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1 Onderzoeksgroep Genetische Virologie, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
2 Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
3 Structural Biology Brussels, Department of Molecular and Cellular Interactions, VIB, B-1050 Brussels, Belgium
Correspondence
Jean-Pierre Hernalsteens
jphernal{at}vub.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Present address: Afdeling Gentechnologie, KU Leuven, Kasteelpark Arenberg 30, B-3001 Heverlee, Belgium.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Autotransporters typically consist of an N-terminal signal peptide, a secreted passenger domain, a linker region and a C-terminal translocator domain (Henderson et al., 2004). The signal peptide directs translocation across the inner membrane (Sijbrandi et al., 2003), the translocator domain thereupon forms a β-barrel structure in the OM and the passenger domain is then extruded to the cell surface, where it may undergo further proteolytic processing (Desvaux et al., 2004; Henderson et al., 2004).
The adhesin involved in diffuse adherence (AIDA-I) is a member of the autotransporter family. It is responsible for the diffuse adherence phenotype of the clinical Escherichia coli O126 : H27 isolate 2787 (Benz & Schmidt, 1989). AIDA-I and other autotransporter proteins have been used for the surface expression of a variety of recombinant proteins, by replacing the authentic passenger by heterologous proteins or peptides, such as T-cell epitopes, the cholera toxin B subunit, or β-lactamase (reviewed by Jose, 2006). Although some studies have shown that this replacement does not abolish secretion across the OM (Konieczny et al., 2000; Lattemann et al., 2000; Maurer et al., 1997), in other cases, replacement of the passenger domain with a similarly sized higher-contact-order structure or with a structure containing disulfide bonds resulted in less efficient secretion compared to the wild-type passenger (Rutherford et al., 2006).
In this study, we examined the use of AIDA-I for the display of the receptor-binding domain of the F17a-G fimbrial adhesin on the bacterial surface. Fimbriae are important virulence factors of pathogenic bacteria, allowing these to colonize specific host tissues (Klemm & Schembri, 2000). F17a fimbriae, for example, are found on some bovine enterotoxigenic E. coli strains (Mainil et al., 2000) and mediate attachment of these bacteria to the intestinal epithelium, by the F17a-G adhesin (Lintermans et al., 1991). Because fimbriae are essential in the first steps of infection, anti-adhesin antibodies can play an important role in the protection against infection, by blocking bacterial adhesion (Langermann et al., 1997).
In a previous study, we fused the receptor-binding domain of the F17a-G adhesin to the translocator subunit of AIDA-I (Van Gerven et al., 2008b). For immunization studies, the fusion protein was expressed under the control of the in vivo inducible pagC promoter, in a Salmonella enterica serovar Typhimurium (S. Typhimurium) vaccine strain. Although expression of the F17a-G–AIDA-I fusion protein was observed in vitro after induction, no antibody response against the F17a-G receptor-binding domain was detected after immunization of mice with the live vaccine strain (our unpublished results). On the other hand, after parenteral immunization of mice with acetone-inactivated cells of the same S. Typhimurium vaccine strain, expressing the same hybrid F17a-G–AIDA-I fusion protein under the control of the strong L-arabinose-inducible PBAD promoter, we observed a fast and efficient IgG antibody response (Van Gerven et al., 2008b). Since surface exposure of antigens on live vaccine strains can be critical for eliciting an antibody response (Garmory et al., 2002; Liljeqvist & Stahl, 1999; Spreng et al., 2006), we investigated in this study the cellular localization of the F17a-G receptor-binding domain in bacterial cells expressing the F17a-G–AIDA-I fusion protein and compared the different methods that were used for this aim in similar studies.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Bacteria were grown at 37 °C on Luria–Bertani (LB) agar plates or in liquid LB medium, supplemented with 100 mg carbenicillin l–1 or 50 mg kanamycin l–1 when required.
