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.
Highlights
Streptococcus species are natural inhabitants of humans and animals
Studying essential genes of Streptococcus mutans number of essential genes that are required for the survival of the bacteria
Many of these genes are conserved in other pathogenic streptococci, and some are unique to S. mutans
Summary
Streptococcus species are natural inhabitants of humans and animals. Under the right conditions many streptococci can cause or exacerbate diverse infections in humans, including lifethreatening illnesses that include meningitis or pneumonia [1]. Oral streptococci have important roles in the initiation and maturation of dental biofilms (plaque), and many are associated with oral and dental health. Oral streptococci that are especially acidogenic and acidtolerant are implicated as primary etiologic agents in the pathogenesis of dental caries (tooth decay) [3]. The organism most often linked to caries development is Streptococcus mutans, which has been shown to have multiple virulence traits, some unique, that imbue the organism with the capacity to initiate and worsen the disease [4]. S. mutans and other oral streptococci can cause infective endocarditis [5]. Investigating the biology of this organism is important for developing therapies that combat dental caries and other diseases associated with S. mutans and related organisms. Expanding the understanding of streptococcal biology will positively impact efforts to prevent or treat many other diseases in humans and animals
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