Department of Molecular Microbiology and Center for Women's Infectious Disease Research, Washington University School of Medicine, St. Louis, Missouri, USA.
J Bacteriol. 2013 Oct;195(19):4484-95. doi: 10.1128/JB.00451-13. Epub 2013 Aug 2.
Enterococci commonly cause hospital-acquired infections, such as infective endocarditis and catheter-associated urinary tract infections. In animal models of these infections, a long hairlike extracellular protein fiber known as the endocarditis- and biofilm-associated (Ebp) pilus is an important virulence factor for Enterococcus faecalis. For Ebp and other sortase-assembled pili, the pilus-associated sortases are essential for fiber formation as they create covalent isopeptide bonds between the sortase recognition motif and the pilin-like motif of the pilus subunits. However, the molecular requirements governing the incorporation of the three pilus subunits (EbpA, EbpB, and EbpC) have not been investigated in E. faecalis. Here, we show that a Lys residue within the pilin-like motif of the EbpC subunit was necessary for EbpC polymerization. However, incorporation of EbpA into the pilus fiber only required its sortase recognition motif (LPXTG), while incorporation of EbpB only required its pilin-like motif. Only the sortase recognition motif would be required for incorporation of the pilus tip subunit, while incorporation of the base subunit would only require the pilin recognition motif. Thus, these data support a model with EbpA at the tip and EbpB at the base of an EbpC polymer. In addition, the housekeeping sortase, SrtA, was found to process EbpB and its predicted catalytic Cys residue was required for efficient cell wall anchoring of mature Ebp pili. Thus, we have defined molecular interactions involved in fiber polymerization, minor subunit organization, and pilus subcellular compartmentalization in the E. faecalis Ebp pilus system. These studies advance our understanding of unique molecular mechanisms of sortase-assembled pilus biogenesis.
肠球菌通常会引起医院获得性感染，例如感染性心内膜炎和导管相关尿路感染。在这些感染的动物模型中，一种长而似毛发的细胞外蛋白质纤维，称为心内膜炎和生物膜相关（Ebp）菌毛，是粪肠球菌的重要毒力因子。对于 Ebp 和其他 sortase 组装菌毛，菌毛相关 sortase 对于纤维形成至关重要，因为它们在 sortase 识别基序和菌毛亚基的菌毛样基序之间创建共价异肽键。然而，尚未在粪肠球菌中研究控制三个菌毛亚基（EbpA、EbpB 和 EbpC）掺入的分子要求。在这里，我们表明 EbpC 亚基菌毛样基序中的一个 Lys 残基对于 EbpC 聚合是必需的。然而，EbpA 掺入菌毛纤维仅需要其 sortase 识别基序（LPXTG），而 EbpB 掺入仅需要其菌毛样基序。只有菌毛尖端亚基的掺入才需要 sortase 识别基序，而基底亚基的掺入仅需要菌毛识别基序。因此，这些数据支持了 EbpA 在 EbpC 聚合物的尖端和 EbpB 在基部的模型。此外，发现管家 sortase SrtA 处理 EbpB，并且其预测的催化 Cys 残基对于成熟 Ebp 菌毛的细胞壁锚定是必需的。因此，我们已经定义了涉及纤维聚合、亚基组织和粪肠球菌 Ebp 菌毛系统中菌毛亚细胞区室化的分子相互作用。这些研究推进了我们对独特的 sortase 组装菌毛生物发生分子机制的理解。
