Investigating Cell-Wall Synthesis in Mycobacterium abscessus

研究脓肿分枝杆菌细胞壁的合成

• 批准号: 9905835
• 负责人: Chidiebere Akusobi
• 金额: $ 5.05万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9905835
• 关键词: Active Sites; Affect; Alleles; Amino Acid Sequence; Amino Acids; Antibiotics; Bacteria; Binding; Biogenesis; Biological Assay; Biological Process; Biology; CRISPR interference; Cell Wall; Cell division; Cells; Cellular Structures; Co-Immunoprecipitations; Complex; Coupled; Data; Defect; Development; Disease; Drug Targeting; Enzymes; Foundations; Genetic; Genus Mycobacterium; Growth; High-Throughput Nucleotide Sequencing; Homologous Gene; Immunocompromised Host; Immunoprecipitation; Impairment; Incidence; Infection; Knowledge; Life Cycle Stages; Lipoprotein Binding; Liquid substance; Lung; Lung diseases; Microscopy; Morphology; Mutagenesis; Mycobacterium abscessus; Mycobacterium tuberculosis; Pathogenicity; Patients; Penicillins; Peptidoglycan; Peptidyltransferase; Physiology; Play; Process; Proteins; Repression; Resistance; Resources; Role; Skin Tissue; Soft Tissue Infections; Solid; Specificity; Structure of parenchyma of lung; Testing; Time; Transmembrane Domain; Work; base; cell growth; crosslink; experimental study; gene synthesis; genome-wide; insight; knock-down; mycobacterial; new therapeutic target; non-tuberculosis mycobacteria; novel therapeutics; overexpression; protein complex; protein function; time use

Project Summary/Abstract Mycobacterium abscessus (Mab) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of illnesses including lung, skin and soft-tissue infections, as well as disseminated disease. Treatment of Mab infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Thus, there is a need to develop new therapies against Mab infection. The mycobacterial cell wall is a popular target for antibiotics as its biogenesis is essential for bacterial growth. While cell wall synthesis has been extensively studied in M. tuberculosis (Mtb), relatively little is known about how Mab builds its cell wall and how this process differs from Mtb’s. To identify components of cell wall synthesis with unique roles in Mab physiology, I performed genome-wide transposon mutagenesis coupled with high throughput-sequencing on Mab. I then compared the essentiality of cell wall enzymes between Mab and Mtb. The data reveals Mab3167c, a predicted penicillin-binding-lipoprotein (PBP-lipo), as being essential in Mab, while its homolog in Mtb is non- essential. Mab3167c is predicted to be a transpeptidase that cross-links segments of the foundational peptidoglycan (PG) layer of the cell wall. My preliminary data shows that repressing PBP-lipo impairs bacterial growth and leads to gross morphological abnormalities in the cell. Given that PG synthesis has not been studied in Mab, nor has the function of PBP-lipo, this proposal seeks to answer two central questions: 1) What is the role of PBP-lipo in Mab PG synthesis? and 2) What cell wall enzymes genetically and physically interact with PBP- lipo in Mab? Aim 1 interrogates the localization and function of PBP-lipo during PG synthesis using time-lapse microscopy. With this approach, I will determine in real time where PBP-lipo localizes in the cell and assess how its depletion influences PG synthesis. Aim 2 seeks to identify the functional genetic and physical network of PBP-lipo in Mab. Previous work from our lab demonstrated that Mtb cell wall enzymes have unique sets of genetic interactions and work in protein complexes to coordinate PG synthesis in a spatially and temporarily coordinated manner. Using CRISPR-interference, I will knock down cell wall enzymes in combination with PBP- lipo to determine which pairs genetically interact. I will also perform immunoprecipitation assays to identify putative binding partners of PBP-lipo. All together, this work will elucidate when and where PBP-lipo functions in the cell as well as illuminate how this enzyme contributes to PG synthesis. Moreover, this work will identify cell wall synthesis genes that genetically interact with PBP-lipo as well as uncover proteins that function in complex with the enzyme. These experiments will help uncover the specificities of Mab PG synthesis and cell wall construction. Ultimately, my findings will not only advance the knowledge of cell wall biology in NTMs, but also provide key insights into new drug targets and inform the development of successful treatments for Mab infection. ! !

项目摘要/摘要 脓肿分枝杆菌(Mab)是一种快速生长的非结核分枝杆菌(NTM)，可引起 各种疾病，包括肺部、皮肤和软组织感染，以及播散性疾病。 单抗感染的治疗是困难的，因为这种细菌对许多类别的 抗生素。因此，有必要开发新的治疗方法来对抗单抗感染。分枝杆菌细胞壁是 抗生素的一个受欢迎的目标，因为它的生物发生是细菌生长所必需的。而细胞壁的合成 在结核分枝杆菌(Mtb)中得到了广泛的研究，但对Mab是如何构建其细胞壁和 这一过程与Mtb的不同之处。识别在Mab中具有独特作用的细胞壁合成的成分 生理学上，我进行了全基因组转座子突变结合高通量测序 Mab.然后，我比较了单抗和结核分枝杆菌细胞壁酶的重要性。数据显示Mab3167c， 一种预测的青霉素结合脂蛋白(PBP-LiPO)，在单抗中是必需的，而它在Mtb中的同源物是非... 必不可少的。Mab3167c被预测为一种转肽酶，它能使基础蛋白的片段发生交叉。 胞壁的肽聚糖(PG)层。我的初步数据显示，抑制PBP-Lipoo会损害细菌 生长，并导致细胞的严重形态异常。鉴于PG的合成还没有被研究过 在Mab中，PBP-lipo也没有功能，该提案试图回答两个核心问题：1)PBP-lipo的作用是什么 PBP-Lipoo在单抗PG合成中的应用以及2)什么细胞壁酶在基因和物理上与PBP相互作用- 脂肪涂抹在Mab里？目的1用时移法研究PBP-Lipoo在PG合成过程中的定位和功能 显微镜。使用这种方法，我将实时确定PBP-lipo在细胞中的位置，并评估如何 它的耗竭会影响PG的合成。目标2试图确定功能性遗传和物理网络 PBP--Mab中的Lipo。我们实验室以前的工作表明，结核分枝杆菌细胞壁酶具有独特的 基因相互作用和蛋白质复合体中协调PG合成的空间和临时工作 协调一致的方式。利用CRISPR干扰，我将结合PBP-击倒细胞壁酶- 用来确定哪些配对在基因上相互作用。我还将进行免疫沉淀分析，以确定 PBP-LIPO的假定结合伙伴。总而言之，这项工作将阐明PBP-Lipo在何时何地发挥作用 以及阐明这种酶如何促进PG的合成。此外，这项工作还将识别细胞 与PBP-Lipoo基因相互作用并发现复合体中起作用的蛋白质的壁合成基因 用这种酶。这些实验将有助于揭示单抗PG合成和细胞壁的特异性 建筑。最终，我的发现不仅将促进非肿瘤细胞的细胞壁生物学知识，而且还将 提供对新药靶点的关键见解，并为单抗感染的成功治疗提供信息。 好了！ 好了！