Investigating Cell-Wall Synthesis in Mycobacterium abscessus

研究脓肿分枝杆菌细胞壁的合成

• 批准号: 10116157
• 负责人: Chidiebere Akusobi
• 金额: $ 5.1万
• 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/10116157
• 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. ! !

项目概要/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)，可导致 多种疾病，包括肺部、皮肤和软组织感染以及播散性疾病。 单克隆抗体感染的治疗很困难，因为该细菌对许多类别的细菌具有内在抵抗力 因此，需要开发针对分枝杆菌细胞壁感染的新疗法。 抗生素的一个流行目标，因为它的生物发生对于细菌生长至关重要，而细胞壁合成则至关重要。 已在结核分枝杆菌 (Mtb) 中进行了广泛研究，但关于 Mab 如何构建其细胞壁和 该过程与 Mtb 有何不同 识别在 Mab 中具有独特作用的细胞壁合成成分。 生理学方面，我进行了全基因组转座子诱变以及高通量测序 然后，我比较了 Mab 和 Mtb 之间细胞壁酶的重要性，数据显示 Mab3167c， 预测的青霉素结合脂蛋白 (PBP-lipo)，在 Mab 中是必需的，而其在 Mtb 中的同源物是非 Mab3167c 预计是一种交联基础片段的转肽酶。 我的初步数据表明，抑制 PBP-lipo 会损害细菌。 鉴于尚未研究 PG 合成，因此可能会影响细胞生长并导致细胞形态异常。 在 Mab 中，也没有 PBP-lipo 的功能，该提案旨在回答两个核心问题：1）作用是什么 Mab PG 合成中 PBP-lipo 的作用？以及 2) 哪些细胞壁酶与 PBP- 发生遗传和物理相互作用？ Mab? Aim 1 使用延时技术询问 PG 合成过程中 PBP-lipo 的定位和功能 通过这种方法，我将实时确定 PBP-lipo 在细胞中的定位并评估其定位方式。 它的消耗影响 PG 合成。目标 2 旨在确定 PG 的功能遗传和物理网络。 Mab 中的 PBP-lipo 之前的工作表明 Mtb 细胞壁酶具有独特的组。 遗传相互作用并在蛋白质复合物中发挥作用，在空间和时间上协调 PG 合成 使用 CRISPR 干扰，我将结合 PBP- 来敲除细胞壁酶。 lipo 来确定哪些对存在遗传相互作用，我还将进行免疫沉淀分析来识别。 PBP-lipo 的假定结合伙伴 总之，这项工作将阐明 PBP-lipo 在何时何地发挥作用。 此外，这项工作还将鉴定细胞。 与 PBP-lipo 遗传相互作用的壁合成基因以及揭示在复杂的功能中发挥作用的蛋白质 这些实验将有助于揭示 Mab PG 合成和细胞壁的特异性。 最终，我的发现不仅将推进 NTM 细胞壁生物学的知识，而且还将推动 NTM 的发展。 提供有关新药物靶点的重要见解，并为单克隆抗体感染的成功治疗方法的开发提供信息。 ！ ！

项目成果

Transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding protein involved in septal peptidoglycan synthesis and antibiotic sensitivity.

• DOI: 10.7554/elife.71947
• 发表时间: 2022-06-06
• 期刊: ELIFE
• 影响因子: 7.7
• 作者: Akusobi, Chidiebere;Benghomari, Bouchra S.;Zhu, Junhao;Wolf, Ian D.;Singhvi, Shreya;Dulberger, Charles L.;Ioerger, Thomas R.;Rubin, Eric J.;Kana, Bavesh D.
• 通讯作者: Kana, Bavesh D.