Flagellin glycosylation in Pseudomonas aeruginosa PAK requires the O-antigen biosynthesis enzyme WbpO

Abstract

Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.

FIGURE 1. Putative biosynthetic pathway for the serotype O6 B-band O-antigen repeat unit

All enzymes are encoded within the B-band O-antigen gene cluster, except RmlB, RmlC, and RmlD, which are encoded in the rmlBDAC locus. UDP-D-GalNAcA, UDP-N-acetyl-D-galacturonic acid; UDP-DGal( 2NAc3OAc)N, UDP-2-acetamido-3-O-acetyl-2-deoxy-D-galactopyranosiduronamide; dTDP–D-Glc, dTDP-D-glucose; dTDP-L-Rha, dTDP-L-rhamnose; D-Gal(2NAc3OAc)N, 2-acetamido-3-O-acetyl-2-deoxy-D-galactopyranosiduronamide; D-GalNFoAN, 2-formamido-2-deoxy-D-galactopyranosiduronamide; D-QuiNAc, N-acetyl-D-quinovosamine; L-Rha, L-rhamnose.

FIGURE 2. SDS-PAGE and Western immunoblot analysis of P. aeruginosa flagellins

Flagellins were isolated from wild-type P. aeruginosa PAK, three PAK mutants, wbpL, wbpP, and wbpO, and wbpO mutant transformed with the plasmid pFV616-26a containing a copy of the wild-type wbpO to complement the mutation. Flagellin isolated from PAO1 was included as negative control. A, SDS-PAGE; B, Western immunoblotting using anti-FliC polyclonal antiserum.

FIGURE 3. Isoelectric focusing of PAK flagellins

Flagellins were resolved based on their pI in an ampholyte mixture of pH 3–7. A, simply blue-stained IEF gel; B, Western immunoblot using anti-FliC antiserum.

FIGURE 4. Biotin-hydrazide glycosylation assay of PAK flagellins

Flagellins isolated from P. aeruginosa PAK and mutant strains were examined for the presence of carbohydrate residues. Negative and positive controls included in the glycosylation assay were purified WbpD and the glycoprotein transferrin, respectively.

FIGURE 5. CE analysis of WbpO-catalyzed reactions

Trace A, NAD⁺ and UDP-D-GlcNAc standards; trace B, UDP-D-GlcNAc converted with WbpO; trace C, NAD⁺ and UDP-D-GalNAc standards; trace D, UDP-D-GalNAc converted with WbpO.

FIGURE 6. CE analysis of WbpP-catalyzed reactions

Trace A, NAD⁺ and UDP-D-GlcNAc standards; trace B, UDP-D-GlcNAc converted to UDP-D-Glc-NAcA by WbpA_O5; trace C, UDP-D-GlcNAc completely converted to UDP-DGlcNAcA by WbpA_O5, followed by the addition of WbpP; trace D, NAD⁺ and UDP-D-GalNAc standards; trace E, UDP-D-GalNAc converted to UDP-D-Gal-NAcA by WbpO; trace F, UDP-D-GalNAc completely converted to UDP-D-Gal-NAcA by WbpO, followed by the addition of WbpP.

FIGURE 7. Effect of ammonium sulfate on the initial rate and overall substrate conversion of WbpO reactions

All reactions contained 1mM UDP-DGlcNAc, 2.5 mM NAD^+, and 4 μg of WbpO in a final volume of 170 μl, with varying concentrations of (NH₄)₂SO₄ as labeled.

FIGURE 8. UDP-D-GlcNAcA and UDP-D-GalNAcA in the saccharide-binding pocket of WbpP

UDP-D-GlcNAcA (yellow; A) and UDP-D-GalNAcA (blue; B) were individually modeled into the active site of WbpP. The saccharide moieties of substrates were positioned in the catalytically productive orientation with respect to the cofactor NAD⁺ (brown) and the catalytic base Tyr-166. Although the saccharide moieties of UDP-D-GlcNAcA and UDP-D-GalNAcA are in different orientations, both substrates interact with a virtually identical set of active site residues (gray; G102/S103, S142, S143, Y166, and N195) to form five hydrogen bonds (green; 3.5 Å or less). In addition, the 2′-N-acetyl group of UDP-D-GlcNAcA forms an extra hydrogen bond with Gln-201. Note that a peptide bond between Gly-102 and Ser-103 is expected to flip in the presence of UDP-D-GlcNAcA to allow the carbonyl oxygen of Gly-102 to interact with 3′-OH of GlcNAcA (41). This figure was prepared using The PyMol Molecular Graphics System (DeLano Scientific, San Carlos, CA).

FIGURE 9. Two alternative pathways for the conversion of UDP-D-GlcNAc into UDP-D-GalNAcA by WbpO and WbpP

Whereas WbpO and WbpP can act in either order, kinetic analysis of WbpO and equilibrium analysis of WbpP suggest a preference for conversion of UDP-D-GlcNAc to UDP-D-GlcNAcA by WbpO before the conversion by WbpP into UDP-D-GalNAcA.