M-Like Proteins ofStreptococcus dysgalactiae

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Abstract
Streptococcus dysgalactiae is one of the most important bacterial species isolated from bovine mastitis. To identify potential virulence factors of this species we prepared chromosomal DNA from strain 8215 and constructed a phage display library. By affinity selection of the library against fibrinogen (Fg), we isolated and characterized a gene, called demA, encoding a protein with the molecular mass of ∼58 kDa, called DemA, displaying both plasma protein binding properties and sequence similarities with the M and M-like proteins of other streptococcal species. Purified recombinant DemA protein was found to completely inhibit Fg-binding to cells ofS. dysgalactiae. A continued sequence analysis revealed that the demA gene was preceded by an open reading frame (dmgA) coding for a putative protein, called DmgA, with high similarities to the Mga proteins of Streptococcus pyogenes. By additional cloning, the correspondingdmgA and demA genes from another strain, called Epi9, were isolated and analyzed. These genes, called dmgBand demB, respectively, revealed a high degree of similarity to the corresponding genes in strain 8215. Increased binding of Fg by cells of strain Epi9, grown in an atmosphere with 10% CO2, was correlated to an enhanced transcription of thedemB gene as shown in a Northern blot. Strain 8215 did not respond to CO2, which could be explained by a nonfunctionaldmgA gene due to insertion of an insertion sequence element. Based on sequence similarities of the described proteins to Mga, M, and M-like proteins and the response to elevated level of CO2, we suggest that the dmg anddem genes are members of a regulon similar to the describedmga regulon in S. pyogenes, which encodes several virulence factors in this species.