Exploration of Bacterial Bottlenecks and Streptococcus pneumoniae Pathogenesis by CRISPRi-Seq.
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ABSTRACT:
SUBMITTER: Liu X
PROVIDER: S-EPMC7855995 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Genome-wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae 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 S. pneumoniae 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.
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Project description:RNases perform indispensable functions in regulating gene expression in many bacterial pathogens by processing and/or degrading RNAs. Despite the pivotal role of RNases in regulating bacterial virulence factors, the functions of RNases have not yet been studied in the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus). Here, we sought to determine the impact of two conserved RNases, the endoribonuclease RNase Y and exoribonuclease polynucleotide phosphorylase (PNPase), on the physiology and virulence of S. pneumoniae serotype 2 strain D39. We report that RNase Y and PNPase are essential for pneumococcal pathogenesis, as both deletion mutants showed strong attenuation of virulence in murine models of invasive pneumonia. Genome-wide transcriptomic analysis revealed that the abundances of nearly 200 mRNA transcripts were significantly increased, whereas those of several pneumococcal small regulatory RNAs (sRNAs), including the Ccn (CiaR-controlled noncoding RNA) sRNAs, were altered in the Δrny mutant relative to the wild-type strain. Additionally, lack of RNase Y resulted in pleiotropic phenotypes that included defects in pneumococcal cell morphology and growth in vitro. In contrast, Δpnp mutants showed no growth defect in vitro but differentially expressed a total of 40 transcripts, including the tryptophan biosynthesis operon genes and numerous 5' cis-acting regulatory RNAs, a majority of which were previously shown to impact pneumococcal disease progression in mice using the serotype 4 strain TIGR4. Together, our data suggest that RNase Y exerts a global impact on pneumococcal physiology, while PNPase mediates virulence phenotypes, likely through sRNA regulation. IMPORTANCE Streptococcus pneumoniae is a notorious human pathogen that adapts to conditions in distinct host tissues and responds to host cell interactions by adjusting gene expression. RNases are key players that modulate gene expression by mediating the turnover of regulatory and protein-coding transcripts. Here, we characterized two highly conserved RNases, RNase Y and PNPase, and evaluated their impact on the S. pneumoniae transcriptome for the first time. We show that PNPase influences the levels of a narrow set of mRNAs but a large number of regulatory RNAs primarily implicated in virulence control, whereas RNase Y has a more sweeping effect on gene expression, altering levels of transcripts involved in diverse cellular processes, including cell division, metabolism, stress response, and virulence. This study further reveals that RNase Y regulates expression of genes governing competence by mediating the turnover of CiaR-controlled noncoding (Ccn) sRNAs.
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Project description:Genome-wide screens have discovered a large set of essential genes in the human pathogen Streptococcus pneumoniae. However, the function of many essential genes is still unknown, hampering vaccine and drug development programs. Based on results from transposon-sequencing (Tn-Seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting all 391 potentially essential genes using CRISPR interference (CRISPRi). Using high-content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi-based depletion. We identified SPD1416 and SPD1417 (named to MurT and GatD, respectively) as essential peptidoglycan synthesis proteins and we show that SPD1198 and SPD1197 (named 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. Our CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets. This RNA-Seq dataset is aimed to show that induction of the CRISPRi system very selectively represses its target gene, firefly luciferase, without other observable transcriptional effects.
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