Investigating Cell-Wall Synthesis in Mycobacterium abscessus

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 抗生素。因此，需要开发针对 Mab 感染的新疗法。分枝杆菌的细胞壁是 抗生素的热门目标，因为其生物发生对于细菌生长至关重要。虽然细胞壁合成具有 在结核分枝杆菌 (Mtb) 中进行了广泛的研究，但人们对 Mab 如何构建其细胞壁和 这个过程与 Mtb 有何不同。鉴定在 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 马布。然后我比较了 Mab 和 Mtb 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 必不可少的。 Mab3167c 预计是一种转肽酶，可交联基础片段 细胞壁的肽聚糖（PG）层。我的初步数据表明，抑制 PBP-lipo 会损害细菌 生长并导致细胞的总体形态异常。鉴于PG合成尚未研究 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合成中的PBP-lipo？ 2) 哪些细胞壁酶与 PBP 在遗传上和物理上相互作用 - Mab 中的脂肪？目标 1 使用延时探究 PG 合成过程中 PBP-lipo 的定位和功能 显微镜。通过这种方法，我将实时确定 PBP-lipo 在细胞中的定位并评估如何定位 它的消耗会影响PG的合成。目标 2 旨在识别功能遗传和物理网络 Mab 中的 PBP-lipo。我们实验室之前的工作表明，结核分枝杆菌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 协调一致的方式。使用 CRISPR 干扰，我将结合 PBP 来敲低细胞壁酶- lipo 来确定哪些对存在遗传相互作用。我还将进行免疫沉淀试验来识别 PBP-lipo 的假定结合伴侣。总之，这项工作将阐明 PBP-lipo 在何时何地发挥作用 细胞并阐明了这种酶如何促进 PG 合成。此外，这项工作将识别细胞 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 与酶。这些实验将有助于揭示 Mab PG 合成和细胞壁的特异性 建设。最终，我的发现不仅将推进 NTM 细胞壁生物学的知识，而且 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。 ！ ！

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. ! !