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In addition, a fusion of only the signal peptide of F17a-G with the translocator domain of AIDA-I was made and put under the control of the PBAD promoter using the same strategy. For this aim, the DNA fragment encoding the signal peptide of the F17a-G gene (position 4200–4283 of accession number AF022140) was amplified by PCR from plasmid pHD52 (Van Gerven et al., 2008a), using primers F17-95 and F17-100, and inserted into plasmid pDONR221 by BP recombination. This gave rise to plasmid pGV5287. An LR recombination between the inserts of the donor vectors pGV5159, pGV5287 and pGV5266 or pGV5267 resulted in pGV5289 and pGV5290, respectively.
Expression of the F17a-G receptor-binding domain fused to AIDA-I.
Recombinant gene expression was induced in E. coli K514 and S. Typhimurium DV6181 at OD600 0.6, by adding L-arabinose to a final concentration of 0.2 % (w/v) and incubating for 1 h. Expression in E. coli BW27784 was induced using L-arabinose concentrations ranging from 0.2 % (w/v) to 0.00002 % (w/v). The presence of the fusion protein in bacterial whole-cell lysates was determined by SDS-PAGE and subsequent Western blotting (Sambrook & Russell, 2001), using a rabbit antiserum that was raised against the F17a-G receptor-binding domain and exhaustively pre-adsorbed against acetone powder (Harlow & Lane, 1988) of E. coli K514 or S. Typhimurium DV6181.
Preparation of OMs.
OM-enriched fractions of bacteria were prepared using the Sarkosyl method (Rizos et al., 2003). The resulting fractions were analysed by SDS-PAGE.
Protease digestion.
Proteinase K digestion of surface-exposed domains was carried out essentially as described by Maier & Myers (2004). Briefly, induced cells were harvested by centrifugation and resuspended in PBS. Cell pellets were washed twice in PBS with 50 µM MgCl2 and incubated for 15 min with 27.5 U proteinase K ml–1 or PBS as a negative control. PMSF was then added (5 mM final concentration) to inhibit the protease. The cells were pelleted and washed four times in PBS containing 5 mM PMSF.
For trypsin digestion, 1 ml of induced cells was washed in PBS and resuspended in 100 µl PBS containing 0.01 M CaCl2 and 0.125 mg trypsin ml–1. After 15 min incubation at 37 °C, 750 µl PBS was added. The cells were subsequently collected by centrifugation and washed three times with PBS.
Whole-cell ELISA.
Display of the F17a-G receptor-binding domain on the bacterial surface was examined in a whole-cell ELISA according to Konieczny et al. (2000), except for the blocking solution, where 10 % (w/v) skimmed milk powder (Bio-Rad) in PBS was used. Different dilutions (1 : 200 to 1 : 3200) of the primary rabbit anti-F17a-G antibody, in PBS supplemented with 5 % milk, were tested. Binding was detected using horseradish-peroxidase-conjugated goat anti-rabbit antibodies (1 : 5000) (Sigma). S. Typhimurium transformed with plasmid pPLHD673 was used as negative control.
Immunofluorescence microscopy.
For immunofluorescence microscopy, bacteria were grown and induced as described above, except that together with L-arabinose, IPTG was added to a final concentration of 1 mM to induce the β-galactosidase expression in E. coli. Cells were subsequently fixed and coated onto poly-L-lysine-treated microscope slides (Pallesen et al., 1995). If required, cells were permeabilized using lysozyme (Hiraga et al., 1998). Non-specific binding was blocked by incubation with 2 % (w/v) BSA for 15 min. The slides were subsequently incubated for 1 h with a 1 : 200 dilution of rabbit antibodies raised against the F17a-G receptor-binding domain, together with a 1 : 500 dilution of an antibody against β-galactosidase raised in mice (Sigma). The slides were subsequently washed and incubated for 1 h with a 1 : 50 dilution of FITC-labelled goat anti-rabbit antibody (Sigma) together with a 1 : 50 dilution of a tetramethyl rhodamine isothiocyanate (TRITC)-labelled goat anti-mouse antibody (Sigma). After extensive washing, the samples were covered with mounting medium [1 mg p-phenylenediamine ml–1, 90 % (v/v) glycerol in PBS; pH 9] and a coverslip (Hiraga et al., 1998). Slides were examined by fluorescence and phase-contrast microscopy, using a Carl Zeiss Axiophot fluorescence microscope. Photography was performed using a Nikon Coolpix 990 digital camera. Brightness and contrast were adjusted in Microsoft Office PowerPoint 2003.
