Bacterial strains, bacteriophages, growth and induction conditions

The E. coli strains used in this work are listed in Table 1. Bacteria carrying plasmids with VHH were grown at 30 °C in Luria-Bertani (LB) liquid medium or on agar plates with the appropriate antibiotic for plasmid selection. LB plates and pre-inoculum media prior to induction contained 2% (w/v) glucose for repression of the lac promoter. The preinocula cultures were started from individual colonies (for single clones) or from a mixture of clones (in case of libraries), freshly grown and harvested from plates, diluted to an initial OD₆₀₀ of 0.5, and grown overnight (o/n) under static conditions. For induction, bacteria (corresponding to an OD₆₀₀ of 0.5) were harvested by centrifugation (4000 xg, 5 min), and grown in the same media with 0.05 mM isopropylthio-β-D-galactoside (IPTG), but without glucose for 3 h with agitation (160 rpm), unless indicated otherwise. For over-expression of soluble VHH in the periplasm, E. coli WK6 cells with the corresponding pCANTAB6-VHH plasmid (Ap^R) were induced with 0.3 mM IPTG for 3 h at 30 °C. Secretion of VHH into the culture media was performed using the hemolysin (Hly) secretion system of E. coli HB2151 cells carrying pVDL9.3 (HlyBD; Cm^R) and the corresponding pEHlyA4SD-VHH (Ap^R) plasmids, induced with 0.3 mM IPTG for 6 h [34,35]. Over-expression of soluble TirM_EHEC with N-terminal His-tag was induced in E. coli BL21 (DE3) cells carrying the pET28a-TirM_EHEC plasmid (Km^R) and grown at 37 °C in LB medium containing 1.0 mM IPTG for 2 h. Further details can be found in Materials and Methods S1.

Plasmids, DNA constructs and oligonucleotides

Plasmids used in this study are summarized in Table 1. DNA manipulation, ligation, transformation and plasmid preparation were performed following standard techniques. Oligonucleotides were synthesized by Sigma Genosys, except those used for VHH amplification, which were from Scandinavian Gene Synthesis (SGS). All DNA constructs were sequenced by Secugen SL. PCR reactions for cloning were performed with proof-reading Vent DNA polymerase (New England Biolabs) or Taq DNA polymerase (Roche). The plasmid pHEA (Cm^R), for in frame fusions to N-terminal PelB signal peptide and E-tagged C-EhaA (amino acids 989-1327 of EhaA from EHEC O157:H7 strain EDL933stx-), was reported previously [33]. The amino acid sequence of the E-tag is: GAPVPYPDLEPA. The plasmid pNeae2 (Cm^R) is a derivative of the pNeae vector [25], encoding Intimin residues 1-659 (from EHEC O157:H7 strain EDL933stx-) followed by the E-tag, the hexahistidine (His) epitope, and a C-terminal myc-tag (EQKLISEED). The details of plasmid constructions are described in Materials and Methods S1.

Immunization and generation of VHH sdAb libraries

Dromedary camel immunization (Camelus dromedarious) was performed with purified TirM_EHEC (~1 mgr) mixed with veterinary vaccine adjuvant (GERBU). Amplification of V_HH genes was done by RT-PCR of the mRNA isolated from ~2x10⁷ lymphocytes from a peripheral blood sample of the immunized animal using standard methods and primers [36]. The camel immunization protocol followed the animal experimentation guidelines published by the Canary Islands Regional Government (Spain) and was approved by the Ethics Commission of the Department of Animal Medicine and Surgery, University of "Las Palmas de Gran Canaria" (Spain). The amplified VHH fragments were digested with SfiI and NotI restriction enzymes and ligated into the same sites of purified pHEA and pNeae2 backbone vectors and finally transformed in E. coli EcM1 cells by electroporation. The size of each library was ~2-3x10⁶ clones, as determined by plating on LB-Cm agar plates with 2% w/v glucose incubated at 30 °C. Further details are provided in t Materials and Methods S1.

Protein extracts, SDS-PAGE and Western blots

Whole cell protein extracts were prepared by harvesting bacteria after induction (1 ml of OD₆₀₀ 1.5), resuspended in 50 µl of 10 mM Tris HCl pH 8.0, mixed with the same volume of SDS-sample buffer (2X) or urea-SDS sample buffer (2X) and boiled for 10 min (pHEA constructs) or 30 min (pNeae constructs) respectively. The boiled samples were sonicated (5 sec; Labsonic B Braun), centrifuged (14,000 xg, 5 min) to pellet insoluble material, loaded onto 8% or 10% SDS-PAGE gels and run using a Miniprotean III electrophoresis system (Bio-Rad). For Western blot, the gels were transferred to a polyvinylidene difluoride membrane (PVDF, Immobilon-P, Millipore) using a semi-dry electrophoresis transfer apparatus (Bio-Rad), the membranes were blocked and incubated with anti-E-tag mAb (Phadia) or anti-c-myc-POD mAb (clone 9E10; Roche), as indicated. Bound anti-E-tag mAb was developed using anti-mouse IgG conjugated with peroxidase (POD) (Sigma). Streptavidin-POD conjugate (Roche) was employed to detect the biotinylated broad range SDS-PAGE protein markers (Bio-Rad). All mAbs and POD conjugates were used in a 1:5000 dilution. Membranes were developed by chemiluminescence and either exposed to an X-ray film (Curix, Agfa) or scanned in a Chemi-Doc XRS (Bio-Rad) and analyzed using the Quantity One software (Bio-Rad). To quantify the total number of V_HH fusions expressed in E. coli, Western blots of whole cell protein extracts and dilutions of a purified E-tagged V_HH of known concentration [37] (hereafter referred to as “unknowns” and “standard”, respectively) were visualized on a ChemiDoc XRS and analyzed using the Quantity One software (Bio-Rad). Composition of buffers and further details are described in Materials and Methods S1.

