Cell envelope integrity in mycobacteria: interplay of lipoglycans, peptidoglycan, and capsular polysaccharides

项目摘要 脂甘露聚糖（LM）和脂阿拉伯甘露聚糖（LAM）是分枝杆菌脂聚糖，对分枝杆菌至关重要。 肺结核以建立感染。这些脂聚糖在质膜中合成， 运输到细胞表面。LM和LAM在分枝杆菌中广泛保守，因此也必须 在致病性和非致病性分枝杆菌物种的生理学中发挥重要作用。然而，在这方面， 它们的生物合成是如何与其他细胞包膜生物合成协调的，以及它们是如何 有助于细胞包膜的完整性。长期目标是确定脂聚糖在我的- cobacterial生理学和识别在创建和维持细胞包膜的过程中的漏洞 结构作为下一步，该提案的目标是从根本上了解脂蛋白的作用- 在耻垢分枝杆菌和M.结核核心假设是， LM/LAM生物合成与肽聚糖和荚膜α-葡聚糖生物合成协调， 有助于聚糖的稳态，确保活跃生长期间细胞表面结构的可塑性和完整性。 其基本原理是，在协调聚糖生物合成的监管过程中发现漏洞， 为细胞靶向抗菌药物的开发奠定基础。以已公布的和初步的 根据申请人实验室的数据，将通过以下两个目标来检验这一假设：1）确定 LM/LAM生物合成在抑制肽聚糖降解中的作用;和2）确定囊层的作用 维持细胞膜完整性的关键在第一个目标中，假设LAM缺陷突变体的细胞形状 将测试由于弱化细胞壁而导致的变形。LAM抑制肿瘤生长的分子机制 将检查肽聚糖水解酶。在第二个目标，假设形态缺陷的 LAM缺陷型突变体可以通过激活SenX 3-RegX 3双组分系统来克服， 将检查α-葡聚糖的生产。这项研究具有创新性，因为它揭示了一种新的 分枝杆菌脂聚糖在维持聚糖体内平衡中的作用，并且因为它结合了 申请人及其合作者在正向遗传学和细胞生物正交代谢标记方面的专业知识 壁生物合成，从概念和执行现状的实质性偏离。拟议 研究是重要的，因为这里获得的数据将形成开发药物的基础， 杀了M结核分枝杆菌通过靶向结核分枝杆菌细胞包膜稳态的调节机制。这样的 药物还可以使细胞被膜对其它抗菌剂更具有渗透性。

PROJECT SUMMARY Lipomannan (LM) and lipoarabinomannan (LAM) are mycobacterial lipoglycans that are critical for Mycobacte- rium tuberculosis to establish infection. These lipoglycans are synthesized in the plasma membrane and trafficked to the cell surface. LM and LAM are widely conserved among mycobacteria, and therefore must also play a fundamental role in the physiology of both pathogenic and nonpathogenic mycobacterial species. However, it remains obscure how their biosynthesis is coordinated with other cell envelope biosyntheses and how they contribute to the integrity of the cell envelope. The long-term goal is to determine the role of lipoglycans in my- cobacterial physiology and identify vulnerabilities in the process of creating and maintaining the cell envelope structure. As the next step, the objective of this proposal is to gain fundamental insights into the role of lipogly- cans in cell envelope integrity in Mycobacterium smegmatis and M. tuberculosis. The central hypothesis is that LM/LAM biosynthesis, in coordination with peptidoglycan and capsular a-glucan biosyntheses, positively con- tributes to glycan homeostasis, ensuring the plasticity and integrity of cell surface structure during active growth. The rationale is that finding vulnerabilities in the regulatory process that coordinates the glycan biosyntheses will lay the foundation for cell envelope-targeting antimicrobial development. Guided by published and preliminary data from the applicant’s laboratory, this hypothesis will be tested by pursuing two aims: 1) Identify the role of LM/LAM biosynthesis in suppressing peptidoglycan degradation; and 2) Determine the role of the capsular layer in maintaining cell envelope integrity. In the first aim, the hypothesis that the cell shape of a LAM-deficient mutant deforms due to the weakened cell wall will be tested. The potential molecular mechanism of LAM suppressing peptidoglycan hydrolases will be examined. In the second aim, the hypothesis that the morphological defect of the LAM-deficient mutant can be overcome by activating the SenX3-RegX3 two-component system and increas- ing the production of a-glucan will be examined. The proposed research is innovative because it reveals a new role of mycobacterial lipoglycans in maintaining glycan homeostasis, and because it combines the synergistic expertise of the applicant and his collaborators in forward genetics and bioorthogonal metabolic labeling of cell wall biosynthesis, a substantive departure from the status quo in both concept and execution. The proposed research is significant because the data obtained here will form the foundation for developing drugs that could kill M. tuberculosis by targeting a regulatory mechanism of mycobacterial cell envelope homeostasis. Such a drug may also make the cell envelope more permeable to other antimicrobials.