Characterization of the arginine deiminase of Streptococcus equi subsp. zooepidemicus.
Streptococcus equi subsp. zooepidemicus is an important cause of infectious diseases in horses and rarely humans. Little is known about the virulence factors or protective antigens of S. equi subsp. zooepidemicus. In the present study, I designed original primers based on an alignment of the gene sagp(arcA) from Streptococcus pyogenes encoding streptococcal acid glycoprotein-arginine deiminase (SAGP/AD) to amplify the S. equi subsp. zooepidemicus counterpart sequence by polymerase chain reaction, and I analyzed the sagp(arcA) gene of the organism. Using chromosomal walking steps, I identified a contiguous eight-gene locus involved in SAGP/AD production. Their open reading frames were found to share significant homologies and to correspond closely in molecular mass to previously sequenced arc genes of S. pyogenes, thus they were designated ahrC.2 (arginine repressor), arcR (CRP/FNR transcription regulator), sagp(arcA) (streptococcal acid glycoprotein-arginine deiminase), putative acetyltransferase gene, arcB (ornithine carbamyl transferase), arcD (arginine-ornithine antiporter), arcT (Xaa-His peptidase), and arcC (carbamate kinase). The SAGP homologue of S. equi subsp. zooepidemicus (SzSAGP), encoded by arcA gene of the bacteria (arcA(SZ)), was successfully expressed in Escherichia coli and purified to homogeneity. When in vitro growth inhibitory activity of the recombinant SzSAGP was tested against MOLT-3 cells, it inhibited the growth of the cells during the 3 days of culture in a dose-dependent manner, accompanied by the induction of apoptotic cell death. The recombinant protein also possessed AD activity. By immunoblot analysis using both anti-SzSAGP-SfbI(H8) and anti-SfbI(H8) sera, I was able to demonstrate that the SzSAGP protein is expressed on the streptococcal surface.
Hong
Kyongsu
K
Laboratories for Bioengineering and Research, JCR Pharmaceuticals Co., Ltd., Kobe, Japan. kou@jcrpharm.co.jp
D017209:Apoptosis / Q000187:drug effects; D001120:Arginine / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003470:Culture Media / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D023061:Gene Order; D006801:Humans; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D018502:Streptococcus equi / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D001120:Arginine; D006867:Hydrolases; C019723:arginine deiminase