SalY of theStreptococcus pyogenesLantibiotic Locus Is Required for Full Virulence and Intracellular Survival in Macrophages

Abstract

Streptococcus pyogenes utilizes numerous mechanisms for evading the host immune response but has only recently been found to survive in the intracellular environment. In this study, we demonstrate the requirement of a putative ABC transporter permease for intracellular survival in macrophages. The highly attenuated S. pyogenes mutant, SalY, was identified from a transposon mutagenesis screen, with over 200-fold attenuation in virulence in a zebrafish invasive-disease model. Sequencing of the region surrounding the insertion identified a locus that is highly conserved in other S. pyogenes genomes and is homologous to an operon involved in lantibiotic production. In vitro analysis demonstrated that the SalY mutant is deficient in intracellular survival in murine macrophages, a phenotype also observed in zebrafish macrophages in vivo. Macrophage crude cell lysates added to bacterial cultures resulted in the death of the SalY mutant but only growth inhibition of the wild-type strain. Specific depletion of zebrafish macrophages in vivo restored the ability of the SalY mutant to cause disease to wild-type levels. The SalY-infected, macrophage-depleted zebrafish exhibit large lesions and invasive dissemination at a rate and level similar to those of the wild type. In contrast, an M protein mutant with a degree of attenuation similar to that of the SalY mutant did not regain full virulence by in vivo depletion of macrophages. The putative SalY ABC transporter may be an example of the ability of S. pyogenes to adapt and evolve new survival strategies that allow dissemination and growth in previously uninhabitable sites.

Effects of the ERES Pathogenicity Region Regulator Ralp3 on Streptococcus pyogenes Serotype M49 Virulence Factor Expression

“…The expression of emm49, encoding for antiphagocytic M protein, and the expression of the whole sal operon is reduced. SalY is required for full virulence and intracellular survival in macrophages (28). The reduced transcript amounts of both genes leads to a decreased protection against phagocytosis and impaired survival in human media.…”

First Streptococcus pyogenes Signature-Tagged Mutagenesis Screen Identifies Novel Virulence Determinants

“…Zebrafish were inoculated intramuscularly as described above, and histology sections were prepared as reported previously (45). Tissue sections from the zebrafish infected with the reporter construct were stained with an antiserum to detect S. pyogenes (Lee Laboratories, Grayson, GA) at a dilution of 1:500 and incubated for 2 h in a humidified atmosphere at room temperature, after which they were washed with PBS and stained with goat anti-rabbit antibody conjugated to Alexa Fluor 488 (Molecular Probes) at a dilution of 1:2,500 for 30 min.…”

Transcriptional Analysis of the Streptococcus pyogenes Salivaricin Locus

“…These results suggest that the salKR promoter requires tight regulation, indicating the importance of regulated expression of the downstream genes encoding the two-component signaling system salKR. Previous studies demonstrated that the SalKR two-component system is required for the full virulence of pathogenic bacteria like S. suis (29) and S. pyogenes (16).…”

“…Genes in the sal locus are important for the full virulence of S. pyogenes strain HSC5 (16). To determine if the identified salKR promoter was affected by host factors, we tested the expression of the salKR promoter (full length with all regulatory sequences) in the wildtype strain in the presence of human serum compared to that in the presence of fetal bovine serum.…”

“…A previous transposon mutagenesis screen identified a mutant with a transposon insertion in the salK gene of the S. pyogenes sal locus (16). The salK gene encodes the putative histidine kinase of the sal locus TCS.…”