Glycosylation of the self-recognizing Escherichia coli Ag43 autotransporter protein

Abstract

Glycosylation is a common modulation of protein function in eukaryotes and is biologically important. However, in bacteria protein glycosylation is rare, and relatively few bacterial glycoproteins are known. In Escherichia coli only two glycoproteins have been described to date. Here we introduce a novel member to this exclusive group, namely, antigen 43 (Ag43), a self-recognizing autotransporter protein. By mass spectrometry Ag43 was demonstrated to be glycosylated by addition of heptose residues at several positions in the passenger domain. Glycosylation of Ag43 by the action of the Aah and TibC glycosyltransferases was observed in laboratory strains. Importantly, Ag43 was also found to be glycosylated in a wild-type strain, suggesting that Ag43-glycosylation may be a widespread phenomenon. Glycosylation of Ag43 does not seem to interfere with its self-associating properties. However, the glycosylated form of Ag43 enhances bacterial binding to human cell lines, whereas the nonglycosylated version of Ag43 does not to confer this property.

FIG. 1.

Glycosylation of the Ag43 passenger domain by the Aah and TibC heptosyl transferases. (A) Western blotting, with antiserum against the Ag43 passenger domain, of α domains liberated from E. coli MS427 hosts by brief heat treatment. (B) Glycan detection. Lanes: 1, LH56 (vector control); 2, LH57 (Ag43⁺); 3, OS64 (Aah⁺ Ag43⁺); 4, OS111 (TibC⁺ Ag43⁺).

FIG. 2.

Glycosylation of Ag43 is qualitatively unaffected by copy number. The product of the chromosomally located flu gene in strain MG1655 is glycosylated by heptosyl transferases encoded by low- or high-copy-number plasmids. (A) Western blotting of passenger domains liberated from E. coli host cells by brief heat treatment and detected with antiserum raised against the Ag43 passenger domain. (B) Glycan detection. Lanes: 1, MG1655; 2, OS123 (Aah⁺) on low-copy plasmid; 3, OS116 (Aah⁺) on medium-copy plasmid; 4, OS135 (TibC⁺) on medium-copy plasmid.

FIG. 3.

Glycosylation of Ag43 does not affect Ag43-Ag43 interaction and the associated cell aggregation. (A) LH56 (vector control); (B) LH57 (Ag43⁺); (C) OS64 (Aah⁺ Ag43⁺).

FIG.4.

MALDI-TOF mass spectroscopy. (A) Trypsin-digested Ag43 passenger domain isolated from a host strain unable to perform glycosylation. (B) Representative enlarged segment of spectrum of trypsin-digested Ag43 passenger domain isolated from a host strain capable of performing protein glycosylation. The indicated peaks correspond to the same peptide with and without heptose. The mass difference represents heptose minus water. Values in angle brackets indicate average masses; all other masses are represented as monoisotopic. Masses are presented as M + H. The asterisk indicates three heptose residues.

FIG.4.

MALDI-TOF mass spectroscopy. (A) Trypsin-digested Ag43 passenger domain isolated from a host strain unable to perform glycosylation. (B) Representative enlarged segment of spectrum of trypsin-digested Ag43 passenger domain isolated from a host strain capable of performing protein glycosylation. The indicated peaks correspond to the same peptide with and without heptose. The mass difference represents heptose minus water. Values in angle brackets indicate average masses; all other masses are represented as monoisotopic. Masses are presented as M + H. The asterisk indicates three heptose residues.

FIG. 5.

Summary of identified tryptic peptides from the Ag43 passenger domain found to be glycosylated with heptose residues by MALDI-TOF mass spectrometry. Potential O-glycosylated amino acids are indicated in blue. (A) Identified glycosylated peptides. Molecular masses (in daltons) are indicated. MI and AVG, monoisotopic and average masses, respectively. Glycosylation status is indicated. Note that several of the peptides are overlapping due to partial tryptic digestion. (B) Primary structure of the passenger domain with glycosylated peptides indicated. (C) Overall picture of Ag43 glycosylation. Glycosylated segments are indicated as black bars. S, signal peptide; α, passenger domain; β, transmembrane domain; aa, amino acids.

FIG. 6.

Ag43-variants from E. coli UTI536 are glycosylated. (A) Western blotting of Ag43 passenger domains liberated from E. coli host cells by brief heat treatment and detected with anti Ag43 passenger domain serum. (B) Glycan detection. Lanes: 1, UTI536; 2, UTI536 DM; 3, UTI536 DM (Ag43-III⁺); 4, UTI536 DM (Ag43-K12⁺). The latter detection analysis was somewhat hampered by the heavy encapsulation of UTI536, which accounts for the capsule material observed in the double mutant.

FIG. 7.

Glycosylated Ag43 promotes adhesion to HEp-2 cells. Interaction of HEp-2 epithelial cell line cells with E. coli strains expressing nonglycosylated Ag43 (LH57, Ag43⁺) (A), Aah heptosyl transferase (OS101, Aah⁺) (B), or glycosylated Ag43 (OS64, Aah⁺ Ag43⁺) (C) is shown.