Host proteins that interact with the BCG cell envelope

项目摘要 牛分枝杆菌卡介苗对成人肺结核的疗效各不相同，但 保护儿童免受结核病和无关感染，并用于膀胱癌治疗。近期 流行病学研究表明，接种卡介苗可降低新冠肺炎死亡率。虽然有多个， 目前正在进行随机对照试验来检验因果关系，迫切需要分子研究来 讨论拟议的卡介苗交叉保护的“训练有素[先天]免疫”机制。受过训练的豁免权可能 也有助于卡介苗对结核病的特殊保护，结核病每年困扰着1000万人。这个 对活体分枝杆菌或灭活成分(如完全弗氏佐剂)的先天免疫反应 [CFA])长期以来一直认为起源于分枝杆菌的细胞膜，特别是壁酸部分 细胞壁上的肽聚糖和海藻糖二聚氰酸酯(TDM)在外膜上。在培训期间 免疫，例如，单核细胞的表观遗传重新编程依赖于先天免疫受体NOD2。 反过来，NOD2又被分枝杆菌及其近亲中发现的N-羟基胞壁酸有效地刺激。 确定卡介苗被膜片段如何与先天性免疫系统相互作用可能会增强我们的 了解异源和结核病特异性保护。这些碎片的分子同一性 在体内被识别的是未知的，哺乳动物结合伙伴的名单可能是不完整的。我们会 通过合成、验证和部署可点击的光亲和探针来解决这些长期存在的问题 要么模仿包膜片段，要么整合到活体卡介苗的肽聚糖或TDM中。作为证明- 在概念性实验中，我们将鉴定与卡介苗多肽和TDM相互作用的宿主蛋白。探头 这种标记活细菌的包膜将允许宿主传递与微生物相关的分子模式 (MAMP)在更具生理相关性的背景下。官能化肽聚糖的平行合成 片段和TDM将允许对与合成的或 纯化的MAMP和那些与活细菌在感染期间释放的MAMP相互作用的MAMP。这些 实验将揭示最早的宿主-卡介苗相互作用可能形成对结核病的保护和无关 像新冠肺炎这样的病症。此外，他们将向社区提供使能工具，用于研究先天的 对非蛋白质佐剂、疫苗和病原体的免疫反应。我们的方法可以扩展到其他 含糖和含脂的MAMPs(例如，脂多糖)。

Project Summary The Mycobacterium bovis BCG vaccine has variable efficacy against adult pulmonary tuberculosis (TB) but protects children against both TB and unrelated infections and is used in bladder cancer treatment. Recent epidemiological studies have associated BCG vaccination with lowered COVID-19 mortality. While multiple, randomized controlled trials are now underway to test causality, molecular studies are urgently needed to address the proposed `trained [innate] immunity' mechanism of BCG cross-protection. Trained immunity may also contribute to BCG's specific protection against TB, a disease that afflicts 10 million people each year. The innate immune response to live mycobacteria or inactivated components (e.g. complete Freund's adjuvant [CFA]) has long thought to originate with the mycobacterial cell envelope, specifically the muramic acid moiety of cell wall peptidoglycan and trehalose dimycolates (TDM) in the outer `myco' membrane. During trained immunity, for example, epigenetic reprograming of monocytes depends on the innate immune receptor Nod2. Nod2, in turn, is potently stimulated by the N-glycolyl muramic acid found in mycobacteria and their relatives. Determining how fragments of the BCG envelope interact with the innate immune system may enhance our understanding of both heterologous and TB-specific protection. The molecular identities of the fragments recognized in vivo are unknown and the list of mammalian binding partners is likely incomplete. We will address these long-standing questions by synthesizing, validating and deploying clickable photoaffinity probes that either mimic envelope fragments or that incorporate into the peptidoglycan or TDM of live BCG. In proof- of-concept experiments, we will identify host proteins that interact with BCG peptidoglycan and TDM. Probes that label the envelope of live bacteria will permit host delivery of microbe-associated molecular patterns (MAMPs) in a more physiologically-relevant context. In parallel, synthesis of functionalized peptidoglycan fragments and TDM will allow head-to-head comparison of host proteins that interact with synthesized or purified MAMPs and those that interact with MAMPs released by live bacteria during infection. These experiments will reveal the earliest host–BCG interactions that may shape protection against TB and unrelated pathologies like COVID-19. Further, they will provide enabling tools to the community for studying the innate immune response to non-protein adjuvants, vaccines, and pathogens. Our approach is extendable to other glycan- and lipid-containing MAMPs (e.g., LPS).