Pilus assembly in Gram-positive bacteria

描述(申请人提供)：菌毛或菌毛存在于许多革兰氏阳性病原体的表面，包括白喉棒状杆菌、肠球菌和链球菌。这些类纤维蛋白聚合物在细菌致病的许多方面发挥着关键作用，如与宿主组织的黏附、侵袭、生物膜的形成和宿主免疫的调节。这些菌毛在细菌细胞壁上的组装是通过一种依赖于分解酶的机制进行的，这一机制首先在白喉的病原体白喉杆菌中得到证实。白喉隐孢子虫根据构成菌毛柄的主要亚基表达三种不同的异三聚体菌毛结构，分别命名为SPAA-型、SPAD-型和SPAH型菌毛。很好的特征是SPAA型 由SpAA-srtA-SpAB-Spac基因座编码的菌毛。这个原型菌毛由构成杆部的SPAA、位于顶端的SPAC和位于菌毛底部的SPAB组成。重要的是，SPAA菌毛介导棒状杆菌对咽上皮细胞的黏附，咽上皮细胞是白喉杆菌感染的主要部位。我们发现，菌毛亚基的聚合需要一种同源转肽酶，即通过赖氨酸介导的转肽酶，称为菌毛蛋白特异性排序酶(SrtA)。产生的聚合物通过管家分类酶SrtF连接到细胞壁。近年来，对革兰氏阳性菌毛的生物学研究取得了很大进展，确定了菌毛在致病机制中的重要作用，以及它们适合于开发针对链球菌病原体的多价疫苗。然而，在我们对(A)菌毛的表面表达是如何控制的，(B)菌毛是如何以特定的顺序组装的，以及(C)在细菌发病机制中，是什么决定了菌毛聚合和细胞壁锚定及其分支的决定方面，我们存在着重大的差距。在目前的提案中，我们使用多学科方法寻求这些基本问题的答案。具体地说，我们试图通过表征SrtA-SrtF与其底物和SarA(一种新的膜结合蛋白，SrtF介导的细胞壁锚定所需的一种新的膜结合蛋白)的相互作用，以及通过发现调节srtF表达的特定反式作用因子，来描述控制菌毛表面组装的机制。其次，我们的目标是通过破译调控菌毛有序组装的分子决定因素和潜在的相互作用，以及揭示参与菌毛生物发生的潜在伴侣和辅助因子，进一步阐明菌毛生物发生的基本机制。最后，我们的目标是建立两个聚合原则，即棒状杆菌菌毛的表面组装显著有助于细菌的致病作用，以及结合菌毛和毒素抗原的疫苗增强保护效果。所产生的结果不仅将使人们清楚地了解革兰氏阳性菌中菌毛组装和菌毛介导的致病机制的基本机制，而且还将提供改善健康的知识。

DESCRIPTION (provided by applicant): Pili or fimbriae are present on the surface of many gram-positive pathogens, including Corynebacterium diphtheriae, enterococci and streptococci. These fiber-like protein polymers play a pivotal role in many facets of bacterial pathogenesis such as adherence to host tissues, invasion, biofilm formation and modulation of host immunity. The assembly of these pili on the bacterial cell wall occurs by a sortase-dependent mechanism, which was first demonstrated in C. diphtheriae, the causative agent of diphtheria. C. diphtheriae expresses three distinct heterotrimeric pilus structures, named SpaA-, SpaD-, and SpaH-type pili according to the major subunit that forms the pilus shaft. Well characterized is the SpaA-type pilus that is encoded by the spaA-srtA- spaB-spaC locus. This archetype pilus is composed of SpaA forming the shaft, SpaC located at the tip and SpaB at the pilus base. Importantly, the SpaA pili mediate corynebacterial adherence to pharyngeal epithelial cells, the major site of C. diphtheriae infection. We showed that polymerization of the pilus subunits requires a cognate transpeptidase enzyme named pilin-specific sortase (SrtA) via lysine-mediated transpeptidation. The resulting polymer is joined to the cell wall by the housekeeping sortase SrtF. Recently, a great deal of progress has been made on the biology of Gram-positive pili, establishing the prominent roles of pili in pathogenesis as well as their suitability for developing multivalent vaccines against streptococcal pathogens. However, there are major gaps in our understanding of (a) how surface expression of pili is controlled, (b) how pilins are assembled in a specific order, and (c) what governs the decision between pilus polymerization and cell wall anchoring and its ramification in bacterial pathogenesis. In the current proposal, we seek answers to these fundamental questions by using a multidisciplinary approach. Specifically, we seek to delineate the mechanism that controls surface assembly of pili by characterizing SrtA-SrtF interactions with their substrates and SarA, a novel membrane bound protein required for SrtF-mediated cell wall anchoring, and by discovering specific trans-acting factors that regulate srtF expression. Secondly, we aim to further elucidate the fundamental mechanisms of pilus biogenesis by deciphering the molecular determinants and the underlying interactions that orchestrate the ordered assembly of the pilus and by uncovering potential chaperones and cofactors involved in pilus biogenesis. Finally, we aim to establish two paradigmatic principles that surface assembly of corynebacterial pili significantly contributes to bacterial pathogenesis and that a vaccine combining pilus and toxin antigens enhances protective efficacy. The results generated will certainly provide not only a clear understanding of the basic mechanisms of pilus assembly and pilus-mediated pathogenesis in Gram-positive bacteria but also knowledge that improves health.