Figure legends
Figure 1. mAb-EspB-B7 binds EspB with high affinity.(A) mAb-EspB-B7 binding affinity to purified EspB was evaluated by ELISA. A 96-well plate coated with EspB was incubated with serially diluted mAb-EspB-B7. mAb-EspB-B7 binding was determined using anti-human IgG HRP-conjugated antibody. Error bars represent ± SD. (B) SPR sensorgrams of mAb-EspB-B7 binding to an EspB-coated chip. mAb-EspB-B7 was added at various concentrations between 10 and 90 nM. Sensorgrams were fitted to the steady-state model.
Figure 2 . mAb-EspB-B7 binds to recombinant and native EspB. (A ) EPEC wild type (WT), ΔescN , ΔespB and ΔespB expressing EspB-His strains were grown under T3SS-inducing conditions for 6 hr. The bacterial pellets and supernatants were separated and analyzed using SDS-PAGE and western blotting with mAb-EspB-B7. EspB expression within the bacteria (pellet) was observed only for the ΔespB + EspB-His strain, while EspB secretion (supernatant) was observed for both WT EPEC and the complemented ΔespB + EspB-His strain. (B ) EPEC WT, ΔescN , ΔespB and ΔespB + EspB-His bacteria were grown under T3SS-inducing conditions for 3 hr. Thereafter, 1×107bacteria were incubated with mAb-EspB-B7, washed, and stained with Alexa Fluor 488 goat anti-human IgG antibody. Flow cytometry analysis was performed on a Gallios instrument (Beckman coulter).
Figure 3. mAb-EspB-B7 binding to EspB under various conditions.mAb-EspB-B7 binding to EspB was evaluated by ELISA (A ) in different media, (B ) under various pH conditions, and (C ) at different NaCl concentrations. Error bars represent ± SD.
Figure 4. mAb-EspB-B7 does not interfere with the EspB-EspD interaction. Supernatants of EPEC ΔespD expressingEspD- 35His were purified using Ni-NTA beads. EPEC ΔespD strain without the pEspD -35His expression vector was used as a negative control. Samples of supernatants (S) and elution (E) fractions were loaded on SDS-PAGE and analyzed by western blotting with mouse anti-His and anti-EspB antibodies (to avoid detection of the human EspB antibody). Analysis of the supernatants confirmed EspB and EspD secretion into the extracellular medium. The co-elution of EspB with EspD-35His was not affected by the absence or presence (100 nM and 200 nM) of mAb-EspB-B7. Low EspB non-specific binding to the Ni-NTA beads was detected (in the absence ofEspD- 35His).
Figure 5. mAb-EspB-B7 epitope mapping. (A) An EspB pepstar peptide array of 78 cyclic peptides (15-residue long peptides with an 11-residue overlap) was examined for mAb-EspB-B7 binding. Image analysis was carried out with Genepix Pro 6.0 analysis software (Molecular Devices) to detect antibody binding; fluorescence signals were normalized showing their relative intensities. The putative binding site of mAb-EspB-B7 along the EspB protein is marked in red. Arrows indicate the signals obtained from peptides #49 and #50, which displayed the highest signal intensities. (B, C, D) mAb-EspB-B7 binding to EspB following pre-incubation with peptide #49 and peptide #49 scrambled (B), peptide #50 and peptide #50 scrambled (C), or peptide #49+50 (D) was evaluated by competitive ELISA and detected using anti-human IgG HRP-conjugated antibody. Peptide #78 was used as a negative control. Error bars represent ± SD.
Figure 6. mAb-EspB-B7 binds EspB homologs in other T3SS-expressing bacteria . (A ) Wild type and mutant EPEC, EHEC,C. rodentium and Salmonella were grown under T3SS-inducing conditions. EPEC, EHEC and C. rodentium mutant strains contain a deletion in the escN gene, while Salmonella contains a deletion in the invA gene, which results in non-functional T3SSs in these mutants. The bacterial cultures were centrifuged, and the supernatants were collected, normalized, and analyzed by SDS-PAGE and western blotting using mAb-EspB-B7. (B ) Amino acid sequence alignment of EspB from EPEC with C. rodentium, EHEC, orSalmonella EspB homologs. Identical, similar and non-identical amino acids are marked in blue, cyan, and red, respectively. The mAb-EspB-B7 epitope is annotated above the amino acids that are part of the epitope.
Figure 7. mAb-EspB-B7 does not inhibit EPEC translocation activity into HeLa cells. (A)  Scheme of the effector translocation assay. Infection of HeLa cells with EPEC was monitored by detecting the degradation profile of JNK, a human kinase that is subjected to cleavage by the EPEC effector, NleD. (B) HeLa cells were infected with wild-type (WT) EPEC in the presence or absence of 400 nM mAb-EspB-B7. After 3 hr, cells were washed, and host cell proteins were extracted and subjected to western blot analysis using anti-JNK and anti-actin (loading control) antibodies. JNK and its degradation fragments are indicated at the right of the gel. Degradation of JNK was evident in the WT EPEC, sample but not in the uninfected sample or in the samples infected with EPEC ΔescN . HeLa cells infected with WT EPEC in the presence of 400 nM mAb-EspB-B7 showed a JNK degradation profile similar to that of WT EPEC in the absence of mAb-EspB-B7.