Bacterial pathogens must adapt to changing in vivo niches in order to colonize and persist within their host, often overcoming nutritional immunity to obtain essential nutrients. Identification of genes required for fitness in the host can provide targets for new therapeutic approaches. The Group A Streptococcus (GAS) is a strict human pathogen typically associated with asymptomatic carriage and self-limiting infection but can sometimes invade sterile sites and causing life-threatening diseases (e.g., toxic shock syndrome, necrotizing fasciitis). There is clear evidence that physiology is linked to pathogenesis for GAS in the human and allows the pathogen to proliferate in a range of host environments. An in vivo Tn-seq screen of GAS fitness in the M1T1 strain 5448 in a murine model of soft tissue infection identified the subcutaneous fitness locus scfCDE encoding a putative ABC importer conserved in Streptococcus and most Firmicutes, where ScfC is the substrate-binding protein, ScfD the membrane permease, and ScfE the ATPase. Although its role has not established, homology suggests it may transport aromatic amino acids such as tryptophan (L-trp). The scfCDE locus also contains two uncharacterized small open reading frames (smORFs) that might play a role. Preliminary data shows that scfDE are required for GAS competitive fitness in murine soft tissue, both scfDE are required for GAS survival in human blood, a ΔscfD permease mutant is highly attenuated in the murine soft tissue model and the fitness defect for scfDE in soft tissue can be replicated in vitro using nutrient-limiting media (CDM, mRPMI). Our published transcriptomic results reveal that scfCDE is repressed in the presence of asparagine, induced during heme stress, and repressed by the global virulence regulator Mga. Still, this locus has yet to be studied in the context of the pathophysiology of GAS and the function of this conserved ABC importer remains enigmatic in Gram-positive pathogens. We hypothesize that scfCDE encodes for an essential ABC importer that is important the ability of GAS to obtain key nutrients in host tissues. The goal in this exploratory R21 is to investigate the basic role of scfCDE in the pathophysiology of GAS, with the expectation that this work will advance our understanding of how this locus contributes to virulence in GAS and other pathogens. In Aim 1, we will investigate the functional role of ScfCDE in GAS by identifying potential substrates that ScfCDE may transport with an emphasis on aromatic amino acids such as tryptophan (L-trp). We will also explore interacting partners and metabolic pathways that scfCDE could be involved in through Tn-seq of the ΔscfD mutant in a low-nutrient medium that mimics the soft tissue phenotype. In Aim 2, we will explore expression of the scfCDE locus by characterizing the transcriptional architecture and expression patterns of scfCDE's under different media conditions. The contribution of the two small ORFs (smORFs) in t...