Identification of a glycoprotein produced by enterotoxigenic Escherichia coli

Abstract

Enterotoxigenic Escherichia coli (ETEC) strain H10407 is capable of invading epithelial cell lines derived from the human ileocecum and colon in vitro. Two separate chromosomally encoded invasion loci (tia and tib) have been cloned from this strain. These loci direct nonadherent and noninvasive laboratory strains of E. coli to adhere to and invade cultured human intestinal epithelial cells. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane protein that is directly correlated with the adherence and invasion phenotypes. TibA is synthesized as a 100-kDa precursor (preTibA) that must be modified for biological activity. Outer membranes of recombinant E. coli expressing TibA or preTibA were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose. The presence of glycoproteins was detected by oxidization of carbohydrates with periodate and labeling with hydrazide-conjugated digoxigenin. Only TibA could be detected as a glycoprotein. Complementation experiments with tib deletion mutants of ETEC strain H10407 demonstrate that the TibA glycoprotein is expressed in H10407, that the entire tib locus is required for TibA synthesis, and that TibA is the only glycoprotein produced by H10407. Protease treatment of intact H10407 cells removes the carbohydrates on TibA, suggesting that they are surface exposed. TibA shows homology with AIDA-I from diffuse-adhering E. coli and with pertactin precursor from Bordetella pertussis. Both pertactin and AIDA-I are members of the autotransporter family of outer membrane proteins and are afimbrial adhesins that play an important role in the virulence of these organisms. Analysis of the predicted TibA amino acid sequence indicates that TibA is also an autotransporter. Analysis of the tib locus DNA sequence revealed an open reading frame with similarity to RfaQ, a glycosyltransferase. The product of this tib locus open reading frame is proposed to be responsible for TibA modification. These results suggest that TibA glycoprotein acts as an adhesin that may participate in the disease process.

FIG. 1

Detection of glycoproteins in outer membranes of recombinant E. coli HB101. (A) Restriction endonuclease map of the tib locus as found in the H10407 genome or in the indicated plasmids. The direction of tib gene transcription is indicated by arrows above the H10407 map. The extent of the tib locus contained by each plasmid is indicated by an open box. The black arrowhead to the left of the pET140 map indicates the direction of transcription from an exogenous promoter found in the plasmid vector. B, BamHI; C, ClaI; E, EcoRI; H, HindIII; Hp, HpaI; N, NruI; S, SalI; Sm, SmaI. (B) Coomassie blue-stained SDS-PAGE (7.5% polyacrylamide) of outer membranes purified from the following strains (by lane): 1, HB101; 2, HB101(pHC79); 3, HB101(pET140); 4, HB101(pET109). Plasmid pET140 expresses the 100-kDa preTibA protein, whereas pET109 expresses the 104-kDa TibA protein. (C) Samples identical to those shown in panel B were transferred to nitrocellulose and then stained for glycoprotein as described in Materials and Methods. The migration of molecular mass standards is shown to the left of panels B and C. The mobility of TibA is shown by an arrow to the right of panels B and C.

FIG. 2

Complementation analysis of an H10407 tib deletion mutant. (A) Restriction endonuclease map of the tib locus as found in H10407, TIB3 (an H10407 tib deletion mutant), or the indicated plasmids. The direction of tib gene transcription is indicated by arrows above the H10407 map. The tib locus sequences deleted in TIB3 are indicated by a thin black line in the TIB3 map. The extent of the tib locus contained by each plasmid is indicated by an open box. The black arrowhead to the right of the pET139 map indicates the direction of transcription from an exogenous promoter found in the plasmid vector. Restriction enzymes are as indicated in Fig. 1. (B) SDS-PAGE (7.5% polyacrylamide) of whole-cell lysates transferred to nitrocellulose and then stained for glycoprotein as described in Materials and Methods. Lanes: 1, H10407; 2, TIB3; 3, TIB3(pET109); 4, TIB3(pET146); 5 and 6, TIB3(pET139) and TIB3(pET146), respectively. Previously, it had been shown that in the absence of the sequences contained on plasmid pET146, plasmid pET139 did not direct the production of either TibA or preTibA (15). The migration of molecular mass standards (kilodaltons) is shown to the left of panel B. The mobility of TibA is shown by an arrow to the right of panel B.

FIG. 3

Protease treatment of intact bacteria. Surface carbohydrates on strain TIB3(pET109) were labeled with DIG after periodate oxidation. Intact bacteria were then incubated with various amounts of trypsin (A) or proteinase K (B). Whole-cell lysates were prepared and then separated by SDS-PAGE (7.5% polyacrylamide). After transfer to nitrocellulose, DIG-labeled glycoproteins were detected by anti-DIG antibodies. Lanes: 1, TIB3(pET109) with 0 μg of protease per ml; 2, TIB3(pET109) with 10 μg of protease per ml; 3, TIB3(pET109) with 50 μg of protease per ml; 4, TIB3(pET109) with 100 μg of protease per ml. The migration of molecular mass standards is shown to the left of each panel. The mobility of TibA is shown by an arrow. In panel A, lane 3 appears to stain more intensely than lanes 2 and 4. In replicates of this experiment, the intensities of staining were identical at each trypsin concentration.

FIG. 4

Alignment of TibA and pertactin precursor carboxy termini. Alignment of the C terminus of TibA (residues 791 to 989) and pertactin precursor (residues 712 to 910) was performed by using Clustalx software. A vertical line indicates positions with identical residues. A colon indicates that one of the following “strong” groups is fully conserved: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, or FYW. A period indicates that one of the following “weaker” groups is fully conserved: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, or HFY.

FIG. 5

Prediction of amphipathic β-sheets (β-AMPH). TibA C terminus from residues 678 to 989 was analyzed for the presence of amphipathic β-sheets (full window 10) by using TopPret software and for surface-exposed amino acids and β-turns by using MacVector software. Solid bars along the top of the figure indicate membrane-spanning β-sheets predicted as certain (1.6 upper cutoff), and open bars indicate membrane-spanning β-sheets predicted as putative (0.4 cutoff). Shaded bars indicate amino acids with high surface probability, and arrows indicate amino acids that are likely to be in β-turns, as predicted by Chou-Fasman (9).