Molecular Mechanisms Underlying Transition to Virulence in Staphylococcus epidermidis

项目摘要 S.表皮是医院获得性血流感染的最常见原因， 迫切需要预防和急性护理点治疗。然而，缺乏确定的毒力 决定因素，关于感染菌株来源的相互矛盾的证据，以及老年人的局限性， 解决菌株来源或功能的技术对制定此类战略构成障碍。因此，我们的目标是 1)S.通过更好地了解感染性菌株的起源， （2）S.通过发现感染的分子机制， 提供潜在的治疗靶点。在目标1中，我们将收集以下人员的血液样本和皮肤拭子样本： 诊断为S.表皮导管相关性血流感染（CRBSI）。使用shotgun 测序和高分辨率计算分析，我们将确定皮肤和感染的共同发生 为了更好地了解感染性菌株的起源，辅助分析将确定 在感染和导管生物膜的多物种组成中富集的基因。在目标2中，我们补充 目的1的基因组分析，以解决这一假设，即皮肤和 感染相关菌株可能是毒力转变的基础。我们将进行元转录组测序 对目标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，我们研究了 S.表皮感染确定的候选感染相关基因 目的1和2中鉴定的基因将使用修饰的CRISPR/Cas敲除。 系统，并在多种生长测定中进行体外和体内表型分析， 条件下，S.表皮真菌作为病原体而繁殖。二次分析（包括基因过表达， 多基因敲除、网络分析）将对鉴定为感染贡献者的基因进行。 这一目标的成功完成将代表CRISPR/Cas系统的首次大规模使用， 葡萄球菌总的来说，我们提出的目标是揭示S的遗传决定因素。表皮 生存作为一个机会致病菌和菌株变异的贡献，这一作用。

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.