Protease accessibility assays

Induced bacteria (1 ml, OD₆₀₀=1.5) were harvested by centrifugation (4000 xg, 3 min) and resuspended in 100 µl of 10 mM Tris HCl pH 8.0. This bacterial suspension was incubated with trypsin (10 µg/ml; Sigma) or with proteinase K (ProtK; 40 µg/ml; Roche) as indicated, for 20 min at 37°C. Next, the trypsin inhibitor (5 µg/ml; Sigma) or the serine proteases inhibitor (PMSF 1 mM; Sigma) was added to stop further proteolysis. The cell suspension was centrifuged (14,000 xg, 1 min), the cell pellet resuspended in 50 µl of 10 mM Tris HCl pH 8.0, lysed with one volume of SDS-sample buffer (2X) or urea-SDS-sample buffer (2X), boiled and analyzed by Western blot.

Purification of TirM_EHEC

TirM_EHEC with a N-terminal His-tag was purified from 500 ml cultures of E. coli BL21(DE3) cells carrying pET28a-TirM_EHEC grown and induced as described above. Bacteria were lysed and the soluble protein extract loaded onto a chromatography column filled with a Cobalt-containing resin (Talon, Clontech). TirM_EHEC was eluted with 150 mM imidazole, dialyzed against HEPES-buffer (20 mM HEPES pH 7.4, 200 mM NaCl, sterile filtered and degassed), and loaded onto a calibrated gel filtration column (HiLoad 16/600 Superdex 75 preparative grade, GE Healthcare). Fractions containing TirM_EHEC were collected and checked for purity by SDS-PAGE. Protein concentration was estimated using the Bicinchoninic acid (BCA) Pierce protein assay kit (Thermo Scientific). Further details are described in Materials and Methods S1.

Protein biotinylation

Biotinamidocaproate N-hydroxysuccinimide ester (Biotin-NHS; Sigma) was re-constituted at 25 mg/ml in dimethylsulfoxide (DMSO, Fluka) and immediately used. Purified protein (0.1-1 mg) [TirM_EHEC, GFP (Upstate, Merck Millipore), BSA (Sigma), anti-E mAb (Phadia)] was mixed with Biotin-NHS (20-fold molar excess) in 1 ml of PBS and incubated for 2 h at RT with slow agitation on a gyratory wheel. The reaction was stopped by addition of Tris-HCl pH 7.5 at final concentration of 50 mM and the samples were placed on ice for 1 h. The reaction mix was loaded onto a pre-packed column for gel filtration chromatography (Sephadex G25 PD-10; GE Healthcare) and the biotinylated protein was eluted in 500-µl fractions with PBS. Protein concentration was estimated using the Bicinchoninic acid (BCA) Pierce protein assay kit (Thermo Scientific).

Enzyme-linked immunosorbent assay (ELISA)

TirM_EHEC or BSA (Sigma) proteins were adsorbed at 4°C o/n onto 96-well immunoplates (Maxisorb; Nunc) at a concentration of 5 µg/ml in PBS. Next, immunoplates were washed in PBS and blocked by incubation with 200 µl of 3% (w/v) Milk -PBS for 2 h at RT. The sdAbs (secreted or purified) were diluted in 3% (w/v) Milk-PBS, added at the indicated concentrations (0.1-100 nM) in duplicates and incubated for 1 h at RT. After incubation, the wells were washed three times with PBS (Immunowash 1575, Bio-Rad) and the bound sdAbs was detected by the addition of anti-c-myc-POD mAb (clone 9E10; Roche; 1:1000), or anti-E-tag mAb (Phadia; 1:1000) followed by anti-mouse-POD (Sigma; 1:1000) for E-tagged sdAb, and incubation of the plates for 1 h at RT. The plates were washed three times with PBS and developed with H₂O₂ and o-phenylenediamine (OPD; Sigma) as previously described [38]. The plates were read at 490 nm using the iMark ELISA plate reader (Bio-Rad).

