目主要集中在鉴定和描述由S。猪。更具体地说，未来五年的研究计划依赖于两个特定的假设：1）S.猪链球菌在感染过程中起着几个关键作用;猪流感病毒具有广泛的病理功能，并代表了一种新的毒力机制。下面列出的与每个假设相关的研究是我过去NSERC研究资助期间所做工作的逻辑延续。* 在我的研究计划的第一部分，我建议研究SspA蛋白酶可能有助于S.猪传染病。我们将首先确定上调sspA基因表达的参数和生长条件（生物膜生长、与宿主细胞共培养、存在血清或亚最小抑制浓度的抗生素等）。然后，使用SspA缺陷突变体以及纯化的重组蛋白酶，我们将研究S. suis SspA对i）先天性免疫组分（抗微生物肽、上皮细胞屏障完整性）的失活，和ii）通过活性TNF-α的脱落和从高分子量激肽原产生缓激肽来放大炎症反应。在我的研究计划的第二部分中，我提出了表征膜囊泡产生的S。并鉴定其病理功能。我们将首先研究S. suis囊泡的产生受到各种环境条件的影响。然后，我们将研究囊泡在毒素传递的作用，通过使用蛋白质组学方法表征其组成。鉴于初步数据显示囊泡具有高DNA酶活性，我们将评估它们允许S.猪逃避中性粒细胞胞外陷阱。最后，S.猪膜囊泡将通过研究其i）激活核因子-kB信号传导途径和ii）通过微血管内皮细胞和巨噬细胞的共培养模型诱导炎症介质分泌的能力来测试。*意义：本研究有两个互补的目的：（i）增加我们对沙门氏菌致病策略的基本认识;猪是抵消宿主防御系统和放大破坏性炎症反应，和（ii）使用这些知识，以确定新的疫苗分子候选人，并设计针对细菌毒力因子的药物，以更有效地控制S.猪传染病。

Streptococcus suis (serotype 2) is a swine pathogen responsible for important economic losses in the swine industry worldwide. Major infections caused by this pathogen include arthritis, septicemia, meningitis and endocarditis. The research program of my laboratory focusses on the identification and characterization of pathogenic strategies developed by S. suis. More specifically, the research proposed for the next five years relies on two specific hypotheses: 1) The cell surface protease SspA of S. suis plays several key roles in the infectious process; and 2) Membrane vesicles secreted by S. suis possess a wide array of pathological functions and represent a new virulence mechanism. The studies related to each hypothesis are outlined below are a logical continuation of the work performed during my past NSERC research funding. ***In the first part of my research program, I propose to investigate the mechanisms by which the SspA protease may contribute to S. suis infections. We will first determine the parameters and growth conditions (biofilm growth, co-culture with host cells, presence of serum or sub-minimal inhibitory concentrations of antibiotics, etc) upregulating the expression of sspA gene. Then, using an SspA-deficient mutant as well as the purified recombinant protease, we will investigate the effects of S. suis SspA on i) inactivation of the innate immune components (antimicrobial peptide, epithelial cell barrier integrity), and ii) amplification of the inflammatory response through shedding of active TNF-alpha and generation of bradykinin from high-molecular-weight kininogens.***In the second part of my research program, I propose to characterize membrane vesicles produced by S. suis and to identify their pathological functions. We will first study the modulation of S. suis vesicle production by various environmental conditions. Then, we will investigate the role of vesicles in toxin delivery by characterizing their composition using a proteomic approach. Given that preliminary data revealed that vesicles possess a high DNase activity, we will evaluate their ability to allow S. suis to escape from neutrophil extracellular traps. Lastly, the inflammatory potential of S. suis membrane vesicles will be tested by investigating their ability to i) activate the nuclear factor-kB signaling pathway, and ii) induce the secretion of inflammatory mediators by a co-culture model of microvascular endothelial cells and macrophages.***Significance: The proposed studies have two complementary objectives: (i) to increase our basic knowledge of the pathogenic strategies used by S. suis to counteract the host defense system and amplify the destructive inflammatory response, and (ii) to use this knowledge to identify new vaccine molecule candidates and to design drugs targeting bacterial virulence factors in view to more efficiently control S. suis infections.