Post-translational regulation of alpha PSM production in Staphylococcus aureus by the small RNA Teg41

项目摘要/摘要 金黄色葡萄球菌既是人类的共生菌，又是一种高度危险的细菌病原体。金黄色葡萄球菌 致病机制由大量分泌的和细胞壁相关的毒力因子介导，包括 许多强大的细胞溶解多肽被称为苯酚可溶性调制素(PSM)。PSM是两亲性的，阿尔法 根据其分类大小不同的螺旋多肽。α类型的PSM大小约为22个氨基酸 近几年来一直是研究的热点。他们被牵连到促成了 社区获得性耐甲氧西林金黄色葡萄球菌(CA-MRSA)菌株的毒力潜力 属于USA300血统。虽然αPSM在金黄色葡萄球菌感染中的作用已经被广泛研究， 在我们对它们是如何在细菌细胞中产生的理解上仍然存在重大差距。一共有五个 大多数金黄色葡萄球菌产生的α多肽(PsMα1-4和δ毒素)。PSMα1-4在 相同的多顺反子转录本(αPSM转录本)，但研究表明，四个 多肽的差异很大。PSmα4(位于转录本的3‘端)通常是最丰富的， 而PSmα3(四种多肽中最有效的)通常是最不丰富的。αPSM中的这一变化 丰度暗示转录后调控。这个项目的长期目标是 了解α在金黄色葡萄球菌中产生的分子机制(S)。在这项提案中，我们 将专门研究小RNA Teg41对αPSM生产和毒力的贡献。 初步研究表明，Teg41在转录后水平上对αPSM的产生有正向影响。我们 我将调查(I)哪一种PSmα1-4肽受Teg41调节，(Ii)Teg41介导的调节是否 通过与αPSM转录本的直接碱基配对促进，以及(Iii)在αPSM生产的哪个阶段，以及如何， Teg41发挥了它的影响力。为了研究这三个目标，我们将使用体外、体内、遗传、 生物化学和分子生物学的方法。我们还将利用一些尖端技术基于 关于高通量DNA测序(SHAPE-SEQ、miR-CATCH和RiBO-SEQ)。这项研究的结果可能 对人类健康有直接影响。干扰Teg41介导的αpsm-生产可能大幅降低 金黄色葡萄球菌的毒力潜力，因此可能成为治疗干预的新靶点。

PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is both a commensal of humans and a highly dangerous bacterial pathogen. S. aureus pathogenesis is mediated by a large repertoire of secreted and cell wall-associated virulence factors, including a number of potent cytolytic peptides called phenol soluble modulins (PSMs). PSMs are amphipathic, alpha helical peptides that vary in size depending on their classification. The α type PSMs are ~22 amino acids in size and have been the focus of intense study in recent years. They have been implicated in contributing to the high virulence potential of community-acquired methicillin resistant S. aureus (CA-MRSA) strains, in particular those of the USA300 lineage. While the role of αPSMs in S. aureus infection has been extensively investigated, significant gaps still exist in our understanding of how they are produced in the bacterial cell. There are five αPSM peptides produced by most S. aureus strains (PSMα1-4 and the δ-toxin). PSMα1-4 are encoded within the same polycistronic transcript (the αPSM transcript), yet studies have shown that the relative levels of the four peptides vary considerably. PSMα4 (located at the 3' end of the transcript) is commonly the most abundant, while PSMα3 (the most potent of the four peptides) is typically the least abundant. This variation in αPSM abundance is suggestive of post-transcriptional regulation. The long-term objective of this project is to understand the molecular mechanism(s) that contribute to αPSM production in S. aureus. In this proposal, we will specifically investigate the contribution of the small RNA Teg41 to αPSM production and virulence. Preliminary studies show that Teg41 positively influences αPSM production at the post-transcription level. We will investigate (i) which of the PSMα1-4 peptides is/are regulated by Teg41, (ii) if Teg41-mediated regulation is facilitated by direct base pairing with the αPSM transcript, and (iii) at what stage in αPSM production, and how, Teg41 exerts its influence. To investigate these three aims, we will use a combination of in vitro, in vivo, genetic, biochemical, and molecular biology approaches. We will also utilize a number of cutting edge techniques based on high throughput DNA sequencing (SHAPE-seq, miR-CATCH, and Ribo-seq). The results from this study could have direct implications for human health. Disrupting Teg41-mediated αPSM-production could dramatically lower the virulence potential of S. aureus and therefore may represent a novel target for therapeutic intervention.