Identification of infection-critical S. aureus traits by TnSeq

描述(申请人提供)：在医院和社区，金黄色葡萄球菌已经成为威胁生命的感染的主要原因。开始于 2005年，美国因耐甲氧西林金黄色葡萄球菌(MRSA)导致的死亡人数超过了可归因于艾滋病毒/艾滋病的死亡人数(18650人对16000人)。这一统计数字仅代表那些由甲氧西林耐药菌株引起的金黄色葡萄球菌死亡--大约一半的死亡是由青霉素耐药、甲氧西林敏感菌株引起的，这使得2005年所有侵袭性金黄色葡萄球菌感染造成的实际死亡人数超过25,000人。在侵袭性感染中，15%(和8%的死亡)是由社区获得性MRSA感染引起的，没有已知的潜在健康风险。越来越多的耐药性，包括对万古霉素的耐药性，以及超强毒力菌株的出现，使得了解金黄色葡萄球菌感染的发病机制和开发新的治疗方法变得更加重要。我们建议使用一种名为TnSeq或TN-seq的新方法来定义金黄色葡萄球菌的关键特性，使其能够在感染相关的环境(血液、眼液、脓肿)中增殖。TnSeq涉及产生高密度转座子插入池(基因组周围每隔35个碱基插入一次)。转座子插入在该池中每个细胞中的位置通过选择性地扩增每个插入连接片段来确定， 然后在单个光敏反应中对这些扩增产物进行测序。突变体池在各种环境条件下生长(例如，与实验室介质相比，血液、眼液或脓肿)，并对得到的输出池进行重新测序和比较。这一过程识别的基因，当突变时，导致金黄色葡萄球菌菌株在一个或另一个生态中生长的能力受损，并在单一反应中以大规模平行的方式对基因组进行采样。我们建议不仅应用这项技术来识别对感染相关环境中的生长至关重要或非常重要的基因，而且还可以揭示金黄色葡萄球菌在感染过程中如何改变其对各种代谢途径的依赖。

DESCRIPTION (provided by applicant): Staphylococcus aureus has emerged as a leading cause of life-threatening infection, both in the hospital and in the community. Beginning in 2005, deaths in the US due methicillin resistant S. aureus (MRSA) exceeded those attributable to HIV/AIDS (18,650 versus 16,000). This statistic represents only those S. aureus deaths caused by methicillin resistant strains - about half as many again were caused by penicillin resistant, methicillin sensitive strains, making the actual number of deaths in 2005 attributable to all invasive S. aureus infections over 25,000. Of invasive infections, 15% (and 8% of deaths) resulted from community acquired MRSA infection contracted with no known underlying health risk. Increasing resistance, including resistance to vancomycin, and emergence of hypervirulent strains, has heightened the importance of understanding the pathogenesis of S. aureus infection and the development of new therapeutics. We propose to use a new approach, termed "TnSeq, or Tn-seq" to define the key properties of S. aureus that enable it to proliferate in infection-related environments (blood, ocular fluids, abscess). TnSeq involves generating a high density transposon insertion pool (with insertions every ~35 bp around the genome). The location of transposon insertions in every cell in this pool is determined by selectively amplifying every insertion junction fragment, then sequencing these amplicons in a single Illumina reaction. The mutant pool is grown out in a variety of environmental conditions (e.g., blood, ocular fluids, or abscesses compared to laboratory medium) and the resultant output pools are resequenced and compared. This process identifies genes that, when mutated, result in a strain of S. aureus compromised in its ability to grow in one or the other ecology, and sample the genome in a massively parallel way in a single reaction. We propose to apply this technology not only for identifying genes that are essential or very important for growth in infection related environments, but also t reveal the manner in which S. aureus shifts its dependence on various metabolic pathways during the course of infection.