Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis

Abstract

Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagic Escherichia coli the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenic E. coli mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.

FIG. 1

Detection of EspD-EspD protein interaction using a yeast two-hybrid system. β-galactosidase assays showed a 10-fold increase in enzymatic activity in strains expressing EspD from the two yeast vectors compared with the single transformants. Error bar represents standard error from the mean of three independent experiments.

FIG. 2

Structural organization of EspD from EPEC. The predicted carboxy-terminal coiled-coil segment (residues 334 to 370) is located downstream of a putative second coiled-coil region (145 to 177) and the two central transmembrane domain regions (180 to 204 and 234 to 256) (33, 38). The a and d position residues within the heptads are indicated, and residues targeted for mutagenesis are highlighted in bold.

FIG. 3

Detection of EspD, EspA, and EspB in EPEC supernatants. DMEM culture supernatants were analyzed by using Western blotting. Wild-type EPEC E2348/69 (lane 1), UMD870(pLCL123) (lane 3), UMD870(pICC72) (lane 4), UMD870(pICC73) (lane 5), and UMD870(pICC74) (lane 6) all demonstrated similar levels of the secreted proteins. EspD was absent from the supernatant of the espD deletion mutant strain UMD870 (lane 2), while UMD870 had reduced supernatant levels of EspA and normal levels of EspB.

FIG. 4

EspA expression and A/E lesion formation on HEp-2 cells produced by EspD coiled-coil mutants. EspA fluorescence (column 1), FAS test actin fluorescence (column 2) and corresponding phase-contrast micrographs (column 3) of wild-type EPEC strain E2348/69 (a), espD deletion mutant strain UMD870 (b), cloned EspD strain UMD870(pLCL123) (c), double EspD coiled-coil mutant strain UMD870(pICC73) (d), and triple EspD coiled-coil mutant strain UMD870(pICC74) (e). The coiled-coil mutants expressed EspA filaments (d, e), but whereas the double mutant produced a positive FAS reaction (actin accumulation at sites of bacterial attachment) (d), the triple mutant produced a barely detectable FAS reaction (e). Note that the espD deletion mutant produced barely detectable EspA filaments (b) and that EspA filaments expressed from strains harboring plasmid pLCL123 (c to e) are of the same length. Bars, 5 μm (column 1) and 20 μm (columns 2 and 3).

FIG. 5

Adhesion of A/E bacteria to HEp-2 cells. Whereas all the strains adhered to HEp-2 cells, there were differences in their ability to produce A/E lesions. A/E adherence of the double coiled-coil mutant [UMD870(pICC73)] was comparable to that of wild-type E2348/69, whereas A/E adherence of the triple mutant [UMD870(pICC74)] was significantly attenuated. The EspD deletion mutant did not produce A/E lesions. Error bars represent standard deviations of three independent experiments.

FIG. 6

Hemolytic activity of the EspD coiled-coil mutants. Hemolytic activity of the double EspD coiled-coil mutant UMD870(pICC73) was comparable to that of wild-type E2348/69 and cloned EspD strain UMD870(pLCL123), but the triple mutant UMD870(pICC74) was significantly attenuated in hemolytic activity. The EspD deletion mutant was nonhemolytic. Error bars represent standard deviations of three independent experiments.

FIG. 7

EspD coiled-coil mutant interaction with RBCs. Following infection, EspA filaments and the RBC membrane were visualized by fluorescence staining with EspA antiserum (green) and wheat germ agglutinin (red), respectively. Wild-type E2348/69 (b), cloned EspD strain UMD870(pLCL123) (d), and the double and triple coiled-coil mutant strains UMD870(pICC73) (e) and UMD870(pICC74) (f) produced EspA filaments which promoted binding of bacteria to the red cell membrane, although the level of binding of the triple mutant was significantly reduced (f). The EspD deletion mutant did not produce EspA filaments and was nonadherent (c). An uninfected RBC monolayer is shown in panel a. Bar, 5 μm.

FIG. 8

Localization of EspD, as shown by Immunofluorescence (a), a corresponding phase-contrast micrograph (b), and a combined fluorescence and phase micrograph (c) showing EspD localization in HEp-2 cells (a and c) and red blood cells (d) infected with wild-type E2348/69. The EspD antibody stained large protein aggregates (a and c, arrows) that were closely associated with adherent bacteria (b and c). A fibrillar arrangement of secreted EspD was indicated both by immunofluorescence (a and c) and immunogold EspD staining (d). Bars, 5 μm (a to c) and 0.2 μm (d).

FIG. 9

(A) Immunogold labeling of negatively stained EPEC culture supernatants, revealing the presence of EspD aggregates (arrows). (B) Occasionally, the EspD aggregates were seen in the vicinity of EspA filaments, which were stained in independent experiments with anti-EspA monoclonal antibodies (arrowhead). Bar, 50 nm.

FIG. 10

Detection of coelution of MBP-EspD-C and secreted EspD by immunoblotting with anti-EspD antiserum. Lane 1, MBP plus supernatant (first eluted fraction); lane 2, MBP plus supernatant (second eluted fraction); lane 3, MBP-EspD-C plus supernatant (fraction 1); lane 4, MBP-EspD-C plus supernatant (fraction 2); lane 5, MBP-EspD-N plus supernatant (fraction 2). The monoclonal EspD antibodies, which were reactive with an amino-terminal EspD epitope, detected coelution of EspD with MBP-EspD-C and the MBP-EspD-N fusion protein.