Table 1.
Strains, plasmids, gene and primers used in this study.
Fig 1.
Ability of recombinant Stx1B to bind to Gb3 receptor in vitro.
(A) Binding of serially diluted Stx1B to Gb3 as determined by ELISA using THE™ His tag antibody. A450: Absorbance at 450 nm. Error bars denote standard deviations. (B) Internalization of FITC-labelled Stx1B into HeLa cells. Golgi apparatus was detected with mouse anti-human Golgin-97 primary antibody and Alexa Fluor 555-conjugated donkey anti-mouse secondary antibody (red). DAPI staining was used to label nuclei (blue). Bars = 10 μm.
Fig 2.
Sequence similarity analysis (A) and binding affinity (B) of 17 S1B binders selected after five rounds of ribosome display.
A: The sequence of the parental ABD wild-type domain was used as a root of the tree and is highlighted as a square, while S1B variants selected for more detailed analysis are highlighted as circles. B: S1B binders-containing cell lysates were incubated with immobilized Stx1B (grey bars) or BSA (white bars) and detected with HRP-conjugated streptavidin. Error bars denote standard deviations.
Fig 3.
(A) SDS PAGE analysis of selected binders S1B9, S1B22 and S1B26 after purification from E. coli cell lysates, stained with Coomassie brilliant blue. (B) ELISA-determined binding of serially diluted biotinylated binders selected against immobilized recombinant Stx1B. The binding was detected by HRP-conjugated streptavidin. (C) Sequence similarity comparison of selected binders with parental non-mutated ABDwt. Randomized sequences between residues 20 and 46 were compared. Randomized positions are indicated in grey.
Fig 4.
SPR analysis of binding of S1B22, S1B26 and ABDwt in fusion with TolA-Avi and H6 at 1 μM concentration to immobilized recombinant Stx1B.
Fig 5.
Determination of binding affinity of selected S1B variants to recombinant Stx1B by SPR analysis (A, B) and MST (C).
(A) Recombinant Stx1B at seven different concentrations was injected over the chip surface with immobilized H6-S1B22-TolA-Avi or H6-S1B26-TolA-Avi. (B) Steady state response (obtained in (A)) was plotted against Stx1B concentration and Steady State Affinity model was applied to calculate the affinity constant. (C) Sixteen serial dilution concentrations of H6-S1B22-TolA-Avi or H6-S1B26-TolA-Avi were mixed with fluorescently labelled Stx1B at 5 nM final concentration. Fluorescence change Kd fit in NanoTemper software was used to calculate Kd. Error bars denote standard deviations.
Fig 6.
Influence of S1B binders on Stx1B transport into HeLa cells.
A, B: Flow cytometric analysis of HeLa cells demonstrating shift in fluorescence intensity (A) and mean fluorescence intensity (MFI; B) upon 1h incubation of HeLa cells with mixtures of S1B22, S1B26 or ABDwt (all in fusion with TolA-Avi and H6) and Alexa Fluor 488-labeled Stx1B. Cont: unstained HeLa cells. Stx1B: HeLa cells incubated with Stx1B-Alexa Fluor 488 alone. C, D: Fluorescence microscopy images of HeLa cells incubated with Alexa Fluor 488 labelled Stx1B (green) with or without pre-incubation with S1B22 and S1B26. DAPI staining (blue) was used to label nuclei. Cells were stained either with PKH26 membrane labeling dye (red; C) and Golgi apparatus was detected with mouse monoclonal Golgin-97 antibody and secondary polyclonal goat anti-mouse antibody conjugated with Alexa Fluor 633 (purple; D). Bars = 10 μm.
Fig 7.
(A) SDS PAGE analysis of lysates of L. lactis cells expressing S1B22, S1B26, ABDwt and H6-ABDwt (ABDwtH), all in fusion with Usp45 secretion signal and the LysM-containing cA domain, and stained with Coomassie brilliant blue. ABD fusion proteins are high-lighted with arrows. (B) Flow cytometric analysis of ABD surface display, detection with FITC-conjugated human serum albumin. The MFI value of ABDwt was compared with that of the control using Student’s t test. *** p<0.001. Cont.: control containing empty plasmid pNZ8148.
Fig 8.
Flow cytometric (A) and whole-cell ELISA (B) analyses of binding of recombinant Stx1B by L. lactis cells displaying S1B variants or ABDwt on their surface.
(A) Alexa488-conjugated Stx1B was used for detection. MFI: Mean fluorescence intensity. (B) Mouse antiStx1B antibody and HRP-conjugated anti mouse antibody were used for detection of Stx1B. A450: Absorbance at 450 nm. Vertical bars denote standard deviation. MFI or A450 values of S1B binders were compared to those of the ABDwt control using Student’s t test. *: p<0.05, ** p<0.01, *** p<0.001.