MHC-ACCESSIBILITY OF SECRETED SALMONELLA PROTEINS

细胞内的细菌病原体通过选择性地分泌蛋白质来适应宿主细胞的环境，这些蛋白质旨在改变宿主细胞的正常结构和代谢机制，从而促进细菌的生存和避免宿主免疫监视。这些分泌的细菌蛋白在吞噬溶酶体内或直接在宿主细胞细胞质内调节它们的作用。因为进入细胞内部就意味着进入宿主的蛋白分解机制，所以我们将这些蛋白统称为MHC类可访问蛋白(CAPS)。对宿主细胞入侵后分泌的CAPS进行鉴定，将为研究宿主细胞的细菌抗原处理和细菌修饰提供宝贵的资源。此外，由于CAP具有进入宿主抗原处理和递送机制的独特途径，它们是潜在的有吸引力的疫苗靶标和有用的载体，用于通过细菌载体疫苗递送外源表位。为了识别鼠伤寒沙门氏菌的CAPS，我们开发了一种新的方法，称为I类表位的弥散性插入(DICE)。DICE使用基于Tn5的可分辨转座子随机分布MHC-1H-2kb限制性卵清蛋白表位SIINFEKL。当解决的插入是在一个基因的框内时，感染后细菌释放的帽被宿主细胞处理，导致携带的表位呈现在H-2kb的背景下。然后，使用H-2kb/SIINFEKL特异性抗体，通过流式细胞仪很容易地分离出在帽内含有表位插入的沙门氏菌菌株。我将把重点放在I类可获得的细菌蛋白质上，并准确地描述它们如何到达受感染细胞的细胞质。这些研究对疫苗的开发和细菌的发病机制具有重要的意义。

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.