Atomic force microscopy (AFM).
Cells were fixed and coated onto poly-L-lysine-treated glass slides as described above. AFM imaging of the cells was conducted in tapping mode in air using the Nanoscope IIIa multimode AFM (Veeco) and RTESP7 phosphorus-doped Si probes (Veeco). Tip-resonance frequencies were readjusted when the tip was lowered to within 30 µm of the sample surface. To minimize the force applied to the cell surface during scanning, the set-point voltage was continually adjusted to the lowest level for which tip-surface contact was maintained.
Permeability measurements.
Overnight bacterial cultures in LB medium supplemented with 1 mM IPTG were diluted 20-fold in the same medium. After 1 h, L-arabinose (final concentration 0.2 %) was added. The β-galactosidase activity was measured at different times, using the colorimetric substrate ONPG. The measurements were performed essentially as described by Miller (1992), but PBS was used instead of Z-buffer and no treatment was performed to permeabilize the cells. The reactions were stopped after 5 min. Before the A420, all samples were centrifuged for 1 min at 15 000 g.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The same induction conditions were followed in the present study.
To determine the cellular localization of the F17a-G receptor-binding domain, we first prepared OM-enriched fractions of non-induced and L-arabinose-induced E. coli K514 and S. Typhimurium DV6181 transformed with plasmid pGV5141. In both strains, the fusion protein was mainly recovered in the Sarkosyl-insoluble fraction, containing the bacterial OM and protein aggregates. Western blot analysis showed that only a small part of the fusion proteins was present in the Sarkosyl-soluble fraction, containing cytoplasmic and periplasmic proteins, and components of the cytoplasmic membrane (Fig. 1). This suggests that the AIDA-I translocator domain is most likely inserted in the OM. As reported by other investigators (Rizos et al., 2003), in our study also some degradation of the fusion proteins was detected, most likely due to partial proteolysis (Fig. 1).
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). S. Typhimurium DV6181 transformed with pPLHD673, which expressed the F17a-G receptor-binding domain in the periplasm, showed no increase in A450 value upon induction (Fig. 2). This confirms that these bacteria are impermeable to the antibodies.
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; Rodriguez-Ortega et al., 2006). After protease digestion, cells were extensively washed and whole-cell lysates were analysed by SDS-PAGE and subsequent Western blotting with an anti-F17a-G antibody. A reduced intensity of the band corresponding to the fusion proteins was observed after Coomassie blue staining for both protease treatments (Fig. 3a and data not shown). The protein pattern of the cells subjected to protease treatment was not significantly altered, suggesting that, most likely, mainly surface-exposed proteins were digested. Western blotting showed that, although the amount of protein was reduced, a fraction of the F17aG–AIDA-I hybrid protein remained intact (Fig. 3b). Possibly, not all expressed fusion proteins are exposed on the surface of the bacteria and consequently the band seen in Western blotting after proteinase K treatment can be due to internal proteins.
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The previously described methods used to determine if the fusion proteins are surface exposed via autotransporters are not completely conclusive. If expression of the fusion protein kills cells and thereby renders these permeable, proteases and antibodies can conceivably enter induced cells. Induction of the PBAD promoter will typically mediate expression in only a fraction of the L-arabinose-treated cells (Siegele & Hu, 1997). By entering into induced and consequently permeabilized cells, proteases can diminish the quantity of intact fusion protein in the protease experiments. If antibodies are able to enter into leaky cells, these can also give rise or contribute to a positive response in the whole-cell ELISA experiments.
