Abstract
Streptococcus pyogenes (Group A Streptococcus, GAS) remains a major public health burden worldwide, infecting over 750 million people leading to over 500,000 deaths annually. GAS pathogenesis is complex, involving genetically distinct GAS strains and multiple infection sites. To overcome fastidious genetic manipulations and accelerate pathogenesis investigations in GAS, we developed a mariner-based system (Krmit) for en masse monitoring of complex mutant pools by transposon sequencing (Tn-seq). Highly saturated transposant libraries (Krmit insertions in ca. every 25 nucleotides) were generated in two distinct GAS clinical isolates, a serotype M1T1 invasive strain 5448 and a nephritogenic serotype M49 strain NZ131, and analyzed using a Bayesian statistical model to predict GAS essential genes, identifying sets of 227 and 241 of those genes in 5448 and NZ131, respectively. A large proportion of GAS essential genes corresponded to key cellular processes and metabolic pathways, and 177 were found conserved within the GAS core genome established from 20 available GAS genomes. Selected essential genes were validated using conditional-expression mutants. Finally, comparison to previous essentiality analyses in S. sanguinis and S. pneumoniae revealed significant overlaps, providing valuable insights for the development of new antimicrobials to treat infections by GAS and other pathogenic streptococci.
Highlights
Streptococcus pyogenes (Group A Streptococcus, GAS) remains a major public health burden worldwide, infecting over 750 million people leading to over 500,000 deaths annually
We recently developed a mariner transposon (Oskar) for stable random mutagenesis in GAS28 and used a complex mutant library for TraSH to identify genes that are important for GAS fitness in an ex vivo model of human blood infection[29]
High saturation mutagenesis was achieved in two divergent GAS strains that are representative of strains with tropism for the throat alone and both the throat and skin, respectively
Summary
Streptococcus pyogenes (Group A Streptococcus, GAS) remains a major public health burden worldwide, infecting over 750 million people leading to over 500,000 deaths annually. Essential genes, defined as those necessary for growth and survival under a given condition, represent attractive targets for the discovery of new therapeutics against bacterial pathogens[10] Identification of these genes on a genomic scale has been of particular interest as it provides candidates for guiding subsequent drug development[11,12]. Essentiality screens have been successfully carried out in two pathogenic streptococci, the oral pathogen S. sanguinis using directed mutagenesis[15] and the respiratory pathogen S. pneumoniae both by TraSH27 and Tn-seq[26]; these analyses have not yet been undertaken for GAS Towards this goal, we recently developed a mariner transposon (Oskar) for stable random mutagenesis in GAS28 and used a complex mutant library for TraSH to identify genes that are important for GAS fitness in an ex vivo model of human blood infection[29]. This work establishes a baseline for future Tn-seq studies of GAS growing in disease-relevant environments and provides invaluable information for the discovery of new targets for antimicrobial drugs as well as the study of GAS pathogenesis on a genome-scale
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