Salmonella Pathogenesis and Processing of Secreted Effectors by Caspase-3

沙门氏菌发病机制和 Caspase-3 对分泌效应子的处理

基本信息

批准号：
8705749
负责人：
Beth A McCormick
金额：
$ 41.16万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8705749
关键词：
Accounting Actins Amino Acid Substitution Amino Acids Animal Model Apoptosis Apoptotic Attenuated Bacteria Binding Biochemical Biochemistry Bioinformatics Biological Biological Assay Biological Phenomena C-terminal Cell Culture Techniques Cells Cellular biology Cleaved cell Clinical Computer Simulation Developed Countries Disease Drug Design Epithelial Epithelial Cells Escherichia coli EHEC Evaluation Gastroenteritis Genes Histologic Imaging Techniques In Vitro Infection Inflammatory Inflammatory disease of the intestine Intestines Investigation Knock-out Link Medical Modeling Molecular Multi-Drug Resistance Mus N-terminal Necrosis Organism Pathogenesis Pathology Pathway interactions Play Positioning Attribute Process Property Protein S Proteins Role Salmonella Salmonella enterica Salmonella infections Salmonella typhimurium Seminal Series Serotyping Severities Shigella Signal Pathway Signal Transduction Site Structure Structure-Activity Relationship Surface Testing Therapeutic Type III Secretion System Pathway Vaccines Virulence Virulent Yersinia actin 2 arm base caspase-3 design enteric pathogen enteropathogenic Escherichia coli foodborne in vivo in vivo Model innovation insight interest intestinal epithelium microbial migration neutrophil novel pathogen polymerization research study

项目摘要

DESCRIPTION (provided by applicant): We have shown that the S. Typhimurium type III secretion system (T3SS) effector SipA is necessary and sufficient for the promotion of active states of intestinal inflammation, a hallmark pathology of salmonellosis (1). SipA is a bi-functional molecule that is not only responsible for the induction of PMN migration across the intestinal epithelium but also plays a role in promoting actin polymerization, a process that facilitates bacterial entry into epithelial cells. We determined that SipA harbors distinct functioal motifs that account for its induction of PMN transcellular signals and the binding to actin (2). In examining the bi-functional properties of SipA, we discovered that SipA contains a caspase-3 (CASP3) recognition and cleavage motif (DEVD) at amino acid position 431, a site precisely located between the two functional domains, termed SipAa (inflammatory domain) and SipAb (actin binding domain). The caspase-3 cleavage motif is physiologically significant, as a single amino acid substitution to a sequence not recognized by CASP3 profoundly attenuates the virulence of this pathogen in both in vitro and in vivo models of salmonellosis. Further analysis of the S. Typhimurium T3SS revealed the presence of CASP3 cleavage motifs in other type three secreted effectors (T3SE) with known bi-functional properties (i.e., SopA, SifA), indicating this phenomenon is not limited to SipA. Based on these observations we speculate that certain effector proteins of the S. Typhimurium T3SS exist in a pro-form, requiring processing by CASP3 to become functional. Thus, the objective of this proposal is to test the novel hypothesis that CASP3 cleavage of T3SS secreted effectors represents a common mechanism by which effector functions are regulated in host cells. We envisage activation of CASP3 cleaves Salmonella effector proteins harboring CASP3 recognition motifs into distinct functional subunits. To test this hypothesis, the aims of this proposal are centered on the mechanism underlying CASP3 cleavage of T3SEs with a particular focus on the temporal, spacial, and biological significance of this novel biological phenomenon. Specific Aim 1 is designed to determine the structure/function relationship of the Salmonella-effector CASP3 motifs. Under this aim, extensive biochemical analysis (using both in vitro and in vivo assays) will be performed to evaluate the extent to which the activity of Salmonella type III secreted effector functional domains requires CASP3 cleavage. In Specific Aim 2, we will begin to evaluate CASP3 activation and its role during infection by S. Typhimurium. In particular, we will employ state-of-the-art imaging techniques combined with sophisticated biochemical and bioinformatics approaches designed to the link temporal induction of CASP3 activity to the cleavage of specific S. Typhimurium effectors. Finally, in Specific Aim 3 we will define the mechanism(s) of activation of CASP3 during infection by S. Typhimurium. Our prior studies have shown that early after infection, the S. Typhimurium effector, SipA, is necessary and sufficient to promote activation of CASP3 but without inducing apoptosis or necrosis. Using a multi- disciplinary approach, involving cell biology, biochemistry and animal modeling, we will explore the molecular mechanism by which SipA triggers the activation of CASP3. Understanding the role that CASP3 plays in controlling T3SS effector function will greatly advance the understanding of Salmonella pathogenesis, and reveal novel insight on co-evolutionary relationships. On a broader spectrum, understanding of this new biological phenomenon will allow us to dissect specific aspects of host-bacterial relationships that have yet to be documented. Speculatively, the ability of the organism to hijack the host CASP3 machinery in order to activate secreted effectors is likely to be a general paradigm employed by enteric pathogens that harbor a T3SS to cause disease.

