Structure-function analysis of polymorphic CDI toxin-immunity protein complexes a

描述（由申请人提供）：细菌已经进化出复杂的策略来相互竞争和交流。最近在大肠杆菌中发现了一种新的细菌间竞争机制，称为接触依赖性生长抑制（CDI）。CDI系统存在于多种革兰氏阴性细菌中，包括许多重要的人类病原体。CDI由CdiB/CdiA双伴侣分泌系统介导。CdiB是预测的外膜蛋白，其是将CdiA外蛋白输出和组装到细胞表面上所需的。CdiA的C-末端区域（CdiA-CT）含有生长抑制活性，并且可能被切割并易位到靶细胞中以抑制生长。CDI系统还编码CdiI免疫蛋白，其结合并抑制其同源CdiA-CT毒素，从而保护CDI+细胞免受自身抑制。值得注意的是，CdiA-CT毒素结构域是多态性的，因此它们相应的CdiI免疫蛋白也是高度可变的。到目前为止，我们已经确定了至少30个不同的毒素免疫家族。不同的CdiA-CT/CdiI家族之间通常存在小于20%的氨基酸序列同一性，这强烈表明每个CdiA-CT/CdiI复合物的蛋白质-蛋白质相互作用是独特的。此外，我们最近发现，一些CdiA-CT结构域与称为“允许因子”的特定靶细胞蛋白相互作用，并且这些结合相互作用是激活递送的毒素所必需的。目前，CdiI蛋白中和和允许因子激活CdiA-CT毒素的机制尚不清楚。我们提出了结构，生化和遗传分析，以深入了解细菌CDI系统编码的复杂的毒素免疫网络。在这项提案中，我们要求PSI网络中心协助解决至少10个不同的CdiA-CT/CdiI复合物的晶体结构。 这一建议代表了一个独特的机会，阐明如何保持特异性结合的毒素免疫对通过进化分歧。 公共卫生相关性：细菌已经进化出复杂的策略来相互竞争和交流。接触依赖性生长抑制（CDI）是近年来在革兰氏阴性菌中发现的一种新的细菌间竞争机制。该提案利用结构，生物化学和遗传分析，以获得对CDI系统编码的复杂毒素免疫网络的机械见解。这项研究将大大增加我们对细菌病原体的进化和生态学的理解，并可能为新型抗菌疗法的开发提供信息。

DESCRIPTION (provided by applicant): Bacteria have evolved complex strategies to compete and communicate with one another. A new mechanism of interbacterial competition, termed contact-dependent growth inhibition (CDI) was recently discovered in Escherichia coli. CDI systems are found in a wide variety of gram-negative bacteria, including many important human pathogens. CDI is mediated by the CdiB/CdiA two-partner secretion system. CdiB is a predicted outer membrane protein that is required for the export and assembly of the CdiA exoprotein onto the cell surface. The C-terminal region of CdiA (CdiA-CT) contains the growth inhibition activity and is presumably cleaved and translocated into target cells to inhibit growth. CDI systems also encode CdiI immunity proteins, which bind and inactivate their cognate CdiA-CT toxins, thereby protecting CDI+ cells from autoinhibition. Remarkably, the CdiA-CT toxin domains are polymorphic, and accordingly their corresponding CdiI immunity proteins are also highly variable. We have identified at least 30 distinct toxin-immunity families to date. There is typically less than 20% amino acid sequence identity between different CdiA-CT/CdiI families, strongly suggesting that the protein-protein interactions underlying each CdiA-CT/CdiI complex are unique. Moreover, we have recently discovered that some CdiA-CT domains interact with specific target cell proteins termed "permissive factors", and these binding interactions are required to activate the delivered toxins. Currently, the mechanisms by which CdiI proteins neutralize and permissive factors activate CdiA-CT toxins are not understood. We propose structural, biochemical and genetic analyses to gain insights into the intricate toxin-immunity network encoded by bacterial CDI systems. In this proposal, we challenge the PSI Network center to assist in solving the crystal structures of at least ten distinct CdiA-CT/CdiI complexes. This proposal represents a unique opportunity to elucidate how specific binding is maintained as toxin-immunity pairs diverge through evolution. PUBLIC HEALTH RELEVANCE: Bacteria have evolved complex strategies to compete and communicate with one another. A new mechanism of interbacterial competition, termed contact-dependent growth inhibition (CDI) has recently been discovered in a wide variety of gram-negative bacterial pathogens. This proposal utilizes structural, biochemical and genetic analyses to gain mechanistic insights into the intricate toxin-immunity network encoded by CDI systems. This research will significantly increase our understanding of the evolution and ecology of bacterial pathogens and could inform the development of novel antimicrobial therapies.