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Journal of Bacteriology, December 2006, p. 8504-8512, Vol. 188, No. 24
0021-9193/06/$08.00+0     doi:10.1128/JB.00864-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Proteolytic Processing Is Not Essential for Multiple Functions of the Escherichia coli Autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I)

Marie-Ève Charbonneau, Frédéric Berthiaume, and Michael Mourez^*

Canada Research Chair on Bacterial Animal Diseases, Université de Montréal, Faculté de Médecine Vétérinaire, 3200 Sicotte, St-Hyacinthe, J2S 7C6, Québec, Canada

Received 15 June 2006/ Accepted 5 October 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Escherichia coli adhesin involved in diffuse adherence (AIDA-I), like many other autotransporter proteins, is released in the periplasm as a proprotein undergoing proteolytic processing after its translocation across the outer membrane. The proprotein is cleaved into a membrane-embedded fragment, AIDAc, and an extracellular fragment, the mature AIDA-I adhesin. The latter remains noncovalently associated with the outer membrane and can be released by heat treatment. The mechanism of cleavage of the proprotein and its role in the functionality of AIDA-I are not understood. Here, we show that cleavage is independent of the amount of AIDA-I in the outer membrane, suggesting an intramolecular autoproteolytic mechanism or a cleavage mediated by an unknown protease. We show that the two fragments, mature AIDA-I and AIDAc, can be cosolubilized and copurified in a folded and active conformation. We observed that the release by heat treatment results from the unfolding of AIDA-I and that the interaction of AIDA-I with AIDAc seems to be disturbed only by denaturation. We constructed an uncleavable point mutant of AIDA-I, where a serine of the cleavage site was changed into a leucine, and showed that adhesion, autoaggregation, and biofilm formation mediated by the mutant are indistinguishable from the wild-type levels. Lastly, we show that both proteins can mediate the invasion of cultured epithelial cells. Taken together, our experiments suggest that the proteolytic processing of AIDA-I plays a minor role in the functionality of this protein.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adherence is a crucial step in the development of infectious diseases. One group of diarrheagenic strains of Escherichia coli is characterized by their pattern of diffuse adherence (DA) on the surface of epithelial cells and therefore called diffusely adhering E. coli (DAEC). One afimbrial adhesin was shown to mediate DA and called adhesin involved in diffuse adherence (AIDA-I) (2). AIDA-I was originally cloned as a plasmid-encoded protein from a DAEC strain isolated in a case of infantile diarrhea. This adhesin has since been shown to play a role in the pathogenesis of E. coli strains causing postweaning diarrhea and edema disease in piglets, two infections that cause major economic losses in farms worldwide (32, 38, 39). AIDA-I has been shown to be one of the adhesins mediating DA, along with the fimbrial adhesin F1845 and others (5). In addition to its adhesive properties, AIDA-I has also been implicated in biofilm formation and autoaggregation of bacterial cells (50). The aidA gene that codes for the autotransporter adhesin is associated with a second gene, named aah for autotransporter adhesin heptosyltransferase, which codes for a cytoplasmic glycosyltransferase (3).

AIDA-I is secreted by the monomeric autotransporter pathway of the type V secretion system (21). To date, most autotransporters identified are implicated in virulence (20), and many, like AIDA-I, are involved in adhesion (17). AIDA-I, a protein of 140 kDa, is produced as a preproprotein, processed at both N-terminal and C-terminal ends (Fig. 1). Its translocation occurs in three steps. First, the preprotein is secreted across the inner membrane, presumably via the sec secretion system, as observed with other autotransporters (6, 46). In this process, an N-terminal signal sequence of 49 amino acids is cleaved, releasing the proprotein in the periplasm. The second step is the insertion of a C-terminal domain of the proprotein in the outer membrane. Lastly, the rest of the proprotein is translocated through the outer membrane, presumably via the membrane-inserted C-terminal domain (1, 53). After the translocation at the cell surface, the proprotein is cleaved into a membrane-embedded domain of 45 kDa, AIDAc, and a 100-kDa domain exposed to the extracellular milieu, the mature AIDA-I adhesin (Fig. 1). This cleavage is not mediated by the known periplasmic or outer membrane proteases (DegP, OmpT, or OmpP) and occurs in E. coli as well as in Salmonella or Shigella strains, suggesting that it is an autocatalytic event (53). However, no protease catalytic site has been identified in the protein.

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FIG. 1. Structural organization of AIDA-I. AIDA-I is synthesized as a preproprotein cleaved at its N and C termini. The three domains resulting from these events are shown. The N-terminal signal sequence (SS) of the preproprotein is cleaved after translocation across the inner membrane, which releases the proprotein in the periplasm. After translocation across the outer membrane the proprotein is proteolytically processed, which separates the polypeptide into an N-terminal domain (mature AIDA-I) exposed to the extracellular milieu and a membrane-embedded C-terminal domain (AIDAc).

