Identification of a major, CsrRS-regulated secreted protein of Group A streptococcus

Abstract

CsrR/CsrS (CovR/CovS) is a two-component regulator of extracellular virulence factors in Group A streptococcus, but the full range of regulated exoproteins is unknown. Since CsrR represses expression of regulated factors, culture supernates of wild-type and CsrR−mutant strains were compared by two-dimensional gel electrophoresis (2DGE) to identify regulated exoproteins. Supernates of ΔcsrRS−mutant, but not wild-type, bacteria contained an abundant 23 kDa protein. The N-terminal sequence of this spot corresponded to a putative open reading frame (ORF) in the streptococcal genome. In a mobility shift assay, phosphorylated CsrR bound to a PCR amplicon that included sequences upstream of this ORF. By primer extension analysis, the ORF (designated mspA, for m_ucoidy-associated s_ecreted p_rotein) was expressed in mid- and late-exponential phase in a ΔcsrRS−mutant. The presence of an in-frame deletion in mspA did not affect colony appearance, mucoidy or in vitro growth, and there was no difference between ΔmspA and wild-type strains in a mouse model of skin infection. MspA is co-regulated with other factors required for dermonecrosis (e.g. capsule, streptolysin S and purogenic exotoxin B); however, deletion of this gene does not affect expression of hyaluronic acid capsule or severity of skin infection in mice.