Host proteins that interact with the BCG cell envelope

项目摘要 牛分枝杆菌BCG疫苗对成人肺结核（TB）具有不同的疗效， 保护儿童免受结核病和无关感染，并用于膀胱癌治疗。最近 流行病学研究已将卡介苗接种与降低COVID-19死亡率联系起来。虽然有多个， 随机对照试验正在进行中，以测试因果关系，迫切需要分子研究， 解决提议的卡介苗交叉保护的“训练[先天]免疫”机制。经过训练的免疫力可以 也有助于BCG对结核病的特异性保护，结核病每年折磨1000万人。的 对活分枝杆菌或灭活成分（例如完全弗氏佐剂）的先天免疫应答 [CFA]）一直被认为起源于分枝杆菌细胞包膜，特别是胞壁酸部分 细胞壁肽聚糖和海藻糖二分枝菌酸盐（TDM）的外部`myco'膜。培训期间 免疫，例如，单核细胞的表观遗传重编程依赖于先天免疫受体Nod 2。 反过来，Nod 2又被分枝杆菌及其亲属中发现的N-羟乙酰胞壁酸有力地刺激。 确定BCG包膜片段如何与先天免疫系统相互作用，可能会增强我们的免疫功能。 了解异源和TB特异性保护。碎片的分子特征 体内识别的结合伴侣是未知的，并且哺乳动物结合伴侣的列表可能是不完整的。我们将 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 其模拟包膜片段或掺入活BCG肽聚糖或TDM中。作为证据- 在概念实验中，我们将鉴定与BCG肽聚糖和TDM相互作用的宿主蛋白。探针 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 （MAMPs）在更生理相关的背景下。平行地，官能化肽聚糖的合成 片段和TDM将允许头对头比较与合成的或 纯化的MAMP和与感染期间由活细菌释放的MAMP相互作用的那些。这些 实验将揭示最早的宿主-BCG相互作用，这可能会形成对结核病和无关疾病的保护。 比如COVID-19此外，他们将为社区提供研究先天性 对非蛋白佐剂、疫苗和病原体的免疫应答。我们的方法可以扩展到其他 含聚糖和脂质的MAMP（例如，LPS）。

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).