Evaluation of Antigenic and Nonantigenic Proteins ofEdwardsiella ictaluriat Two Culture Temperatures and in Enriched and Minimal Media using Two-Dimensional Polyacrylamide Gel Electrophoresis

Abstract

Proteins in whole-cell lysates (WCLs) of two Edwardsiella ictaluri isolates grown in brain–heart infusion broth (BHI) and defined minimal medium broth (MM19) were analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In denatured 2D-PAGE, there were no differences in the expression of proteins between virulent isolates 92-266 and 93-146 grown in BHI or between growth temperatures of 25°C and 30°C for 92-266 grown in BHI. Comparisons of growth of 93-146 in BHI and MM19 showed that there were differences in protein expression between the culture media. Pooled convalescent catfish serum (CCS) recognized 39 bands and 89 spots in Western blots of WCL of E. ictaluri grown in BHI and MM19. The same antigenic bands and spots recognized in BHI-grown WCLs were detected in the MM19-grown WCL, with the exception of an additional 57-kDa protein that was only expressed and recognized in the MM19 WCL. Two other antigenic spots of 58 and 71 kDa had differences in their level of expression between the MM19 and BHI WCLs. Comparisons of Western blots with total-protein-stained 2D-gels showed that each of the antigenic bands was resolved into multiple spots that included both antigenic and nonantigenic proteins. In native 2D-PAGE (N2D-PAGE), there was no difference in expression of proteins between isolates 93-146 or 92-266 grown in BHI, or between growth temperatures for isolate 92-266 grown in BHI. The MM19-grown 93-146 WCL had differences in protein separation compared with BHI-grown WCLs. Five antigenic spots were recognized by CCS in N2D-PAGE Western blots of BHI-grown WCLs. The same five antigenic spots were identified in the MM19-grown WCL along with seven additional spots. These results demonstrate that antigenic protein bands of E. ictaluri separated by SDS–PAGE may actually consist of one or more antigenic and nonantigenic proteins. In addition, culture medium affected the expression of antigenic proteins in the isolate tested. Recognition of the unique 57-kDa protein by CCS demonstrates that it is expressed during an E. ictaluri infection and, because this protein was not expressed in BHI, indicates that the MM19 medium may be more analogous to the in vivo environment.