After its outer membrane cleavage, the mature AIDA-I remains noncovalently associated with the cell surface and can be released after a brief heating at 60°C. To date, no role has been attributed to this cleavage. Other autotransporters have been shown to be cleaved. The Shigella flexneri autotransporter protein VirG mediating intracellular spreading through actin polymerization is cleaved by a specific outer membrane protease, SopA/IcsP (10). The cleavage has been shown to be dispensable for its function but could be involved in the polar localization of this protein on the bacterial cell surface (8, 10, 16). The Haemophilus influenzae autotransporter adhesin Hap is also cleaved after translocation (52). Inhibition of the autoproteolytic cleavage of Hap increased bacterial autoaggregation and adhesion, suggesting that the cleavage acts as a regulation mechanism (15, 23, 24). These proteins, however, are significantly different from AIDA-I, in sequence and function; thus, the mechanism and function of the cleavage of AIDA-I remain obscure.

TibA, another adhesin autotransporter cloned from an enterotoxigenic strain of E. coli, is similar to AIDA-I both in sequence and in function: both proteins have almost identical repetitive amino acid sequences in their extracellular domain (12), and both can mediate biofilm formation, bacterial autoaggregation, and cell adhesion (51). Both are modified by the addition of heptose residues, and their corresponding glycosyltransferases can be functionally exchanged (30, 35). Two major differences exist between these two proteins, however: TibA is not cleaved after secretion, and it mediates the invasion of epithelial cells. It is therefore tempting to speculate that the outer membrane cleavage of AIDA-I could be one reason for the functional difference between AIDA-I and TibA. Interestingly, AIDA-I and TibA have recently been grouped with the E. coli autotransporter Ag43 (43) in a new class of virulence factors tentatively named self-associating autotransporters (28). Like TibA and AIDA-I, Ag43 is glycosylated and mediates adhesion to cultured epithelial cells (49), has a similar 19-amino-acid repeat, and promotes autoaggregation (44). Furthermore, like AIDA-I, Ag43 is cleaved by an unknown mechanism after its outer membrane translocation and is not known to mediate invasion (7).

In this study, we characterized the outer membrane cleavage of AIDA-I. We observed that this cleavage occurs at a rate independent of the level of expression of AIDA-I and that it is dramatically reduced by a single point mutation at the cleavage site. When AIDA-I is cleaved, we observed that the mature AIDA-I is strongly associated with AIDAc, as the two polypeptides can be cosolubilized by detergents and copurified in a native conformation. Lastly, using the uncleaved mutant of AIDA-I, we showed that cleavage at the cell surface is not essential for the stability of AIDA-I or its function, including the ability to invade an epithelial cell line, an activity which was not previously described.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and plasmids. The Escherichia coli K-12 strains C600 (F^– thr-1 leuB6 thi-1 lacY1 supE44 rfbD1 fhuA21) and UT5600 [F^– ara-14 leuB6 secA6 lacY1 proC14 tsx-67 (ompT-fepC)266 entA403 trpE38 rfbD1 rpsL109 xyl-5 mtl-1 thi-1], obtained from New England Biolabs, were used in this study. We amplified by PCR the aah gene from E. coli strain 2787 (2), kindly provided by John M. Fairbrother, Université de Montréal, using primers Aah-1F and Aah-1R (Table 1) and cloned the resulting fragment in the empty vector pTRC99a (Pharmacia Biotech) to produce plasmid pTRC-aah. We then amplified by PCR the whole operon including the aah and the aidA genes from E. coli strain 2787 using the Expand Long Template PCR system (Roche) with primers A-1F and A-1R (Table 1). The amplified fragment was then digested with XbaI and AgeI and cloned at the same sites in the pTRC-aah plasmid, generating the construct pAg, containing the whole aah-aidA operon. The S846L, S846G, S846C, A847R, A847I, and A847T mutations were introduced in pAg by oligonucleotide-directed mutagenesis performed with the QuikChange II site-directed mutagenesis kit (Stratagene) using the primers listed in Table 1 and their corresponding complementary oligonucleotides. A six-histidine and glycine tag (HisG) was introduced in the N-terminal part of AIDA-I in construct pAg and pAgS846L, the construct allowing expression of AIDA-I bearing the S846L mutation, by site-directed mutagenesis using the primer listed in Table 1 and its complementary oligonucleotide, generating plasmids pAgH and pAgHS846L, respectively. All constructions were verified by restriction mapping and sequencing.

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TABLE 1. Primers used in this study

Bacterial and cell culture growth conditions. Bacteria containing the different plasmids were grown on an LB agar plate or in liquid LB medium containing 100 µg · ml^–1 of ampicillin. Bacterial cultures were grown at 30°C. Growth was followed by measuring turbidity as optical density at 600 nm (OD₆₀₀). At an OD₆₀₀ of 0.8 the cultures were induced overnight with 10 µM of isopropyl-ß-D-thiogalactopyranoside (IPTG), unless indicated otherwise. This low concentration of IPTG was used to limit the toxicity associated with the overexpression of AIDA-I.

