The hemolytic Streptococcus pyogenes can use a variety of heme compounds as an iron source. In this study, we investigate hemoprotein utilization by S. pyogenes. We demonstrate that surface proteins contribute to the binding of hemoproteins to S. pyogenes. We identify an ABC transporter from the iron complex family named sia for streptococcal iron acquisition, which consists of a lipoprotein (siaA), membrane permease (siaB), and ATPase (siaC). The sia transporter is part of a highly conserved, iron regulated, 10-gene operon. SiaA, which was localized to the cell membrane, could specifically bind hemoglobin. The operon's first gene encodes a novel bacterial protein that bound hemoglobin, myoglobin, heme-albumin, and hemoglobin-haptoglobin (but not apo-haptoglobin) and therefore was named Shr, for streptococcal hemoprotein receptor. PhoZ fusion and Western blot analysis showed that Shr has a leader peptide and is found in both membrane-bound and soluble forms. An M1 SF370 strain with a polar mutation in shr was more resistant to streptonigrin and hydrogen peroxide, suggesting decreased iron uptake. The addition of hemoglobin to the culture medium increased cell resistance to hydrogen peroxide in SF370 but not in the mutant, implying the sia operon may be involved in hemoglobin-dependent resistance to oxidative stress. The shr mutant demonstrated reduced hemoglobin binding, though cell growth in iron-depleted medium supplemented with hemoglobin, whole blood, or ferric citrate was not affected, suggesting additional systems are involved in hemoglobin utilization. SiaA and Shr are the first hemoprotein receptors identified in S. pyogenes; their possible role in iron capture is discussed.

Group A streptococcus (GAS) is a common pathogen of the human skin and mucosal surfaces capable of producing a variety of diseases. In this study, we investigated regulation of iron uptake in GAS and the role of a putative transcriptional regulator named MtsR (for Mts repressor) with homology to the DtxR family of metal-dependent regulatory proteins. An mtsR mutant was constructed in NZ131 (M49 serotype) and analyzed. Western blot and RNA analysis showed that mtsR inactivation results in constitutive transcription of the sia (streptococcal iron acquisition) operon, which was negatively regulated by iron in the parent strain. A recombinant MtsR with C-terminal His 6 tag fusion (rMtsR) was cloned and purified. Electrophoretic mobility gel shift assays demonstrated that rMtsR specifically binds to the sia promoter region in an iron-and manganese-dependent manner. Together, these observations indicate that MtsR directly represses the sia operon during cell growth under conditions of high metal levels. Consistent with deregulation of iron uptake, the mtsR mutant is hypersensitive to streptonigrin and hydrogen peroxide, and55 Fe uptake assays demonstrate that it accumulates 80% ؎ 22.5% more iron than the wild-type strain during growth in complete medium. Studies with a zebrafish infection model revealed that the mtsR mutant is attenuated for virulence in both the intramuscular and the intraperitoneal routes. In conclusion, MtsR, a new regulatory protein in GAS, controls iron homeostasis and has a role in disease production.

We found that the global regulatory two-component signal transduction system CovRS mediates the ability of group A streptococcus (GAS) to grow under two stresses encountered during infection: iron starvation and the presence of LL-37. We also showed that CovRS regulates transcription of the multimetal transporter operon that is important for GAS growth in a low concentration of iron.

SUMMARY The important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) produces many virulence factors that are regulated by the two-component signal transduction system CovRS (CsrRS). Dissemination of GAS infection originating at the skin has been shown to require production of streptokinase, whose transcription is repressed by CovR. In this work we have studied the interaction of CovR and phosphorylated CovR (CovR-P) with the promoter for streptokinase, Pska. We found that, in contrast to the other CovR-repressed promoters, Pska regulation by CovR occurs through binding at a single ATTARA consensus binding sequence (CB) that overlaps the −10 region of the promoter. Binding of CovR to other nearby consensus sequences occurs upon phosphorylation of the protein, but these other CBs do not contribute to the regulation of Pska by CovR. Thus, binding at a specific site does not necessarily indicate that site is involved in regulation by CovR. In addition, at Pska, CovR binding to the different sites does not appear to involve cooperative interactions, which simplifies the analysis of CovR binding and gives us insight into the modes of interaction that occur between CovR and its specific DNA binding sites. Finally, the observation that regulation of transcription from Pska occurs at a very low concentration of phosphorylated CovR may have important implications for the regulation of virulence gene expression during group A streptococcal infection.

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