Structural basis for alginate secretion across the bacterial outer membrane

Abstract

Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 Å crystal structure of AlgE, which reveals a monomeric 18-stranded β-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 (ΔT8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or ΔL2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with ΔT8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane

“…Unlike AlgE, an 18-strand outer membrane protein that is responsible for exopolysaccharide alginate secretion, no narrow pore constriction is found in the ␤ barrel of PgaA, and no extracellular or periplasmic loops occlude the lumen (Figs. 1C and 2A) (29). However, on the extracellular side, the PgaA ␤-barrel is mainly closed by four long extracellular loops, eL1, eL4, eL6, and eL8 (Fig.…”

“…For instance, the export of the tightly surface-associated capsular polysaccharides across the outer membrane occurs via a novel type of octameric ␣-helical pore formed by Wza and its otholog KpsD (41)(42)(43), whereas the exopolysaccharide alginate secretin AlgE forms an 18-strand ␤-barrel pore with an arginine-rich highly electropositive pore constriction acting as a selectivity filter for the negatively charged alginate polymer (29). Apart from this, the extracellular loop L2 and a long periplasmic loop T8 of the AlgE ␤-barrel seem to play important roles in regulating alginate secretion ( Fig.…”

“…film Formation-In many bacterial outer membrane proteins, long extracellular loops play important roles in regulating substrate translocation (29). To investigate the functional roles of the extracellular loops of PgaA in dPNAG secretion, a pgaA deletion mutant (E. coli.…”

“…The expression of the E. coli pgaABCD operon is tightly regulated. For example, the RNA-binding protein CsrA (carbon storage regulator A) negatively regulates pgaA translation, pgaABCD transcription, and mRNA stability (16,26,27 29 solved the crystal structure of AlgK. In combination with functional analyses, they proposed a mechanism for alginate secretion across the bacterial outer membrane; Little et al (23,24) reported the crystal structures of PgaB and its complex with ␤-1,6-(GlcNAc) 6 and disclosed the catalytic mechanism of the N-deacetylase PgaB on PNAG polymers and the manner of periplasmic translocation of PNAG.…”

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

“…An adaptation of the current method has been used for reconstitution and functional characterization of two membrane proteins in Pseudomonas aeruginosa, a common CF pathogen. Reconstitution of purified AlgE outer membrane protein coupled with iodide efflux measurements were used to support a model for anionic alginate secretion through this transporter 26 . Reconstitution and iodide efflux measurements were applied to the purified Wzx protein, which allowed a model to be proposed that suggests an H + -dependent antiport mechanism for lipid-linked oligosaccharide translocation across the bacterial inner membrane by this protein 27 .…”