Bacterial Immunosuppression: Host Target Identification Through Chemical Probes

描述（由申请人提供）：在革兰氏阴性菌中，N-酰基-L-高丝氨酸内酯（AHL）作为群体感应（QS）信号分子产生，以促进多细胞性和毒力。对于铜绿假单胞菌，细菌利用N-3-氧代-十二烷酰基-L-高丝氨酸内酯（C12）和N-丁酰基-L-高丝氨酸内酯来进行其QS控制的活性。与铜绿假单胞菌产生的其他致病性促进因子相比，C12已被证明在哺乳动物细胞中发挥广谱生物活性，包括宿主免疫调节，并且被发现在囊性纤维化患者的铜绿假单胞菌感染中具有病理学相关性。我们的实验室最近报告的证据表明，免疫抑制性质的C12的能力，选择性地破坏调节NF-？B在由TLR激动剂、TNF或全细菌（铜绿假单胞菌）激活的巨噬细胞和其它细胞类型中起作用。有趣的是，尽管抑制NF-？作为B依赖性免疫系统效应物，哺乳动物细胞暴露于C12导致内质网（ER）应激反应、蛋白激酶p38通路和细胞凋亡的激活。值得注意的是，与TLR激动剂和其他病原体相关分子模式（PAMP）不同，C12介导的哺乳动物细胞活化显示通过与目前已知的PAMP识别受体途径不同的机制发生。目前，哺乳动物细胞能够以这种自相矛盾的方式对C12作出反应的机制尚不清楚。在细菌中，C12通过与LuxR型蛋白相互作用发挥其作用;然而，在哺乳动物细胞中不存在明显的LuxR同源物。因此，哺乳动物细胞C12受体蛋白的鉴定应该提供对AHL介导的细菌免疫抑制机制的生物化学理解的深入了解，并导致模式识别受体家族的新成员的鉴定。此外，这些蛋白质的发现可能导致发现用于预防和/或对抗由铜绿假单胞菌引起的感染的新的治疗途径，这是特别重要的，因为迫切需要创新的治疗和预防策略来靶向革兰氏阴性病原体。为了发现C12靶蛋白，我们设计了一个研究计划，重点是利用合成的C12类似物作为化学探针。更具体地，将使用可点击的基于C12的化学探针通过基于点击化学的蛋白质组学介导的方法鉴定C12的哺乳动物细胞受体。然后，为了验证C12的哺乳动物细胞受体，将使用荧光显微镜、RNAi和功能性生物化学测定的组合，或者蛋白质过表达。通过这些研究，我们希望验证C12靶蛋白，并开始提供这些蛋白与C12生物活性之间的功能联系。

DESCRIPTION (provided by applicant): In Gram-negative bacteria N-acyl-L-homoserine lactones (AHLs) are produced as quorum-sensing (QS) signaling molecules to promote multicellularity and virulence. With respect to Pseudomonas aeruginosa, N-3- oxo-dodecanoyl-L-homoserine lactone (C12) and N-butanoyl-L-homoserine lactone are utilized by the bacteria to conduct its QS-controlled activities. In contrast to other pathogenicity-promoting factors produced by P. aeruginosa, C12 has been demonstrated to exert a broad spectrum of biological activities in mammalian cells, including host immunomodulation, and was found to be pathologically relevant in P. aeruginosa infections of cystic fibrosis patients. Our laboratories have recently reported evidence linking the immunosuppressive nature of C12 to its ability to selectively disrupt the regulation of NF-?B functions in macrophages and other cell types activated by TLR agonists, TNF or whole bacteria (P. aeruginosa). Interestingly, despite inhibition of NF-?B-dependent immune system effectors, exposure of mammalian cells to C12 resulted in activation of the endoplasmic reticulum (ER) stress response, protein kinase p38 pathway and apoptosis. Notably, unlike TLR agonists and other pathogen-associated molecular patterns (PAMPs), C12-mediated activation of mammalian cells was shown to occur through mechanism(s) distinct from currently known PAMP recognition receptor pathways. At present, the mechanisms by which mammalian cells are able to respond to C12 in such a paradoxical manner are unknown. In bacteria, C12 exerts its effects through interaction with LuxR-type proteins; however, no obvious LuxR homologue exists in mammalian cells. Thus, identification of mammalian cell C12 receptor proteins should provide great insight into a biochemical understanding of AHL-mediated mechanisms of bacterial immunosuppression and lead to the identification of new members of the pattern recognition receptor family. Moreover, the uncovering of such proteins may lead to the discovery of new therapeutic avenues for preventing and/or combatting infections caused by P. aeruginosa, which is of particular significance, as innovative therapeutic and prophylactic strategies are desperately needed for targeting Gramnegative pathogens. To discover C12 target proteins, we have designed a research program focused on the utilization of synthetic C12 analogues as chemical probes. More specifically, clickable C12-based chemical probes will be employed to identify the mammalian cell receptor(s) of C12 via a click chemistry-based, proteomics-mediated approach. To then validate mammalian cell receptor(s) of C12, a combination of fluorescence microscopy, RNAi and functional biochemical assays, or alternatively, protein overexpression, will be used. From these studies, we hope to validate C12 target proteins and begin to provide functional links between these proteins and the biological activities of C12.