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

原噬菌体介导的毒力驱动社区获得性 MRSA 感染的机制

• 批准号: 10405561
• 负责人: Robert James Ulrich
• 金额: $ 19.92万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-18 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10405561
• 关键词: Abscess; Address; Advisory Committees; Affect; Antibiotics; Automobile Driving; Award; Bacteria; Bacterial Genes; Bacterial Genome; Bacterial Infections; Bacteriophage Genetics; Bacteriophages; Candidate Disease Gene; Centers for Disease Control and Prevention (U.S.); Cessation of life; Child; Clonal Deletion; Communities; Complement; Complex; Development; Disease; Disease Outbreaks; Engineering; Epidemic; Evolution; Foundations; Gene Expression; Gene Expression Profile; Genes; Genetic Complementation Test; Genetic Materials; Genetic Transcription; Genetic Variation; Goals; Growth; Hospitalization; Hospitals; In Vitro; Infection; Infectious Skin Diseases; Knock-out; Knowledge; Laboratories; Leucocidin; Link; Lysogeny; Measures; Mediating; Medical Genetics; Mentors; Methyltransferase; Modeling; Molecular; Mosaicism; Mouse Strains; Mus; Mutation; New York; Pathogenesis; Pathway interactions; Phenotype; Phylogenetic Analysis; Physicians; Play; Production; Prophages; Regulation; Regulatory Pathway; Research; Research Training; Role; Scientist; Signal Transduction; Skin Tissue; Soft Tissue Infections; Staphylococcus aureus; Staphylococcus aureus infection; Testing; Toxin; Toxin Conjugates; Training; United States; Up-Regulation; Variant; Virulence; Virulence Factors; Virulent; Virus; Work; alpha Toxin; base; career; clinical effect; clinically relevant; clinically significant; combat; contagion; cytotoxicity; fitness; genome sequencing; in vivo; insight; methicillin resistant Staphylococcus aureus; mutant; neutrophil; new therapeutic target; novel; novel therapeutics; pathogen; public health emergency; skin abscess; success; transcriptome sequencing; whole genome

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.

项目摘要 已知社区获得性耐甲氧西林金黄色葡萄球菌（CA-MRSA）可引起严重的细菌感染。 感染和传播迅速，造成突发公共卫生事件。CA-MRSA通常含有 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 毒力和菌株适应性在很大程度上是未知的。在本提案中，我们将通过利用 流行性CA-MRSA克隆以鉴定噬菌体编码基因和细菌-噬菌体相互作用机制 潜在的CA-MRSA感染。我们最近描述了一种进化的CA-MRSA菌株（USA 300-BKV）， 严重皮肤感染的流行病涉及纽约布鲁克林的一个主要健康儿童社区。 测序显示USA 300-BKV分散前的主要变化是获得了一个 原噬菌体含有新基因的嵌合块（mΦ11）。我们设计了等基因菌株， 与含有野生型Φ11的菌株或对照菌株相比， 没有噬菌体。然而，mΦ11并不编码任何已知的毒力因子，而mΦ11的存在确实编码任何已知的毒力因子。 不影响体外生长、细胞毒性、外蛋白产生或转录谱。随后初步 研究表明，缺失mΦ11编码的甲基转移酶（MTase）会使皮肤变小， 与对照菌株相似。基于这些观察，我们假设1）一个mΦ11编码的 MTase在感染过程中被激活，导致毒力增加，2）MTase和/或额外的mΦ11基因 通过调节细菌毒力因子增强CA-MRSA毒力。为了验证这些假设，我们将 鉴定负责增强毒力的噬菌体基因（特异性目标1），通过1）补充 将MTase导入缺失克隆中，以确认MTase的功能相关性，2）构建噬菌体诱导 阻遏物突变体以评估体内诱导的效果，和3）在USA 300-BKV中产生缺失克隆， 检查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（具体目标2），我们将1）比较mΦ11溶原菌与 在小鼠皮肤感染期间的野生型CA-MRSA，2）使用RNA测序进行体内转录谱分析 以鉴定另外的mΦ11候选调节靶标，和3）删除和补充候选调节靶标， 目标，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。 我们期望独立但互补的特异性目的将揭示原噬菌体编码的机制， 引起CA-MRSA流行的临床相关菌株的毒力。研究结果将扩大我们的 理解噬菌体与宿主细菌基因组的相互作用，并加强噬菌体与宿主细菌基因组相互作用的范例。 以更复杂的方式影响毒力，而不是作为简单的毒素载体。重要的是，识别 原噬菌体编码的基因和有助于CA-MRSA传染的机制有望为爆发提供信息 监测措施，并发现新的治疗目标，以打击这种实质性的病原体。