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Journal of Bacteriology, November 2007, p. 8353-8356, Vol. 189, No. 22
0021-9193/07/$08.00+0     doi:10.1128/JB.00620-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Pseudomonas aeruginosa Psl Is a Galactose- and Mannose-Rich Exopolysaccharide{triangledown} ,{dagger}

Luyan Ma,1 Haiping Lu,1 April Sprinkle,1 Matthew R. Parsek,2 and Daniel J. Wozniak1*

Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina,1 Department of Microbiology, University of Washington, Seattle, Washington2

Received 21 April 2007/ Accepted 4 July 2007


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ABSTRACT
 
The Pseudomonas aeruginosa polysaccharide synthesis locus (psl) is predicted to encode an exopolysaccharide which is critical for biofilm formation. Here we used chemical composition analyses and mannose- or galactose-specific lectin staining, followed by confocal laser scanning microscopy and electron microscopy, to show that Psl is a galactose-rich and mannose-rich exopolysaccharide.


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TEXT
 
Pseudomonas aeruginosa is an opportunistic human pathogen that can cause life-threatening infections in cystic fibrosis patients and individuals with a compromised immune system. This bacterium can form biofilms on surfaces such as the mucus plugs of the cystic fibrosis lung, contaminated catheters, and contact lenses (4, 6, 17). Bacteria in a biofilm are less susceptible to antimicrobial agents and are protected from the host immune response, giving rise to chronic infections that are notoriously difficult to eradicate (14, 20).

Exopolysaccharides (EPS) are an important component of the microbial biofilm extracellular matrix, since they contribute to overall biofilm architecture and to the resistance phenotype of bacteria in biofilms (2, 7, 18, 22). The psl gene cluster contains 15 cotranscribed genes (pslA to pslO) encoding proteins predicted to synthesize the Psl EPS, which is important to initiate and maintain biofilm structure by providing cell-cell and cell-surface interactions (3, 5, 8, 13, 15). In the present study, we used a Psl-inducible P. aeruginosa strain, WFPA801 ({Delta}psl pBAD-psl), and several psl mutants to study the chemical composition of the Psl EPS. This revealed that the Psl EPS is composed mainly of mannose and galactose and that Psl is indeed a matrix component of the biofilm. The results of the chemical composition analysis were confirmed using lectins that specifically detect the sugar structures in Psl EPS.

In a previous report, we showed that increased psl expression resulted in enhanced Congo red binding and increased biofilm formation, leading us to conclude that Psl is likely an extracellular matrix component of P. aeruginosa biofilms (13). In these studies, we utilized strain WFPA801 ({Delta}psl pBAD-psl), which has been described previously (see Table S1 in the supplemental material) (13). In WFPA801, psl transcription is increased sevenfold over wild-type levels with 2.0% arabinose (13). Since induced WFPA801 has significantly increased levels of Psl EPS, we used this strain to isolate the soluble EPS matrix material for further analyses. Bacteria were grown on cellophane sheets (Sigma) on Jensen's (9) agar plates with 2.0% arabinose as described previously (24, 25). After a 24-h incubation, the biomass was collected and resuspended in 0.9% NaCl. This cell suspension was centrifuged, and the supernatant was subsequently frozen and lyophilized. This EPS sample was used for carbohydrate monomer composition analysis, which was performed as previously outlined (24). The composition analyses showed that the WFPA801 EPS preparation contained galactose, mannose, and glucose, as well as trace amounts of xylose, rhamnose, and N-acetylglucosamine (GlcNAc) (Table 1). The results of three experiments showed that the EPS from WFPA801 was composed mainly of galactose and mannose. These two sugars were also found in the EPS sample purified from strain PAO1 by the above method (27% galactose and 6% mannose). These results suggest that Psl is a galactose- and mannose-rich EPS.


