Characteristics of Vibrio cholerae proteinases: potential, candidate vaccine antigens.
Vibrio cholerae extracellular proteinases (proteases) have been studied as potential candidate antigens for acellular cholera vaccines. Proteinases from V. cholerae NCTC 10732 were prepared from batch culture either by ammonium sulphate precipitation and G100 Sephadex gel filtration or by isoelectric focusing (IEF). Proteinase activity was at a maximum level after 24 h, coincident with the late exponential phase and early stationary phase. Three major IEF peaks of activity were resolved with specific activities in the range 17.2-195 EU ml(-1 )mg(-1). Sodium dodecyl sulphate-polyacrylamide gel electrophoreses (SDS-PAGE) of these fractions revealed 42, 45, 57 and 75 kDa bands in which proteinase activity was demonstrable. Peptide digest analysis suggested different catalytic specificities for each proteinase fraction. Metalloproteinase and serine proteinase inhibitors, alpha(2)-macroglobulin (alpha(2)-M), the thiol proteinase inhibitor and N-ethylmaleimide inhibited the proteinases. The proteinases nicked Escherichia coli heat-labile toxin to yield catalytically active sub-units, confirmed by the measurement of intrinsic ADP-ribosylation activity. The possible value of these putative V. cholerae antigens in an acellular vaccine is discussed.
Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK.
D E S
DE
Stewart-Tull
Carol R
CR
Bleakley
Tamara S
TS
Galloway
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology*; D001427:Bacterial Toxins / Q000737:chemistry; D001428:Bacterial Vaccines / Q000737:chemistry / Q000276:immunology*; D002364:Caseins / Q000737:chemistry; D002772:Cholera Toxin / Q000737:chemistry; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000032:analysis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D004768:Enterotoxins / Q000737:chemistry; D029968:Escherichia coli Proteins; D005353:Fibronectins / Q000737:chemistry; D006868:Hydrolysis; D007070:Immunoglobulin A / Q000737:chemistry; D007525:Isoelectric Focusing; D007781:Lactoferrin / Q000737:chemistry; D008033:Limulus Test; D009439:Neuraminidase / Q000737:chemistry / Q000276:immunology; D010455:Peptides / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D014734:Vibrio cholerae / Q000201:enzymology* / Q000254:growth & development / Q000276:immunology*
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D002364:Caseins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D005353:Fibronectins; D007070:Immunoglobulin A; C002087:N,N-dimethylcasein; D010455:Peptides; D011480:Protease Inhibitors; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; C027538:heat-labile enterotoxin, E coli; D000242:Cyclic AMP; D009439:Neuraminidase; D010450:Endopeptidases; D007781:Lactoferrin