Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical School, Houston, Texas, United States of America.
PLoS One. 2013 Jul 11;8(7):e68813. doi: 10.1371/journal.pone.0068813. Print 2013.
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.
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、与宿主纤维蛋白原、胶原蛋白和血小板的黏附以及实验性心内膜炎和尿路感染模型中具有重要作用。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 Western blot、免疫电子显微镜，并使用 ebpR 进行转染以增加菌毛产量，我们确定 EbpA 是尖端菌毛，EbpB 是菌毛基部的锚定蛋白，后者通过管家分选酶 SrtA 附着在细胞壁上。我们还证实 EbpC 和 Bps 分别是主要菌毛和菌毛特异性分选酶，这两者都是菌毛聚合所必需的。有趣的是，缺失 ebpA 会增加菌毛长度并减少菌毛数量，而 ebpA 的反向过表达则恢复了野生型水平，这表明 EbpA 在菌毛聚合的起始和终止中具有双重作用。接下来，我们研究了每个菌毛亚基对生物膜形成和尿路感染的贡献。缺失 ebpA 或 ebpC 显著降低了生物膜形成（P<0.001），而 ebpB 则是可有可无的；在实验性尿路感染的肾脏 CFU 中也观察到了类似的结果（ΔebpA、ΔebpC，P≤0.0093；ΔebpB，无统计学意义，每个与 OG1RF 相比）。因此，我们的数据提供了这些普遍存在的粪肠球菌菌毛的重要结构和功能信息，并且基于它们在生物膜和感染中的重要性，表明 EbpA 和 EbpC 可能是基于抗体的治疗方法的潜在靶点。
