Genome sequencing and transcriptome profiling have helped establish the genetic determinants expressed by a pathogen during infection; however, revealing genes that are "functionally required" for survival and fitness within the host is key to understanding pathophysiology. We have been applying a powerful genetic approach, transposon-sequencing (Tn-seq), to identify en masse genes important for fitness using relevant models of in vivo infection in the strict human pathogen Streptococcus pyogenes (Group A Streptococcus, GAS). GAS causes a wide array of disease manifestations ranging from self-limiting superficial infections of the skin & throat to severe invasive diseases of soft tissues & other normally sterile sites. Colonization of epithelia, followed by infection of the sub-epithelial tissue represents initial events during GAS infections. Using a near-saturation mariner (Krmit) transposon mutant library of the globally disseminated M1T1 GAS 5448, we performed Tn-seq to define essential (non-mutable) genes and assessed the genetic determinants of increased and decreased fitness during in vitro growth in rich media. We have now used Tn-seq to investigate the genes important for fitness and survival in lesion formation during soft tissue infection. The 5448 Krmit mutant library was inoculated subcutaneously into immunocompetent hairless mice and fitness was monitored by Tn-seq during abscess (24 h) and subsequent ulcerative lesion (48 h) formation in vivo. After comparison to our in vitro Tn-seq dataset, we were able to define and validate GAS 5448 genes required for fitness in lesions. One category of emphasis were genes annotated as “unknown function” that we renamed subcutaneous fitness (scf) genes. A two-gene operon (scfAB) encoding putative membrane proteins was identified in the screen and was found to be conserved amongst many important Gram-positive pathogens. Defined GAS 5448 mutants in scfAB were outcompeted by wild type GAS in vivo, were attenuated for lesion formation in vivo, and exhibited reduced survival in human blood. Continuing Tn-seq studies are exploring different GAS disease-relevant environments in vitro (minimal media, metals), ex vivo (blood, phagocytes, biofilm), and in vivo (colonization, invasive sites). By comparing and contrasting our Tn-seq screens, we can begin to reveal key pathways and genes that play a functional role during GAS infections as potential new therapeutic targets.