HEp-2 cells (ATCC CCL-23) were grown at 37°C with 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 10 mM sodium pyruvate (Sigma), bovine growth serum (HyClone), 2.5 µg · ml^–1 of amphotericin B (Fungizone), and 100 µg · ml^–1 of penicillin-streptomycin (Gibco).

SDS-PAGE and immunoblotting. Protein samples were diluted in twice-concentrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer containing ß-mercaptoethanol and denatured by being heated at 100°C for 10 min. The samples were then separated by SDS-PAGE on 10% acrylamide gels. The gels were either stained with Coomassie blue or transferred to polyvinylidene fluoride membranes (Millipore). Immunodetection was performed with a serum raised against the heat-extracted mature AIDA-I (a generous gift of M. Ngeleka, University of Saskatchewan), diluted 1:10,000 in blocking buffer (5% skim milk, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Triton X-100). A goat anti-rabbit horseradish peroxidase-conjugated antibody (Sigma) was used as a secondary antibody, according to the instructions of the manufacturer. Alternatively, an anti-HisG horseradish peroxidase-coupled antibody (Invitrogen) was used diluted 1:5,000 in the blocking buffer for the detection of proteins containing the HisG tag. Immune complexes were revealed using a 3,3',5,5'-tetramethylbenzidine solution (Sigma).

Preparation of membrane fractions. Subcellular fractionation was performed essentially as previously described (48). Briefly, bacterial cultures, normalized at the same OD₆₀₀, were harvested and resuspended in 950 µl of 10 mM Tris-HCl, pH 8, 0.75 mM sucrose. Lysozyme (0.4 mg · ml^–1 final concentration) and EDTA, pH 8 (10 mM final concentration), were added, and the sample was kept on ice for 30 min. The resulting spheroplasts were collected and lysed in Tris-buffered saline (TBS; 50 mM Tris-HCl, pH 8, 150 mM NaCl) containing protease inhibitor (Complete Mini; Roche) using an ultrasonic processor. The lysates were centrifuged for 1 h in an ultracentrifuge at 250,000 x g. The pellets, containing bacterial membranes, were resuspended in TBS and submitted to SDS-PAGE and immunoblotting, as described above.

Analysis of AIDA-I outer membrane processing. E. coli C600 harboring plasmid pTRC99a or pAg was grown to an OD₆₀₀ of 0.4 and induced for 3 h with 10 µM or 50 µM of IPTG. Normalized cultures were centrifuged for 10 min at 12,000 x g in microcentrifuge tubes, and the pellets were resuspended in 100 µl of phosphate-buffered saline (PBS). Alternatively, the bacterial cultures were grown to an OD₆₀₀ of 0.4 and induced with 10 µM of IPTG. Aliquots (200 µl) were taken every 10 min after induction for the following 2 h. The samples were centrifuged for 10 min at 12,000 x g in microcentrifuge tubes, and the pellets were resuspended in 20 µl of PBS. All samples were processed by SDS-PAGE and immunoblotting, as described above.

Extraction of the cleaved mature AIDA-I. Overnight cultures from E. coli C600 or UT5600 harboring empty vector pTRC99a, pAg, or pAgS846L were normalized at the same OD₆₀₀ in 25 ml of LB broth. Bacteria were harvested and resuspended in 375 µl of 10 mM sodium phosphate buffer, pH 7. In order to release the cleaved mature AIDA-I, the samples were heated at various temperatures between 30°C and 60°C for 20 min or treated for 1 hour with SDS (0.1%), Triton X-100 (1%), Tween 20 (0.01%), 10 mM glycine-HCl, pH 2, 100 mM sodium carbonate, pH 11, or urea (0.1 M and 2 M, final concentrations). The treated samples were centrifuged for 5 min at 12,000 x g in microcentrifuge tubes. A 10-µl aliquot from the supernatant was submitted to SDS-PAGE and stained with Coomassie blue, as described above.

Purification of AIDA-I. Heat extracts or membrane fractions were prepared as described above, except that 1 liter of overnight cultures of E. coli C600 harboring pAgH or pAgHS846L was used and all volumes were modified accordingly. The membranes were resuspended in TBS containing 1% Triton X-100, incubated for 1 hour, and centrifuged for 1 h in an ultracentrifuge at 250,000 x g. The histidine-tagged AIDA-I contained in the solubilized membranes or the heat extracts was purified using an ÁKTA purifier system with a 1-ml His Trap HP column (Amersham Biosciences), according to the instructions of the manufacturer. The protein concentration was calculated from the absorbance at 280 nm using a computed extinction coefficient estimated to be 116,090 M^–1 cm^–1 for the whole AIDA-I (mature AIDA-I and associated AIDAc) and 32,780 M^–1 cm^–1 for the mature AIDA-I alone, based on the number of tryptophan and tyrosine residues (45). The purity of the purified proteins was confirmed by SDS-PAGE and staining with Coomassie blue.