Magnetic Cell Sorting (MACS)

Induced E. coli cells (equivalent to a final OD₆₀₀ of 5.0) were harvested by centrifugation (4000 xg, 3 min), washed three times with 2 ml PBS (sterile filtered and degassed), and resuspended in a final volume of 1 ml of PBS. Biotinylated TirM_EHEC (at concentrations of 50 nM or 250 nM, as indicated) was added to 100 µl of bacteria, the final volume was adjusted to 200 µl with PBS-BSA (PBS supplemented with 0.5% w/v BSA, sterile filtered and degassed), and incubation was carried out for 1 h at RT. After incubation, bacteria were washed three times with 1 ml of PBS-BSA, resuspended in 100 µl of the same buffer containing 20 µl of anti-biotin paramagnetic beads (Miltenyi Biotec) and incubated at 4 °C for 20 min. Next, bacteria were washed three times with 1 ml of PBS-BSA, resuspended in 500 µl of the same buffer, of which 10 µl was kept aside to calculate the input bacteria before the procedure, while the rest (490 µl) was applied onto a MACS MS column (Miltenyi Biotec), previously equilibrated with 500 µl of PBS-BSA and placed on the OctoMACS Separator (Miltenyi Biotec). The flow through of unbound cells was collected and the column was washed three times with 500 µl of PBS-BSA. The wash was combined with the flow-through as “Unbound fraction”. Next, the column was removed from the OctoMACS Separator and placed onto a new collection tube, 2 ml of LB was added and the cells were eluted out. This fraction was labeled as the “Bound fraction”. Serial dilutions of Unbound and Bound fractions were plated to determine CFU and to harvest Bound bacteria.

Flow cytometry analysis

For standard flow cytometry, induced bacterial cells (equivalent to a final OD₆₀₀ of 1.0; ~10⁹ CFU) were harvested by centrifugation (4,000 xg, 3 min), washed twice with 500 µl of PBS (filter-sterilized) and resuspended in a final volume of 400 µl of PBS. Next, 190 µl of this cell suspension (~3x10⁸ CFU) was incubated with the primary antibody or antigen (as indicated) and PBS was added to adjust the total volume to 200 µl. The primary antibodies (for assay of expression levels) were anti-E-tag mAb (1:200; Phadia) or anti-c-myc mAb (1:200; 9B11 clone; Cell Signalling), while biotinylated antigens (GFP, TirM_EHEC, BSA) were used at 50 nM for assay of antigen binding, unless otherwise indicated. The samples were incubated at RT for 1h. After incubation, the cells were washed once with 500 µl of PBS, and resuspended either in 500 µl of PBS containing 1 µl of anti-mouse-IgG1 conjugated to Alexa 488 Fluor (2 mg/ml, Invitrogen) or in 200 µl of PBS containing 30 µl of 1:200 dilution of Streptavidin-phycoerythrin (PE) (0.5 mg/ml, Beckman Coulter). The mixture was incubated 30 min at 4 °C in the dark. The cells were washed once with 500 µl of PBS and resuspended in a final volume of 1 ml in PBS. For each experiment at least 100,000 cells were analyzed in a cytometer (Gallios, Beckman Coulter).

Affinity determination by flow cytometry analysis

Induced E. coli cells (equivalent to final OD₆₀₀ of 1) were centrifuged (4000 xg, 3 min), washed twice with 1 ml of PBS (filter-sterilized) and resuspended in a final volume of 1 ml of PBS. Next, 50 µl of this cell suspension (~3x10⁷ CFU) was incubated at room temperature for 90 min with a fixed amount of biotinylated TirM_EHEC (2 pmols; ~30 ng) and increasing volumes of PBS (from 0.1 to 1.5 ml) to attain a final concentration range between 20 nM to 1 nM. After incubation, cells were centrifuged (4000 xg, 3 min), washed twice with 1 ml of PBS (filter-sterilized) and labeled with Streptavidin-PE as described for standard flow cytometry. After a final washing step with PBS, the mean fluorescence intensity (MFI) of Phycoerythrin (PE) was quantified in a cytometer (Gallios, Beckman Coulter). Data of MFI (relative values to maximum MFI) obtained from the cytometer were plotted against the concentration of TirM_EHEC to obtain the dissociation constant (K_D). Curve was fitted according to non-linear least squares regression method and one site - specific binding saturation kinetics model using the data analysis tool in Prism software (GraphPad).

Secretion of sdAbs into E. coli culture media

Secretion of Ab fragmemts to E. coli culture media with the hemolysin secretion system has been reported previously [34,35]. Induced cultures of 10 ml E. coli HB2151 cells transformed with pVDL9.3 and pEHlyA4SD-V_HH derivative were centrifuged (10000 xg, 10 min, 4°C) and the culture supernatants were adjusted to PBS 1X by adding 1/10th volume of PBS 10X. The amount of secreted V_HH-HlyA fusion (~5 µg/ml) was estimated by densitometric analysis of silver-stained SDS-polyacrylamide gels loaded with 10 µl samples of culture supernatants and dilutions of BSA standards of known concentration (Thermo Scientific). When needed, the V_HH-HlyA fusions in culture supernatants were concentrated with 3-kDa or 10-kDa centrifugal filter units (Amicon Ultra-15).

Purification of sdAbs from the periplasm of E. coli

Soluble sdAbs with hexahistidine and myc tags in their C-termini were induced in E. coli WK6 cells carrying pCANTAB6-VTIR1 or pCANTAB6-Vgfp. Cells were pelleted by centrifugation (4000 xg, 12 min, 4°C) from 1 L cultures, resuspended in 22.5 ml Periplasmic Extraction buffer [50 mM Sodium phosphate pH 7.4, 200 mM NaCl, 5 mM EDTA and 1 mg/ml polymyxin B sulphate (Sigma)] and stirred at 4°C for 2 h using a magnetic stirrer. The periplasmic extract was obtained by ultracentrifugation (40000 xg, 30 min, 4°C) and dialyzed o/n at 4°C against 5 L of PN2 buffer (50 mM sodium phosphate pH 7.4, 200 mM NaCl). Dialyzed extract was loaded onto a Cobalt-containing affinity resin (Talon, Clontech), washed, and bound protein eluted in PN2 with 150 mM imidazole. Eluted sdAb was dialyzed, concentrated, and loaded onto a calibrated gel filtration column (HiLoad 16/600 Superdex 75 preparative grade, GE Healthcare) as described previously for TirM_EHEC. The fractions corresponding to the monomeric sdAb were collected and concentrated in a 3-kDa centrifugal filter unit (Amicon Ultra-15). Protein concentration was estimated using the Bicinchoninic acid (BCA) Pierce protein assay kit (Thermo Scientific).

