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

CsrR/CsrS (CovR/CovS) is a two-component regulator of extracellular virulen ce factors in Group A streptococcus, but the full range of regulated exopro teins is unknown. Since CsrR represses expression of regulated factors, cul ture supernates of wild-type and CsrR(-) mutant strains were compared by tw o-dimensional gel electrophoresis (2DGE) to identify regulated exoproteins. Supernates of Delta csrRS(-) mutant, but not wild-type, bacteria contained an abundant 23 kDa protein. The N-terminal sequence of this spot correspon ded to a putative open reading frame (ORF) in the streptococcal genome. In a mobility shift assay, phosphorylated CsrR bound to a PCR amplicon that in cluded sequences upstream of this ORF. By primer extension analysis, the OR F (designated mspA, for mucoidy-associated secreted protein) was expressed in mid- and late-exponential phase in a Delta csrRS(-) 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 Delta mspA 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 expre ssion of hyaluronic acid capsule or severity of skin infection in mice. (C) 2001 Academic Press.