Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum.,PLoS Pathogens

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Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum.
PLoS Pathogens ( IF 5.5 ) Pub Date : 2019-12-20 , DOI: 10.1371/journal.ppat.1008232
Zoltán Attila Nagy ₁ , Dávid Szakács ₁ , Eszter Boros ₁ , Dávid Héja _{1,

2} , Eszter Vígh ₁ , Noémi Sándor ₃ , Mihály Józsi ₃ , Gábor Oroszlán ₄ , József Dobó ₄ , Péter Gál ₄ , Gábor Pál ₁

Affiliation

Ecotin is a serine protease inhibitor produced by hundreds of microbial species, including pathogens. Here we show, that ecotin orthologs from Escherichia coli, Yersinia pestis, Pseudomonas aeruginosa and Leishmania major are potent inhibitors of MASP-1 and MASP-2, the two key activator proteases of the complement lectin pathway. Factor D is the key activator protease of another complement activation route, the alternative pathway. We show that ecotin inhibits MASP-3, which is the sole factor D activator in resting human blood. In pathway-specific ELISA tests, we found that all ecotin orthologs are potent lectin pathway inhibitors, and at high concentration, they block the alternative pathway as well. In flow cytometry experiments, we compared the extent of complement-mediated opsonization and lysis of wild-type and ecotin-knockout variants of two E. coli strains carrying different surface lipopolysaccharides. We show, that endogenous ecotin provides significant protections against these microbicidal activities for both bacteria. By using pathway specific complement inhibitors, we detected classical-, lectin- and alternative pathway-driven complement attack from normal serum, with the relative contributions of the activation routes depending on the lipopolysaccharide type. Moreover, in cell proliferation experiments we observed an additional, complement-unrelated antimicrobial activity exerted by heat-inactivated serum. While ecotin-knockout cells are highly vulnerable to these activities, endogenous ecotin of wild-type bacteria provides complete protection against the lectin pathway-related and the complement-unrelated attack, and partial protection against the alternative pathway-related damage. In all, ecotin emerges as a potent, versatile self-defense tool that blocks multiple antimicrobial activities of the serum. These findings suggest that ecotin might be a relevant antimicrobial drug target.

中文翻译：

Ecotin是一种丝氨酸蛋白酶的微生物抑制剂，可阻断人类血清中多种依赖补体和独立的杀微生物活性。

Ecotin是一种由数百种微生物（包括病原体）产生的丝氨酸蛋白酶抑制剂。在这里，我们表明，大肠杆菌，鼠疫耶尔森菌，铜绿假单胞菌和利什曼原虫的大肠素是直链同源物，是补体凝集素途径的两个关键激活蛋白酶MASP-1和MASP-2的有效抑制剂。因子D是另一种补体激活途径（另一种途径）的关键激活蛋白酶。我们表明，生态素抑制MASP-3，这是人类血液中唯一的D因子激活剂。在特定于途径的ELISA测试中，我们发现所有Ecotin直向同源物都是有效的凝集素途径抑制剂，并且在高浓度下，它们也阻断了替代途径。在流式细胞仪实验中我们比较了两种携带不同表面脂多糖的大肠杆菌菌株的补体介导的调理作用和野生型和生态素敲除变体裂解的程度。我们表明，内源性生态素为两种细菌的这些杀微生物活性提供了重要的保护。通过使用途径特异性补体抑制剂，我们从正常血清中检测到经典，凝集素和替代途径驱动的补体攻击，其中活化途径的相对贡献取决于脂多糖的类型。此外，在细胞增殖实验中，我们观察到由热灭活的血清产生的另外的，与补体无关的抗菌活性。虽然敲除Ecotin的细胞极易受到这些活动的影响，野生型细菌的内源性生态素可提供针对凝集素途径相关和与补体无关的攻击的完全保护，并提供针对替代途径相关损伤的部分保护。总之，Ecotin可以作为一种强大的，多功能的自卫工具出现，它可以阻止血清的多种抗菌活性。这些发现表明，抑菌素可能是相关的抗菌药物靶标。

更新日期：2019-12-21

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