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  		TABLE 1. Chemical composition analysis of Psl EPS purified from surface-grown WFPA801 cultured with 2% arabinose

Support for the Psl chemical composition data comes from two additional lines of investigation. The psl operon contains 15 genes, which encode enzymes that have similarity to those involved in polysaccharide synthesis in other bacteria (5, 8, 15). The pslH- and pslI-encoded proteins exhibit homology to galactosyltransferases and mannosyltransferases, respectively. We used overlap extension PCR (21) with the appropriate primers (see Table S1 in the supplemental material) to generate clones that were used to create nonpolar, in-frame deletions of pslH (strain WFPA818) and pslI (strain WFPA819). The biofilm formation capacities of these strains were compared with those of wild-type and psl-deficient strains in a rapid attachment assay (Table 2). Loss of either PslH or PslI function results in a profound attachment defect, similar to that observed with the psl null strain WFPA800 (Table 2). The attachment defect of WFPA818 and WFPA819 was restored when a plasmid expressing either pslH (pMA10) or pslI (pMA11) was introduced into the respective strain (Table 2). Plasmids pMA10 and pMA11 were obtained by PCR amplification of pslH and pslI using primers pslH5 and pslH6 or pslI5 and pslI6, respectively (see Table S1 in the supplemental material). Collectively, these data indicate that PslH and PslI are key proteins for Psl EPS synthesis. In a prior transposon mutagenesis screen, pslH and pslI mutants also exhibited reduced biofilm formation (5).


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  		TABLE 2. Rapid attachment assays with P. aeruginosa strains PAO1, WFPA800 ({Delta}psl), WFPA818 ({Delta}pslH), and WFPA819 ({Delta}pslI) and complemented strainsa

Additional support for the psl operon encoding a galactose and mannose-containing EPS was obtained with lectin staining. We identified two lectins, HHA and MOA, which bind specifically to Psl EPS. HHA, which can detect either 1,3- or 1,6-linked mannosyl units in polysaccharides (1, 10), is derived from the plant Hippeastrum hybrid. MOA is a mushroom lectin specific for Gal{alpha}1,3Gal and Gal{alpha}1,3Galß1,4GlcNAc/Glc moieties present on the ends of glycan chains (12, 23). Fluorescently labeled (fluorescein isothiocyanate [FITC]) HHA and MOA lectins (EY Laboratories, Inc.) were evaluated by staining planktonically grown WFPA801 (Fig. 1A and B, top panels, respectively). WFPA801 cells pregrown in Jensen's medium with 2% arabinose were rinsed once with phosphate-buffered saline (PBS) before staining with 100 µg/ml FITC-labeled lectins. Staining was allowed to progress for 2 h at room temperature; samples were washed three times with PBS and then imaged with a Zeiss 510 confocal laser-scanning microscope (Carl Zeiss, Jena, Germany). The 63x/1.3 water objective was used for all image acquisition. For both lectins, fluorescent signals were associated with the WFPA801 cell surface. FITC-HHA- and FITC-MOA-stained PAO1 samples gave signals similar to, albeit weaker than, those for WFPA801 (Fig. 1A and B, bottom panels, respectively). Importantly, no fluorescent signals were observed with the psl-deficient WFAP800 bacteria (Fig. 1A and B, second row), which indicates that lectins HHA and MOA stain Psl EPS specifically. Additionally, no staining was observed when we used either lectin on planktoncially grown WFPA818 ({Delta}pslH) or WFPA819 ({Delta}pslI) (Fig. 1A and B, third and fourth rows, respectively). This result supports the data in Table 2 showing that Psl EPS synthesis requires both the PslH and PslI enzymes.


Figure 1
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  		FIG. 1. Mannose lectin HHA and galactose lectin MOA specifically stain the Psl EPS. (A) HHA-FITC staining of planktonic P. aeruginosa WFPA801, WFPA800, WFPA818 ({Delta}pslH), WFPA819 ({Delta}pslI), and PAO1 cultured in Jensen's medium with 2% arabinose. (B) MOA-FITC staining of planktonic P. aeruginosa cells cultured in Jensen's medium with 2% arabinose and depicted as in panel A.