描述（由申请人提供）：我们已经证明，鼠伤寒沙门氏菌 III 型分泌系统 (T3SS) 效应子 SipA 对于促进肠道炎症的活跃状态是必要且充分的，肠道炎症是沙门氏菌病的标志性病理学 (1)。 SipA 是一种双功能分子，不仅负责诱导 PMN 跨肠上皮迁移，而且还在促进肌动蛋白聚合（促进细菌进入上皮细胞的过程）中发挥作用。我们确定 SipA 具有独特的功能基序，这些基序解释了其诱导 PMN 跨细胞信号以及与肌动蛋白的结合 (2)。在通过检查 SipA 的双功能特性，我们发现 SipA 在氨基酸位置 431 处包含 caspase-3 (CASP3) 识别和切割基序 (DEVD)，该位点精确位于两个功能域之间，称为 SipAa（炎症域）和SipAb（肌动蛋白结合域）。 caspase-3 切割基序具有重要的生理意义，因为对 CASP3 不识别的序列进行单个氨基酸取代可显着减弱该病原体在沙门氏菌病体外和体内模型中的毒力。对鼠伤寒沙门氏菌 T3SS 的进一步分析揭示了其他具有已知双功能特性的三型分泌效应子 (T3SE)（即 SopA、SifA）中存在 CASP3 裂解基序，表明这种现象并不限于 SipA。基于这些观察，我们推测鼠伤寒沙门氏菌 T3SS 的某些效应蛋白以原形式存在，需要 CASP3 的处理才能发挥作用。因此，本提案的目的是测试新的假设，即 T3SS 分泌效应器的 CASP3 裂解代表宿主细胞中调节效应器功能的常见机制。我们设想 CASP3 的激活会将含有 CASP3 识别基序的沙门氏菌效应蛋白裂解成不同的功能亚基。为了检验这一假设，本提案的目的集中在 T3SE 的 CASP3 裂解的机制上，特别关注这种新生物现象的时间、空间和生物学意义。具体目标 1 旨在确定沙门氏菌效应子 CASP3 基序的结构/功能关系。在此目标下，将进行广泛的生化分析（使用体外和体内测定）以评估沙门氏菌 III 型分泌效应功能域的活性需要 CASP3 裂解的程度。在具体目标 2 中，我们将开始评估 CASP3 激活及其在鼠伤寒沙门氏菌感染期间的作用。特别是，我们将采用最先进的成像技术与复杂的生化和生物信息学方法相结合，旨在将 CASP3 活性的时间诱导与特定鼠伤寒沙门氏菌效应子的裂解联系起来。最后，在具体目标 3 中，我们将定义鼠伤寒沙门氏菌感染期间 CASP3 的激活机制。我们之前的研究表明，感染后早期，鼠伤寒沙门氏菌效应子 SipA 对于促进 CASP3 的激活是必要且充分的，但不会诱导细胞凋亡或坏死。我们将采用细胞生物学、生物化学和动物模型等多学科方法，探索SipA触发CASP3激活的分子机制。了解 CASP3 在控制 T3SS 效应器功能中所起的作用将极大地促进对沙门氏菌发病机制的理解，并揭示关于共同进化关系的新见解。在更广泛的范围内，对这种新生物现象的理解将使我们能够剖析尚未记录的宿主与细菌关系的特定方面。据推测，生物体劫持宿主 CASP3 机制以激活分泌效应器的能力可能是携带 T3SS 的肠道病原体引起疾病的一般范例。