title: Characteristics of Vibrio cholerae proteinases
Creators: name: Bleakley, Carol R.
Publisher: name: University of Glasgow
Year: 1984

description: V. cholerae extracellular proteases were isolated, characterized and their role in adherence and as vaccine additives considered. Gel filtration of the crude protease produced one large peak of pro tease activity quite distinct from the major protein bando Isoelectric focussing of the crude protease or the pooled G100 protease peak (G100/P) yielded three peaks of protease activity. These were termed IEF-1 which was a broad peak with a pi of 4.5-5.5+0.1, IEF-2 with a pi of 6.5 0.1 and IEF-3 with a pi of 9.0 0.1. The three IEF proteases were shown by peptide digest analysis to have distinct catalytic activities. Gelatin-PAGE of the crude protease, G100/P, IEF-1, IEF-2 and IEF-3 showed that each preparation contained at least three areas of protease activity, suggesting either the presence of several proteases in each preparation or autocatalytic degradation of a single protease which had produced multiple activities on the PAGE gel. Characterisation of the three IEF proteases by inhibitors revealed that IEF-1 was inhibited by metalloprotease and serine protease inhibitors, IEF-2 was inhibited by metallo, serine and thiol protease inhibitors and IEF-3 was inhibited by only metalloprotease inhibitors. This suggested that more than one protease was present in IEF-1 and IEF-2 as it is unusual for a single protease to be inhibited by more than one class of protease inhibitor. IEF-3 was only inhibited by metallo- protease inhibitors but showed multiple protease activities in gelatin-PAGE suggesting either several metalloproteases or autodegradation of one metalloprotease which has then displayed electrophoretic heterogeneity. The crude protease, G100/P and IEF-3 all showed muclnase and haemagglutinating activity. Fibronectin and lactoferrin, two host substances that may be involved at the level of mucosal defence were tested for their susceptibility to V. cholerae proteaseso Fibronectin was found to be digested after 1 h and further digestion occurred after 24 h. No digestion of lactoferrin was seen even after a 24 h incubation with V. cholerae proteases. IgA is the main immunoglobulin present in mucosal secretions and so is the major antibody involved in mucosal defenceo V. cholerae proteases were shown to digest IgA specifically with the loss of three peptide bands. Immunoelectrophoresis showed that the V. cholerae protease(s) was not a typical IgA protease as no distinct Fc and Fab fragments were producedo The antigenicity of the IgA remained but the single precipitin arc was shortened following protease treatment. The binding of V. cholerae enterotoxin to ileal segments was found not to be affected by enzyme treatment of the tissue. However it is interesting that treatment of the cholera enterotoxin with the protease-containing mixture G100/P caused an increase in toxin activity, peptide fragments with similar molecular weights to A1 and A2 following protease treatment. The effect of V. cholerae proteases on the binding of the vibrios to ileal segments in vitro was assessed by a radioactive adherence assay. Three different types of tissue preparation were used. Experiments which involved the use of tissue segments where both the mucosal and serosal surface of the ileum were present during the assay procedure showed no effect on the number of vibrios binding following enzyme treatment. V. cholerae was found to adhere to the serosal surface of the ileum and the numbers adhering to the serosal surface increased following protease treatment of the tissue segment. Adherence of Vo cholerae to the mucosal surface was found to decrease following protease treatment of the tissue segments and neuraminidase had no effect on the adherence of vibrios to either the mucosal or serosal surfaces. The use of the protease-containing mixture G100/P as a vaccine to produce active protection against V. cholerae was tested in guinea-pigs. Initial experiments were done by giving guinea-pigs one parenteral vaccination. These guinea-pigs were found only to be significantly protected if given a vaccine containing both protease plus neuraminidase. Guinea-pigs given one parenteral vaccination and one parenteral booster dose with various concentrations of protease and neuraminidase were highly protected when the vaccine contained 80 mug of protease and 0.02 I.U, of neuraminidase. A parenteral vaccination followed by an oral booster dose of 80 mug of protease and 0.02 I.U. of neuraminidase significantly protected guinea-pigs and for the first time protease alone (80 (mug) was a protective vaccine. In conclusion, it is apparent that V. cholerae produces several proteases which play important roles in the pathogenesis of cholera.