Membrane permeability of E. coli cells
Permeabilization of E. coli cells by the expression of the F17a-G–AIDA-I fusion protein was further confirmed using ONPG, a histochemical β-galactosidase substrate that does not enter readily into intact non-permeabilized cells. The results are shown in Fig. 4. Induction of the expression of the F17a-G–AIDA-I fusion protein encoded by the plasmid pGV5141, by the addition of L-arabinose, results in rapid permeabilization of the bacteria. Expression of the translocator domain of AIDA-I without the F17a-G fusion, encoded by pGV5289, led to a much slower but still significant permeabilization of the cells, while no permeabilization was observed by the expression of the F17a-G receptor-binding domain in the periplasm of K514(pPLHD673).
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and Fig. 5(h) shows that only a fraction of the fusion proteins is presented on the surface of the bacteria, since after permeabilization the intensity of the fluorescence increases. Although some cells are permeable (positive for β-galactosidase), surface expression can be observed on a substantial number of intact bacteria.
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showed that AIDA-I and other autotransporters are localized to the bacterial pole. This restriction of autotransporters to the pole is, however, dependent on the presence of a complete LPS, consistent with known effects of LPS composition on membrane fluidity (Jain et al., 2006). In the study reported here, immunofluorescence was carried out in E. coli K514, which, like other strains derived from E. coli K-12, lacks complete LPS, so, as expected, no polar localization is seen.
AFM enables the high-resolution imaging of the bacterial cell surface in three dimensions, without staining or shadowing, by mechanically scanning a sharp tip mounted on a flexible cantilever over the sample surface (Ivanova et al., 2002). AFM imaging confirmed the leakiness of bacterial cells, since the height of induced K514(pGV5141) cells was much lower than the height of non-induced cells (Fig. 6a, b).
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; Maurer et al., 1999). Although Maurer and coworkers stated that the length of linker between the translocator domain and the foreign passenger that was used in their study was sufficient for autodisplay (Maurer et al., 1999), other investigators used longer linkers (Maurer et al., 1997; Rizos et al., 2003; Rutherford et al., 2006). To examine the effect of the length of the linker, we constructed two new fusion proteins, one having a linker corresponding to 163 residues of the AIDA-I linker (Maurer et al., 1997; Rizos et al., 2003), and one having a linker of 157 residues (Rutherford et al., 2006), both followed by nine cloning-derived amino acid residues of the attachment site of the Gateway vector. Expression levels (data not shown) and counts of viable bacteria in induced and non-induced cultures were, however, similar to those of the fusion protein containing the previously used linker of 75 amino acid residues: non-induced K514(pGV5141), K514(pGV5264) and K514(pGV5265) cultures reached cell densities of 7x108, 4x108 and 3x108 c.f.u. ml–1, respectively, and induced cultures 1x105, 4x105 and 3x105 c.f.u. ml–1. Expression of the AIDA-I translocator domain, without the F17a-G receptor-binding domain, did not, on the other hand, alter the viability of cells, since counts of viable cells of induced and non-induced K514(pGV5289) or K514(pGV5290) cultures did not differ. This indicates that presumably the expression of the AIDA-I translocator domain itself is not toxic for the bacteria, but rather the fusion to the F17a-G receptor-binding domain, which could possibly create translocation problems.
Effect of expression level on viability of cells
To find out whether the fusion protein is lethal for bacteria after production in excessive quantities, the expression level of the PBAD promoter must be strictly controlled. However, the expression level of the PBAD promoter is not dependent on the concentration of L-arabinose, but shows an all-or-none response. Rather than varying the level of gene expression in individual cells of the culture, the concentration of L-arabinose in the medium determines the fraction of cells that are fully induced (Siegele & Hu, 1997). The all-or-none expression from PBAD is due to the arabinose-dependent expression of the arabinose transporter araE, and expression of heterologous genes from PBAD in individual cells can be regulated by L-arabinose after placing the araE gene under the control of an arabinose-independent promoter (Khlebnikov et al., 2000).