Surface Plasmon Resonance affinity determination

SPR measurements were performed using a Biacore 3000 instrument (GE Healthcare). All proteins solutions were dialyzed against HEPES-buffer [20 mM HEPES 200 mM NaCl (pH 7.4) sterile filtered and degassed] at 4°C o/n. Biotinylated TirM_EHEC (0.1 µg/ml) was immobilized on a Streptavidin SA chip (GE Healthcare) at 150 response units (RU) at a flow rate of 10 µl/min in HEPES-buffer containing 0.005% (v/v) of the surfactant Polysorbate 20 (P20, GE Healthcare). For determination of binding kinetics, dilutions of purified sdAb (analyte) from 32 nM to 200 pM were flown at 30 µl/min in HEPES-buffer and sensograms were generated. The biotinylated TirM_EHEC surface on the Streptavidin SA chip was regenerated after every cycle using three injections of 10 µl of 10 mM Glycine-HCl (pH 1.7). Sensograms with different concentrations of anaylte were overlaid, aligned and analyzed with BIAevaluation 4.1 software (GE Healthcare). All data were processed using a double-referencing method [39].

Comparison of EhaA and Intimin β-domains for display of sdAbs on E. coli

To test the potential of Intimin β-domain for the display of Ab fragments and compare it with the display capacity of EhaA β-domain, we employed sdAbs derived from heavy-chain antibodies (HCAbs) of camelids (e.g. dromedaries, llamas) also known as VHH (for VH of HCAbs) or nanobodies [40]. These sdAbs have a small size (ca. 12-14 kDa), high stability and solubility, as well as excellent antigen binding properties with high affinity and specificity. In addition, their sequence identity with human VH makes them attractive for therapy [41]. Initial assays were conducted using a model VHH clone binding GFP (Vgfp) [42] fused to the β-domains of EhaA and Intimin for comparison of the two systems before cloning of an immune library of sdAbs. Vgfp was cloned in pHEA vector (Table 1) in frame with the N-terminal SP of PelB [43] and the C-terminal fragment of EhaA (residues 989-1327; named as C-EhaA), bearing its native β-barrel with α-helix linker, and including the E-tag epitope between the VHH and C-EhaA (Figure 1A) [33]. The Vgfp sequence was cloned in pNeae2 (Table 1) in frame with the N-terminal fragment of Intimin (residues 1-659; named as Neae), comprising its N-terminal SP, the periplasmic LysM domain (expected to bind the peptidoglycan), its native β-barrel with C-terminal linker, and the first Ig-like domain (D0) (Figure 1B). Vector pNeae2 incorporated the E-tag and myc-tag epitopes flanking the VHH (Figure 1B). Both E. coli display vectors contained unique SfiI and NotI restriction sites flanking the VHH in the same frame as those of conventional phagemids (e.g. pHEN6, pCANTAB6) [44]. The resulting fusion proteins were referred to as VHHA (fusions to C-EhaA) and NVHH (fusions to Neae).

The expression of VgfpA and NVgfp fusions in E. coli K-12 cells (strain UT5600; Table 1) was analyzed by Western blot after induction with 0.05 mM IPTG at 30 °C for 3 h (see Materials and Methods). Discrete protein bands corresponding to VgfpA and NVgfp were detected with anti-E or anti-myc monoclonal Abs (mAbs) in whole cell protein extracts from the induced cells (Figure 1C and Figure 1D). Both fusion proteins showed a shift in their electrophoretic mobility, characteristic of native OMPs with correctly folded β-barrels, which are resistant to SDS denaturation at low temperatures and hence migrate faster than the unfolded polypeptides due to the compact structure of the β-barrel [17,45]. This heat-modifiable mobility was observed in both VgfpA and NVgfp, having the expected mobility according to their molecular weight (MW) after boiling (ca. 52 kDa and 84 kDa, respectively) and a faster mobility in non-boiled samples (Figure 1C and 1D). Interestingly, NVgfp was resistant to 2% SDS and 4 M urea at low temperatures (i.e. 22 °C) and required boiling in this buffer to unfold, as previously reported for full-length Intimin and its β-domain [25]. The major protein bands detected with anti-E mAb in the boiled samples corresponded to full-length VgfpA and NVgfp fusions (Figure 1C and 1D; labeled with arrows). Detection of NVgfp with anti-myc mAb confirmed the integrity of its C-terminal end (Figure 1D). Minor bands of lower MW were also detected in Western blot with anti-E mAb, which likely represent proteolytic fragments of the full-length fusions (Figure 1C and 1D; labeled with asterisks). The induced E. coli cultures expressing VgfpA or NVgfp fusions showed only a slight decrease in their growth rate compared to control cultures having the empty vector (pAK-Not), or expressing the β-domains C-EhaA (pHEA) or Neae (pNeae2), and reached final optical densities at 600 nm (OD₆₀₀) identical to controls (Figure S1).

