Genetic Determinants of the hVISA Mechanism

描述(由申请人提供)：导致异型万古霉素中间体(HVISA)表达的基因改变目前无法表征。HVISA机制研究中的一个主要问题是缺乏等基因的万古霉素敏感和hVISA，特别是相关或等基因的真实临床菌株。我们现在已经鉴定了一个克隆性hVISA和万古霉素敏感的金黄色葡萄球菌临床菌株集。这项建议的具体目的是确定：(1)在临床上发生的hVISA和表达万古霉素敏感MIC升高的这种菌株的克隆的基因组变化；(2)由于hVISA机制获取和导致万古霉素敏感MIC升高的基因改变而发生的转录组变化；以及(3)在表达万古霉素敏感MIC升高的hVISA和金黄色葡萄球菌中发生的转录组变化。第一个目标将通过对暂时分离的克隆菌株集进行454基因组测序，其中包括表达各种低水平和高敏感性万古霉素MIC的hVISA和非hVISA，然后是基因组注释和比较，或者Nimblegene比较基因组测序。在这个过程中，PI将接受454测序以及基因组注释管道Xgi、Alpheus和实验GenVar的培训。第二和第三个目标将通过比较和对比一组克隆的hVISA、表达高敏感性万古霉素MIC的非hVISA菌株和表达低水平敏感万古霉素MIC的非hVISA菌株的转录来实现。还将通过应用称为葡萄球菌微阵列元数据库(SAMMD)的调查转录组工具来加强这些目标。在这一过程中，PI将接受关于当前NIAID-PFGRC-TIGR支持的金黄色葡萄球菌微阵列版本4和微阵列分析软件的培训。这些研究目标旨在产生巨大的数据集，使这种由分数资助的PI具有传统NIH资助的竞争力。

DESCRIPTION (provided by applicant): Genetic alterations leading to hetero-vancomycin intermediate (hVISA) expression presently defies characterization. A major problem in hVISA mechanistic studies has been the lack of isogenic vancomycin-susceptible and hVISA, especially related or isogenic real-world clinical strains. We have now characterized a clonal hVISA and vancomycin-susceptible S. aureus clinical strain set. The specific aims of this proposal are to determine: (1) genomic alterations that occur in a clinical hVISA and clones of this strain expressing elevated susceptible vancomycin MICs; (2) transcriptome alterations that occur as a result of hVISA mechanism acquisition and genetic alterations leading to elevated vancomycin susceptible MICs; and (3) transcriptome alterations due to vancomycin induction that occur in hVISA and S. aureus strains expressing elevated vancomycin susceptible MICs. The first aim will be accomplished by either 454-based genomic sequencing of a temporally isolated clonal strain set that includes hVISA and non-hVISA expressing varied low-level and elevated susceptible vancomycin MICs, followed by genome annotation and comparison, or Nimblegene comparative genomic sequencing. During this process the PI will be trained on 454-sequencing as well as the genome annotation pipelines XGI, Alpheus, and the experimental GenVar. The second and third aims will be accomplished by comparing and contrasting the transcriptomes of a clonal set of hVISA, non-hVISA strains expressing elevated susceptible vancomycin MICs and non-hVISA strains expressing low-level susceptible vancomycin MICs, isolated following growth with and without vancomycin. These aims will also be enhanced by applying an investigative transcriptome tool referred to as the Staphylococcus microarray meta-database (SAMMD). During this process the PI will be trained on the current NIAID-PFGRC-TIGR-supported S. aureus microarrays version 4 and microarray analysis software. These research objectives are designed to produce enormous datasets that will make this SCORE-funded PI competitive for traditional NIH funding.