Fig 5.

EscA interacts with the secretin protein, EscC. (A) Representative mass spectra for peptides recovered from a SILAC experiment with EscA-2HA. In the right panel, open and filled triangles indicate the expected mass/charge ratio (m/z) of light and heavy forms, respectively, of a peptide (VASLESITSGTLLR) from the specifically bound EscC from pulldowns with EscA-2HA and 2HA, respectively. The light peptide of EscC is present at a ratio of >2.0 higher than the heavy peptide, indicating a specific interaction with EscA. In the left panel, open and filled triangles indicate the expected m/z of light and heavy forms, respectively, of a peptide (AGTYATTLPAGR) from the nonspecifically bound FepA protein from pulldowns with EscA-2HA and 2HA, respectively. (B) EPEC ΔescA ΔescC strain cultures expressing EscC-2HA, EscA-HSV, or both proteins were grown under T3S-inducing conditions. Whole-cell extracts were subjected to immunoprecipitation using an antibody directed against HSV. Whole-cell extracts were analyzed by Western blotting probing for EscA (using anti-HSV antibody) and EscC (using anti-HA antibody). (C) Complementation of the EPEC ΔescA ΔescC strain by the EscC-2HA and EscA-HSV constructs. Protein secretion profiles of WT EPEC, a ΔescA ΔescC strain, and a ΔescA ΔescC strain complemented with EscC-2HA and EscA-HSV in trans. Secreted proteins were concentrated from supernatants of bacterial cultures grown in DMEM and analyzed by Coomassie staining of an SDS–12% PAGE gel. The locations of the translocators EspA, EspB, and EspD are indicated at the right of the gel. Also indicated is the location of EPEC EspC, which is not secreted via the LEE-encoded T3SS.