缅甸蟒免疫调节肽Cb-CATH1抗耐甲氧西林金黄色葡萄球菌感染的机制研究

Infections caused by MRSA (methicillin-resistant Staphylococcus aureus) have drawn great attention in clinical settings worldwhidely, due to high mortality and continuously increasing detection rate. Immunotherapy is a promising treatment for drug-resistant bacterial infections. Cb-CATH1, an immunomodulatory peptide identified from Python bivittatus, exhibited significant protection in MRSA infection mice models, and reduced the bacterial load in mice peritoneal cavity and spleen, by modulating chemokine and cytokine secretion of macrophages/monocytes. The protection was lost in neutropenic and macrophage/monocyte-deficient mice models. Cb-CATH1 markedly potentiated the neutrophil’s bactericidal effect against MRSA, but did not improve the phagocytosis of FITC-MRSA by mouse macrophages, indicating that Cb-CATH1 might protect from MRSA by primarily modulating neutrphils’ trafficking and functions. However, the detailed mechanism underlying such immunomodulation is still not yet clear. In current project, we will continue to study the function and mechanism of Cb-CATH1 in inducing macrophage and neutrophil chemotaxis and regulating the inflammatory response in vivo, and furthermore, to uncover the Cb-CATH1’s regulating effects on neutrophils, including its activation and adhesion, and the neutrophil extracellular traps strategy. Combining the transmembrane localization analysis of Cb-CATH1 in PMN and the signal pathway study, we expect to understand the function and mechanism of Cb-CATH1 in regulating neutrophils and protecting from MRSA infection. Meanwhile, the current study will help complementing the theory of function and mechanism of cathelicidins in boa immune system.

耐甲氧西林金黄色葡萄球菌（MRSA）严重威胁人类健康。免疫疗法治疗耐药菌感染极具前景。我们自缅甸蟒体内发现的免疫调节肽Cb-CATH1可通过调节巨噬细胞（Mø）趋化因子与细胞因子分泌水平、提高中性粒细胞（PMN）的吞噬杀菌率显著降低MRSA感染小鼠死亡率，但对Mø或PMN敲除小鼠无效，对Mø吞噬率亦无影响，表明Cb-CATH1可能通过对PMN的诱导趋化和功能调控实现抗MRSA感染功效，这在免疫调节肽研究中尚属首次发现，作用机制尚不明确。本课题提出在in vivo水平考察Cb-CATH1选择性诱导趋化靶细胞、调控炎症反应的功能与机制；研究其对PMN活化粘附、呼吸爆发及胞外陷阱策略的影响规律；结合PMN内Cb-CATH1的跨膜定位与信号通路研究，揭示其对 PMN功能的调控机理与抗MRSA感染的作用机制，同时完善cathelicidins家族免疫调节肽在蟒蛇免疫系统中的功能与机制理论。

结论.(1) 首次从爬行纲蛇亚目蟒科缅甸蟒物种体内获得6条脊椎动物重要的cathelicidin序列(CATHPb1-6)，并对其in vitro功能活性及毒性进行了分析研究。CATHPb1,2,4对一些临床分离的耐药菌表现出很强的广谱快速杀菌活性；CATHPb1-4能显著抑制病原菌生物膜的形成，并有效消除已经形成的成熟生物膜，防止细菌耐药性的产生；CATHPb1,2,4,6具有很强的免疫调节活性，in vitro显著诱导小鼠腹腔巨噬细胞及人血中性粒细胞分泌趋化因子，抑制炎性细胞因子的表达，降低炎症反应。结合毒理及初级成药性评价，CATHPb1综合表现出较好的活性与成药性。.(2) 无论感染前(-24 h)还是感染后(+4 h)给药，或通过不同给药方式，CATHPb1均表现出很强的抗感染治疗效果，可显著降低MRSA、VRSA感染引起的小鼠死亡率及小鼠腹腔积液及脾脏中的活菌数，且5 mg/kg的剂量效果远优于20 mg/kg的头孢哌酮/舒巴坦(CPZ/SBT)。腹腔注射和皮下注射被证明是CATHPb1抗金黄色葡萄球菌感染治疗的两种比较理想的给药方式。.(3) CATHPb1具备两亲性α螺旋结构，可通过破坏细菌细胞膜完整结构引起细菌死亡，发挥in vitro抗菌活性。此外，还可通过调控MAPK/NF-κB信号通路，抑制VRSA感染引起的小鼠腹腔炎症反应。其免疫调节抗感染活性依赖于中性粒细胞和巨噬细胞， CATHPb1预处理能显著提高中性粒细胞的杀菌率，但对巨噬细胞吞噬率无影响，说明其可能主要通过对中性粒细胞的诱导趋化和功能调控实现in vivo抗病原菌感染功效。 .(4) CATHPb1 in vivo可显著诱导中性粒细胞趋化，及呼吸爆发；在细胞水平上可显著促进中性粒细胞胞外陷阱的形成、增强NETs诱捕病原菌的能力、同时提高NETs骨架抗病原菌核酸酶降解的稳定性。