A single amino-acid change between the antigenically different extracellular serine proteases V2 and B2 from Dichelobacter nodosus

Abstract

Dichelobacter nodosus (Dn), the causative organism of ovine footrot, secrets three distinct types of extracellular serine proteases which have been implicated in virulence. Southern analyses have shown that the proteases are encoded by three separate genes, and the genes encoding an acidic protease V5 and a basic protease have already been characterised from virulent Dn strain 198. The gene encoding the third protease type, as represented by acidic protease V2, was isolated from an EcoRI-BamHI library of strain 198 genomic DNA by probing with a polymerase chain reaction (PCR) fragment generated with oligodeoxyribonucleotides based on protease V2 amino acid (aa) sequences. A further clone from an RsaI library was isolated to complete the 5' region of the gene to yield an ORF of 1803 bp encoding a protein precursor of 601 aa. The acidic protease V2 gene, aprV2, shows the same precursor structure as the bprV and aprV5 genes with 72% and 69% similarity at the nucleotide (nt) level and with 73% and 69% similarity at the aa level, respectively. As monoclonal antibodies consistently distinguish the virulent (V) and benign (B) forms of this protease, the gene encoding the acidic protease B2 from benign Dn strain 305 was isolated using the PCR and characterized to investigate the molecular basis for this difference in antigenicity. A 2-bp substitution in a single codon was identified which appeared to be responsible for a change of epitope.