Use of surface-enhanced laser desorption ionization protein chip system to analyze streptococcal exotoxin B activity secreted by Streptococcus pyogenes

• Published in: Journal of microbiological methods 2001-08, Vol.46 (2), p.87-97
• Main Authors: Boyle, Michael D.P, Romer, Terence G, Meeker, Amanda K, Sledjeski, Darren D
• Format: Article
• Language: English
• Subjects: Bacterial diseases; Bacterial Proteins - analysis; Bacterial Proteins - biosynthesis; Bacteriology; Biological and medical sciences; Culture Media; Cysteine Endopeptidases - analysis; Cysteine Endopeptidases - biosynthesis; Enzyme Precursors - analysis; Enzyme Precursors - biosynthesis; exotoxin B; Experimental bacterial diseases and models; Fundamental and applied biological sciences. Psychology; Infectious diseases; Kinetics; Mass Spectrometry; Medical sciences; Microbiology; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Protein chip system; Streptococcal exotoxin B; Streptococcus pyogenes; Streptococcus pyogenes - enzymology; Streptococcus pyogenes - growth & development
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/S0167-7012(01)00279-2

Ciphergen surface-enhanced laser desorption ionization (SELDI) protein chip technology was used to analyze the secretion and autoactivation of the Streptococcus pyogenes cysteine protease SpeB. This method allowed rapid identification of both the zymogen form of the protein Mr∼41,000 and the fully active enzyme Mr∼28,500. SpeB production in culture supernatants was demonstrated to be growth-phase regulated and SpeB positive and negative variants of a blood passaged S. pyogenes isolate could readily be distinguished. In kinetic studies of the autoactivation of the zymogen form of SpeB, the sequential generation of four intermediates was detected before the accumulation of the fully active enzyme. The methods described enabled enhanced speed, use of lower sample volumes and concentrations, and a more complete molecular characterization of SpeB than allowed by existing methods of analysis using SDS-PAGE and Western immunoblotting.