Aeromonas Surface Glucan Attached through the O-Antigen Ligase Represents a New Way to Obtain UDP-Glucose

We previously reported that A. hydrophila GalU mutants were still able to produce UDP-glucose introduced as a glucose residue in their lipopolysaccharide core. In this study, we found the unique origin of this UDP-glucose from a branched α-glucan surface polysaccharide. This glucan, surface attached through the O-antigen ligase (WaaL), is common to the mesophilic Aeromonas strains tested. The Aeromonas glucan is produced by the action of the glycogen synthase (GlgA) and the UDP-Glc pyrophosphorylase (GlgC), the latter wrongly indicated as an ADP-Glc pyrophosphorylase in the Aeromonas genomes available. The Aeromonas glycogen synthase is able to react with UDP or ADP-glucose, which is not the case of E. coli glycogen synthase only reacting with ADP-glucose. The Aeromonas surface glucan has a role enhancing biofilm formation. Finally, for the first time to our knowledge, a clear preference on behalf of bacterial survival and pathogenesis is observed when choosing to produce one or other surface saccharide molecules to produce (lipopolysaccharide core or glucan)

Bacterial strains, and plasmids used.

<p>Bacterial strains, and plasmids used.</p

Sephadex G-50 gel permeation chromatography elution profiles of the water soluble (carbohydrate) material isolated by mild acid degradation of the LPS preparations from <i>A. hydrophyla</i> AH-3 (wild type) (A), AH-3Δ<i>rmlB</i> (B), AH-3Δ<i>wecP</i> (C) and AH-3Δ<i>glgA</i> (D) mutants.

<p>Sephadex G-50 gel permeation chromatography elution profiles of the water soluble (carbohydrate) material isolated by mild acid degradation of the LPS preparations from <i>A. hydrophyla</i> AH-3 (wild type) (A), AH-3Δ<i>rmlB</i> (B), AH-3Δ<i>wecP</i> (C) and AH-3Δ<i>glgA</i> (D) mutants.</p

Primers used in the construction of chromosomal in-frame deletion mutants.

a<p>Italic letters show overlapping regions.</p>b<p>Underlined letters show <i>BglII</i> restriction site.</p

Enzymatic average values for <i>A. hydrophila</i> crude extracts and purified proteins.

<p><i>A. hydrophila</i> strains were grown under the conditions mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035707#s4" target="_blank">Materials and Methods</a>.</p>a<p>Activity was measured in the pyrophosphorolysis direction.</p><p>Values are the average of three independent experiments +/– standard deviation.</p><p>ND, not determined.</p

LPS of <i>Aeromonas hydrophila</i> AH-3.

<p>(A) Chemical structure of the LPS-core of <i>A. hydrophila</i> strain AH-3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035707#pone.0035707-KnirelYA2" target="_blank">[36]</a>. The O34-antigen is linked to the Gal residue (shown in italics) of the LPS core <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035707#pone.0035707-KnirelYA2" target="_blank">[36]</a>. (B) Silver-stained SDS-PAGE of purified LPS from <i>A. hydrophila</i> strain AH-3 (lane 1) and GalU mutant AH-2886 (lane 2) and structures of the two LPS-core bands of AH-2886 mutant <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035707#pone.0035707-Vilches1" target="_blank">[3]</a>. All monosaccharides are in the pyranose form. Kdo, 3-deoxy-d-<i>manno</i>-oct-2-ulosonic acid; l-α-d-Hep, d-α-d-Hep, l-<i>glycero</i>- and d-<i>glycero</i>-α-d-<i>manno</i>-heptose; Glc, glucose; GlcN, glucosamine; Gal, galactose. C = LPS core, O = O34-antigen LPS.</p

Silver-stained Tricine SDS-PAGE of the LPS from <i>A. hydrophyla</i> AH-3 (1), AH-3Δ<i>galU</i>:<i>glgA</i> double mutant (2), AH-3Δ<i>galU</i>:<i>glgA</i> plus pBAD33-GlgA from AH-3 (3), and AH-3Δ<i>galU</i>:<i>glgA</i> plus pBAD33-GlgA_Ec from <i>E. coli</i> (4).

<p>Silver-stained Tricine SDS-PAGE of the LPS from <i>A. hydrophyla</i> AH-3 (1), AH-3Δ<i>galU</i>:<i>glgA</i> double mutant (2), AH-3Δ<i>galU</i>:<i>glgA</i> plus pBAD33-GlgA from AH-3 (3), and AH-3Δ<i>galU</i>:<i>glgA</i> plus pBAD33-GlgA_Ec from <i>E. coli</i> (4).</p

Negative ions ESI-MS of the core oligosaccharide LPS.

<p>(A) Core oligosaccharide obtained by acid release from the purified LPS of AH-3Δ<i>galU</i>:<i>glgA</i>. double mutant. (B) Core oligosaccharide obtained from the slow LPS migration band from AH-3Δ<i>galU</i>:<i>glgA</i> harbouring the AH-3 <i>glgA</i> (pBAD33-GlgA). anhKdo, an anhydro form of Kdo; M_r, calculated molecular mass (Da).</p

Biofilm values of several <i>Aeromonas</i> strains using the method of Pratt and Kolter [35].

<p>Biofilm values of several <i>Aeromonas</i> strains using the method of Pratt and Kolter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035707#pone.0035707-Pratt1" target="_blank">[35]</a>.</p