Antifungal Activity of the Enterococcus faecalis Peptide EntV Requires Protease Cleavage and Disulfide Bond Formation
Antifungal Activity of the Enterococcus faecalis Peptide EntV Requires Protease Cleavage and Disulfide Bond Formation
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DOI：
10.1128/mbio.01334-19
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发表时间：
2019-07-01
期刊：
影响因子：
6.4
通讯作者：
Garsin, Danielle A.
中科院分区：
文献类型：
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作者：
Brown, Armand O.;Graham, Carrie E.;Garsin, Danielle A.
粪肠球菌是一种革兰氏阳性细菌，白色念珠菌是一种多形性真菌，它们是微生物组的常见组成部分，也是越来越成问题的感染原因。有趣的是，我们之前发现这两个物种相互拮抗毒力，并且E. faecalis对白色念珠菌的抑制是由EntV特异性介导的。EntV是一种由EntV （ef1097）基因座编码的细菌素，通过抑制菌丝形态发生来降低白色念珠菌的毒力和生物膜的形成。在本报告中，我们研究了env抗真菌活性所需的翻译后修饰。首先，我们发现粪肠杆菌分泌的明胶酶（GelE）负责将EntV切割成68个氨基酸的活性形式，并且该过程不需要丝氨酸蛋白酶SprE。此外，我们证明了在EntV中形成的二硫键是抗真菌活性所必需的。通过化学处理或基因改造使EntV对白色念珠菌失去活性。此外，我们还确定了这种二硫键的可能催化剂，即粪肠球菌基因组中称为DsbA的先前未被表征的硫氧还蛋白。DsbA的缺失，或其氧化还原活性半胱氨酸的破坏，导致env抗真菌活性的丧失。最后，我们发现二硫键的形成不是解理的先决条件；在没有DsbA的情况下，EntV裂解正常进行。总之，我们提出了一个模型，在分泌后，EntV经过DsbA的二硫键形成和GelE的裂解，以产生能够抑制白色念珠菌的肽。粪肠球菌和白色念珠菌是最重要和最有问题的病原体，通常是无害的共生生物，但在免疫功能低下的宿主中可引起危险的感染。事实上，这两种微生物都被疾病控制和预防中心列为严重的全球公共卫生威胁，原因是抗菌素耐药性的日益普遍。考虑到目前可用的治疗方法很少，抗真菌药物耐药性的上升尤其令人担忧。EntV是一种具有抗真菌特性的肽，它或类似的化合物可以单独或与现有药物联合开发成一种治疗替代方案。然而，要做到这一点，需要了解EntV的哪些特性是其抗真菌活性所必需的。在这项工作中，我们研究了env的翻译后加工以及抑制白色念珠菌所需要的修饰，以填补这方面的知识空白。
Enterococcus faecalis, a Gram-positive bacterium, and Candida albicans, a polymorphic fungus, are common constituents of the microbiome as well as increasingly problematic causes of infections. Interestingly, we previously showed that these two species antagonize each other's virulence and that E. faecalis inhibition of C. albicans was specifically mediated by EntV. EntV is a bacteriocin encoded by the entV (ef1097) locus that reduces C. albicans virulence and biofilm formation by inhibiting hyphal morphogenesis. In this report, we studied the posttranslational modifications necessary for EntV antifungal activity. First, we show that the E. faecalis secreted enzyme gelatinase (GelE) is responsible for cleaving EntV into its 68-aminoacid, active form and that this process does not require the serine protease SprE. Furthermore, we demonstrate that a disulfide bond that forms within EntV is necessary for antifungal activity. Abrogating this bond by chemical treatment or genetic modification rendered EntV inactive against C. albicans. Moreover, we identified the likely catalyst of this disulfide bond, a previously uncharacterized thioredoxin within the E. faecalis genome called DsbA. Loss of DsbA, or disruption of its redox-active cysteines, resulted in loss of EntV antifungal activity. Finally, we show that disulfide bond formation is not a prerequisite for cleavage; EntV cleavage proceeded normally in the absence of DsbA. In conclusion, we present a model in which following secretion, EntV undergoes disulfide bond formation by DsbA and cleavage by GelE in order to generate a peptide capable of inhibiting C. albicans.IMPORTANCE Enterococcus faecalis and Candida albicans are among the most important and problematic pathobionts, organisms that normally are harmless commensals but can cause dangerous infections in immunocompromised hosts. In fact, both organisms are listed by the Centers for Disease Control and Prevention as serious global public health threats stemming from the increased prevalence of antimicrobial resistance. The rise in antifungal resistance is of particular concern considering the small arsenal of currently available therapeutics. EntV is a peptide with antifungal properties, and it, or a similar compound, could be developed into a therapeutic alternative, either alone or in combination with existing agents. However, to do so requires understanding what properties of EntV are necessary for its antifungal activity. In this work, we studied the posttranslational processing of EntV and what modifications are necessary for inhibition of C. albicans in order to fill this gap in knowledge.