Far-UV CD spectroscopy. Far-UV circular dichroism (CD) spectra from 10 µg · ml^–1 of the wild-type and S846L protein purified from Triton X-100 extracts were recorded in 10 mM sodium phosphate, pH 7, in a 1-cm-path-length cuvette between 190 and 260 nm, using a spectropolarimeter (Jasco Spectroscopic Co. Ltd.; model J-810). Multiple spectra were obtained at 10°C intervals between 30°C and 70°C using the temperature control unit. For each spectrum, three accumulations were averaged and the contribution of buffer to the measured ellipticity was subtracted. The CD spectra of 125 µg · ml^–1 wild-type protein purified from heat extracts were obtained similarly in a 0.1-cm-path-length cuvette. For the wild-type and S846L protein purified from Triton X-100 extracts, thermal denaturation was followed by monitoring the ellipticity at 218 nm of a 35-µg · ml^–1 solution of protein and varying the temperature between 30°C and 70°C at a speed of 5°C per min. Ellipticities were converted to mean residual ellipticities.

Autoaggregation assay and biofilm formation. The autoaggregation assay was performed as described before (50). Overnight cultures from E. coli C600 harboring empty vector pTRC99a, pAg, or pAgS846L were normalized at an OD₆₀₀ of 1.5 in 10 ml of LB in a 16- x 125-mm borosilicate tube. The cultures were vortexed for 10 seconds and left at 4°C for 3 h. A 200-µl sample was taken 1 cm below the surface at the beginning of the assay and after 180 min. For inhibition of autoaggregation, a 250-µl sample of the Triton X-100-solubilized product or the heat-extracted product was added prior to the 3-hour incubation. Biofilm formation was monitored as described before (41), and normalized cultures of E. coli C600 harboring empty vector pTRC99a, pAg, or pAgS846L were grown without agitation for 24 h at 30°C in M9 medium containing 0.005% proline, leucine, and threonine and 100 µg · ml^–1 ampicillin in 96-well polyvinyl chloride plates (Falcon). Biofilms were stained for 15 min with crystal violet (1%), and the fixed dye was solubilized by addition of ethanol-acetone (80:20). The absorbance of the dye solution was measured at 595 nm.

Adhesion assay. The adhesion assay was performed as previously described (2). Briefly, HEp-2 cells were grown to confluence in 24-well plates in DMEM without antibiotic. The cells were inoculated with 10⁶ CFU per well of E. coli C600 harboring empty vector pTRC99a, pAg, or pAgS849L for 3 h. Cells were washed with PBS, and attached bacteria were recovered with 100 µl of Triton X-100 (1%) in order to be plated on LB agar for numbering. As a control, wells containing only 1 ml of DMEM were inoculated with the same amount of bacteria. Adherence to HEp-2 cells was calculated by dividing the number of adherent bacteria by the number of bacteria found in the inoculum after 3 h of incubation.

Invasion assay. HEp-2 cells were infected with E. coli C600 harboring empty vector pTRC99a, pAg, or pAgS846L for 3 h, as described above. The cells were then washed with PBS and incubated for 2 h with DMEM containing 100 µg · ml^–1 of gentamicin and 100 µg · ml^–1 of penicillin-streptomycin in order to kill extracellular bacteria. Cells were then lysed with 100 µl of Triton X-100 (1%) in order to recover the intracellular bacteria, which were plated on LB agar for numbering. As a control, bacteria were inoculated in 1 ml of medium alone and antibiotics were added. No bacteria were recovered from these wells. In other controls, cytochalasin D (0.1-µg · ml^–1 final concentration) was added in the medium during the infection and the gentamicin treatment. The gentamicin treatment was reduced to 1 hour to limit the toxic effect of cytochalasin D. The presence of cytochalasin D had no effect on bacterial adhesion to HEp-2 cells (data not shown).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Outer membrane cleavage of AIDA-I is independent of its expression level. The processing of AIDA-I after outer membrane translocation is thought to be autoproteolytic (53), but it is not known if the cleavage is intermolecular or intramolecular. To address this question, we evaluated the extent of autoproteolysis under different conditions of induction, in order to assess if AIDA-I cleavage varied with protein density. Similar experiments were used to characterize the intermolecular cleavage of Hap (14, 15). We used plasmid pAg, which contains the aidA and aah genes in their original operon organization but under the control of an IPTG-inducible promoter. We induced the expression of AIDA-I at the beginning of the exponential phase of growth, and samples were collected every 10 min for the following 2 h to prepare whole-cell extracts. As shown in Fig. 2A, in the earlier time points, only the mature cleaved AIDA-I is visible, not the uncleaved proprotein. This suggests that cleavage occurs even when a small amount of the adhesin is present at the cell surface. We performed a second experiment where bacteria were induced at the beginning of the exponential phase of growth with various concentrations of IPTG before normalized whole-cell extracts were obtained. As shown in Fig. 2B, the predominant form of AIDA-I at all levels of induction, including without induction, is the mature AIDA-I, not the uncleaved proprotein. Together, these results suggest that cleavage occurs independently of the amount of protein at the bacterial surface. This observation is consistent with an intramolecular mechanism of autoproteolysis. It should be noted, however, that this result would also be consistent with a cleavage mediated by an unknown surface protease.

