Proteolytic processing is not essential for multiple functions of the Escherichia coli autotransporter adhesin involved in diffuse adherence (AIDA-I)

Abstract

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.

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).

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.

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.

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.

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.

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.

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.