Molecular Mechanisms Underlying Transition to Virulence in Staphylococcus epidermidis

项目摘要 表皮葡萄球菌是医院获得性血流感染的最常见原因和新的治疗策略 预防和急性护理点疗法是迫切需要的。然而，由于缺乏已确定的毒力 决定因素，关于感染菌株来源的相互矛盾的证据，以及较老的 解决菌株来源或功能的技术对开发此类战略构成了障碍。因此，我们的目标是 1)通过更好地了解感染菌株的来源，促进表皮葡萄球菌的预防 2)通过发现感染的分子机制来推进表皮葡萄球菌的治疗， 提供潜在的治疗靶点。在目标1中，我们会收集血液样本和皮肤拭子样本。 诊断为表皮葡萄球菌导管相关血流感染(CRBSI)的患者。使用猎枪 测序和高分辨率计算分析，我们将确定皮肤和感染的共同出现 个体内的菌株，以更好地了解传染性菌株的来源。辅助分析将确定 导管生物被膜感染基因丰富，构成多物种。在目标2中，我们补充 Aim 1的基因组分析旨在解决以下假设：皮肤和皮肤之间的调节性差异，而不是基因差异 感染相关菌株可能是毒力转换的基础。我们将进行元翻译测序 对来自目标1的样本进行分析，以确定与感染相关的基因。最后，在目标3中，我们调查了 表皮葡萄球菌感染的分子机制。已确定的感染相关候选基因 根据文献和在AIMS 1和2中确定的基因将使用改进的CRISPR/CAS被击倒 系统，并在体外和体内进行各种平行生长试验的表型分析 表皮葡萄球菌作为病原体生长的条件。二次分析(包括基因过度表达， 将对被确认为感染因素的基因进行多基因敲除、网络分析)。 这一目标的成功实现将是#年CRISPR/CAS系统的首次大规模使用 葡萄球菌。总体而言，我们提出的目标旨在揭示表皮葡萄球菌的遗传决定因素。 作为一种机会性病原体存活以及菌株变异对这一作用的贡献。

Project Summary S. epidermidis is the most common cause of hospital-acquired blood stream infections and new strategies for prevention and acute, point of care therapeutics are critically needed. However, a lack of identified virulence determinants, conflicting evidence regarding the source of infectious strains, and the limitations of older technologies to resolve strain origin or function present barriers to developing such strategies. Thus, we aim to 1) advance prevention of S. epidermidis BSIs through a better understanding of the origin of infectious strains and 2) advance treatment of S. epidermidis BSIs through discovery of the molecular mechanisms of infection, providing potential therapeutic targets. In Aim 1, we will collect blood samples and skin swab samples from patients with a diagnosed S. epidermidis catheter-related blood stream infection (CRBSI). Using shotgun sequencing and high-resolution computational analyses, we will determine co-occurrence of skin and infection strains within an individual to better understand the origin of infectious strains. Ancillary analyses will identify genes enriched in infection and multi-species composition of catheter biofilms. In Aim 2, we complement genomic analyses of Aim 1 to address the hypothesis that regulatory, not genic differences between skin and infection-associated strains may underlie virulence transition. We will perform metatranscriptomic sequencing on the samples from Aim 1 to identify genes correlated with infection. Finally, in Aim 3, we investigate the molecular mechanisms underlying S. epidermidis infections. Candidate infection-associated genes identified from the literature and genes identified in Aims 1 and 2 will be knocked down using a modified CRISPR/Cas system and subjected to in vitro and in vivo phenotypic profiling in a variety of growth assays that parallel conditions in which S. epidermidis thrives as pathogen. Secondary analyses (including gene over-expression, multi-gene knockdowns, network analyses) will be conducted for genes identified as contributors to infection. Successful completion of this aim would represent the first large-scale use of the CRISPR/Cas system in staphylococci. Overall, our proposed aims are designed to reveal the genetic determinants of S. epidermidis’ survival as an opportunistic pathogen and the contribution of strain variation to this role.