Molecular basis for CesT recognition of type III secretion effectors in enteropathogenic Escherichia coli
Fig 7
Disruption of the Tir CesT-extension motifs reduces host translocation and pedestal formation.
(A) Sequence alignment of Tir with other T3SS effectors identifies the CesT-extension motif present within the CesT-binding regions of Tir and that of the highly translocated effectors NleH1, NleH2, and EspZ. Sequence alignment figures were generated using ESPript 3.0 [67], Φ represents the three hydrophobic residues found in the conserved β-motif, the purple star represents the residues mutated to glutamate, and residues highlighted in purple and cyan correlate to those in the Tir32-80 structure (Fig 2D). (B) The His6-Tir23-550 I38E, His6-Tir23-550 I500E, and His6-Tir23-550 I38E I500E variants were co-expressed with CesT-FLAG and the soluble cell lysates (input, white circle) and Ni2+ pull-down (elution, grey circle) fractions were analyzed by SDS-PAGE and immunoblotting for the ability of the Tir CesT-extension motif variants to co-purify CesT-FLAG. EPEC strains (C) grown in T3SS inducing conditions and (D) used to infect HeLa cells were analyzed by immunoblotting of secreted and whole cell lysate, and translocated fractions, respectively. The Δtir mutant was trans-complemented with pWSK29-PLEE5 and expressing Tir, Tir I38E, Tir I500E, and Tir I38E I500E. DnaK and GAPDH were used as loading controls. (E) Immunofluorescence microscopy of EPEC infected HeLa cells stained with phalloidin to detect F-actin (red), DAPI to detect nuclei (blue), and bacteria containing a GFP-plasmid (green). The Δtir mutant strain complemented with pWSK29-PLEE5 and expressing Tir, Tir I38E, Tir I500E, and Tir I38E I500E were analyzed. Representative images of infected HeLa cells with each strain are shown with quantitation in (F). White scale bar represents 20 μm and error bars represent standard deviation of the mean. Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparison test. ***P ≤ 0.0001, *P ≤ 0.05.