Liu Xue, Gallay Clement, Kjos Morten, Domenech Arnau, Slager Jelle, van Kessel Sebastiaan P, Knoops Kèvin, Sorg Robin A, Zhang Jing-Ren, Veening Jan-Willem
Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, Groningen, The Netherlands.
Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China.
Mol Syst Biol. 2017 May 10;13(5):931. doi: 10.15252/msb.20167449.
Genome-wide screens have discovered a large set of essential genes in the opportunistic human pathogen However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn-seq), we refined the list of essential genes in serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high-content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi-based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp-proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP-dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.
全基因组筛选在这种机会性人类病原体中发现了大量必需基因。然而，许多必需基因的功能仍然未知，这阻碍了疫苗开发和药物发现。基于转座子测序（Tn-seq）的结果，我们完善了2型菌株D39中的必需基因列表。接下来，我们通过CRISPR干扰（CRISPRi）创建了一个针对348个潜在必需基因的敲低文库，并显示其中254个（73%）具有生长表型。通过高内涵显微镜筛选，我们寻找在基于CRISPRi的基因敲减后细胞形态具有清晰表型的未知功能必需基因。我们发现SPD_1416和SPD_1417（分别重新命名为MurT和GatD）对肽聚糖合成至关重要，而SPD_1198和SPD_1197（分别重新命名为TarP和TarQ）负责磷壁酸（TA）前体的聚合。这些知识使我们能够重建独特的肺炎球菌TA生物合成途径。CRISPRi还被用于揭示必需的Clp蛋白水解系统在感受态发育调控中的作用，我们发现ClpX是负责ClpP依赖性抑制感受态的必需ATP酶。CRISPRi文库为肺炎球菌基因和途径的表征提供了一个有价值的工具，并揭示了几个有前景的抗生素靶点。
