Identification of infection-critical S. aureus traits by TnSeq

通过 TnSeq 鉴定感染关键的金黄色葡萄球菌性状

基本信息

批准号：
8564610
负责人：
Michael S Gilmore
金额：
$ 23.01万
依托单位：
MASSACHUSETTS EYE AND EAR INFIRMARY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-15 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8564610
关键词：
AIDS/HIV problem Abscess Antibiotic Resistance Antibiotics Antisense RNA Bacteremia Biology Blood Cattle Cause of Death Cells Cessation of life Communities Contracts Cues DNA Resequencing Data Dependence Development Ecology Environment Eye Genes Genome Goals Growth Health Hospitals Human Immune In Vitro Individual Infection Infection prevention Laboratories Libraries Life Liquid substance Location Mediating Messenger RNA Metabolic Pathway Methicillin Methicillin Resistance Microbe Modeling Multi-Drug Resistance Mus Mutagenesis Mutate Nature Nosocomial Infections Output PAWR protein Pathogenesis Pathway interactions Penicillin Resistance Process Proliferating Property Proteins Public Health Reaction Research Project Grants Research Proposals Resistance Risk Role Sampling Simulate Site Staphylococcus aureus Surface Technology Testing Validation Vancomycin Virulence aqueous base density fitness follow-up in vivo Model inhibitor/antagonist insight methicillin resistant Staphylococcus aureus microbial mutant new technology next generation novel strategies novel therapeutics public health relevance research study resistant strain statistics structural biology trait

项目摘要

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.

描述（由申请人提供）：金黄色葡萄球菌已成为医院和社区中危及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艾滋病毒/艾滋病造成的死亡人数（18,650 人对 16,000 人）。这一统计数据仅代表由耐甲氧西林菌株引起的金黄色葡萄球菌死亡，大约一半的死亡人数是由耐青霉素、甲氧西林敏感菌株引起的，因此 2005 年由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 例。在侵袭性感染中，15%（以及 8% 的死亡）是由社区获得性 MRSA 感染造成的，且没有已知的潜在健康风险。耐药性的增加，包括对万古霉素的耐药性和高毒力菌株的出现，提高了了解金黄色葡萄球菌感染发病机制和开发新疗法的重要性。我们建议使用一种称为“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环境（血液、眼液、脓肿）中增殖。 TnSeq 涉及生成高密度转座子插入池（基因组周围每约 35 bp 插入一次）。该池中每个细胞中转座子插入的位置是通过选择性扩增每个插入连接片段来确定的，然后在一次 Illumina 反应中对这些扩增子进行测序。突变体池在各种环境条件下生长（例如，与实验室培养基相比，血液、眼液或脓肿），并对所得输出池进行重新测序和比较。该过程识别出当突变时会导致金黄色葡萄球菌菌株在一种或另一种生态中生长能力受损的基因，并在单个反应中以大规模并行方式对基因组进行采样。我们建议应用这项技术不仅可以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。