Efficient translocon and effector secretion is dependent on EscU auto-cleavage. (A): Immunoblot demonstrating EscU variant cleavage status within whole cell lysates. The blots were imaged separately to get representative signals for the auto-cleavage products. A longer exposure was used for the 39 kDa protein species. (B) Left: trans-complementation of ΔescU with pJLT21 restores EspA (~20 kDa), EspB and EspD (co-migrate ~30 kDa) secretion to wild type levels (the identity of these dominant protein species has been previously determined using protein sequencing , and here by immunoblotting [right panel]). EspC is an abundant type 5 secreted protein. Bovine serum albumin (BSA) was added to collected secreted protein fractions as a carrier protein to assist in the precipitation of proteins. A molecular weight standard is in the left most lane. Right: immunoblot analyses of secreted protein and whole cell lysate fractions from bacterial strains used in panel A (as indicated). The respective secreted protein fractions were diluted 20 fold prior to SDS-PAGE. (C) Left: secreted protein fractions derived from ΔescNΔescU double mutant strains with the indicated plasmids. Right: Immunoblot analysis of secreted protein fractions. DnaK, an abundant non-secreted cytoplasmic protein, was used as a gel loading control (when needed) or to assess cytoplasmic contamination of secreted fractions or non-specific bacterial lysis. All samples were diluted 20 fold as in panel B. All experiments within the panels were performed twice and representative images are shown.
Thomassin et al. BMC Microbiology 2011 11:205 doi:10.1186/1471-2180-11-205