The accessibility of VgfpA and NVgfp fusions to the external milieu was initially compared by incubation of intact E. coli cells with externally added proteases. Trypsin digested full-length VgfpA leaving some weakly detectable proteolytic fragments with a size similar to C-EhaA and Vgfp domains (Figure 1C, lane 3). The NVgfp fusion was resistant to Trypsin digestion (Figure S2) but was sensitive to Proteinase K (ProtK) (Figure 1D, lane 3). Nevertheless, ProtK digestion of NVgfp fusion left a resistant fragment comprising Neae. Hence, both the β-domains display the sdAb to the extracellular milieu, in a way that is accessible to externally added proteases but C-EhaA fusions are more sensitive to digestion than Neae fusions. Resistance to proteolysis was previously observed for full-length Intimin [25].

Surface display of VgfpA and NVgfp was assessed by flow cytometry (Figure 2). Induced E. coli cells harboring pVgfpA, pNVgfp, or pAK-Not (control) were stained with anti-E or anti-myc mAbs followed by anti-mouse IgG-Alexa488 (Figure 2, left panels). E. coli cells expressing VgfpA or NVgfp were positively bound by anti-E mAb, though cells expressing NVgfp were also positively bound with the anti-myc mAb. Control E. coli cells with pAK-Not were negative for both mAbs. Importantly, the presence of a single peak in the flow cytometry histograms indicated that most E. coli cells were expressing a homogenous level of the fusion proteins. The mean fluorescence intensity (MFI) of cells with anti-E-tag mAb suggested a higher expression and display level of NVgfp than VgfpA (~3-fold).

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Figure 2. E. coli cell surface display and antigen binding activity of VgfpA and NVgfp fusions.

Fluorescent flow cytometry analysis of induced E. coli UT5600 cells bearing the indicated plasmids: pAK-Not (control), pVgfpA, and pNVgfp. Histograms show the fluorescence intensity of bacteria stained with anti-E or anti-myc mAbs (as indicated) and secondary anti-mouse IgG-Alexa 488 (left panels) or incubated with biotinylated antigens (GFP or BSA, as labeled) and secondary Streptavidin-phycoerythrin (PE) (right panels).

https://doi.org/10.1371/journal.pone.0075126.g002

The antigen-binding activity of the surface displayed Vgfp sdAb using both display systems was compared by flow cytometry after incubation of E. coli cells expressing the fusions with 50 nM biotin-labeled GFP (positive antigen) or biotin-labeled BSA (negative antigen), followed by incubation with Streptavidin-Phycoerythrin (PE) conjugate (Streptavidin-PE) (Figure 2, right panels). This analysis showed the specific binding of the E. coli cells expressing VgfpA or NVgfp fusions to GFP, whereas control E. coli cells did not bind GFP. None of these cells bound BSA. Therefore, the sdAb is functional when displayed on the surface of E. coli cells with the β-domains of EhaA and Intimin. Nevetheless, the MFI of GFP binding was higher in E. coli cells expressing NVgfp than in those with VgfpA (ca. 8-fold) (Figure 2, right panels). The 3-fold difference in expression level of NVgfp does not appear to be sufficient to account for this difference in binding, suggesting that the sdAb may have a higher antigen-binding activity when fused to the β-domain of Intimin (see Discussion).

Selection of antigen binding sdAbs from an immune library displayed on E. coli cells

To test the effectiveness of the E. coli display systems for the expression of a VHH library and selection of antigen-binding clones, an immune library against the soluble extracellular fragment of the translocated intimin receptor (tir) from EHEC (named TirM_EHEC, corresponding to residues 252-360 of full-length Tir_EHEC) [46], was generated and cloned in vectors pHEA and pNeae2. TirM_EHEC binds to the C-terminal Ig-like and lectin-like domains (D2-D3) of full-length Intimin_EHEC [30,47] but not to the β-domain of Intimin (Neae) used for in this study. The VHH library against TirM_EHEC was obtained by immunization of a dromedary with purified recombinant his-tagged TirM_EHEC and subsequent amplification of the VHH gene segments from ~2x10⁷ lymphocytes isolated from a peripheral blood sample (Materials and Methods). The amplified VHH gene segments were cloned into the SfiI and NotI sites of pHEA and pNeae2 vectors, generating two E. coli display immune libraries of similar size (~2-3x10⁶ clones). This relatively small library size is reported to be sufficient for having a good representation of the repertoire of V_HH genes in the peripheral blood of camelids after immunization and to select the sdAbs of higher affinity raised against the antigen using conventional phage display [48]. The E. coli strain EcM1 (Table 1) was used as host for cell display. This strain is derived from the reference wild type K-12 strain (MG1655) with a deletion in the operon encoding type 1 fimbriae (ΔfimA-H) [49]. Sequencing of 40 clones picked randomly from each library confirmed the cloning of different VHH sequences in frame with the β-domains of EhaA and Intimin (data not shown).

