Department of Microbiology and Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States of America.
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.
PLoS One. 2020 Dec 30;15(12):e0244031. doi: 10.1371/journal.pone.0244031. eCollection 2020.
Ecotin, first described in Escherichia coli, is a potent inhibitor of a broad range of serine proteases including those typically released by the innate immune system such as neutrophil elastase (NE). Here we describe the identification of ecotin orthologs in various Campylobacter species, including Campylobacter rectus and Campylobacter showae residing in the oral cavity and implicated in the development and progression of periodontal disease in humans. To investigate the function of these ecotins in vitro, the orthologs from C. rectus and C. showae were recombinantly expressed and purified from E. coli. Using CmeA degradation/protection assays, fluorescence resonance energy transfer and NE activity assays, we found that ecotins from C. rectus and C. showae inhibit NE, factor Xa and trypsin, but not the Campylobacter jejuni serine protease HtrA or its ortholog in E. coli, DegP. To further evaluate ecotin function in vivo, an E. coli ecotin-deficient mutant was complemented with the C. rectus and C. showae homologs. Using a neutrophil killing assay, we demonstrate that the low survival rate of the E. coli ecotin-deficient mutant can be rescued upon expression of ecotins from C. rectus and C. showae. In addition, the C. rectus and C. showae ecotins partially compensate for loss of N-glycosylation and increased protease susceptibility in the related pathogen, Campylobacter jejuni, thus implicating a similar role for these proteins in the native host to cope with the protease-rich environment of the oral cavity.
Ecotin 最初在大肠杆菌中被描述，是一种强效的广谱丝氨酸蛋白酶抑制剂，包括中性粒细胞弹性蛋白酶（NE）等固有免疫系统释放的蛋白酶。在这里，我们描述了在各种弯曲杆菌属物种中发现的 ecotin 同源物，包括居住在口腔中并与人类牙周病的发展和进展有关的直肠弯曲杆菌和展示弯曲杆菌。为了研究这些 ecotins 在体外的功能，我们从大肠杆菌中重组表达和纯化了来自直肠弯曲杆菌和展示弯曲杆菌的同源物。使用 CmeA 降解/保护测定、荧光共振能量转移和 NE 活性测定，我们发现来自直肠弯曲杆菌和展示弯曲杆菌的 ecotins 抑制 NE、因子 Xa 和胰蛋白酶，但不抑制空肠弯曲杆菌的丝氨酸蛋白酶 HtrA 或其在大肠杆菌中的同源物 DegP。为了进一步评估 ecotin 在体内的功能，我们用来自直肠弯曲杆菌和展示弯曲杆菌的同源物补充了大肠杆菌 ecotin 缺陷突变体。使用中性粒细胞杀伤测定，我们证明，在表达来自直肠弯曲杆菌和展示弯曲杆菌的 ecotins 后，大肠杆菌 ecotin 缺陷突变体的低存活率可以得到挽救。此外，直肠弯曲杆菌和展示弯曲杆菌的 ecotins 部分补偿了相关病原体空肠弯曲杆菌中 N-糖基化的缺失和蛋白酶易感性的增加，这表明这些蛋白质在天然宿主中发挥类似的作用，以应对口腔中富含蛋白酶的环境。
