Exploration of Bacterial Bottlenecks and Streptococcus pneumoniae Pathogenesis by CRISPRi-Seq.
Exploration of Bacterial Bottlenecks and Streptococcus pneumoniae Pathogenesis by CRISPRi-Seq.
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CRISPRI-SEQ的细菌瓶颈和肺炎链球菌发病机理的探索。
DOI：
10.1016/j.chom.2020.10.001
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发表时间：
2021-01-13
影响因子：
30.3
通讯作者：
Veening JW
中科院分区：
文献类型：
--
作者：
Liu X;Kimmey JM;Matarazzo L;de Bakker V;Van Maele L;Sirard JC;Nizet V;Veening JW
肺炎链球菌是一种机会性人类病原体，可引起包括肺炎在内的侵袭性疾病，在合并感染甲型流感病毒（IAV）时具有更大的健康风险。为了促进体内发病机制的研究，我们开发了一种可诱导的CRISPR干扰系统（CRISPR -seq），可以在一个测序步骤中进行全基因组适应度测试。我们应用crispr -seq来评估瓶颈并鉴定在小鼠肺炎模型中重要的肺炎球菌基因。关键的瓶颈发生在48小时内，很少有细菌引起全身感染。在IAV重复感染期间不存在这一瓶颈，有助于鉴定肺炎球菌致病相关基因。体内最重要的基因包括编码腺苷酸琥珀酸合成酶的purA和生产胶囊所需的cps操纵子。令人惊讶的是，crispr -seq显示溶肺素和编码s-腺苷蛋氨酸合成酶的metK在体外是必需的，但在体内是必不可少的，没有健康相关的作用。这突出了crispr -seq比基于转座子的基因筛选的优势，因为所有基因，包括必需基因，都可以检测潜在的发病机制。Liu等人开发了crispr -seq，实现了肺炎链球菌体内全基因组适应度的一次测序测试。crispr -seq揭示了小鼠肺炎球菌感染期间没有在流感病毒合并感染中观察到的瓶颈，从而能够鉴定必需基因。通过测试所有基因，包括必需基因，crispr -seq具有广泛的用途。
Streptococcus pneumoniae is an opportunistic human pathogen that causes invasive disease, including pneumonia, with greater health risks upon influenza A virus (IAV) co-infection. To facilitate pathogenesis studies in vivo, we developed an inducible CRISPR interference system (CRISPRi-seq) that enables genome-wide fitness testing in one sequencing step. We applied CRISPRi-seq to assess bottlenecks and identify pneumococcal genes important in a murine pneumonia model. A critical bottleneck occurs at 48 hours with few bacteria causing systemic infection. This bottleneck is not present during IAV superinfection, facilitating identification of pneumococcal pathogenesis-related genes. Top in vivo essential genes included purA, encoding adenylsuccinate synthetase, and the cps operon required for capsule production. Surprisingly, CRISPRi-seq indicated no fitness-related role for pneumolysin and metK, encoding S-adenosylmethionine synthetase, was essential in vitro but dispensable in vivo. This highlights advantages of CRISPRi-seq over transposon-based genetic screens as all genes, including essential genes, can be tested for pathogenesis potential. Liu et al. developed CRISPRi-seq to enable in vivo genome-wide fitness testing of Streptococcus pneumoniae in one sequencing step. CRISPRi-seq revealed a bottleneck during murine pneumococcal infection not observed upon influenza virus co-infection, enabling identification of essential genes. By testing all genes, including essential genes, CRISPRi-seq has broad utility.
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