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

• 批准号: 8660637
• 负责人: Michael S Gilmore
• 金额: $ 20.5万
• 依托单位: MASSACHUSETTS EYE AND EAR INFIRMARY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-15 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8660637
• 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

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。年开始 2005年，美国因耐甲氧西林S.金黄色葡萄球菌（MRSA）超过了艾滋病毒/艾滋病（18，650对16，000）。这个统计数据只代表那些S。由耐甲氧西林菌株引起的金黄色葡萄球菌死亡-大约一半是由耐青霉素、甲氧西林敏感菌株引起的，使得2005年的实际死亡人数可归因于所有侵入性金黄色葡萄球菌。 超过25000例金黄色葡萄球菌感染。 在侵入性感染中，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。耐药性的增加，包括对万古霉素的耐药性，以及超强毒株的出现，提高了理解S. 金黄色葡萄球菌感染和新疗法的发展。我们建议使用一种新的方法，称为“TnSeq，或Tn-seq”来定义S的关键属性。 金黄色葡萄球菌，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（在基因组周围每约35 bp插入一次）。通过选择性扩增每个插入连接片段来确定该池中每个细胞中转座子插入的位置， 然后在单个Illumina反应中对这些扩增子进行测序。突变体库在各种环境条件下生长（例如，血液、眼液或与实验室培养基相比的渗出物），并对所得输出池进行重新测序和比较。这一过程确定了基因，当突变时，导致一个菌株的S。金黄色葡萄球菌在一种或另一种生态中生长的能力受到损害，并在单一反应中以大规模平行的方式对基因组进行采样。我们建议将这项技术不仅应用于鉴定在感染相关环境中生长所必需或非常重要的基因，而且还揭示了S。金黄色葡萄球菌在感染过程中改变其对各种代谢途径的依赖。