RESUMO
Streptococcus dysgalactiae, a Lancefield group C streptococcus, is commonly isolated from bovine mastitis. We recently identified a putative regulon in two S. dysgalactiae strains, 8215 and Epi9, consisting of two consecutive genes, dmg and dem, coding for a possible regulatory protein and an M-like protein with fibrinogen- and IgG-binding-properties, respectively. During these studies a short sequence homologous to an IS element was found to be inserted in the dmg gene of strain 8215. The present investigation describes the complete sequence of this IS-like element, named ISSdy1, which consists of 1218 bp and contains two ORFs, flanked by imperfect repeats. The nucleotide sequence of the IS-like element shows 82% identity to the previously reported sequence of IS199 from Streptococcus mutans V403. The deduced amino acid sequences of the ORFs also revealed high homology to transposases from IS elements in Enterococcus faecium, Escherichia coli, and Shigella dysenteriae, all belonging to the IS3 family. We studied the distribution of ISSdy1 in 57 S. dysgalactiae isolates using PCR analysis with specific primers derived from the IS element. Ninety-eight percent of the isolates contained the ISSdy1 element. Surprisingly, in the majority of studied strains a copy of the IS-like element was found to be inserted in the dmg gene, a putative virulence regulator.
Assuntos
Proteínas de Bactérias/genética , Doenças dos Bovinos/microbiologia , Elementos de DNA Transponíveis/genética , Mastite Bovina/microbiologia , Streptococcus/genética , Streptococcus/patogenicidade , Virulência/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Bovinos , DNA/análise , DNA/isolamento & purificação , Primers do DNA/química , Feminino , Dosagem de Genes , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Streptococcus/classificaçãoRESUMO
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 approximately 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 of S. 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 corresponding dmgA and demA genes from another strain, called Epi9, were isolated and analyzed. These genes, called dmgB and 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% CO(2), was correlated to an enhanced transcription of the demB gene as shown in a Northern blot. Strain 8215 did not respond to CO(2), which could be explained by a nonfunctional dmgA 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 CO(2), we suggest that the dmg and dem genes are members of a regulon similar to the described mga regulon in S. pyogenes, which encodes several virulence factors in this species.
Assuntos
Antígenos de Bactérias , Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Streptococcus/genética , Streptococcus/patogenicidade , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Ligação Competitiva , Proteínas de Transporte/metabolismo , Bovinos , Clonagem Molecular , Primers do DNA/genética , Feminino , Fibrinogênio/metabolismo , Expressão Gênica , Genes Bacterianos , Mastite Bovina/microbiologia , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Streptococcus/metabolismo , Streptococcus pyogenes/genética , Virulência/genéticaRESUMO
Protein MIG, from Streptococcus dysgalactiae, binds alpha2-macroglobulin and immunoglobulin G (IgG). MIG-derived fusion proteins with one to five IgG-binding repeats differed up to 72,000- fold in avidity for goat IgG, indicating a considerable cooperativity of the repeats. Significant sequence variation in the IgG-binding repeats was recognized. Protein MIG interacted with goat IgG1 via both the Fc and Fab parts.
