LOCALIZED VIRULENCE FACTOR ASSEMBLY IN ENTEROCOCCUS FAECALIS: COORDINATION AND TA

粪肠球菌局部毒力因子组装：协调和 TA

• 批准号: 8090834
• 负责人: Kimberly Ann Kline
• 金额: $ 9万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2011-10-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8090834
• 关键词: Affect; Animal Model; Antibiotic Resistance; Antimicrobial Cationic Peptides; Award; Bacteremia; Bacteria; Binding; Biochemical; Biogenesis; Biological Assay; Biological Process; Blood; Catheters; Cell Cycle; Cell Wall; Cell surface; Cells; Clinical; Communicable Diseases; Complex; Computer Analysis; Coupled; Cystitis; Cytoplasmic Tail; Data; Disease; Disease model; Doctor of Philosophy; Endocarditis; Enterococcus; Enterococcus faecalis; Environment; Enzymes; Epidemiology; Ethics; Extracellular Protein; Faculty; Fimbriae Proteins; Genes; Genetic Materials; Genome; Genomics; Goals; Gonorrhea; Gram-Negative Bacteria; Gram-Positive Bacteria; Heart; Human; Immunoelectron Microscopy; Infection; Infection prevention; Mass Spectrum Analysis; Measures; Mediating; Membrane; Membrane Proteins; Meningitis; Mentors; Mentorship; Methods; Microbial Biofilms; Microscopic; Modeling; Molecular; Multiprotein Complexes; Mutation; Neisseria gonorrhoeae; Nosocomial Infections; Oral; Organism; Pathogenesis; Pathway interactions; Patients; Pattern; Peptide Hydrolases; Peptides; Physiology; Pilum; Polymers; Population; Positioning Attribute; Prevalence; Process; Protein Secretion; Proteins; Public Health; Reading; Recruitment Activity; Regulation; Research; Research Personnel; Resistance; Resort; Role; Scientist; Site; Sorting - Cell Movement; Surface; Techniques; Testing; Time; Training; Universities; Urinary tract; Urinary tract infection; Virulence; Virulence Factors; Washington; Work; Wound Infection; Writing; antimicrobial; antimicrobial drug; base; chemical genetics; comparative; design; experience; extracellular; human disease; human tissue; in vivo; interest; member; methicillin resistant Staphylococcus aureus; microbial; mutant; new therapeutic target; novel; pathogen; polymerization; post-doctoral training; programs; protein transport; skills; sortase; tool; vector

DESCRIPTION (provided by applicant): Enterococci are one of the leading causes of nosocomial infections and cause a variety of disease states including endocarditis, bacteremia, meningitis, wound infections, and urinary tract infections. Enterococcal and other Gram positive infections are becoming increasingly difficult to treat due to a rising prevalence of resistance to antibiotics of last resort. E. faecalis also serves as a vector for the spread of antibiotic resistance determinants through conjugal transfer to other bacterial species. Thus, it is of great interest to understand fundamental biological processes of this organism with the aim of identifying novel therapeutic targets. It is becoming increasingly evident that focally localized assembly of surface-exposed proteins is a general strategy among Gram positive pathogens and is strongly associated with microbial virulence. However, little is known about how the focal surface protein assembly machinery itself localizes. Therefore, the objective of this project is to determine the mechanisms underlying localized virulence factor assembly and assess whether these sites can be specifically targeted by antimicrobials. My research aims to clarify the conceptual details and implications of localized virulence factor assembly for Gram positive infection and therapy using two complementary approaches. First, I will determine the molecular components that guide focal localization and retention at single domains using classical and chemical genetic methods to manipulate localization patterns of secretion and sorting proteins, coupled with biochemical and microscopic assays for focal interactions. Second, I will assess the functional consequence of perturbing focal localization for virulence factor assembly read out at the single cell level, the bacterial population level, and in models of disease. The virulence factor and model organism of choice for these studies is the sortase-assembled pilus of E. faecalis because of their outstanding clinical importance. A more thorough understanding of focally localized virulence factor assembly will be achieved in 3 aims designed to: 1) define structural components of the localized complex, 2) elucidate subcellular assembly mechanisms, and 3) characterize the role of localization in virulence factor function and virulence. The accessibility of localized sites of Enterococcal secretion and sorting to the extracellular environment and its critical role in processing and secreting virulence factors make it a likely site for both localized interaction between the bacterium and the host (thus supporting virulence) and for targeting by antivirulence and antimicrobial agents (thus representing a vulnerable target). Therefore, this research has implications for both fundamental bacterial processes and also for host-pathogen interactions and therapy of an important disease agent. I obtained a Masters in Public Health and a PhD at Northwestern University, training with Dr. Hank Seifert on the contribution of pili to disease caused by the Gram-negative bacterium Neisseria gonorrhoeae. I chose to couple these two degrees because I wanted to approach the problem of gonorrhea from a multi-dimensional standpoint of understanding bacterial strategies for pathogenesis as well as implications of those particular strategies for human disease. I capitalized on my expertise in molecular and epidemiological perspectives on infectious disease with existing expertise in the Hultgren lab in the structural basis of pilus assembly and function, mammalian models of pathogenesis, and post-genomic analysis of bacterial pathogens. Working at Washington University has also enabled me to draw on the mentorship and experience of multiple scientists, including the previous chair of the department, Staffan Normark, an expert on Gram positive pilus assembly, protein secretion, and cell wall biogenesis. I have developed fruitful collaborative and mentorship relationships with current faculty members as well, including Dr. Amanda Lewis who has provided scientific expertise as a Gram positive microbiologist and professional guidance as the newest member of our faculty. I have been able to develop a new line of research within the Hultgren lab that synthesizes my experience with all of my mentors and aims to elucidate the general role of coordinated protein secretion and trafficking in both basic Gram positive physiology as well as Gram positive infections of humans. I continue to deliberately focus on fundamental concepts as they relate to disease. I plan to utilize the remaining time of my mentored training to become expert in biochemical techniques important for the continued expansion of my research interests. During my postdoctoral period, I have received formal and informal training in writing and oral presentation, lab management and mentoring skills, and in the ethical conduct of research. I will continue to develop these skills during the remainder of my postdoctoral training. After acquiring this expertise, I will transition into an independent faculty position, pursuing this research program full time at a major research university. A K99/R00 award is thus ideally suited for my current situation and would be a decided advantage in both providing me additional mentored time to acquire specific technical training and facilitating my transition into an independent investigator. PUBLIC HEALTH RELEVANCE STATEMENT: Enterococcus faecalis is a bacterium that causes infections in sites ranging from the heart to the blood to the urinary tract, most often in already ill and hospitalized patients. This project will investigate the way these bacteria builds "tools" to attach to human tissues during infection. The goal of these studies is to find new antimicrobial agents that can target these bacterial "tools" and help prevent infection in humans.