The expression and display of the VHH libraries with the β-domains of EhaA (VHHA) and Intimin (NVHH) were analyzed by flow cytometry with anti-E and anti-myc mAbs, revealing a fairly homogeneous expression of both libraries in E. coli EcM1 (Figure 3A). The MFI with anti-E mAb indicated a similar expression level of VHHA and NVHH libraries, in contrast to the significantly lower expression of VgfpA observed previously. Western blot analysis of whole-cell protein extracts from induced cultures revealed major protein bands with the expected size for full-length VHHA and NVHH fusions, upon boiling in SDS or SDS-urea buffer, respectively, and which have heat-modifiable electrophoretic mobility indicating the correct folding of their β-barrels (Figure 3B). Quantification of the Western blot signals with anti-E mAb using ~1.5x10⁸ bacteria (0.15 units of OD₆₀₀) expressing VHHA or NVHH fusions, was carried out using a standard curve generated with a purified E-tagged VHH of known concentration (Figure S3), and allowed an estimation of ~6.5x10³ molecules of VHHA and ~7.8x10³ molecules of NVHH per bacterium.

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Figure 3. E. coli cell surface display of VHHA and NVHH immune libraries.

(A) Fluorescent flow cytometry analysis of induced E. coli EcM1 cells expressing VHHA or NVHH immune libraries anti-TirM_EHEC (as indicated). Control cells carried the empty vector pAK-Not. Histograms show the fluorescence intensity of bacteria stained with anti-E or anti-myc mAbs (as labeled) and secondary anti-mouse IgG-Alexa 488. (B) Western blots of whole-cell protein extracts from induced E. coli EcM1 cells expressing VHHA or NVHH immune libraries anti-TirM_EHEC (as indicated). Protein extracts were prepared in SDS (VHHA library) or SDS-urea (NVHH library) sample buffers and boiled (+) or not boiled (-) before SDS-PAGE. Western blots were developed with anti-E mAb. The positions of full-length fusions are labeled with arrows. The mass of protein markers (in kDa) is shown on the left.

https://doi.org/10.1371/journal.pone.0075126.g003

The growth of E. coli cultures expressing VHHA or NVHH libraries was only slightly delayed compared to a control with pAK-Not and the cultures reached similar OD₆₀₀ after induction (Figure S4). Plating of the induced cultures to determine the number of colony forming units (CFU) per OD₆₀₀ gave ~1.0x10⁹ CFU/OD₆₀₀ in the control, ~ 0.9 x10⁹ CFU/OD₆₀₀ in the VHHA library, and ~0.6x10⁹ CFU/OD₆₀₀ in the NVHH library. Hence, although growth is not affected, expression of NVHH fusions appear to reduce the viability of E. coli cells compared to control cultures. Nevertheless, since the CFU/OD₆₀₀ after expression of NVHH fusions remains within the same order of magnitude as the control, the diversity of the sdAb library is not compromised. Cell toxicity for the expression of Intimin constructs has been reported previously in some E. coli K-12 strains [22].

E. coli EcM1 cells expressing VHHA and NVHH libraries were screened to isolate clones binding to TirM_EHEC by magnetic cell sorting (MACS). In MACS, cells are incubated with a biotinylated target protein (e.g. antigen, mAb, etc.), washed to remove the unbound protein, and incubated with a suspension of paramagnetic microbeads coupled to Streptavidin or a mAb binding biotin (anti-biotin microbeads). The cell mixture is then passed through a small ferromagnetic column held in a magnetic holder, which retains cells coated with anti-biotin microbeads whereas unbound cells are washed out of the column (Figure 4A). Elution of bound cells is carried out with buffer or fresh culture media (e.g. LB) upon removal of the column from the magnet. The CFU washed out of the column (Washed fraction) and eluted (Bound fraction) are determined by plating. The MACS conditions for capturing E. coli cells expressing VHHA or NVHH fusions were established using biotin-labeled anti-E mAb. An initial input of 0.1 units of OD₆₀₀ (~6-9x10⁷ CFU of each culture) was incubated with 50-250 nM of biotinylated anti-E mAb allowing the recovery of a total of ~2-4x10⁷ CFU in the Washed and Bound fractions. The Bound fractions contained ~95-99% of the CFU in the VHHA and NVHH libraries, and only ~0.2-0.5% in control E. coli cells with pAK-Not. Using these conditions, the VHHA and NVHH libraries were incubated with biotinylated TirM_EHEC (250 nM) for the first selection step (MACS1) and ~0.3-0.6% of the total CFU from both libraries were collected in the Bound fractions (Table 2). The colonies grown from the Bound fractions of each library were pooled independently and their plasmids purified and electroporated into fresh E. coli EcM1 cells to obtain VHHA and NVHH sublibraries (≥2x10⁶ transformants). This step was added to avoid multiple rounds of induction of the same bacterium, which may favor the amplification of clones with reduced levels of expression of the fusions.

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Figure 4. Magnetic cell sorting of E. coli display libraries VHHA and NVHH with biotinylated antigen TirM_EHEC.

