Molecular Assembly on the Cell Surface of Actinomyces

项目摘要 牙菌斑代表了已知折磨人类的最复杂的微生物群落或生物膜之一。 生物膜相关疾病，例如牙携带、齿龈炎、牙周炎和念珠菌病，影响了大量的 所有年龄组的人都患有这种疾病，由于缺乏有效的治疗方法，这种疾病继续造成巨大的经济负担。的 牙菌斑的形成开始于早期细菌定殖者附着到牙釉质上， 产生粘附基质，然后吸引中期和晚期定殖体。放线菌属关键在于早期 在生物膜发育中发挥突出作用的定殖体，凭借其直接相互作用的能力，不仅 与牙齿表面，但也与一些早期和中期殖民者。因此，我们的研究 已经专注于解剖粘附特性，即菌毛和非菌毛蛋白，指示这些 相互作用及其在口腔放线菌表面组装的机制-最丰富的 人口腔中的放线菌。在过去的补助金期间，我们确定了主要的共同汇总因素 名为CafA，它介导A.口腔链球菌相互作用。值得注意的是，CafA被发现在一个 由菌毛轴FimA构成的独特菌毛结构，尽管cafA与2型菌毛没有遗传联系 基因簇fimB-fimA-srtC 2。值得注意的是，我们发现菌毛尖端粘附素CafA的空间定位是 这是CafA介导的细菌共聚集所必需的，并且该过程需要管家分选酶SrtA。 我们还发现了一种名为SafA的小膜蛋白，它在放线菌目中是保守的， 对SrtA膜结合至关重要。对srtA本质的研究揭示了 两个保守的途径，SrtA催化的细胞壁锚定和LytR-CpsA-Psr（LCP）介导 糖基化，在细胞壁上锚定糖蛋白GspA，其本身对于A.单声道和多声道的形成 物种生物膜和膜完整性。因此，我们认为SrtA是分子组装的支点， 放线菌的细胞表面。利用生物化学，遗传电子显微镜和结构方法，我们 目的是通过检验菌毛劫持和多微生物相互作用的机制来检验这一中心假设 在A. oris，阐明SrtA调节的机制， CafA空间定位和CafA介导的共聚集，并检查CafA的糖基化机制。 细胞壁锚定的GspA有助于生物膜形成和膜完整性。养护 分选酶介导的革兰氏阳性菌表面组装及srtA抑制剂必需性的利用 因此，在其他革兰氏阳性病原体中的筛选放大了我们关于分选酶SrtA如何 通过粘附因子的表面展示调节多微生物相互作用。

PROJECT SUMMARY Dental plaque represents one of the most complex microbial communities or biofilms known to afflict man. Oral biofilm-related diseases, e.g. dental carries, gingivitis, periodontitis, and candidosis, impact a large population of all age groups and continue to impose a huge economic burden due to the lack of effective therapies. The development of dental plaque begins with the attachment of early bacterial colonizers to the tooth enamel, generating an adhesive matrix that then attracts intermediate and late colonizers. Actinomyces spp. are key early colonizers that play a prominent role in biofilm development by virtue of their ability to directly interact not only with the tooth surface but also with a number of both early and intermediate colonizers. Therefore, our studies have focused on dissecting the adhesive properties, i.e. fimbriae and non-fimbrial proteins, dictating these interactions and the mechanism of their assembly on the surface of Actinomyces oris – the most abundant Actinomyces in the human oral cavity. During the past grant period, we identified the major co-aggregation factor named CafA, which mediates A. oris interaction with oral streptococci. Remarkably, CafA is found at the tip of a distinct fimbrial structure made of the pilus shaft FimA, although cafA is not genetically linked to the type 2 fimbrial gene cluster fimB-fimA-srtC2. Significantly, we found that spatial positioning of the pilus tip adhesin CafA is essential for CafA-mediated bacterial coaggregation and this process requires the housekeeping sortase SrtA. We also discovered a small membrane protein named SafA, conserved in the Actinomycetales order, which is critical for SrtA membrane association. Investigations into the essential nature of srtA revealed the convergence of two conserved pathways, SrtA-catalyzed cell wall anchoring and LytR-CpsA-Psr (LCP)-mediated glycosylation, on the cell wall anchored glycoprotein GspA, itself critical for A. oris formation of mono- and multi- species biofilms and membrane integrity. Thus, we propose that SrtA is the fulcrum for molecular assembly on the cell surface of Actinomyces. Using biochemical, genetic electron microscopic, and structural approaches, we aim to test this central hypothesis by examining the mechanism of pilus hijacking and polymicrobial interactions mediated by the major co-aggregation factor CafA in A. oris, elucidating the mechanism of SrtA modulation of CafA spatial positioning and CafA-mediated coaggregation, and examining the glycosylation mechanism of the cell wall anchored GspA that contributes to biofilm formation and membrane integrity. The conservation of sortase-mediated surface assembly in Gram-positive bacteria and the utilization of srtA essentiality for inhibitor screens in other Gram-positive pathogens thus magnify the significance of our studies on how sortase SrtA modulates polymicrobial interactions via surface display of adhesive factors.