猪链球菌2型半胱氨酸蛋白酶ApdS切割宿主防御肽cathelicidin LL-37介导其免疫逃逸的分子机制

Host defense peptides (HDPs, also known as antimicrobial peptides) are the first line of innate immune defense against microbial pathogens. Pathogenic bacteria have evolved multiple strategies to resist and escape from the HDPs-mediated killing. Streptococcus suis serotype 2 (SS2) is an important emerging zoonotic pathogen. The mechanisms used by SS2 to escape from HDPs-mediated killing are not clear. Our previous study found that the cysteine protease ApdS which is a surface protein of SS2 can efficiently cleave HDP cathelicidin LL-37. Deletion of the apdS gene significantly reduced the resistance of SS2 to LL-37. Thus we speculate that the LL-37 cleavage mediated by ApdS is a novel immune evasion strategy used by SS2. In this study, we will determine the proteolytic activity and cleavage site of LL-37 acted by ApdS protease. Subsequently, the apdS gene deleted mutant will be used to investigate the role of ApdS protease in LL-37 resistance of SS2. We will also reveal the impact of ApdS protease on assisting SS2 to escape from the killing by neutrophils. Then, we will explore the differences of membrane damage in SS2 when respectively attacked by the LL-37 and LL-37/ApdS (its major fragment after ApdS cleavage). Lastly, we will analyze the differences of the immunomodulatory properties of LL-37 and LL-37/ApdS on neutrophils. The aim of this study is to clarify the immune evasion mechanism of SS2 mediated by the cysteine protease ApdS. This study will improve comprehension of the pathogenic mechanism of SS2, and contribute to developing novel strategies to prevent and control this important infectious disease.

宿主防御肽（HDPs）是宿主抵御病原菌入侵的第一道免疫防线，病原菌可通过多种机制抵抗和逃避HDPs的杀伤。猪链球菌2型（SS2）是一种重要的人兽共患病原菌，其HDPs免疫逃逸机制尚不清楚。本项目前期研究发现，SS2表面的半胱氨酸蛋白酶ApdS能切割HDP cathelicidin LL-37，且缺失该基因会显著降低SS2对LL-37的抵抗力。我们推测ApdS切割LL-37是SS2的一种新的免疫逃逸策略。本项目拟鉴定ApdS对LL-37的切割活性和位点，揭示ApdS在SS2抵抗、逃逸LL-37和嗜中性粒细胞杀伤中的作用，解析LL-37和ApdS切割LL-37后肽段在致SS2细胞膜损伤机制的差异，并探讨两者在调节嗜中性粒细胞免疫功能上的差异，阐明ApdS介导SS2免疫逃逸的分子机制。本项目从免疫逃逸的角度入手，不仅会增进对SS2致病机制的理解，也对发展新的SS2防治策略具有重要意义。

猪链球菌是一种全球分布的人畜共患病原菌，感染猪和人类会导致脑膜炎和败血症等疾病，对人类健康和公共卫生构成严重威胁。猪链球菌为了成功入侵并在宿主体内传播，必须克服先天免疫系统。宿主防御肽cathelicidin LL-37通过直接穿透细菌细胞膜和刺激嗜中性粒细胞的免疫功能来阻止入侵病原菌，而嗜中性粒细胞是对抗猪链球菌的主要效应细胞。然而，对猪链球菌-LL-37的生物学关系以及猪链球菌如何适应和逃避LL-37介导的免疫攻击知之甚少。本研究中确定了宿主防御肽LL-37为猪链球菌半胱氨酸蛋白酶ApdS的靶标，阐明了由ApdS介导的猪链球菌与LL-37以及人嗜中性粒细胞之间的相互作用。猪链球菌感染会刺激人嗜中性粒细胞中LL-37的大量产生，而当猪链球菌暴露于LL-37时，apdS基因的转录表达随后上调。ApdS蛋白酶快速靶向并切割LL-37，削弱其对猪链球菌的杀菌活性；这归因于截短肽中二级结构的螺旋含量降低，从而导致了截短肽对于猪链球菌细胞膜透化能力的降低。通过对LL-37和ApdS切割后的截短肽作用猪链球菌的差异蛋白质组学分析，二者会引起双组分调控系统、氨基酸代谢、氮代谢、ABC转运系统和肽聚糖合成等途径相关蛋白表达差异。此外，ApdS参与了猪链球菌逃逸嗜中性粒细胞及其胞外陷阱的杀伤，主要是由于LL-37的截短减弱了其趋化性，并抑制了其胞外诱捕网形成和活性氧生成的能力。通过C57BL/6 小鼠感染模型，证实了ApdS缺陷会减弱猪链球菌的毒力及其在小鼠血液和脏器的存活力，说明ApdS在猪链球菌体内定植和致病性中发挥重要作用。总之，本项目的研究发现揭示了猪链球菌的一种新的免疫抑制策略：通过ApdS蛋白酶对LL-37的切割来减弱先天宿主防御。该研究结果从一个全新的角度解读了猪链球菌的免疫逃逸机理，丰富了对猪链球菌致病机制的理解，为猪链球菌新型药物设计提供新的方向。

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