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

基本信息

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
项目类别：
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

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

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项目摘要

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（由申请人提供）：肠球菌是医院感染的主要原因之一，并引起多种疾病状态，包括心内膜炎，菌血症，脑膜炎，伤口感染和尿路感染。由于对持续性的抗生素抗生素的发生率上升，肠球菌和其他革兰氏阳性感染越来越难以治疗。粪肠球菌还用作抗生素耐药性决定因素通过结合转移到其他细菌物种的载体。因此，了解该生物体的基本生物学过程是非常有趣的，目的是确定新颖的治疗靶标。越来越明显的是，表面暴露蛋白的局部局部组装是革兰氏阳性病原体的一般策略，并且与微生物毒力密切相关。但是，关于焦点表面蛋白质组装机械本身如何定位的焦点表面蛋白质组装机器知之甚少。因此，该项目的目的是确定局部毒力因子组装的基础机制，并评估这些位点是否可以由抗微生物剂特异性靶向。我的研究旨在使用两种互补方法阐明局部毒力因子组装对革兰氏阴性感染和治疗的概念细节和含义。首先，我将使用经典和化学遗传学方法来确定指导焦点定位和保留在单个结构域中的分子成分，以操纵分泌和分类蛋白质的定位模式，并与生化和微观测定法进行局灶性相互作用。其次，我将评估在单细胞水平，细菌种群水平和疾病模型中读取毒力因子组装局部摄动焦点定位的功能后果。这些研究的毒力因子和模型有机体是粪肠球菌的分类酶菌毛，因为它们具有出色的临床重要性。在旨在：1）定义局部复合物的结构成分的3个目标中将对局部局部毒力因子组装进行更透彻的理解，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。肠球菌分泌的局部位点以及对细胞外环境的分类及其在处理和分泌毒力因子中的关键作用，使其成为细菌与宿主之间局部相互作用的可能位点（从而支持毒力）（从而支持毒力），并靶向抗病毒和抗生素和抗菌体（从而代表一个易于范围的目标）。因此，这项研究对基本细菌过程以及宿主 - 病原体相互作用和重要疾病剂的治疗都有意义。我在西北大学获得了公共卫生硕士学位和博士学位，并与汉克·塞弗特（Hank Seifert）博士培训有关革兰氏阴性细菌淋病的疾病造成的疾病的贡献。我之所以选择这两个学位，是因为我想从了解发病机理的细菌策略以及这些特定策略对人类疾病的影响的多维角度来解决淋病问题。我利用了我在传染病的分子和流行病学观点方面的专业知识，并在霍尔格伦实验室中具有现有专业知识的结构基础，在菌毛组装和功能的结构基础上，发病机理的哺乳动物模型以及细菌病原体的基因组分析。在华盛顿大学工作还使我能够借鉴多个科学家的指导和经验，包括该系先前的主席Staffan Normark，Staffan Normark是革兰氏正pilus组装，蛋白质分泌和细胞壁生物发生的专家。我也与现任教职员工建立了富有成果的合作和指导关系，其中包括阿曼达·刘易斯（Amanda Lewis）博士，他作为我们教职员工的最新成员提供了科学专业知识，并作为革兰氏积极的微生物学家和专业指导。我已经能够在Hultgren实验室内开发一项新的研究系列，该研究综合了我与所有导师的经验，并旨在阐明协调蛋白质分泌和贩运在基本革兰氏阴性阳性生理学以及革兰氏阳性感染中的一般作用。我继续故意专注于与疾病有关的基本概念。我计划利用我的受过指导培训的剩余时间，成为生化技术专家，对于继续扩展我的研究兴趣很重要。在博士后期间，我在写作和口头表现，实验室管理和指导技能以及研究道德行为方面接受了正式和非正式的培训。在其余的博士后培训中，我将继续发展这些技能。在获得了这一专业知识后，我将过渡到一个独立的教师职位，并在一所主要的研究大学全日制攻读这项研究计划。因此，K99/R00奖项非常适合我当前的状况，这是为我提供更多的指导时间来获得特定的技术培训并促进我向独立调查员的过渡。公共卫生相关性声明：粪肠球菌是一种细菌，可引起从心脏到血液到血液再到尿路的部位感染，最常见于已经病和住院的患者。该项目将研究这些细菌在感染过程中建立“工具”以附着在人体组织上的方式。这些研究的目的是找到可以针对这些细菌“工具”并帮助防止人类感染的新抗菌剂。