alpha-Mannan hydrolysing enzymes as drivers of mycobacterial cell surface diversity.

研究目的：正在流行的结核病是传染病中的第二大杀手。在全球范围内，冲突和气候变化导致的大规模移民和生活条件恶化给遏制这一病原体的努力带来了难以置信的压力。在实际层面上，临床医生被越来越无效的药物和糟糕的诊断选择夹在中间。在这两个领域都迫切需要开发创新技术，但尚未得到满足。目前也没有有效的结核病疫苗。分枝杆菌成功的一个关键是它们不寻常的细胞壁。这种细菌的“皮肤”保护生物体免受环境的侵害，但在它与宿主的相互作用中也起着关键作用。形成这种结构的脂质和糖的化学性质不同，这意味着我们的免疫系统已经调整到可以感知它的存在。然而，细菌不会向宿主呈现一个静态的细胞壁，而是调节它来逃避或控制免疫。这种错综复杂的相互作用为开发新的治疗方法、诊断方法和疫苗提供了机会。为了实现这一潜力，我们缺少的关键数据是对分枝杆菌用来调节其细胞壁的分子工具包的理解。我们已经确定了一组由细菌产生的酶，这些酶可以降解含有甘露糖的结构，作为这一过程的关键组成部分。这些酶切割细胞壁，从而雕刻出驱动宿主-病原体相互作用的免疫原性分子。在这项拨款提案中，我们的目标是：1)确定驱动分枝杆菌细胞表面变异的关键机制。2)表征可用于产生免疫活性分子的新酶。3)生成甘露糖基化糖脂表现改变的分枝杆菌菌株，并了解这对其生物学的影响。一种关键的甘露糖基化糖脂，脂阿拉伯糖甘露聚糖，是如何加工和分泌的问题，是分枝杆菌生物学中一个重要而长期存在的问题。现在，我们第一次收集了初步数据和专业知识来回答这个问题。迫切需要这项工作的成果来帮助解决正在发生和日益恶化的结核病大流行问题。该项目将受益于BBSRC在莫伊尼汉实验室能力建设方面的前期投资。我们在肽聚糖回收和分枝杆菌细胞壁相关技术方面的专业知识与我们丰富的初步数据相结合，意味着我们在解决这一主题方面具有独特的优势。该资助没有大型设备需求，由于与Moynihan博士的BBSRC David Phillips奖学金重叠，PI时间部分被覆盖。莫伊尼汉博士还将利用他的经验将抗分枝杆菌细胞壁活性酶商业化。1)对分枝杆菌细胞壁生物学的基本认识。这一建议将回答分枝杆菌领域一个长期存在的问题。2)可能应用于分枝杆菌诊断或疫苗开发。这笔拨款产生的数据将提供一个工具包，用于在体外修饰LAM以改善其检测，或产生具有改变免疫特性的细菌菌株，这将对疫苗开发产生影响。

PURPOSE OF RESEARCH:The on-going tuberculosis pandemic is the second greatest killer of people amongst infectious diseases. Globally, mass migration and worsening living conditions caused by conflict and climate change are putting incredible strain on efforts to contain this pathogen. At a practical level, clinicians are caught between increasingly ineffective medicines and poor diagnostic options. There is an urgent and unmet need for development of innovative technologies in both areas. An effective vaccine against tuberculosis is also not available.A key to the success of mycobacteria is their unusual cell wall. This bacterial "skin" protects the organism from the environment, but also plays a pivotal role in how it interacts with its host. The chemically distinct nature of the lipids and sugars that form this structure mean that our immune systems have been tuned to sense its presence. The bacteria, however, do not present a static cell wall to the host but instead modulate it to evade or control immunity. This intricate interplay provides an opportunity for the development of new therapeutics, diagnostics, and vaccines. To achieve this potential, the key data we are missing is an understanding of the molecular tool kit that mycobacteria use to modulate their cell wall.We have identified a set of enzymes produced by the bacteria that degrade mannose containing structures as a critical component of this process. These enzymes cleave the cell wall, thereby sculpting the immunogenic molecules that drive the host-pathogen interaction. In this grant proposal we aim to:1) Identify key mechanisms driving mycobacterial cell surface variability.2) Characterise new enzymes that can be used to generate immune-active molecules.3) Generate new mycobacterial strains with altered mannosylated glycolipid presentation and understand the impact this has on their biology.TIMELINESSThe question of how a key mannosylated glycolipid, lipoarabinomannan, is processed and secreted is an important and long-standing problem of fundamental importance to mycobacterial biology. Now, for the first time, we have assembled the preliminary data and expertise to answer this question. The outcomes of this work are urgently required to help address the on-going and worsening tuberculosis pandemic.VALUE FOR MONEYThis project will benefit from prior investment of the BBSRC in capacity building in the Moynihan laboratory. Our expertise in peptidoglycan recycling and techniques pertaining to the mycobacterial cell wall when combined with our wealth of preliminary data mean that we are uniquely primed to address this topic. The grant has no large equipment requests and PI time is partially covered due to overlap with Dr. Moynihan's BBSRC David Phillips Fellowship. This grant will also benefit from Dr. Moynihan's experience commercialising enzymes active against mycobacterial cell walls.OUTCOMES1) Fundamental understanding of mycobacterial cell wall biology. This proposal will answer a long-standing question in the mycobacterial field. 2) Likely applications in mycobacterial diagnostics or vaccine development. The data generated in this grant will provide a toolkit to either modify LAM in vitro to improve its detection or generate bacterial strains with altered immunological properties which will have impacts on vaccine development.