Glycosylation of the Escherichia coli TibA self-associating autotransporter influences the conformation and the functionality of the protein

Abstract

The self-associating autotransporters (SAATs) are multifunctional secreted proteins of Escherichia coli, comprising the AIDA-I, TibA and Ag43 proteins. One of their characteristics is that they can be glycosylated. Glycosylation of AIDA-I and Ag43 have been investigated, but not that of TibA. It is still not clear whether glycosylation of the SAATs affect their structure or their functionality. Therefore, we have looked at the effects of glycosylation on the TibA adhesin/invasin. TibA is glycosylated by TibC, a specific glycosyltransferase, and the two genes are encoded in an operon. In this study, we have found that the glycosylation of TibA is not limited to the extracellular functional domain, as previously observed with AIDA-I and Ag43. We have determined that unglycosylated TibA is not able to promote the adhesion of bacteria on cultured epithelial cell, even though it is still able to promote invasion, biofilm formation and autoaggregation of bacteria. We have purified the glycosylated and unglycosylated forms of TibA, and determined that TibA is less stable when not glycosylated. We finally observed that glycosylation affects the oligomerisation of TibA and that unglycosylated TibA is locked in a conformation that is not suited for adhesion. Our results suggest that the effect of glycosylation on the functionality of TibA is indirect.

Figure 1. Effect of glycosylation on the expression level of TibA.

Whole cell extracts of E. coli strain C600 bearing an empty vector (-), or a plasmid allowing the expression of glycosylated TibA (TibA/TibC) or unglycosylated TibA (TibA) were separated by SDS-PAGE and revealed by immunoblotting with an anti-AIDA-I antibody (A) or an anti-His antibody (B). The intensity of the bands probed with the α-His antibody was quantified using ImageJ and values were normalized to the amount of glycosylated TibA (lower panel). Experiments were done five times and ANOVA and Dunnett post-tests were used to identify significant (*; p<0.05) and non-significant (ns) differences with glycosylated TibA. (C) Outer membrane extracts of E. coli strain C600 bearing an empty vector (-), or a plasmid allowing the expression of glycosylated TibA (TibA/TibC) or unglycosylated TibA (TibA) were revealed by immunoblotting using the anti-His antibody.

Figure 2. Localization of the peptides in TibA.

Glycosylated (filled circle) and unglycosylated (empty circle) peptides from Table 1 are localized on a schematic representation, drawn to scale, of TibA highlighting the signal sequence (residues 1 to 54), the passenger domain (residues 54 to 393), the junction region (residues 393 to 627), the proline-rich domain (residues 627 to 677) and the membrane-embedded domain (residues 677 to 989).

Figure 3. Effect of glycosylation on the functionality of TibA.

(A) Autoaggregation assay: E. coli C600 bearing an empty vector (-), a plasmid allowing the expression of glycosylated TibA (TibA/TibC) or unglycosylated TibA (TibA) were normalized to an OD₆₀₀ of 1.5 and left standing. OD₆₀₀ at the top of the culture was measured at the beginning of the assay and after 2 h. Results are shown in percentage of initial OD₆₀₀. (B) Adhesion assay: Bacteria were inoculated onto monolayers of confluent Hep-2 cells. After 3 h, the adhering bacteria were recovered, plated and counted. Results represent the percentage of adhered bacteria compared to glycosylated TibA. (C) Invasion assay: After adhesion, extracellular bacteria were killed by addition of gentamicin and invaded bacteria were recovered, plated and counted. Results represent the percentage of gentamicin resistant bacteria compared to glycosylated TibA. (D) Biofilm formation assay: Biofilms were stained with crystal violet and the amount of fixed dye was determined by measuring OD₅₉₅. Experiments were done three times in duplicate and ANOVA and Dunnett post-tests were used to identify significant (*; p<0.05) and non-significant (ns) differences with glycosylated TibA.

Figure 4. Effect of glycosylation on the generation of cytoplasmic and extracytoplasmic stress.

Reporter E. coli strains SR1364 (A) and SR1458 (B) were transformed with an empty vector (-), a plasmid allowing the expression of glycosylated TibA (TibA/TibC) or unglycosylated TibA (TibA/TibC^C358R) and the β-galactosidase activity was measured. Experiments were done three times in duplicate and ANOVA and Dunnett post-tests were used to identify significant (*; p<0.05) and non-significant (ns) differences with glycosylated TibA.

Figure 5. Effect of glycosylation on protease susceptibility.

E. coli strain C600 bearing an empty vector (-), or a plasmid allowing the expression of glycosylated TibA (TibA/TibC) or unglycosylated TibA (TibA) were pelleted and resuspended in TBS in the presence or absence of trypsin or proteinase K (0.5 µg/ml, 1 µg/mL and 5 µg/mL). After 30 min, whole cell extracts were prepared and revealed by immunoblotting with an anti-His antibody. MW, molecular weight.

Figure 6. Effect of glycosylation on purified TibA.

TibA was purified from E. coli strain C600 bearing plasmids allowing expression of glycosylated and unglycosylated TibA and solubilized in TBS 1% n-octyl-β-D-glucoside. (A) Far-UV CD spectrum of glycosylated TibA (black) and unglycosylated TibA (red) are shown between 205 and 260 nm. (B) Proteins were also subjected to a thermal denaturation followed by a renaturation monitored by far-UV CD. Temperature was increased from 25°C and 80°C at a rate of 5°C per minute and the ellipticities at 218 nm were recorded for glycosylated TibA (black) and unglycosylated TibA (red). Right after the denaturation, temperature was decreased from 80°C to 25°C at a rate of 5°C per minute and the ellipticities were recorded at 218 nm for glycosylated TibA (gray) and unglycosylated TibA (orange).

Figure 7. Effect of glycosylation on the modulation of conformation by salt.

Far-UV CD spectrum of glycosylated TibA (A) and unglycosylated TibA (B) are shown between 205 and 260 nm. The purified proteins were solubilized in TBS 1% n-octyl-β-D-glucoside (bOG). Prior to CD measurement, the proteins were diluted to a final bOG concentration of 0.3%, below the CMC, in TBS (black line) or in TBS 500 mM NaCl (dotted line). (C) E. coli strain C600 bearing an empty vector (-), or a plasmid allowing the expression of glycosylated TibA (TibA/TibC) or unglycosylated TibA (TibA) were resuspended in TBS (black bars) or in TBS 500 mM NaCl (white bars) and autoaggregation assays were performed.