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 non-mucoid strains that do not rely on alginate as the principal biofilm polysaccharide. In-frame deletion and complementation studies of individual psl genes revealed that 11 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: [See text]. 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.

Figure 1

Structure of the psl operon and confirmation of Psl-specific antisera. A. Map of the psl operon in PAO1 and selected mutants used in this study. Genes pslA-O are shown in boxes (not to scale), with the color corresponding to the putative functions assigned to the gene products, shown in the lower panel. Functions were assigned using bioinformatics by three independent sources (Friedman and Kolter, 2004b; Jackson et al., 2004; Stover et al., 2000). Angled lines represent the extent of deleted sequence, and black arrows indicate transcriptional start sites (not to scale). B. Crude polysaccharide extracts from PAO1, WFPA800, WFPA826, and WFPA801 were blotted on nitrocellulose, probed with α-Psl, and detected by chemiluminescence using a HRP-conjugated secondary antibody. Extract from WFPA801 was diluted 1:50 in order to remain within range of detection.

Figure 2

Mutagenesis of psl genes reveals that pslACDEFGHIJKL are necessary for Psl synthesis and P. aeruginosa attachment. A. Solid surface attachment of psl single mutants correlates with Psl production. Crystal violet staining, read at 540 nm, shown as a percentage of PAO1. The promoter deletion strain WFPA800 is notated as Δpr in both A and B. Values are mean ± SEM from two experiments each in triplicate. Psl production by α-Psl immunoblot is shown below each bar. B. Psl is produced by eleven of fifteen psl single mutants. ELISA performed using plates coated with crude polysaccharide extracts and probed with α-Psl, read at 450 nm. Values are mean ± SEM from two experiments each in triplicate; dashed line shows background signal of extracts derived from the Δpr strain, WFPA800. The mass of Psl is normalized to OD₆₀₀ equivalents used to generate the extracts.

Figure 3

Fractionation of carbohydrate extracts reveals a psl-dependent polysaccharide. A. Typical elution profile of crude carbohydrate extract of the growth medium (1 L) of P. aeruginosa psl-inducible strain WFPA801 on a Sephadex G-50 column. Cells were grown in M63 medium (1 L) with addition of l-arabinose (0.4 %). Fractions A, B, and C are as indicated. Fractions B and C contain polysaccharide with ∼3-5 and ∼1-2 pentasaccharide repeating units, respectively. Fraction C was used for detailed NMR analysis. B. Elution profile of crude carbohydrate extracts of the growth medium of P. aeruginosa psl-inducible strain WFPA801 (closed circles) and psl promoter deletion strain WFPA800 (open circles) on a Sephadex G-50 column. Cells were grown in 0.5 L LBNS with addition of 2.0% l-arabinose. A high MW fraction (HMW) and two low MW fractions (LMW 1 and LMW 2) are as indicated. C. Antiserum reactivity with WFPA801 and WFPA800 cell-associated polysaccharide fractions from B screened by immunoblot analysis.

Figure 4

Partial 2D HSQC spectrum of Psl and the integration data of signals, characteristic for a single pentasaccharide repeating unit (structure I) and its oligomers. I, integral value.

Figure 5

Pathways involved in synthesis of Psl sugar nucleotide precursors. Terminal steps of GDP-d-Man, UDP-d-Glc, and dTDP-l-Rha synthesis pathways shown. Sugar nucleotides are shown in boxes and enzymes deleted in this study are underlined. In the Psl repeating unit s`, larger box, p indicates the pyranosyl form of the monosaccharide residue. PGI, phosphoglucose isomerase.

Figure 6

dTDP-l-Rha and UDP-d-Glc are essential for Psl production. Deletion of rmlC or galU results in defective surface attachment due to loss of Psl, while loss of rmd does not affect attachment despite a reduction in Psl. Crystal violet staining, read at 540 nm, shown as a percentage of PAO1. The promoter deletion strain WFPA800 is notated as Δpr. Values are mean ± SEM from two or three experiments. Psl production by α-Psl immunoblot shown below each bar.

Figure 7

WbpW is the PMI/GMP in Psl synthesis in the absence of PslB. A. Deletion of pslB and wbpW results in an attachment defect and loss of Psl. Crystal violet staining, read at 540 nm, shown as a percentage of PAO1. The promoter deletion strain WFPA800 is notated as Δpr in both A and B. Values are mean ± SEM from three experiments. Psl production by α-Psl immunoblot shown below each bar. B. Psl is produced by a wbpW mutant but not by a pslB wbpW double mutant. ELISA performed using plates coated with crude polysaccharide extracts and probed with α-Psl, read at 450 nm. Values are mean ± SEM from two experiments each in triplicate; dashed line shows background signal of extracts derived from the Δpr strain, WFPA800. The mass of Psl is normalized to OD₆₀₀ equivalents used to generate the extracts.

Figure 8

Attachment phenotypes of pslB and wbpW single and double mutants. Cells were allowed to attach to vertically oriented glass coverslips for 4 h, then fixed and visualized by SEM. A, PAO1; B, WFPA800; C, pslB; D, wbpW; E, pslB wbpW. Larger images taken at 170×, inset images taken at 2500×. White boxes indicate the magnified region of the air-liquid interface shown in inset.

Figure 9

PslB is the PMI/GMP in A-band LPS synthesis in the absence of WbpW. A. A-band LPS is detectable in a wbpW mutant but not in a pslB wbpW double mutant. LPS was purified from P. aeruginosa grown on plates 12 h at 37°C. Western blot analysis of PAO1, WFPA800 (notated as Δpr), pslB, wbpW, and pslB wbpW, using the A-band-specific mAb N1F10. B. B-band LPS is unaffected in mutant P. aeruginosa strains. Western blot analysis of strains from A, using the B-band-specific mAb MF15-4.