Dataset Information

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.

ABSTRACT: A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

SUBMITTER: Shields RC

PROVIDER: S-EPMC7082069 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.

Shields Robert C RC Walker Alejandro R AR Maricic Natalie N Chakraborty Brinta B Underhill Simon A M SAM Burne Robert A RA

PLoS pathogens 20200309 3

A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We ...[more]

PMID: 32150575

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Project description:The Streptococcus mutans lrgAB and cidAB operons have been previously described as a potential model system to dissect the complexity of biofilm development and virulence of S. mutans Herein, we have attempted to further characterize the Cid/Lrg system by focusing on CidB, which has been shown to be critical for the ability of S. mutans to survive and persist in a nonpreferred oxygen-enriched condition. We have found that the expression level of cidB is critical to oxidative stress tolerance of S. mutans, most likely by impacting lrg expression. Intriguingly, the impaired aerobic growth phenotype of the cidB mutant could be restored by the additional loss of either CidA or LrgA. Growth-dependent expression of cid and lrg was demonstrated to be tightly under the control of both CcpA and the VicKR two-component system (TCS), regulators known to play an essential role in controlling major catabolic pathways and cell envelope homeostasis, respectively. RNA sequencing (RNA-Seq) analysis revealed that mutation of cidB resulted in global gene expression changes, comprising major domains of central metabolism and virulence processes, particularly in those involved with oxidative stress resistance. Loss of CidB also significantly changed the expression of genes related to genomic islands (GI) TnSmu1 and TnSmu2, the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system, and toxin-antitoxin (T/A) modules. Taken together, these data show that CidB impinges on the stress response, as well as the fundamental cellular physiology of S. mutans, and further suggest a potential link between Cid/Lrg-mediated cellular processes, S. mutans pathogenicity, and possible programmed growth arrest and cell death mechanisms.The ability of Streptococcus mutans to survive a variety of harmful or stressful conditions and to emerge as a numerically significant member of stable oral biofilm communities are essential elements for its persistence and cariogenicity. In this study, the homologous cidAB and lrgAB operons, previously identified as being highly balanced and coordinated during S. mutans aerobic growth, were further characterized through the functional and transcriptomic analysis of CidB. Precise control of CidB levels is shown to impact the expression of lrg, oxidative stress tolerance, major metabolic domains, and the molecular modules linked to cell death and lysis. This study advances our understanding of the Cid/Lrg system as a key player in the integration of complex environmental signals (such as oxidative stress) into the regulatory networks that modulate S. mutans virulence and cell homeostasis.

Dataset Information

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.

Publications

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.

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