Abstract
The public health impact of Streptococcus pyogenes (group A Streptococcus, GAS) as a top 10 cause of infection-related mortality in humans contrasts with its benefit to biotechnology as the main natural source of Cas9 nuclease, the key component of the revolutionary CRISPR-Cas9 gene editing platform. Despite widespread knowledge acquired in the last decade on the molecular mechanisms by which GAS Cas9 achieves precise DNA targeting, the functions of Cas9 in the biology and pathogenesis of its native organism remain unknown. In this study, we generated an isogenic serotype M1 GAS mutant deficient in Cas9 protein and compared its behavior and phenotypes to the wild-type parent strain. Absence of Cas9 was linked to reduced GAS epithelial cell adherence, reduced growth in human whole blood ex vivo, and attenuation of virulence in a murine necrotizing skin infection model. Virulence defects of the GAS Δcas9 strain were explored through quantitative proteomic analysis, revealing a significant reduction in the abundance of key GAS virulence determinants. Similarly, deletion of cas9 affected the expression of several known virulence regulatory proteins, indicating that Cas9 impacts the global architecture of GAS gene regulation.
Highlights
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are recognized as an adaptive immune system that allows prokaryotic organisms to defend against plasmids, bacteriophages and transposons (Barrangou et al, 2007)
The discovery and molecular characterization of RNAprogrammable Cas9 nuclease emerged from basic research on the type II CRISPR-Cas system from GAS and has provided a revolutionary biotechnological tool for genome engineering, with promising potential to develop novel strategies to fight and cure many diseases (Le Rhun et al, 2019)
Despite the attention that GAS Cas9 has received and the major health problem that GAS infections continue to exert on the public health, the native biological role of Cas9 and its contribution for GAS pathogenesis has yet to be reported
Summary
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are recognized as an adaptive immune system that allows prokaryotic organisms to defend against plasmids, bacteriophages and transposons (Barrangou et al, 2007). CRISPR-Cas systems are widely distributed in many bacterial and archaeal genomes (Makarova et al, 2015; Burstein et al, 2016), and are evolutionarily classified in two main classes, with class II as the most representative and uniquely driven by the nuclease Cas (Makarova et al, 2015). Type II CRISPR-Cas systems occur only in bacteria, and not in archaea (Haft et al, 2005). A variety of important human pathogens possess a type II CRISPR-Cas system, including bacterial species that cause acute or chronic infections (Louwen et al, 2014). Cas Role in Streptococcus pyogenes investigation support the notion that endogenous bacterial factors involved in stress responses and virulence gene regulation might interact to modulate the expression of CRISPR-Cas genes. Mutants in stress adaptation regulatory proteins RelAQ down-regulate cas genes in Enterococcus faecalis (Yan et al, 2009), deletion of the osmotic regulator OmpR represses cas gene expression in Yersinia pestis (Gao et al, 2011), and Escherichia coli two-component regulatory system (TCS) BaeSR modulates cas genes expression in response to cell envelope stress (Perez-Rodriguez et al, 2011)
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