Role for sagA and siaA in quorum sensing and iron regulation in Streptococcus pyogenes

Abstract

Streptococcus pyogenes is a ubiquitous and versatile pathogen that causes a variety of infections with a wide range of severity. The versatility of this organism is due in part to its capacity to regulate virulence gene expression in response to the many environments that it encounters during an infection. We analyzed the expression of two potential virulence factors, sagA and siaA (also referred to as pel and htsA, respectively), in response to conditions of varying cell densities and iron concentrations. The sagA gene was up-regulated in conditioned medium from a wild-type strain but not from sagA-deficient mutants, and the gene was also up-regulated in the presence of streptolysin S (SLS), the gene product of sagA, thus indicating that this gene or its product is involved in density-dependent regulation of S. pyogenes. By comparison, siaA responded in a manner consistent with a role in iron acquisition since it was up-regulated under iron-restricted conditions. Although siaA expression was also up-regulated in the presence of SLS and in conditioned media from both wild-type and sagA-deficient mutants, this up-regulation was not growth phase dependent. We conclude that sagA encodes a quorum-sensing signaling molecule, likely SLS, and further support the notion that siaA is likely involved in iron acquisition.

FIG. 1.

Growth kinetics of MGAS166 in various conditioned media (A) and various concentrations of SLS (B) and ferric chloride (C). Each experiment represents an average of three independent cultures. Error bars indicate standard deviations.

FIG. 2.

Average normalized expression of siaA (A) and sagA (B) in MGAS166 from duplicate cultures grown in nonconditioned medium at 2, 4, and 6 h postinoculation. Both siaA and sagA expression levels were normalized with gyrA. Error bars indicate standard deviations.

FIG. 3.

Expression of siaA and sagA during growth of MGAS166 in conditioned medium from MGAS166 (A), SBNH5(ΔSLS) (B), and NZ131:sagAΔcat (C) relative to nonconditioned medium as determined by real-time PCR analysis. Statistical significance (P < 0.05) as determined by a single factor ANOVA is indicated by the asterisk. Error bars indicate standard deviations.

FIG. 4.

Expression of siaA (A) and sagA (B) of MGAS166 in the presence of 5.0 μg/ml of SLS relative to nonconditioned medium as determined by real-time PCR analysis. Statistical significance (P < 0.05) as determined by a single factor ANOVA is indicated by the asterisk. Error bars indicate standard deviations.

FIG. 5.

Expression of siaA (A) and sagA (B) during growth of MGAS166 in low (1.0 μg/ml) relative to high (1,000 μg/ml) iron concentrations as determined by real-time PCR analysis. Statistical significance (P < 0.05) as determined by a single factor ANOVA is indicated by the asterisk. Error bars indicate standard deviations.