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FIG. 2. Outer membrane cleavage of AIDA-I is independent of its level of expression. A. Whole-cell extracts were obtained from an exponential culture of C600 harboring plasmid pAg (WT) or pTRC99a, an empty vector (-). The cultures were induced with 10 µM of IPTG, and samples of identical volumes were taken each 10 min after induction for 2 h. B. Whole-cell extracts were obtained from an exponential culture harboring plasmid pAg (WT) or an empty vector (-). The cultures were either uninduced or induced with 10 or 50 µM of IPTG for 3 hours and then normalized at an OD600 of 1.2. Proteins separated by SDS-PAGE were probed by immunoblotting with antiserum against cleaved mature AIDA-I, which allows detection of the proprotein (140 kDa) and the cleaved mature AIDA-I (100 kDa). MM, molecular mass.

Release of the cleaved mature AIDA-I. Previous studies have shown that the cleaved mature AIDA-I remains associated with the outer membrane by noncovalent interactions and can be released by treatment at 60°C (4). We tried to release the adhesin by other techniques known to extract proteins peripherally associated with membranes, including treatment with SDS (0.1%), Triton X-100 (1%), Tween 20 (0.01%), Tris-glycine (pH 2), and carbonate (pH 11) and treatment with urea (0.1 M or 2 M). Except for Triton X-100 (1%), all these treatments failed to release the mature adhesin (data not shown). We also performed heat extraction at various temperatures between 30°C and 60°C, and we observed that only a small amount of adhesin could be released at temperatures below 42°C, while this amount increased dramatically above 50°C (data not shown). Taken together, these results suggest that the cleaved mature AIDA-I strongly interacts with the outer membrane and we could not witness its release in a wide variety of conditions.

Copurification of mature AIDA-I with AIDAc. As described above, the cleaved mature AIDA-I can be released by heat treatment or with Triton X-100 (1%). We wanted to purify the proteins released under both conditions to compare their conformations. We introduced a six-histidine and glycine (HisG) tag at the N terminus of the mature form of AIDA-I and used immobilized metal affinity chromatography (IMAC) for purification. The result of the purification starting from heat extracts is shown in Fig. 3A, and the purification from Triton X-100 extracts is shown in Fig. 3B. While the product of the purification from the heat extract was a single polypeptide, we were surprised to observe three major protein bands in the purification from Triton X-100 extracts, one with a molecular mass of 100 kDa that corresponded to the mature AIDA-I and another with a molecular mass of 140 kDa that could correspond to the uncleaved proprotein. The identity of both proteins was confirmed by an immunoblot assay with antiserum against the cleaved mature AIDA-I (data not shown). The third protein could not be detected with these antibodies or with a serum raised against the HisG tag (data not shown). Its molecular mass of approximately 45 kDa was consistent with the size of AIDAc. We assessed the modification of mobility, under denaturing electrophoresis, when the samples were left at room temperature or incubated at 100°C. As shown in Fig. 3C, a clear difference in electrophoretic mobility for the third protein can be observed between the two samples. This property is characteristic of outer membrane proteins folded in a ß-barrel conformation, including AIDAc (18, 34), thus confirming the identity of the third protein. Overall, these results suggest that the Triton X-100 extraction cosolubilized the mature AIDA-I and AIDAc and that the two polypeptides were copurified during IMAC. This is consistent with the strong interaction that we observed between the cleaved mature AIDA-I and the outer membrane.

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FIG. 3. Cosolubilization and copurification of AIDAc and mature AIDA-I. A. Coomassie blue-stained SDS-polyacrylamide gels of the heat extract of C600 harboring plasmid pAgH (lane 1) and of the purification product from this heat extract using an immobilized nickel column (lane 2). B. Coomassie blue-stained SDS-polyacrylamide gels of total membranes preparations of C600 harboring plasmid pAgH solubilized with Triton X-100 1% (lane 1) and of the purification product from this detergent extract using an immobilized nickel column (lane 2). The purified fraction shows three protein bands that are thought to be the uncleaved proprotein (Pro.), the cleaved mature AIDA-I, and the integral outer membrane protein, AIDAc. C. The mobility of AIDAc is different when the sample of purified protein is boiled at 100°C and when it is left at room temperature (RT) prior to electrophoresis. MM, molecular mass.

