Salmonella Pathogenesis and Processing of Secreted Effectors by Caspase-3

描述（由申请人提供）：我们已经表明，S。鼠伤寒III型分泌系统（T3 SS）效应子SipA对于促进肠道炎症的活动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迁移穿过肠上皮，而且还在促进肌动蛋白聚合中发挥作用，这是一种促进细菌进入上皮细胞的过程。我们确定SiPA具有不同的功能基序，这些基序解释了其诱导PMN跨细胞信号和与肌动蛋白结合的原因（2）。在 通过研究SipA的双功能特性，我们发现SipA在氨基酸位置431处含有半胱天冬酶-3（CASP 3）识别和切割基序（DEVD），该位点精确地位于两个功能结构域之间，称为SipAa（炎症结构域）和SipAb（肌动蛋白结合结构域）。半胱天冬酶-3切割基序具有生理学意义，因为对不被CASP 3识别的序列的单个氨基酸取代在沙门氏菌病的体外和体内模型中都显著减弱了该病原体的毒力。对S.鼠伤寒T3 SS揭示了在具有已知双功能特性的其他三型分泌效应子（T3 SE）中存在CASP 3切割基序（即，SopA，SifA），表明这种现象不仅限于SipA。基于这些观察，我们推测，某些效应蛋白的S。鼠伤寒T3 SS以前体形式存在，需要CASP 3处理才能发挥功能。因此，该提议的目的是测试T3 SS分泌的效应物的CASP 3切割代表在宿主细胞中调节效应物功能的常见机制的新假设。我们设想CASP 3的激活将携带CASP 3识别基序的沙门氏菌效应蛋白切割成不同的功能亚基。为了验证这一假设，该提案的目的是集中在T3 SE的CASP 3切割的机制，特别关注这种新的生物学现象的时间，空间和生物学意义。特异性目的1旨在确定沙门氏菌效应子CASP 3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），以评价沙门氏菌III型分泌的效应子功能结构域的活性需要CASP 3切割的程度。在具体目标2中，我们将开始评估CASP 3激活及其在S.鼠伤寒特别是，我们将采用最先进的成像技术，结合复杂的生物化学和生物信息学方法，旨在将CASP 3活性的时间诱导与特异性S.鼠伤寒效应子。最后，在具体目标3中，我们将定义在S.鼠伤寒我们先前的研究表明，感染后早期，S。鼠伤寒沙门氏菌效应子SiPA是促进CASP 3活化所必需的，但不诱导凋亡或坏死。我们将采用多学科方法，涉及细胞生物学、生物化学和动物模型，探索SiPA触发CASP 3激活的分子机制。了解CASP 3在控制T3 SS效应子功能中的作用将大大推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，对这种新的生物现象的理解将使我们能够解剖尚未被记录的宿主-细菌关系的特定方面。推测，生物体劫持宿主CASP 3机制以激活分泌的效应物的能力可能是具有T3 SS的肠道病原体引起疾病的一般范例。

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.