Mechanisms of Prophage-Mediated Virulence Driving Community-Acquired MRSA Contagion

项目总结 社区获得性耐甲氧西林金黄色葡萄球菌(CA-MRSA)可引起严重细菌 感染和传播迅速，造成突发公共卫生事件。CA-MRSA通常包含 噬菌体遗传物质，但除非噬菌体编码已知的分泌毒素，否则它们对 毒力和菌株适合性在很大程度上是未知的。在本计划中，我们将通过利用 流行CA-MRSA克隆鉴定噬菌体编码基因(S)及细菌-噬菌体相互作用机制(S) 潜在的CA-MRSA传染病。我们最近鉴定了一种进化的CA-MRSA菌株(USA300-BKV)，导致 严重的皮肤感染流行，涉及纽约布鲁克林一个主要健康的儿童社区。 测序显示，在USA300-BKV扩散之前的主要变化是获得了一种 含有新基因的嵌合体的前噬菌体(mΦ11)。我们对同基因菌株进行了改造，结果显示mΦ11 与含有野生型Φ11的菌株或对照菌株相比，小鼠产生的皮肤脓肿明显更大 不含噬菌体。然而，mΦ11不编码任何已知的毒力因子，mΦ11的存在编码了 不影响体外生长、细胞毒性、外源蛋白生产或转录图谱。随后的预赛 研究表明，mΦ11编码的甲基转移酶(MTase)的缺失减少了皮肤的大小 脓肿达到对照菌株的水平。基于这些观察，我们假设1)mΦ11编码 MTase在感染期间被激活以导致毒力增加和2)MTase和/或额外的mΦ11基因(S) 通过调节细菌毒力因子增强CA-MRSA毒力。为了检验这些假设，我们将 通过1)互补鉴定毒力增强(特异靶1)的噬菌体基因(S 将MTase插入缺失克隆以确认MTase的功能相关性，2)构建噬菌体诱导 3)在USA300-BKV中建立缺失克隆，以 检测临床遗传背景对皮肤感染表型的影响。为了定义噬菌体- 介导的毒力机制(特定目标2)，我们将1)将mΦ11溶原菌产生的α毒素与 野生型CA-MRSA在小鼠皮肤感染中的作用，2)使用RNA测序进行体内转录分析 确定额外的mΦ11候选监管目标，以及3)删除和补充候选监管目标 靶点，重点是已知的毒力和调控途径，用于在小鼠皮肤感染模型中进行测试。 我们预计，独立但互补的特定目标将揭示出一种前噬菌体编码的机制 引起CA-MRSA流行的临床相关菌株的毒力。结果将扩大我们的 理解噬菌体与宿主-细菌基因组的相互作用并加强噬菌体 以比充当简单的毒素携带者更复杂的方式影响毒力。重要的是，识别 噬菌体编码基因(S)和导致CA-MRSA感染的机制有望通知暴发 监测措施，并发现新的治疗目标，以对抗这种实质性的病原体。

PROJECT SUMMARY Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is known to cause severe bacterial infections and spreads rapidly, creating outbreaks that are public health emergencies. CA-MRSA often contain bacteriophage genetic material, but unless the phage encodes for a known secreted toxin, their contribution to virulence and strain fitness is largely unknown. In this proposal, we will fill this knowledge gap by leveraging an epidemic CA-MRSA clone to identify phage-encoded gene(s) and mechanism(s) of bacteria-phage interaction underlying CA-MRSA contagion. We recently characterized an evolved CA-MRSA strain (USA300-BKV) causing an epidemic of severe skin infection involving a community of predominantly healthy children in Brooklyn, NY. Sequencing revealed the major change antecedent to the dispersion of USA300-BKV was acquisition of a prophage containing a mosaic block of novel genes (mΦ11). We engineered isogenic strains and showed mΦ11 produced significantly larger skin abscesses in mice than strains containing wild type Φ11 or control strain without phage. However, mΦ11 does not encode for any known virulence factors and the presence of mΦ11 did not affect in vitro growth, cytotoxicity, exoprotein production, or transcriptional profiles. Subsequent preliminary studies showed that deletion of a mΦ11-encoded methyltransferase (MTase) decreased the size of the skin abscesses to that of control strain. Based on these observations, we hypothesize that 1) a mΦ11-encoded MTase is activated during infection to cause increased virulence and 2) MTase and/or additional mΦ11 gene(s) enhance CA-MRSA virulence through regulation of bacterial virulence factors. To test these hypotheses, we will identify the bacteriophage gene(s) responsible for enhanced virulence (Specific Aim 1) by 1) complementing MTase into the deletion clone to confirm the functional relevance of MTase, 2) constructing a phage induction repressor mutant to evaluate the effect of induction in vivo, and 3) creating deletion clones in USA300-BKV to examine the effect of the clinical genetic background on the skin infection phenotype. To define the phage- mediated virulence mechanism (Specific Aim 2), we will 1) compare alpha toxin production of mΦ11 lysogens to wild type CA-MRSA during mouse skin infection, 2) perform in vivo transcription profiling using RNA sequencing to identify additional mΦ11 candidate regulatory targets, and 3) delete and complement candidate regulatory targets, with a focus on known virulence and regulatory pathways, for testing in a mouse skin infection model. We expect the independent but complementary Specific Aims will reveal a prophage-encoded mechanism of virulence in a clinically relevant strain causing an epidemic of CA-MRSA. The results will broaden our understanding of phage interactions with the host-bacterial genome and strengthen the paradigm that phages impact virulence in more complex ways than acting as simple toxin carriers. Importantly, identifying the prophage-encoded gene(s) and mechanisms contributing to CA-MRSA contagion promises to inform outbreak surveillance measures and discover novel therapeutic targets to combat this substantial pathogen.