Like many gram-negative bacterial pathogens, the plant pathogen Envinia amylovora requires a type III secretion system (T3SS) to establish infection in the host. This work investigates the factors required for type III secretion of the E. amylovora pathogenicity factor DspE and for its interaction with the specific chaperone DspF. DspE N-terminal fragments fused to the CyaA translocation reporter were used to identify a minimal secretion and translocation signal within the first 51 amino acids, and an optimal translocation signal within 150 N-terminal amino acids. Mutagenesis of portions of the translocation signal revealed that residues 2-10 were required for protein translocation, and that mutagenesis of a segment containing a highly conserved stretch of 5 serines led to diminished translocation levels. Yeast two-hybrid assays, in-vitro pull-down assays, and site-directed mutagenesis were used to map the chaperone binding domain of DspE to residues 51-100, with a crucial role for residues 71-85, although the DspE-specific chaperone DspF and the N-terminal chaperone binding domain were not required for translocation of an N-terminal DspE-CyaA fusion.

Type III secretion chaperones have a highly conserved tertiary structure, but few analyses have been completed to determine which residues are critical for which function. One site postulated, but never tested, to be important in chaperone-effector interaction is the helix-binding groove. A homology-based model of DspF was used to identify 11 amino acids with putative localization in the helix-binding groove. Site-directed mutagenesis of DspF identified four highly-conserved residues required for virulence in DspF, and three of these residues were required for interaction with the N-terminus of DspE. This study supports the significance of polar residues of the helix-binding groove in chaperone-effector interactions.

T3SS chaperones may interact with multiple substrates in the bacterial cell, including regulatory proteins, multiple effectors, and components of the secretion apparatus. A directed yeast two-hybrid screen identified E. amylovora secreted proteins Eop1 and Eop3 and homologs to the type III secretion apparatus proteins HrcU and HrcQ as candidate interactors of DspF. Annotation of the unpublished Erwinia genome confirmed or revealed the presence of putative chaperone genes next to eop1 and eop3, but not other effectors such as eop4. Deletion of dspF positively affected cAMP accumulation mediated by translocation of Eop1- and Eop3-CyaA, but not Eop4-CyaA. Deletion of the putative Eop1 chaperone, esc1, negatively affected translocation of Eop1-CyaA but not of DspE-CyaA, indicating that Esc1 chaperone action is highly specific. Deletion of Eop1, Eop3, and the corresponding putative chaperone genes had no significant effect on virulence, although Ea1189Δ ^dspFΔesc1 showed slightly increased levels of early growth on pear compared with Ea1189ΔdspF.