To assess the conformation of the purified protein, we subjected it to far-UV CD spectroscopy (27). As shown in Fig. 4A, the protein purified from the Triton X-100 extraction has a secondary structure rich in ß sheets, as suggested by the minimum of molar ellipticity observed at 218 nm. This richness in ß sheets is consistent with the predicted and observed ß-barrel structures of outer membrane proteins, including the solved structure of the translocation domain of the NalP autotransporter (40). It is also consistent with the predicted and observed ß-helical structures of extracellular domains of autotransporters (13, 26, 42). This result therefore strongly suggests that the purified protein is correctly folded. Furthermore, the Triton X-100-solubilized extracts could inhibit AIDA-I-mediated bacterial autoaggregation (data not shown). Surprisingly, the protein purified in similar conditions by IMAC, but from a heat extract, is mostly devoid of secondary structure, as evidenced by the negative ellipticity at 200 nm (Fig. 4B). Consistent with this result, the heat extracts had very weak inhibitory activity (data not shown). CD spectra of the proteins were recorded at 10°C intervals between 30°C and 70°C. As shown in Fig. 4A, a dramatic shift in spectrum occurred with the Triton X-100-extracted purified protein when the temperature reached 50°C, with the resulting spectrum losing a significant portion of its structure. Since integral outer membrane proteins are notoriously heat stable, including AIDAc (33, 34), this melting is most likely due to the denaturation of the cleaved mature AIDA-I. After heating at 60°C for a few minutes, immediately cooling the sample down to 30°C did not allow its renaturation, as judged by CD, suggesting that thermal denaturation of the mature AIDA-I is not reversible (data not shown). By contrast, the CD spectrum of the heat-extracted purified protein did not vary between 30°C and 70°C (Fig. 4B), and even at 90°C (data not shown), strengthening the conclusion that this protein was already denatured. Taken together, these results suggest that heat extraction of the cleaved mature AIDA-I causes its denaturation, whereas treatment with Triton X-100 preserves its conformation.

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FIG. 4. Thermal denaturation of AIDA-I monitored by far-UV CD. The figure shows far-UV CD spectra of pure protein obtained from solubilization with 1% Triton X-100 (A and C) or from heat extraction at 60°C (B). Multiple spectra were recorded at 10°C temperature intervals between 30°C and 70°C (light gray shades correspond to lower temperatures and dark gray shades correspond to higher temperatures) with wild-type protein (A and B) or the S846L uncleaved mutant (C). The ellipticities at 218 nm of the wild-type (WT) protein (open squares) or the S846L mutant (filled squares) were recorded continuously with the temperature varying between 30°C and 70°C at a rate of 5°C per min (D). MRE, mean residual ellipticity.

Modification of amino acid at the outer membrane cleavage site. In order to characterize the role of AIDA-I outer membrane cleavage, we constructed mutants of the cleavage site by site-directed mutagenesis. A previous study had demonstrated that this C-terminal processing occurs between residues serine 846 and alanine 847 (53). We modified serine 846 by a cysteine (S846C), a glycine (S846G), or a leucine (S846L) or alanine 847 by an isoleucine (A847I), a threonine (A847T), or an arginine (A847R). We then assessed the presence of the mature form of AIDA-I in the membrane fraction by Western blotting using a serum raised against the cleaved mature domain (Fig. 5). The S846C, A847I, and A847R mutants could not be observed. This suggests that the protein was most likely unstable and rapidly degraded. It should be noted that A847I was previously described as an unprocessed mutant and could be observed, although poorly expressed, in an E. coli strain deficient in outer membrane proteases (36). The sensitivity to point mutations of the cleavage region is surprising, as one could have assumed that it resides in a flexible loop. On the contrary, our results suggest that the cleavage site resides in a structured part of the protein.

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FIG. 5. Effect of modification of the outer membrane cleavage site of AIDA-I. Membrane fractions were prepared from overnight cultures of C600 induced with 10 µM IPTG at an OD600 of 0.8 and harboring pAg (wild type [WT]) or its mutated variants, constructed as described in the text. Proteins in the membrane fractions were separated and probed as described for Fig. 2. MM, molecular mass.

The cleavage was altered to various extents in the other mutants. It was partially inhibited for S846G and A847T and almost abolished for S846L. We examined the proteins that could be heat extracted, using SDS-PAGE and Coomassie blue staining. As explained above, the heat treatment at 60°C releases only the noncovalently associated cleaved mature AIDA-I, whereas the uncleaved proprotein remains associated with the outer membrane and cannot be extracted. As shown in Fig. 6, a small amount of cleaved mature AIDA-I from S846L could be heat extracted, but it was a considerably lesser amount than wild-type AIDA-I. The heat extract of S846L showed two distinct major bands, one with the normal apparent molecular mass of 100 kDa and a second with a slightly lower mass. The latter disappeared in heat extracts obtained from E. coli strain UT5600, which lacks the OmpT and OmpP outer membrane proteases. This result suggests that when the normal autoproteolytic cleavage is inhibited, the adhesin can be cleaved at an alternative site by other outer membrane proteases such as OmpT and OmpP. This observation is consistent with previous reports that a cleavage site for OmpT exist in the surface-exposed portion of AIDAc (53). This site, however, was observed only when cleaved mature AIDA-I had been removed. Our results demonstrate that this cleavage site is also accessible even in the presence of the adhesin when the autocatalytic cleavage is inhibited.

