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）是一种迅速生长的非结核分枝杆菌（NTM） 各种各样的疾病，包括肺，皮肤和软组织感染以及传播疾病。 MAB感染的治疗很困难，因为细菌对许多类别具有抗性 抗生素。那是有必要开发针对MAB感染的新疗法。分枝杆菌细胞壁是 一个流行的抗生素靶标，因为其生物发生对于细菌生长至关重要。而细胞壁合成具有 在结核分枝杆菌（MTB）中广泛研究了，关于mab如何建造其细胞壁和 这个过程与MTB的不同。识别在mAb中具有独特作用的细胞壁合成组件 生理学，我进行了全基因组的转座子诱变，并在高吞吐量上进行 mab。然后，我比较了mAb和MTB之间细胞壁酶的重要性。数据揭示了MAB3167C， 预测的青霉素结合脂蛋白（PBP-LIPO）在mAb中是必不可少的，而其MTB中的同源物是非 - 基本的。 MAB3167C被预测是一种交叉链接段的转肽酶 细胞壁的肽聚糖（PG）层。我的初步数据表明，反映PBP-LIPO会损害细菌 生长并导致细胞中的形态异常。鉴于PG合成尚未研究 在mab中，PBP-Lipo的功能也没有，该提议试图回答两个中心问题：1） MAB PG合成中的PBP-LIPO？ 2）哪种细胞壁在遗传和物理上与PBP- mab中的Lipo？ AIM 1使用延时质疑PB-LIPO在PG合成过程中的定位和功能 显微镜。通过这种方法，我将实时确定PBP-LIPO在细胞中定位并评估如何 它的耗竭会影响PG合成。 AIM 2试图确定功能性遗传和物理网络 mab中的pbp-lipo。我们实验室的先前工作表明，MTB细胞壁酶具有独特的集合 遗传相互作用和蛋白质复合物中的工作，以在空间和临时的 协调的方式。使用CRISPR干预，我将结合PBP-将细胞壁酶击倒 LIPO确定哪种对遗传相互作用。我还将执行免疫沉淀测定法以识别 PBP-LIPO的推定结合伙伴。总之，这项工作将阐明pbp-lipo在何时何地在 细胞以及该酶如何促进PG合成。而且，这项工作将识别细胞 壁合成基因与PBP-LIPO以及发现在复合物中起作用的蛋白质相互作用 与酶。这些实验将有助于发现MAB PG合成和细胞壁的规格 建造。最终，我的发现不仅会推进NTM中细胞壁生物学的知识，而且还会推进 提供有关新药物靶标的关键见解，并为成功的MAB感染的成功疗法提供开发。 呢 呢