MHC-ACCESSIBILITY OF SECRETED SALMONELLA PROTEINS

细胞内细菌病原体通过选择性分泌旨在改变宿主细胞的正常结构和代谢机制的蛋白质来适应其宿主细胞环境，从而促进细菌存活并避免宿主免疫监视。这些分泌的细菌蛋白在吞噬溶酶体内或直接在宿主细胞质内介导其作用。由于进入细胞内部意味着进入宿主的蛋白水解机制，我们将这些蛋白质统称为MHC类可降解蛋白（CAP）。识别响应于宿主细胞入侵而分泌的CAP将是研究细菌抗原加工和宿主细胞的细菌修饰的宝贵资源。此外，由于CAP具有独特的进入宿主抗原加工和呈递机制的途径，因此它们代表了潜在的有吸引力的疫苗靶标和用于通过细菌载体疫苗递送外源表位的有用载体。为了鉴定鼠伤寒沙门氏菌中的CAP，我们开发了一种新的方法，称为I类表位的播散插入（DICE）。DICE使用可解析的基于Tn 5的转座子来随机分布MHC-IH-2Kb限制性卵清蛋白表位SIINFEKL。当解析的插入在基因内的框内时，感染后从细菌释放的CAP被宿主细胞加工，导致在H-2Kb的背景下呈递携带的表位。然后使用H-2Kb/SIINFEKL特异性抗体通过流式细胞术容易地分离CAP内含有表位插入的沙门氏菌菌株。我将集中在细菌的蛋白质，是第一类可访问和精确描绘他们如何可能到达受感染细胞的细胞质。这些研究对疫苗开发和细菌发病机制具有重要意义。

Intracellular bacterial pathogens adapt to their host cell environment by the selective secretion of proteins designed to alter the normal structural and metabolic machinery of the host cell, thus promoting bacterial survival and avoidance of host immune surveillance. These secreted bacterial proteins mediate their effects within the phago-lysosome or alternatively, directly within the host cell cytoplasm. Because access to the interior of the cell infers access to the proteolytic machinery of the host, we have collectively labeled these proteins MHC Class Accessible Proteins (CAPs). The identification of CAPs secreted in response to host cell invasion, would be an invaluable resource for the study of bacterial antigen processing and bacterial modification of the host-cell. In addition, since CAPs possess unique access to the host's antigen processing and presentation machinery, they represent potentially attractive vaccine targets and useful vehicles for the delivery of foreign epitopes by bacterial carrier vaccines. To identify CAPs in Salmonella typhimurium, we developed a novel approach, termed Disseminated Insertion of Class I Epitopes (DICE). DICE uses a resolvable Tn5-based transposon to randomly distribute the MHC-I H-2Kb-restricted ovalbumin epitope, SIINFEKL. When the resolved insertion is in-frame within a gene, CAPs released from the bacteria after infection are processed by the host cell, resulting in the presentation of the carried epitope in the context of H-2Kb. Salmonella strains containing epitope insertions within CAPs are then easily isolated by flow cytometry using H-2Kb/SIINFEKL specific antibodies. I will focus on bacterial proteins that are class I accessible and delineate precisely how they may reach the cytoplasm of infected cells. These studies have important implications for vaccine development and bacterial pathogenesis.