描述（由申请方提供）：肠球菌是医院感染的主要原因之一，可引起多种疾病，包括心内膜炎、菌血症、脑膜炎、伤口感染和尿路感染。肠球菌和其他革兰氏阳性菌感染正变得越来越难以治疗，因为对最后手段抗生素的耐药性越来越普遍。E.粪肠球菌还充当抗生素抗性决定簇通过接合转移至其它细菌物种而传播的载体。因此，了解这种生物体的基本生物学过程以确定新的治疗靶点是非常有趣的。越来越明显的是，表面暴露的蛋白质的局部组装是革兰氏阳性病原体中的一般策略，并且与微生物毒力密切相关。然而，很少有人知道的焦点表面蛋白组装机器本身的本地化。因此，本项目的目的是确定局部毒力因子组装的机制，并评估这些位点是否可以被抗菌剂特异性靶向。 我的研究旨在阐明局部毒力因子组装对革兰氏阳性感染和使用两种互补方法进行治疗的概念细节和影响。首先，我将确定的分子组成部分，指导局灶性定位和保留在单域使用经典和化学遗传学方法来操纵分泌和分选蛋白质的定位模式，再加上生化和显微镜测定局灶性相互作用。第二，我将评估功能的影响扰乱局灶性定位的毒力因子组件读出在单细胞水平，细菌种群水平，并在疾病模型。这些研究选择的毒力因子和模式生物是E.由于其突出的临床重要性。对局灶性定位的毒力因子组装的更透彻理解将在3个目标中实现：1）定义定位复合物的结构组分，2）阐明亚细胞组装机制，3）表征定位在毒力因子功能和毒力中的作用。肠球菌分泌和分选的局部位点对细胞外环境的可及性及其在加工和分泌毒力因子中的关键作用使其成为细菌与宿主之间的局部相互作用（因此支持毒力）和抗毒力剂和抗微生物剂的靶向（因此代表易受攻击的靶标）的可能位点。因此，这项研究对基本的细菌过程以及宿主-病原体相互作用和重要疾病因子的治疗都有意义。 我在西北大学获得了公共卫生硕士学位和博士学位，与汉克·塞弗特博士一起培训皮利对革兰氏阴性细菌淋病奈瑟菌引起的疾病的贡献。我选择将这两个学位结合起来，因为我想从多维度的角度来理解淋病的问题，即理解细菌致病的策略以及这些特定策略对人类疾病的影响。我利用了我在传染病分子和流行病学方面的专业知识，以及Hultgren实验室在菌毛组装和功能的结构基础、哺乳动物发病机制模型和细菌病原体的后基因组分析方面的现有专业知识。在华盛顿大学工作也使我能够借鉴多位科学家的指导和经验，包括该部门的前任主席Staffan Normark，他是革兰氏阳性菌毛组装，蛋白质分泌和细胞壁生物发生的专家。我已经开发了富有成效的合作和指导关系，以及与当前的教师，包括博士阿曼达刘易斯谁提供了科学的专业知识作为革兰氏阳性微生物学家和专业指导作为我们的教师的最新成员。我已经能够在Hultgren实验室内开发一种新的研究路线，该实验室综合了我与所有导师的经验，旨在阐明协调蛋白质分泌和贩运在基本革兰氏阳性生理学以及人类革兰氏阳性感染中的一般作用。我继续刻意关注与疾病相关的基本概念。我计划利用我的指导培训的剩余时间，成为生化技术的专家，这对继续扩大我的研究兴趣很重要。在博士后期间，我接受了正式和非正式的写作和口头陈述，实验室管理和指导技能，以及研究道德行为方面的培训。我将继续发展这些技能在我的博士后培训的其余部分。在获得这些专业知识后，我将过渡到一个独立的教师职位，在一所主要的研究型大学全职从事这项研究计划。因此，K99/R 00奖项非常适合我目前的情况，并且在为我提供额外的指导时间以获得特定的技术培训和促进我向独立调查员的过渡方面具有决定性的优势。 公共卫生相关声明：粪肠球菌是一种细菌，可引起从心脏到血液再到泌尿道的感染，最常见于已经患病和住院的患者。该项目将研究这些细菌在感染过程中如何构建附着在人体组织上的“工具”。这些研究的目标是找到新的抗菌剂，可以针对这些细菌“工具”，并帮助预防人类感染。