To test whether expression of the fusion proteins at intermediate levels can improve cell viability, we transformed the plasmids pGV5264 and pGV5265 into E. coli BW27784, a strain that expresses araE under the control of a synthetic constitutive promoter (Khlebnikov et al., 2001). The resulting strains BW27784(pGV5264) and BW27784(pGV5265) were induced with 0.2 % to 0.00002 % (w/v) L-arabinose for 1 h. Reduced expression of the fusion protein was seen in Western blotting when using decreasing L-arabinose concentrations (data not shown). Subsequently, viable cells were counted by plating. Induction with 0.0002 % of L-arabinose gave rise for both strains to viable cell counts of about 1x107 c.f.u. ml–1, versus about 2x108 c.f.u. ml–1 for non-induced cultures. Addition of higher concentrations of L-arabinose decreased cell viability even more. Induction with 0.00005 % (w/v) L-arabinose in contrast gave no difference in viability between induced and non-induced cells.
These results are consistent with the ones presented by Covone et al. (1998), who found that a S. Typhimurium strain, expressing a gene encoding an E. coli heat-labile enterotoxin mutant using a high-copy-number plasmid, underwent lysis in stationary phase, a phenomenon that was not observed with a medium-copy-number plasmid. In addition, despite the promise of surface display to optimize heterologous antigen production and immunogenicity, the work of Georgiou et al. (1996) strongly suggested that high-level surface display of any heterologous antigen perturbs the integrity of the OM, resulting in leakage of periplasmic contents.
Conclusion
In the literature, the authentic passenger domain of AIDA-I and other autotransporter proteins was frequently replaced with heterologous proteins or peptides to express a variety of recombinant proteins at the bacterial surface (reviewed by Jose, 2006). Display of the FimH receptor-binding domain as an autotransporter hybrid in a live-attenuated Salmonella strain was also reported. In that study, the Ag43 autotransporter was used and surface expression was demonstrated using immunofluorescence (Kjaergaard et al., 2002).
In our study, induction of the F17a-G–AIDA-I fusion protein permeabilized the bacterial cells. Therefore, both protease treatment and whole-cell ELISA are not sufficient to validate surface exposure of F17a-G. Immunofluorescence microscopy with one antibody directed against the F17a-G receptor-binding domain and one directed against the cytoplasmic protein β-galactosidase showed that in a substantial fraction of the intact cells the receptor-binding domain of F17a-G was indeed surface-exposed.
Colonization capabilities and in vivo antigen expression levels of vaccine strains affect their efficacy to induce immune responses (Bumann, 2001). The lack of antibody response in our previous study, where live-attenuated S. Typhimurium vaccine strains expressing the F17a-G–AIDA-I fusion protein under the control of the in vivo inducible PpagC promoter were used as immunogens, can thereby, possibly, be explained by the additional attenuation due to the expression of high amounts of the fusion protein.
Given that the inherent toxicity of heterologous antigens is undoubtedly complex, with specific toxicity mechanisms dependent upon a given protein and its expression level, it is conceivable that the autotransporter fusion proteins studied by other investigators did not permeabilize bacterial cells (Kjaergaard et al., 2002; Lattemann et al., 2000; Rizos et al., 2003). However, experiments proving surface exposure should be meticulously and prudently interpreted. If the expressed protein interferes with the integrity of the cells, artefacts can easily arise. Double immunofluorescence microscopy, using a second antibody directed against an internal bacterial protein, as applied in this study, allows the simultaneous visualization of antigen expression and permeability in individual cells.
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ACKNOWLEDGEMENTS |
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Edited by: T. Palmer
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 17 July 2008;
revised 30 October 2008;
accepted 4 November 2008.
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), E. coli K514 harbouring pGV5141 (
), E. coli K514 harbouring pGV5289 (
) and E. coli K514 harbouring pPLHD673 (x) incubated with ONPG was determined by A420 measurements.
