3.6 | mAb-EspB-B7 specificity towards EPEC EspB homologs
To assess the specificity of mAb-EspB-B7 toward EspB homologs in other bacterial pathogens, bacterial cultures grown under T3SS-inducing conditions were centrifuged, and supernatants and pellets were analyzed by SDS-PAGE and western blotting using mAb-EspB-B7. The following WT bacteria and T3SS-mutant strains were cultured: EPEC; enterohemorrhagicE. coli (EHEC), which causes a more severe disease than EPEC in humans; C. rodentium , an EPEC-related mouse pathogen; andSalmonella enterica serovar Typhimurium, which utilizes two T3SSs for virulence. The strongest signal was observed for the WT EPEC supernatant; a significant, but less strong, signal was also detected for C. rodentium , and an even less strong signal, for EHEC (Figure 6A). However, no signals were detected in the supernatants ofSalmonella or any of the T3SS mutant strains. Sequence alignment of EPEC and C. rodentium EspB proteins revealed high conservation between the proteins and full conservation of the mAb-EspB-B7 epitope sequence. Sequence alignment between EPEC and EHEC EspB proteins revealed 80% similarity at the mAb-EspB-B7 epitope region (Figure 6B).