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FIG. 6. Processing of AIDA-I in a protease-deficient strain. Strain C600 (ompT+ ompP+) or UT5600 (ompT ompP) harboring an empty vector (–), pAg (wild type [WT]), or pAgS846L (S846L) was grown overnight after induction with 10 µM IPTG at an OD600 of 0.8. The cultures were concentrated in 10 mM sodium phosphate buffer, pH 7, and heated at 60°C for 20 min. The heat extracts, containing the cleaved mature AIDA-I, were recovered from bacterial pellets by centrifugation. Proteins in these heat extracts were then separated by SDS-PAGE, and the gels were stained with Coomassie blue. The asterisk represents a second cleavage site in AIDA-I resulting from cleavage by OmpT/OmpP (see text for details). MM, molecular mass.

The stability of the protein is not influenced by the lack of outer membrane cleavage. To assess the conformation and the stability of the uncleaved S846L mutant, we purified it and tested its conformation and thermal denaturation, monitored by CD spectroscopy as described above. As shown in Fig. 4C, the purified protein is correctly folded and its far-UV CD spectrum is almost identical to that of wild-type protein. We followed more carefully the thermal denaturation of S846L and wild-type AIDA-I by monitoring the ellipticities of the proteins at 218 nm continuously while changing the temperature (Fig. 4D). The resulting curves are almost indistinguishable and yield melting temperatures of 50.3 ± 0.1°C and 51.6 ± 0.2°C for the wild-type protein and S846L, respectively. These results strongly suggest that the S846L mutant has the same stability and structure as the wild-type protein (27). A limited proteolysis with proteinase K on whole cells expressing S846L or wild-type AIDA-I also indicated that the stabilities of the two proteins are very similar (data not show).

Lack of outer membrane cleavage does not influence the functionality of AIDA-I. We used the S846L construct to test the role of AIDA-I outer membrane cleavage in its functionality. We first assessed if this cleavage is important for the adhesion mediated by AIDA-I. We compared the adhesive properties of the mutant with those of the wild-type protein in an adhesion assay on HEp-2 cells. As shown in Fig. 7A, S846L had a slightly lower adhesion to cells but it was not statistically significant. We then tested if the outer membrane processing is important for the capacity to mediate bacterial autoaggregation. As shown in Fig. 7B, our results clearly indicate that cleavage is not essential for bacterial autoaggregation mediated by AIDA-I. Lastly, we assessed the effect of the processing on the ability to mediate biofilm formation (Fig. 7C). Again, our results showed that cleavage did not influence the ability to mediate biofilm formation.

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FIG. 7. Effect of the lack of AIDA-I outer membrane cleavage on functionality. A. Adhesion assay. C600 bearing an empty vector (-), plasmid pAg (wild type [WT]), or plasmid pAgS846L (S846L) was inoculated onto a monolayer of confluent HEp-2 cells in a 24-well plate with 106 CFU per well. After 3 h, the cells were washed with PBS and adhering bacteria were recovered, plated, and counted. The adhesion is calculated by dividing the CFU of adhering bacteria recovered by the CFU found in the inoculum after 3 h of incubation. B. Autoaggregation assay. Cultures of C600 harboring an empty vector (-), plasmid pAg (WT), or pAgS846L (S846L) were standardized in 10 ml to an OD600 of approximately 1.5 in a culture tube and left standing at 4°C. A 200-µl sample was taken 1 cm below the surface at the beginning of the assay and after 180 min, and its OD600 was measured. C. Biofilm formation. Normalized cultures of C600 harboring an empty plasmid (-), plasmid pAg (WT), or plasmid pAgS846L (S846L) were grown in M9 minimal medium for 24 h at 30°C in microtiter plates. Biofilms were stained with crystal violet. The fixed dye was solubilized with a mixture of ethanol and acetone (80:20), and the absorption of the solution was measured at 595 nm. D. Invasion assay. After 3 h of adhesion performed as described above, fresh medium containing gentamicin, penicillin, and streptomycin was added. The cells were incubated for 2 additional hours, and the surviving bacteria were recovered, plated, and counted. Experiments were performed at least three times in duplicate (A and D) or triplicate (B and C), and the values, representing means ± standard errors of the means, show typical results.

AIDA-I mediates invasion of HEp-2 cells independently of its outer membrane processing. As mentioned above, AIDA-I shares many similarities with the TibA adhesin/invasin. The major differences between these two proteins were that TibA lacks C-terminal processing and has the capacity to mediate invasion. We therefore asked whether the S846L mutation could confer invasive properties on AIDA-I. To test this hypothesis, we performed gentamicin protection invasion assays (11). As seen in Fig. 7D, S846L could indeed invade HEp-2 cells, but surprisingly, we also observed that wild-type AIDA-I could mediate invasion, to an extent similar to that of S846L. It should be noted, however, that the invasion that we observed was markedly inferior to that conferred by TibA (11). The addition of cytochalasin D, an inhibitor of actin polymerization, reduced 93.3% ± 2.5% (n = 4) of the invasion mediated by wild-type AIDA-I and 94.3% ± 1.8% (n = 4) of invasion mediated by S846L. This inhibition confirms that bacteria are not spontaneously resistant to gentamicin and are internalized inside HEp-2 cells by a mechanism involving actin rearrangement.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Many autotransporters are cleaved after their translocation across the outer membrane, such as the serine protease autotransporters of Enterobacteriaceae (9, 19), the actin-polymerizing surface protein VirG (10), or the H. influenzae Hap adhesin (14). The mechanisms of cleavage of autotransporters and the functions that this cleavage serves are diverse. The cleavage might be autoproteolytic (14) or due to a specific outer membrane protease (10). This processing can be required to deliver a cytotoxic polypeptide in the extracellular milieu (37), to keep a protein localized to the bacterial pole (8), or to control adherence and autoaggregation by regulating the expression level of an adhesin (15). By contrast, the outer membrane processing of the AIDA-I autotransporter is poorly understood.

