Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection

Abstract

The Group A Streptococcus remains a significant human pathogen causing a wide array of disease ranging from self-limiting to life-threatening invasive infections. Epithelium (skin or throat) colonization with progression to the subepithelial tissues is the common step in all GAS infections. Here, we used transposon-sequencing (Tn-seq) to define the GAS 5448 genetic requirements for in vivo fitness in subepithelial tissue. A near-saturation transposon library of the M1T1 GAS 5448 strain was injected subcutaneously into mice, producing suppurative inflammation at 24 h that progressed to prominent abscesses with tissue necrosis at 48 h. The library composition was monitored en masse by Tn-seq and ratios of mutant abundance comparing the output (12, 24 and 48 h) versus input (T ₀) mutant pools were calculated for each gene. We identified a total of 273 sub cutaneous fitness ( scf) genes with 147 genes (55 of unknown function) critical for the M1T1 GAS 5448 fitness in vivo; and 126 genes (53 of unknown function) potentially linked to in vivo fitness advantage. Selected scf genes were validated in competitive subcutaneous infection with parental 5448. Two uncharacterized genes, scfA and scfB, encoding putative membrane-associated proteins and conserved among Gram-positive pathogens, were further characterized. Defined scfAB mutants in GAS were outcompeted by wild type 5448 in vivo, attenuated for lesion formation in the soft tissue infection model and dissemination to the bloodstream. We hypothesize that scfAB play an integral role in enhancing adaptation and fitness of GAS during localized skin infection, and potentially in propagation to other deeper host environments.

Author summary

The WHO ranks the Group A Streptococcus (GAS) in the top 10 leading causes of morbidity and mortality from infectious diseases worldwide. GAS is a strict human pathogen causing both benign superficial infections as well as life-threatening invasive diseases. All GAS infections begin by colonization of an epithelium (throat or skin) followed by propagation into subepithelial tissues. The genetic requirements for M1T1 GAS 5448 within this niche were interrogated by in vivo transposon sequencing (Tn-seq), identifying 273 sub cutaneous fitness ( scf) genes with 108 of those previously of “unknown function”. Two yet uncharacterized genes, scfA and scfB, were shown to be critical during GAS 5448 soft tissue infection and dissemination into the bloodstream. Thus, this study improves the functional annotation of the GAS genome, providing new insights into GAS pathophysiology and enhancing the development of novel GAS therapeutics.