Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production

Abstract

Exopolysaccharides contribute significantly to attachment and biofilm formation in the opportunisitc pathogen Pseudomonas aeruginosa. The Psl polysaccharide, which is synthesized by the polysaccharide synthesis locus (psl), is required for biofilm formation in nonmucoid strains that do not rely on alginate as the principal biofilm polysaccharide. In-frame deletion and complementation studies of individual psl genes revealed that eleven psl genes, pslACDEFGHIJKL, are required for Psl production and surface attachment. We also present the first structural analysis of the psl-dependent polysaccharide, which consists of a repeating pentasaccharide containing d-mannose, d-glucose, and l-rhamnose: false[→3false)−normalβ−normalD−Manp2↑1α−D−Manp−false(1→3false)−normalβ−normalD−Manp−false(1→3false)−normalα−normalL−Rhap−false(1→3false)−normalβ−normalD−Glcp−false(1→false]normaln− In addition we identified the sugar nucleotide precursors involved in Psl generation and demonstrated the requirement for GDP-d-mannose, UDP-d-glucose, and dTDP-l-rhamnose in Psl production and surface attachment. Finally, genetic analyses revealed that wbpW restored Psl production in a pslB mutant and pslB promoted A-band LPS synthesis in a wbpW mutant, indicating functional redundancy and overlapping roles for these two enzymes. The structural and genetic data presented here provide a basis for further investigation of the Psl proteins and potential roles for Psl in the biology and pathogenesis of P. aeruginosa.

PslG, a self-produced glycosyl hydrolase, triggers biofilm disassembly by disrupting exopolysaccharide matrix

“…Addition of 2% arabinose decreased the biofilm biomass of PAO1/pHERD20T-pslG to a level similar to ΔpslG mutant (Supplementary information, Table S1 and Figure 1A). In contrast, the biofilm biomass was increased in Psl-inducible strain WFPA801 (psl promoter was replaced by araC-P BAD , Supplementary information, Table S1) due to elevated Psl production (Figure 1A), where the entire psl gene operon was induced by the P BAD promoter [20,23]. Given that the growth of PAO1 was not affected by PslG expression and arabinose concentration (Supplementary information, Figure S1A), these results suggested that the overexpression of PslG might decrease the production of Psl in PAO1.…”

“…Deletion of pslG led to loss of Psl production and reduction of biofilm formation in P. aeruginosa PAO1 strain [23]. However, the biofilm biomass was significantly reduced in PAO1/pHERD20T-pslG, where expression of the full-length PslG was driven by P BAD promoter in plasmid pHERD20T and was induced by arabinose.…”

“…PslG is an essential protein for the synthesis of Psl, which is predicted to be a periplasmic protein resembling β-D-xylosidases [23,29]. Deletion of pslG led to loss of Psl production and reduction of biofilm formation in P. aeruginosa PAO1 strain [23].…”

“…There are 12 co-transcribed psl genes (pslABCDEFGHIJKL) essential for Psl production [23]. Psl, a repeating pentasaccharide containing D-mannose, D-glucose and L-rhamnose [23], acts as 'molecular glue' to promote bacterial cell-cell and cell-surface interactions and it can form a fiber-like web to enmesh bacterial communities [6,7,17]. This exopolysaccharide also confers resistance of P. aeruginosa biofilms to antibiotics and phagocytic cells [9,[24][25][26].…”

“…P. aeruginosa cells often become mucoid upon prolonged colonization of the CF lung due to the overproduction of alginate that provides an advantage for P. aeruginosa in the airway of CF patients [13], while non-mucoid P. aeruginosa strains produce little alginate and use either Pel or Psl as the primary matrix exopolysaccharide [17,[20][21][22]. There are 12 co-transcribed psl genes (pslABCDEFGHIJKL) essential for Psl production [23]. Psl, a repeating pentasaccharide containing D-mannose, D-glucose and L-rhamnose [23], acts as 'molecular glue' to promote bacterial cell-cell and cell-surface interactions and it can form a fiber-like web to enmesh bacterial communities [6,7,17].…”