Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
PLoS Pathog. 2019 May 20;15(5):e1007773. doi: 10.1371/journal.ppat.1007773. eCollection 2019 May.
Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.
中性粒细胞衍生的 DNA 组成的细胞外纤维网络，覆盖着抗菌分子，被称为中性粒细胞胞外诱捕网（NETs），被认为是先天免疫的一种生理性杀菌机制。NETs 的形成也被归类为一种称为 NETosis 的细胞死亡模型。尽管人们对针对病原体的 NETs 形成进行了深入研究，但特定细菌来源的毒力因子在这个过程中的作用，尽管已经假设，但仍知之甚少。我们的研究目的是确定牙龈蛋白酶（负责 P. gingivalis 毒力的半胱氨酸蛋白酶）在这种主要牙周病原体诱导的 NETosis 过程中的作用。我们表明，由 P. gingivalis 触发的 NETosis 依赖于牙龈蛋白酶，因为与野生型菌株（W83）形成鲜明对比的是，牙龈蛋白酶缺失突变株仅轻微诱导 NETs 的形成。此外，使用纯化的牙龈蛋白酶证明了蛋白酶对 NETosis 的直接作用。值得注意的是，NETosis 的诱导依赖于牙龈蛋白酶的催化活性，因为酶的无活性形式显示出触发 NETs 形成的能力降低。从机制上讲，牙龈蛋白酶诱导的 NETosis 依赖于蛋白酶激活受体-2（PAR-2）的蛋白水解激活。有趣的是，P. gingivalis 和纯化的 Arg 特异性牙龈蛋白酶（Rgp）都诱导了 NETs，这些 NETs不仅缺乏杀菌活性，反而刺激了原本易被 NETs 杀死的细菌物种的生长。这种保护是通过 NETs 的杀菌成分的蛋白水解来执行的。总之，牙龈蛋白酶在 NETosis 中发挥双重作用：它们是 NETs 形成的有效直接诱导剂，但同时，它们的活性防止了 P. gingivalis 的捕获和随后的杀伤。这可以解释一个悖论，即尽管牙周袋中大量中性粒细胞和 NETs 的积累，牙周病原体和相关的共生菌仍在这种环境中茁壮成长。