(A) General scheme summarizing the steps followed during MACS of an E. coli display library of sdAb with a biotinylated antigen. E. coli cells binding the biotinylated antigen are captured in a MACS column held in a magnet, while E. coli cells that do not bind the antigen are washed out of the column. Elution of bound bacteria is done with fresh LB media upon column removal from the magnet. The CFU in the Washed and Bound fractions are determined by plating. (B) Fluorescent flow cytometry analysis of IPTG-induced E. coli EcM1 cells expressing VHHA (top panel) or NVHH (bottom panel) immune libraries, or their respective sublibraries enriched after the indicated round of MACS with biotinylated TirM_EHEC. Histograms show the fluorescence intensity of bacteria incubated with biotinylated TirM_EHEC and secondary Streptavidin-PE.

https://doi.org/10.1371/journal.pone.0075126.g004

Next, the VHHA and NVHH sublibraries were subjected to a new round of selection with biotinylated TirM_EHEC using conditions identical to those used in MACS1. Bacteria harvested from Bound fractions were pooled, their plasmids purified and transformed for the following rounds of MACS. Antigen concentration was reduced to 50 nM in the following MACS. The percentage of E. coli bacteria recovered in the Bound fractions showed a significant increase from the initial 0.3-0.6% to over 70% in MACS3 of NVHH and MACS4 of VHHA (Table 2), suggesting an enrichment of antigen binding clones in both libraries. Bacteria from the different rounds of selection were analyzed by flow cytometry to test their binding to biotinylated TirM_EHEC (50 nM) (Figure 4B), which demonstrated an enrichment of E. coli cells binding to TirM_EHEC along the selection rounds, from ~0.2% positives in the original libraries to ~45% after MACS4 of the VHHA library and more than 75% positives after MACS2 and MACS3 of the NVHH library. No significant binding to biotinylated BSA was detected by flow cytometry in these pools (data not shown). The expression levels of the VHHA and NVHH fusions in the bacterial pools obtained after MACS was similar to those of the original libraries (data not shown).

Fifty colonies from the final round of selection of each library were randomly picked for plasmid isolation and DNA sequencing. A VHH sequence, named as VTIR1, was found in all NVHH clones and in 36 VHHA clones, the rest being different VHH sequences. Flow cytometry analysis confirmed the specific binding of biotinylated TirM_EHEC (50 nM) by E. coli cells displaying VTIR1 fused to EhaA and Intimin β-domains whereas these cells did not bind to biotinylated BSA (Figure 5A and 5B). Similar to the situation observed with Vgfp clone, the MFI of E. coli cells displaying VTIR1 was higher in the NVTIR1 fusion than with VTIR1A fusion, although both were expressed at similar levels (Figure S5). We conducted a flow cytometry screening of the remaining 14 non-VTIR1 clones from MACS4 of VHHA library, identifying three other VHH sequences that bound to biotinylated TirM_EHEC, referred to as VTIR2, VTIR3, VTIR4 (Figure 5A, and data not shown). VTIR4 did not bind biotinylated BSA, but VTIR2 and VTIR3 clones exhibited non-specific binding to biotinylated BSA and were not further analyzed (Figure 5A and 5B). We sought for additional VHH sequences binding TirM_EHEC in the NVHH library by screening 96 colonies picked randomly after the first round of selection (MACS1), in which a higher diversity of binders with low and high affinities could be expected. PCR screening with a specific primer hybridizing the complementarity determining region 3 (CDR3) of VTIR1 enabled us to identify 17 VTIR1 clones out of these 96 colonies. This number is higher than the 1.3% positives found by flow cytometry after MACS1 of the NVHH library (Figure 4B) but fits better with the percentage of clones recovered from this population after MACS2 (Table 2). This could indicate a lower sensitivity of flow cytometry to actually discriminate positive binders from the population. Flow cytometry screening of the remaining clones allowed the identification of two additional VHH sequences that specifically bound biotinylated TirM_EHEC, VTIR4 (2 clones), which was found previously in the VHHA library, and VTIR5 (1 clone) (Figure 5B). The MFI of these clones with 50 nM biotinylated TirM_EHEC was low compared to VTIR1 (Figure 5B) and increased at higher antigen concentrations (200 nM) (data not shown) suggesting that these clones had a lower affinity for TirM_EHEC. The CDR3 amino acid sequences of the selected VHH are shown in Table 3. The sdAbs encoded by VTIR1, VTIR4, VTIR5 and one unrelated V_HH binding α-amylase as a control, were secreted into E. coli culture media as soluble fragments with the hemolysin system [34,35], and used in ELISA plates coated with TirM_EHEC and BSA (Materials and Methods). This experiment showed that soluble VTIR1, VTIR4, and VTIR5 sdAbs, lacking the β-domains of EhaA and Intimin, also bound specifically to TirM_EHEC (Figure 6). VTIR1 was the clone with an apparent higher affinity, as could be inferred from its enrichment in both the E. coli display libraries.

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Figure 5. Binding to TirM_EHEC by E. coli cells displaying selected clones from VHHA and NVHH libraries.

Fluorescent flow cytometry analysis of induced E. coli EcM1 cells bearing the indicated plasmids selected from (A) the VHHA library: pVTIR1A, pVTIR2A, pVTIR3A; and (B) from the NVHH library: pNVTIR1, pNVTIR4, pNVTIR5. Histograms show the fluorescence intensity of bacteria incubated with biotinylated antigens (TirM_EHEC or BSA, as labeled) and secondary Streptavidin-PE.

https://doi.org/10.1371/journal.pone.0075126.g005

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Figure 6. ELISA of sdAbs selected by E. coli display against TirM_EHEC.

