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. 2015 Jul 10;10(7):e0131905.
doi: 10.1371/journal.pone.0131905. eCollection 2015.

The Polymerization of Aeromonas hydrophila AH-3 O-Antigen LPS: Concerted Action of WecP and Wzy

Susana Merino  1 , Victor Gonzalez  1 , Juan M Tomás  1
Affiliations

The Polymerization of Aeromonas hydrophila AH-3 O-Antigen LPS: Concerted Action of WecP and Wzy

Susana Merino et al. PLoS One. .

Abstract

The repeat units of heteropolymeric O antigen are synthesized at the cytosolic side of the inner bacterial membrane via the Wzx/Wzy-dependent assembly pathway. After being translocated across the membrane by Wzx, each repeat unit is polymerized by Wzy to form a glycan chain. In this study, we demonstrate the need of the corresponding enzyme transferring the initial HexNAc to undecaprenol phosphate (lipid carrier), together with the corresponding O-antigen polymerase (Wzy), to produce the Aeromonas hydrophila O:34-antigen. We suggest, the concerted action of WecA or P enzyme (UDP-HexNAc: polyprenol-P HexNAc-1-P transferase) and Wzy is involved in the mechanism responsible for the A. hydrophila O-antigen polymerization.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Chemical structure of Aeromonas hydrophila LPS.
O34-antigen LPS [17] (A) and core LPS [18] (B).
Fig 2
Fig 2. Polyacrylamide gels showing the migration of LPS from AH-3ΔwecP mutant and its complementation.
The LPS samples were separated on SDS-PAGE (A) and SDS-Tricine-PAGE (B) and visualized by silver staining. Shown are LPS samples from A. hydrophila AH-3 (WT) (Lane 1), AH-3ΔwecP (Lane 2), AH-3ΔwecP + pBAD-WecPAh (Lane 3), AH-3ΔwecP + pBAD-WecAEc (Lane 4), AH-3ΔwecP + pBAD-WecA-WzyEEc (Lane 5), AH-3ΔwecP-gne double mutant (Lane 6), AH-3ΔwecP-gne double mutant + pBAD-WecA-WzyEEc (Lane 7), AH-3ΔwecP-gne double mutant + pBAD-WecAEc (Lane 8), AH-3ΔwecP-wzy double mutant (Lane 9), AH-3Δwzy double mutant (Lane 10), AH-3ΔwecP-wzy double mutant + pBAD-WecA-WzyEEc (Lane 11), and AH-3ΔwecP-wzy double mutant + pBAD-WzyAh (Lane 12) All the strains harbouring pBAD plasmids were grown under induced conditions (+ arabinose) as indicated in Materials and Methods section.
Fig 3
Fig 3. Sephadex G-50 (S) elution profile of the LPS carbohydrate portion.
Sephadex G-50 (S) elution profile of the LPS carbohydrate portion from A. hydrophila AH-3 mutants and its complementation, obtained by mild acid degradation. PS, high-molecular-mass polysaccharide; OS, core oligosaccharide; OS1 and OS2, short-chain polysaccharides containing one and two repeating units attached to the core.
Fig 4
Fig 4. Gas-liquid chromatograms and monosaccharide contents.
A) Gas-liquid chromatograms and B) Monosaccharide contents of the LPS fractions from A. hydrophila AH-3ΔwecP mutant and its complementation, determined by GLC. The identity of the polysaccharides is as follow: 6dTal, 6-deoxy-L-talose; Man, D-Mannose; Gal, D-galactose; Glc, D-glucose; GlcNAc, N-acetyl-D-glucosamine; GalNAc, N-acetyl-D-galactosamine; Hep, L-D-heptoses and D-D-heptoses.
Fig 5
Fig 5. Survival of A. hydrophila strains in non-immune human serum (NHS).
The strains carrying pBAD plasmids were grown under inducing conditions (+ arabinose)
Fig 6
Fig 6. Schematic diagram for plasmids construction.
Construction of pBAD-WecP-WzyAh from A. hydrophila AH-3 and described plasmid pBAD33-WecPAh (A), and pBAD-WecA-WzyEc from E. coli DH5α and described plasmid pBAD33-WecAEc (B). The detailed methodology is fully described in the Materials and Methods section.
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