Abstract
A large group of bacterial virulence autotransporters including AIDA-I from diffusely adhering E. coli (DAEC) and TibA from enterotoxigenic E. coli (ETEC) require hyperglycosylation for functioning. Here we demonstrate that TibC from ETEC harbors a heptosyltransferase activity on TibA and AIDA-I, defining a large family of bacterial autotransporter heptosyltransferases (BAHTs). The crystal structure of TibC reveals a characteristic ring-shape dodecamer. The protomer features an N-terminal β-barrel, a catalytic domain, a β-hairpin thumb, and a unique iron-finger motif. The iron-finger motif contributes to back-to-back dimerization; six dimers form the ring through β-hairpin thumb-mediated hand-in-hand contact. The structure of ADP-D-glycero-β-D-manno-heptose (ADP-D,D-heptose)-bound TibC reveals a sugar transfer mechanism and also the ligand stereoselectivity determinant. Electron-cryomicroscopy analyses uncover a TibC-TibA dodecamer/hexamer assembly with two enzyme molecules binding to one TibA substrate. The complex structure also highlights a high efficient hyperglycosylation of six autotransporter substrates simultaneously by the dodecamer enzyme complex.
Highlights
Protein glycosylation is one of the most abundant post-translational modifications in all domains of life (Spiro, 2002)
In a separate parallel study (Lu et al, 2014) we showed that autotransporter adhesion heptosyltransferase (AAH) is a bona fide heptosyltransferase belonging to a large bacterial autotransporter heptosyltransferase (BAHT) family
Co-expression of TibC or AAH together with AIDA-I in E. coli BL21 cells resulted in a tight adhesion of the bacteria to HeLa cells (Figure 1A) and AIDA-I glycosylation (Figure 1B)
Summary
Protein glycosylation is one of the most abundant post-translational modifications in all domains of life (Spiro, 2002). Recent studies have appreciated protein glycosylation in bacteria, which is often associated with pathogen virulence and immune modulation (Szymanski and Wren, 2005; Abu-Qarn et al, 2008; Nothaft and Szymanski, 2010). Flagella glycosylation is found in many bacteria species including Campylobacter jejuni, Helicobacter pylori, Clostridium spp. and Pseudomonas aeruginosa, contributing to bacterial locomotion or virulence (Schirm et al, 2003; Nothaft and Szymanski, 2010). We and others have shown that secreted effectors from enteropathogenic Escherichia coli (EPEC) and related enteric pathogens harbor an arginine N-acetylglucosamine transferase activity that modifies host death-domain proteins and is essential for bacterial colonization in infected mice (Li et al, 2013; Pearson et al, 2013).
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