Contribution of individual Ebp Pilus subunits of Enterococcus faecalis OG1RF to pilus biogenesis, biofilm formation and urinary tract infection.
Contribution of individual Ebp Pilus subunits of Enterococcus faecalis OG1RF to pilus biogenesis, biofilm formation and urinary tract infection.
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DOI：
10.1371/journal.pone.0068813
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Murray BE
中科院分区：
文献类型：
--
作者：
Sillanpää J;Chang C;Singh KV;Montealegre MC;Nallapareddy SR;Harvey BR;Ton-That H;Murray BE
心内膜炎和生物膜相关菌毛（Ebp）操纵子是粪肠球菌核心基因组的一个组成部分，已被证明对生物膜形成、粘附宿主纤维蛋白原、胶原和血小板以及实验性心内膜炎和尿路感染模型非常重要。在这里，我们创建了菌毛亚基和分选酶的单缺失和双缺失突变体;接下来，通过结合蛋白质印迹，免疫电镜，并使用ebpR反式增加菌毛生产，我们确定EbpA作为尖端菌毛蛋白和EbpB作为菌毛基部的锚，后者通过管家分选酶SrtA附着在细胞壁上。我们还证实了EbpC和Bps作为主要的菌毛蛋白和菌毛蛋白特异性分选酶，分别为菌毛聚合所需。有趣的是，菌毛长度增加，皮利的数量减少，删除ebpA，而控制过度表达的ebpA在反式恢复野生型水平，这表明一个双重作用，EbpA在菌毛聚合的启动和终止。我们接下来研究了每个菌毛蛋白亚基对生物膜形成和UTI的贡献。在ebpA或ebpC缺失的情况下观察到生物膜形成显著减少（P<0.001），而发现ebpB缺失;在实验性UTI的肾脏CFU中观察到类似的结果（ΔebpA，ΔebpC，P≤0.0093; ΔebpB，无显著性，均与OG 1 RF相比）。因此，我们的数据提供了重要的结构和功能信息，这些无处不在的E。粪皮利，并基于其在生物膜和感染中的重要性，建议EbpA和EbpC作为基于抗体的治疗方法的潜在靶标。
The endocarditis and biofilm-associated pilus (Ebp) operon is a component of the core genome of Enterococcus faecalis that has been shown to be important for biofilm formation, adherence to host fibrinogen, collagen and platelets, and in experimental endocarditis and urinary tract infection models. Here, we created single and double deletion mutants of the pilus subunits and sortases; next, by combining western blotting, immunoelectron microscopy, and using ebpR in trans to increase pilus production, we identified EbpA as the tip pilin and EbpB as anchor at the pilus base, the latter attached to cell wall by the housekeeping sortase, SrtA. We also confirmed EbpC and Bps as the major pilin and pilin-specific sortase, respectively, both required for pilus polymerization. Interestingly, pilus length was increased and the number of pili decreased by deleting ebpA, while control overexpression of ebpA in trans restored wild-type levels, suggesting a dual role for EbpA in both initiation and termination of pilus polymerization. We next investigated the contribution of each pilin subunit to biofilm formation and UTI. Significant reduction in biofilm formation was observed with deletion of ebpA or ebpC (P<0.001) while ebpB was found to be dispensable; a similar result was seen in kidney CFUs in experimental UTI (ΔebpA, ΔebpC, P≤0.0093; ΔebpB, non-significant, each vs. OG1RF). Hence, our data provide important structural and functional information about these ubiquitous E. faecalis pili and, based on their demonstrated importance in biofilm and infection, suggest EbpA and EbpC as potential targets for antibody-based therapeutic approaches.
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