AIDA-I is cleaved in bacteria lacking the protease OmpT, OmpP, or DegP, suggesting an autoproteolytic mechanism (53). Autoproteolytic processing has been studied in many autotransporters that bear typical serine protease catalytic domains in their extracellular domains (9, 14, 19). Other autotransporters, including Ag43, have a putative aspartyl protease active site in their extracellular domain (22, 43). A catalytic site in AIDA-I that could mediate such a proteolytic processing has not been identified. In this study, we observed that outer membrane cleavage occurs independently of the level of expression of AIDA-I, suggesting that AIDA-I is cleaved by an intramolecular mechanism. To our knowledge, an intramolecular mechanism of autotransporter autoproteolysis has not yet been reported, contrasting with previous studies where autocatalytic processing was studied and found to be intermolecular (14). It should be noted that intramolecular autoproteolysis can also occur in the absence of a typical protease catalytic site as a result of conformational stress upon protein folding (31). Further work is required to characterize the mechanism of proteolysis of AIDA-I.

Proteolytic processing of a protein often achieves a specific biological function that appears when the resulting polypeptidic fragments are separated from one another. Surprisingly, in this study we could not find conditions that could separate the cleaved mature AIDA-I from AIDAc in a folded conformation. Indeed, we found the heat-extracted cleaved mature AIDA-I to be mostly unfolded. Triton X-100 solubilization of the mature AIDA-I coextracted AIDAc, and both polypeptides could then be copurified and shown to be folded. We observed that the folded AIDA-I became denatured at temperatures above 50°C. This explains why the heat extraction, which is performed at 60°C, results in the release of a mostly unfolded mature protein. In previous studies, the mature forms of AIDA-I or Ag43 were purified from heat extracts by gel filtration rather than the IMAC step that we used (7, 29). Presumably, the former procedure can separate correctly folded from denatured protein whereas the latter does not. However, by preventing denaturation in the first place, our procedure is likely to yield a higher amount of native protein. It should be noted that, in the conditions that we used, we could not solubilize AIDA-I with Triton X-100 when it is unglycosylated (M.-E. Charbonneau and M. Mourez, unpublished data), arguing that detergent per se is not solubilizing AIDAc or other integral outer membrane proteins and suggesting that the solubilization relies on the presence of carbohydrates in AIDA-I.

Our results suggest a strong association between the cleaved fragments resulting from a proteolytic processing after outer membrane translocation. This was previously observed with Ag43 (7) and puts into question the role of this cleavage. In order to address this issue, we engineered mutations at the cleavage site. Using the S846L mutant, we could clearly show that cleavage is not essential for the functionality of the protein in in vitro assays, including the capacity to mediate invasion of epithelial cells, an activity that was not previously described for AIDA-I. The in vivo relevance of AIDA-I-mediated invasion remains to be established, but it should be noted that a low level of entry of many pathogenic strains of E. coli has previously been reported, including for DAEC (25). It is often hypothesized that such mechanisms might promote persistence in target tissues (47).

In summary, the results of our study show a consistent picture of the outer membrane processing of AIDA-I and suggest either that it is fortuitous and has no further function or that it serves a purpose that is not reproduced in our in vitro assays. A closer examination of the mechanism of action of AIDA-I using new assays is warranted to investigate the latter hypothesis.

 
We are greatly indebted to M. Ngeleka for the generous gift of serum raised against AIDA-I and to M. Jacques, J.-M. Betton, and H. Le Moual for their comments.

This work was supported by financial contributions from the Groupe de Recherche et d'Etudes sur les Maladies Infectieuses du Porc (GREMIP), the Natural Sciences and Engineering Research Council of Canada (NSERC discovery grant 262746), the Canada Research Chair program, and the Canada Foundation for Innovation (CFI project 201414).

 
* Corresponding author. Mailing address: Canada Research Chair on Bacterial Animal Diseases, Université de Montréal, Faculté de Médecine Vétérinaire, 3200 Sicotte, St-Hyacinthe, J2S 7C6, Québec, Canada. Phone: (450) 773-8521, ext. 8430. Fax: (450) 778-8108. E-mail: m.mourez{at}umontreal.ca.

Published ahead of print on 13 October 2006.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, December 2006, p. 8504-8512, Vol. 188, No. 24
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