Systematic Phenotypic Analysis of the Gram-positive Envelope

革兰氏阳性包膜的系统表型分析

基本信息

批准号：
8594970
负责人：
Jason M. Peters
金额：
$ 4.92万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-12-01 至 2016-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8594970
关键词：
Affinity Chromatography Animal Model Anthrax disease Antibiotic Resistance Antibiotic susceptibility Antibiotics Area Bacillus anthracis Bacillus subtilis Bacteria Biological Assay Biological Process Cell Shape Cell Wall Cell physiology Cells Chemicals Clostridium difficile Communication Communities Complex Culture Media Cytolysis Data Data Set Databases Defect Detergents Environment Escherichia coli Experimental Models Family Gene Deletion Genes Genetic Genetic Epistasis Genome Genomics Gram-Negative Bacteria Gram-Positive Bacteria Growth Homeostasis Human Human Microbiome Individual Large-Scale Sequencing Lead Lipids Listeria monocytogenes Listeriosis Maps Measures Mediating Membrane Metals Methodology Microbiology Orphan Osmolar Concentration Osmotic Shocks Pathway interactions Penicillin-Binding Proteins Penicillins Peptidoglycan Permeability Phenotype Phosphate Carriers Play Pneumonia Polymers Process Proteins Pseudomembranous Colitis Regulon Research Research Personnel Resources Role Shapes Sigma Factor Site Specificity Streptococcus pneumoniae Stress System Teichoic Acids Thick Time Vancomycin base cell envelope deletion analysis deletion library environmental stressor gene function high throughput screening high throughput technology human disease insight microbiome mutant novel pathogen periplasm protein protein interaction public health relevance research study response retinal rods

项目摘要

DESCRIPTION (provided by applicant): The bacterial cell is encased by an envelope that is in direct and constant contact with the environment. Thus, the envelope is the site where environmental fluctuations (e.g., changes in osmolarity) are first sensed by the bacterium. Also, components of the envelope mediate communication among other bacteria as well as commensal or pathogenic interactions between bacteria and their human hosts. These interactions play critical roles in complex environments such as the human gut microbiome. Finally, many of the most successful antibiotics in widespread use target the envelope (e.g., penicillin and vancomycin), and the efficacy of antibiotics with cytoplasmic targets is often reduced by issues with envelope permeability. For these reasons, the envelope is an area of intense research focus. Research approaches that investigate a single gene product or pathway have been immensely important in defining individual components of the envelope, but are limited in determining connections between pathways and are, by definition, low-throughput. Our lab (the Carol Gross lab at UCSF) previously took a systematic approach to investigate envelope function in the Gram-negative bacterium Escherichia coli by exposing a gene deletion library to a panel of environmental stressors that targeted the envelope. Using this dataset, we were able to make significant advances in our understanding of peptidoglycan (PG) synthesis, a key component of the bacterial cell wall. However, the envelope of Gram-positive bacteria, such as Bacillus subtilis, is fundamentally different from that of E. coli. The B. subtilis envelope lacs an outer membrane (and, thus, a distinct periplasmic space), includes teichoic acid polymers that are absent in E. coli, and contains a layer of PG that is several times thicker than found in E. coli. In this study, I will adapt the high-throughput approach previously used by our lab to investigate the B. subtilis envelope. First, I will systematically identify phenotypes for single deletion mutants of all non-essential genes in B. subtilis and a subset of double deletions. Then, I will use these datasets to investigate specific envelope processes such as proteolytic cascades that regulate ECF sigma factors, poorly characterized targets of the envelope-related two-component systems, functional redundancy amongst penicillin-binding proteins, and pathways involving the C55 undecaprenol phosphate carrier lipid. Finally, I will use a novel phenotype I discovered during construction of the B. subtilis deletion library to determine the function of ylaN, a gene involved in cell shape. These high-throughput methodologies will accelerate the assignment of phenotypes and functions to the large number of uncharacterized putative envelope genes in B. subtilis. The discoveries made in this study will likely extend to important Gram-positive human pathogens, as well as human commensals in the gut microbiome.

描述（由申请人提供）：细菌细胞被包膜包裹，与环境直接且持续接触。因此，包膜是细菌首先感知环境波动（例如渗透压的变化）的部位。此外，包膜的成分还介导其他细菌之间的通讯以及细菌与其人类宿主之间的共生或致病相互作用。这些相互作用在人类肠道微生物组等复杂环境中发挥着至关重要的作用。最后，许多广泛使用的最成功的抗生素都针对包膜（例如青霉素和万古霉素），而针对细胞质的抗生素的功效往往会因包膜通透性问题而降低。由于这些原因，信封是一个研究热点领域。研究单个基因产物或途径的研究方法对于定义包膜的各个组成部分非常重要，但在确定途径之间的联系方面受到限制，并且根据定义，通量较低。我们的实验室（加州大学旧金山分校的卡罗尔·格罗斯实验室）之前采用了一种系统方法，通过将基因删除文库暴露于一组针对包膜的环境压力源来研究革兰氏阴性细菌大肠杆菌的包膜功能。使用该数据集，我们能够在对肽聚糖（PG）合成（细菌细胞壁的关键组成部分）的理解方面取得重大进展。然而，革兰氏阳性菌（例如枯草芽孢杆菌）的包膜与大肠杆菌的包膜有根本不同。枯草芽孢杆菌的包膜具有外膜（因此具有独特的周质空间），包含大肠杆菌中不存在的磷壁酸聚合物，并且含有一层比大肠杆菌中厚数倍的 PG 层。在这项研究中，我将采用我们实验室以前使用的高通量方法来研究枯草芽孢杆菌包膜。首先，我将系统地鉴定枯草芽孢杆菌中所有非必需基因的单缺失突变体和双缺失子集的表型。然后，我将使用这些数据集来研究特定的包膜过程，例如调节 ECF sigma 因子的蛋白水解级联、包膜相关双组分系统的特征不明的靶标、青霉素结合蛋白之间的功能冗余以及涉及 C55 十一烯醇磷酸盐的途径载体脂质。最后，我将使用我在枯草芽孢杆菌缺失文库构建过程中发现的一种新表型来确定 ylaN（一种与细胞形状有关的基因）的功能。这些高通量方法将加速枯草芽孢杆菌中大量未表征的假定包膜基因的表型和功能分配。这项研究的发现可能会扩展到重要的革兰氏阳性人类病原体，以及肠道微生物组中的人类共生体。