ELISA against TirM_EHEC of sdAbs secreted into culture media as E-tagged HlyA fusions from the indicated VTIR clones and one a negative control (Vamy) [35]. The plot shows the average OD values at 490 nm with standard error from duplicate experimental samples obtained with the secreted sdAbs at the indicated concentrations. ELISA were developed with anti-E-tag mAb and anti-mouse-POD. ELISA signals against a control antigen (BSA) are subtracted from the represented values.

https://doi.org/10.1371/journal.pone.0075126.g006

Characterization of VTIR1 sdAb and determination of its affinity by surface plasmon resonance and E. coli display

The VTIR1 clone was produced in the periplasm of E. coli WK6 cells as soluble sdAb with C-terminal His- and myc-tags and purified by metal-affinity chromatography followed by gel-filtration chromatography (Materials and Methods). As a control, Vgfp was also expressed and purified in the same manner. Both sdAbs behave as monomers with an apparent mass of ~15 kDa in gel filtration chromatography (Figure S6A). The binding activity of the purified VTIR1 was confirmed in ELISA (Figure S6B). In order to determine the apparent equilibrium dissociation constant (K_D) between VTIR1 and TirM_EHEC their interaction was studied in surface plasmon resonance (SPR) experiments with a Streptavidin (SA) sensor chip coated with biotinylated TirM_EHEC (Materials and Methods). The change in resonance units (RU) was recorded with time at different concentrations of purified VTIR1 from 0.2 to 32 nM showing a clear binding to TirM_EHEC that reached the steady state equilibrium in ~220 s for the two highest concentrations used, but not for the lower concentrations (Figure 7A). No binding was observed when Vgfp (40 nM) was flown over this sensor surface, or when VTIR1 (40 nM) was flown over a SA flow cell lacking biotinylated TirM_EHEC (data not shown). Injection of buffer to evaluate the dissociation of VTIR1 (labeled with an arrow in Figure 7A) showed no loss of RU for > 200 s, indicating that VTIR1 remained stably bound to TirM_EHEC over long periods of time. The absence of dissociation of VTIR1 from TirM_EHEC prevented determination of its kinetic constants (k_on and k_off) from the sensograms. Since reaching the steady state at the lower concentrations of VTIR1 would require very long injection times (>1 h), which are not practical due to the higher amounts of Ab needed and the possibility of conformational changes of the antigen bound to the sensor chip, in order to have an estimation of the apparent K_D between VTIR1 and TirM_EHEC we plotted the RU values obtained with the different concentrations of VTIR1 at 220 s, the time at which two concentrations had reached the steady state. This plot provided an apparent K_D of ~2.2 x 10^-9 M (Figure 7B). Nevertheless, the actual K_D between VTIR1 and TirM_EHEC should be below this estimated value (indicating an even higher affinity of VTIR1 for TirM_EHEC) since the steady state equilibrium has not been reached at the lower concentrations.

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Figure 7. Determination of the equilibrium dissociation constant (K_D) of sdAb VTIR1 by SPR and E. coli display.

(A) SPR sensograms monitoring real-time association and dissociation of purified sdAb VTIR1 (at the indicated concentrations) to biotinylated TirM_EHEC immobilized onto a Streptavidin-SA sensor chip. The increase in resonance units (RU) is recorded along time (in seconds). Dissociation of VTIR1 is evaluated by injection of buffer at the time indicated with an arrow. (B) RU values at 220 seconds (labeled with a rectangle in A) are plotted versus the different concentrations of VTIR1. The curve was fitted by non-linear least squares regression. (C) The K_D of VTIR1 was estimated by flow cytometry analysis of E. coli cells expressing NVTIR1 incubated with different concentrations of biotinylated TirM_EHEC (1-20 nM) under equilibrium conditions. The mean fluorescent intensities (MFI) of bacteria, after labeling with Streptavidin-PE, were plotted versus the concentration of TirM_EHEC used in the assays. The curve was fitted by non-linear least squares regression.

https://doi.org/10.1371/journal.pone.0075126.g007

Flow cytometry analysis under equilibrium conditions has been used to estimate the apparent K_D of Abs and Anticalins displayed on the surface of yeast and E. coli cells [14,23,50]. Thus, we tested whether the affinity of VTIR1 could also be estimated by flow cytometry analysis of E. coli cells with this sdAb on their surface and incubated with biotinylated TirM_EHEC under conditions expected to be close to the equilibrium. We chose the Intimin display system given its superior MFI signals in flow cytometry with the antigen. E. coli EcM1 cells displaying NVTIR1 (~3x10⁷ CFU) were incubated for 90 min with a fixed amount of biotinylated TirM_EHEC (2 pmols) in two-fold increasing volumes of PBS (from 0.1 to 1.5 ml) to reach a final concentration range from 20 nM to 1 nM. After this incubation, cells were washed and labeled with Streptavidin-PE as previously described. The relative MFI of the cells was plotted against the antigen concentration used and the curve fitted by non-linear least squares regression, giving an estimated apparent K_D of 1.7 x10^-9 M (Figure 7C). This value is consistent with the estimated apparent K_D determined by SPR analysis, indicating that these E. coli display systems could also be used to estimate the K_D of selected sdAbs before purification.