Identification of Bacterial Resistance Mechanisms to Antimicrobial Chemokines

描述（由申请人提供）：抗菌趋化因子（AMCs）是宿主防御肽家族中尚未研究的成员，它们在保护多种生物体免受细菌感染方面共同发挥重要作用。了解AMCs如何识别和结合细菌靶标是理解先天免疫系统基本机制的关键，也是设计基于病原体和先天免疫系统之间数百万年共同进化的新型抗菌疗法的关键。这一应用验证了不同细菌不受AMC影响的假设，并且鉴定调节AMC敏感性的细菌蛋白将揭示可能为抗菌治疗提供新靶点的过程。由于与AMC结合的差异可能是细菌敏感性的关键决定因素，我们开发了一种基于流式细胞术的测定方法来测量AMC与细菌细胞的结合。目的1涉及利用该试验筛选数千种耶尔森氏菌转座子突变体，以鉴定与野生型细菌相比具有高AMC结合表型的突变体。与脂多糖生物合成相关的基因似乎在AMC避免和抗性中起主要作用，Aim 2涉及这些基因在假结核杆菌和鼠疫杆菌中对AMC避免的贡献的详细表征。Aim 3测试由于特定突变导致的AMC结合增加是否与对AMC以及其他宿主防御肽杀伤的敏感性增加相关。了解AMCs和其他宿主防御肽如何识别和结合细菌，以及细菌如何逃避宿主防御肽的抗菌活性，可能会导致增强先天免疫系统的新药物靶点。通过同时靶向对这些多肽产生抗性所需的脂多糖生物合成等过程，可以显著增强AMCs抗感染的治疗作用。

DESCRIPTION (provided by applicant): Antimicrobial chemokines (AMCs) are understudied members of the family of host defense peptides, which together play an essential role in protecting a wide variety of organisms from bacterial infection. Understanding how AMCs recognize and bind to bacterial targets is key to understanding the basic mechanisms of the innate immune system, as well as designing new antimicrobial therapies that build on millions of years of co-evolution between pathogens and the innate immune system. This application tests the hypothesis that different bacteria are not uniformly affected by AMCs, and identifying bacterial proteins that modulate AMC susceptibility will uncover processes that could provide novel targets for antimicrobial therapy. Since differences in binding to AMCs could be a critical determinant of bacterial susceptibility, we have developed a flow cytometry based assay to measure AMC binding to bacterial cells. Aim 1 involves utilizing this assay to screen thousands of Yersinia transposon mutants to identify those with high AMC binding phenotypes compared to wild type bacteria. Genes associated with lipopolysaccharide biosynthesis appear to play a major role in AMC avoidance and resistance, and Aim 2 involves a detailed characterization of the contributions of these genes to AMC avoidance in both Y. pseudotuberculosis and Y. pestis. Aim 3 tests whether increases in AMC binding due to specific mutations correlate with increased sensitivity to killing by AMCs, as well as by other host defense peptides. Understanding how AMCs and other host defense peptides recognize and bind bacteria, as well as how bacteria evade the antimicrobial activity of host defense peptides, could lead to novel drug targets that enhance the innate immune system. The therapeutic use of AMCs to fight infections may be significantly enhanced by simultaneously targeting processes such as lipopolysaccharide biosynthesis that are required for resistance to these peptides. PUBLIC HEALTH RELEVANCE: This project involves determining how bacterial pathogens resist the body's innate immune defenses. These defenses include a family of peptides called antimicrobial chemokines. These peptides can kill bacteria directly, in addition to their other roles in the immune system. New treatments that interfere with bacterial defense mechanisms could render pathogens less able to cause disease.