Quorum Sensing Regulation of a Major Facilitator Superfamily Transporter Affects Multiple Streptococcal Virulence Factors

Abstract

Cell-cell signaling mediated by Rgg-family transcription factors and their cognate pheromones is conserved in Firmicutes, including all streptococci. In Streptococcus pyogenes, or group A strep (GAS), one of these systems, the Rgg2/3 quorum sensing (QS) system, has been shown to regulate phenotypes, including cellular aggregation and biofilm formation, lysozyme resistance, and macrophage immunosuppression. Here, we show the abundance of several secreted virulence factors (streptolysin O, SpyCEP, and M protein) decreases upon induction of QS. The main mechanism underlying the changes in protein levels appears to be transcriptional, occurs downstream of the QS circuit, and is dysregulated by the deletion of an Rgg2/3 QS-regulated major facilitator superfamily (MFS) transporter. Additionally, we identify this MFS transporter as the factor responsible for a previously observed increase in aminoglycoside sensitivity in QS-induced cells. IMPORTANCE The production of virulence factors is a tightly regulated process in bacterial pathogens. Efforts to elucidate the mechanisms by which genes are regulated may advance the understanding of factors influencing pathogen behavior or cellular physiology. This work finds expression of a major facilitator superfamily (MFS) transporter, which is governed by a quorum sensing (QS) system, impacts the expression of multiple virulence factors and accounts for QS-dependent antibiotic susceptibility. Although the mechanism underlying this effect is not clear, MFS orthologs with high sequence similarity from S. pneumoniae and S. porcinus were unable to substitute indicating substrate specificity of the GAS MFS gene. These findings demonstrate novel associations between expression of a transmembrane transporter and virulence factor expression and aminoglycoside transport.

Keywords: M protein; SpyCEP; Streptococcus pyogenes; major facilitator superfamily transporter; streptolysin O.

FIG 1

Reduced hemolysis in QS-ON cells is due to streptolysin O. Supernatants from late log phase cultures grown in the presence of reverse (rev) or C8 peptide (100 nM final) were collected and analyzed for (A) hemolytic activity against sheep red blood cells, and (B) the presence of streptolysin O by Western blot. Data shown are the mean and SD of three biological replicates (A) and are representative of at least three experiments (A and B). ****, P ≤ 0.0001 by unpaired t test.

FIG 2

Reduction of streptolysin O in response to QS induction is dependent on a major facilitator superfamily protein, Spy49_0460. Supernatants from late log phase cultures were collected and concentrated, and Western blotting with anti-SLO antisera was performed. Reverse (rev) or C8 peptide was added to a final concentration of 100 nM at t = 60 min. (A) Genetic loci regulated by Rgg2/3 QS. (B) SLO accumulates in supernatants from mutants lacking spy49_0460. (C) Complementation of Δspy49_0460 with MFS homologues from GAS, S. porcinus (SPO), or S. pneumoniae (SPN) expressed from the P_shp3 promoter. (D) Complementation of ΔBGC with GAS MFS protein spy49_0460. (E) SLO in supernatants collected from additional WT GAS strains after induction of quorum sensing with C8 peptide: NZ131 (M49), 5448 (M1), MGAS315 (M3), MGAS10394 (M6), HSC5 (M14). After blotting, membranes were stained with India ink to assess protein loading and photographed (see Fig. S3). All experiments were performed at least three (A to D) or two (E) times.

FIG 3

Transcriptional changes of several target genes in response to QS induction. Growth and light production of WT and Δspy49_0460 strains containing luciferase reporters were monitored. Reverse (rev) or C8 peptide was added to a final concentration of 100 nM at t = 60 min. (A) P_nga (streptolysin O). (B) P_spyCEP. (C) P_emm49 (M protein). (D) P_syncat (constitutive). Data shown represent one of five (A, B, D) or six (C) biological replicates tested over three different experiments; aggregated data from all replicates are shown in Fig. S5.

FIG 4

SpyCEP and M49 protein are reduced in a Spy49_0460-dependent manner in response to QS induction. Whole cell lysates from late log cultures grown in the presence of reverse (rev) or C8 peptide at a final concentration of 100 nM were analyzed by Western blot using (A) anti-SpyCEP or (B) anti-M antisera. After blotting, membranes were stained with India ink to assess protein loading and photographed. Experiments were performed twice.

FIG 5

Expression of spy49_0460 leads to increased aminoglycoside sensitivity. Kanamycin sensitivity was determined using a broth dilution assay in which mid log phase reverse (rev) or C8 peptide-treated bacteria were back-diluted to a low OD600 to inoculate cultures of ranging concentrations of antibiotic. (A) WT and Δrgg2; (B) WT and Δrgg3; (C) WT and Δspy49_0460. WT data are the same in panels A to C. †, significant difference between WT treated with rev versus C8 peptide; *, significant difference between C8-treated samples. Panels D to F show complementation of Δspy49_0460 with GAS (D), S. porcinus (SPO, E), or S. pneumoniae (SPN, F) MFS transporter genes expressed from a P_shp3 promoter. Vector data are the same in panels D to F. (G) Complementation of the ΔBGC mutant with P_shp3-spy49_0460. Panels H to J show expression of GAS (H), S. porcinus (SPO, I), or S. pneumoniae (SPN, J) MFS transporter genes in S. porcinus. Vector data are the same in panels H to J. Data shown are mean and standard deviation from at least three biological replicates. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 by unpaired t test.