The scfCDE Operon Encodes a Predicted ABC Importer Required for Fitness and Virulence during Group A Streptococcus Invasive Infection

Abstract

As a strict human pathogen, Streptococcus pyogenes (group A Streptococcus, or GAS) causes a wide range of infections, from superficial to life-threatening diseases, upon dissemination. Thus, it is necessary to gain a better understanding of how GAS successfully overcomes host-mediated challenges and infects various host niches. We previously identified subcutaneous fitness (scf) genes in the clinically relevant wild-type (WT) GAS M1T1 5448 strain that are critical for fitness during murine soft-tissue infection at both 24 h and 48 h postinfection. The uncharacterized locus scfCDE was transcribed as an operon and is predicted to encode an ABC importer for nutrient uptake (e.g., amino acids). Individual scfCDE deletion mutants grew comparably to WT 5448 in rich medium but exhibited reduced fitness during competitive growth in murine soft tissue and in nutrient-limiting chemically defined medium (CDM). A deletion of the permease gene scfD resulted in a monoculture growth defect in CDM that could be rescued by addition of excess peptides, suggesting a role as an amino acid importer. Interestingly, the ΔscfC substrate-binding and ΔscfD permease mutants, but not the ΔscfE ATPase mutant, were highly attenuated in murine soft tissue. Moreover, all three genes were required for GAS survival in human blood, indicating their impact is not limited to superficial infections. As such, scfCDE plays an integral role in enhancing GAS adaptation during localized infection as well as dissemination to deeper host environments. Since scfCDE is conserved throughout Firmicutes, this work may contribute to the development of therapeutic strategies against GAS and other Gram-positive pathogens.

Phosphotransferase System Uptake and Metabolism of the β-Glucoside Salicin Impact Group A Streptococcal Bloodstream Survival and Soft Tissue Infection

“…2A). To assess whether licT and bglPB were cotranscribed in an operon, total RNA was isolated from WT 5448 cells grown to late exponential phase in THY-rich medium at 37°C and converted to cDNA (see Materials and Methods) (41). RT-PCR of the resulting cDNA using locus-specific primer pairs (Table 2) found transcriptional linkage between licT and bglP, bglP and bglB, and licT and bglB (Fig.…”

“…Since GAS is a strict human pathogen whose virulence factors have evolved to specifically recognize human cells and proteins (62,63), the phenotype discrepancy between human blood and the murine model may be a result of host specificity. Our recent study involving the ATPase of a putative ABC transporter, ScfE, also showed attenuated phenotypes in human blood that were not recapitulated in murine models (41). Additionally, GAS would need to circumvent epithelial barriers during murine subcutaneous infection in order to reach the bloodstream.…”

“…Four independent colonies of each strain were grown static overnight in 10 ml of THY with the appropriate antibiotic, if applicable, at 37°C with 5% CO 2 . RNA isolation was carried out as previously described (41). Briefly, overnight cultures were inoculated into fresh media and grown to late logarithmic phase (ca.…”

“…First-strand cDNA synthesis was done using 500 to 800 ng of total RNA with Moloney Murine Leukemia Virus (M-MulV) reverse transcriptase (New England BioLabs) with primer bglB-cDNA-R (Table 2), following the manufacturer's instructions. Subsequent PCR was carried out as previously described (41). Primers (Table 2) within the 5= start and 3= end of each licT-bglPB ORF were used as controls, as well as samples containing full-length primers, but without RNA or RT enzyme.…”

“…5= rapid amplification of cDNA ends (RACE; Invitrogen) was performed according to the manufacturer's instructions, as previously described (41). Briefly, total RNA was used to generate cDNA with licT-GSP1 within licT (Table 2).…”