To further confirm that Psl EPS contains galactose and mannose, we used gold-labeled lectin staining in conjunction with transmission electron microscopy (Fig. 2). We first used gold-labeled MOA to stain planktonically grown WFPA801 cells cultured under Psl-overproducing conditions. Staining and washing were conducted as described above, except that the gold-labeled lectins (EY laboratories, Inc.) were used at a concentration of 5 µg/ml in PBS. Gold-labeled-lectin-stained samples were fixed by 2% formaldehyde and 0.2% glutaraldehyde, thin sectioned, and observed by transmission electron microscopy (Philips 400, operated at 80 kv). The results showed that the gold-labeled MOA lectin particles localized to extracellular material (Fig. 2A). We also used MOA gold-labeled lectin to stain the biofilm of WFPA801 grown in a flow cell as previously described (13). Following the staining, the fixed biofilm was sectioned from the top to the surface side. Here, the MOA-gold particles bound mostly to material located between cells, although some gold-labeled lectin particles were located on or closely associated with the cell surface (Fig. 2B and C). MOA-gold-stained PAO1 biofilm exhibited a result similar to that for WFPA801 overexpressing Psl (Fig. 2D). As a control, the MOA-gold-treated WFPA800 sample showed little staining (Fig. 2E). The HHA gold-labeled lectin staining of WFPA801 biofilms showed results similar to those of the MOA lectin staining (Fig. 2, panels F to H). The HHA gold-labeled lectin stained the material between the bacterial cells, on the top of the biofilm (T in panel F), and between the cells and the surface (S in panel H). The material recognized by the lectins appeared to hold the bacterial cells together and to the surface. To determine if the two lectins detect the same EPS structure, we subjected the WFPA801 biofilm to double staining. The MOA and HHA gold-labeled lectins could be distinguished based on the size of the attached particle (5-nm gold for HHA and 10-nm gold for MOA). The results showed that the two lectins recognized the same material (Fig. 2I and J). Collectively, these data indicate that both HHA and MOA lectins bind specifically to the Psl EPS and provide additional independent evidence that mannose and galactose are components of the Psl EPS.


Figure 2
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  		FIG. 2. Gold-labeled lectin staining reveals that Psl encodes a mannose- and galactose-rich EPS. Cells were stained with 10-nm-size MOA-gold particles (panels A to E), 5-nm-size HHA-gold particles (panels F to H), or both MOA- and HHA-gold particles (panels I to J). All strains were cultured in Jensen's medium with 2% arabinose. Planktonically grown cells of P. aeruginosa WFPA801 (A), biofilm-grown cells of WFPA801 cultured in a flow cell for 20 h (B and C), biofilm-grown cells of PAO1 cultured in a flow cell for 20 h (D), biofilm-grown cells of WFPA800 cultured in a flow cell for 46-h (E), biofilm-grown cells of WFPA801 cultured in a flow cell for 20 h (F to H), and biofilm-grown cells of WFPA801 cultured in a flow cell for 20 h (I and J) are shown. T, top of the biofilm; S, side of the biofilm next to the surface. Arrows in panels I and J indicate the 5-nm-gold-labeled HHA lectin. Bar = 0.25 µm.

In this study, we showed that psl gene products synthesize an EPS containing mainly galactose and mannose, as well as glucose and trace amount of rhamnose, xylose, and GlcNAc. EPS prepared from WFPA801 contained a large amount of galactose, which has not been found in EPS preparations from PAO1 (5, 15, 24). This might be due to differences in purification protocols and the fact that Psl is being overproduced in strain WFPA801 grown in arabinose-containing medium. According to the MOA lectin staining results, Psl EPS contains a Gal{alpha}1,3Gal structure and possibly a Gal{alpha}1,3Galß1,4GlcNAc/Glc structure at the chain end. Consistent with the lectin staining, our preliminary linkage analyses of this Psl preparation showed a high percentage of 3-linked galactose (data not shown). HHA staining revealed that there are Man{alpha}1,3Man or Man{alpha}1,6Man structures in the Psl EPS. Man{alpha}1,3Man may be a main component, since little 6-linked mannose was detected when the Psl preparations were subjected to linkage analyses (data not shown) (5). More strikingly, HHA and MOA double staining indicates that the Gal{alpha}1,3Gal moiety and the Man{alpha}1,3Man structure colocalize to the same material found on and surrounding P. aeruginosa cells. Moreover, the HHA and MOA lectins detected sugar structures that are not present in the current published P. aeruginosa lipopolysaccharides (11, 19). Overall, our data indicate that the psl gene cluster synthesizes a mannose- and galactose-rich EPS that plays an important structural role in P. aeruginosa biofilms and provide useful information for future investigations aimed at resolving the Psl EPS structure.


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ACKNOWLEDGMENTS
 
We acknowledge Ken Grant, Micromed at WFUHS, for his assistance with microscopy.

This work was supported by Cystic Fibrosis Foundation grant MA06F0, Public Health Service grants AI061396 and HL58334 (D.J.W), and in part by the Department of Energy-funded (DE-FG09-93ER-20097) Center for Plant and Microbial Complex Carbohydrates at the University of Georgia.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1064. Phone: (336) 716-2016. Fax: (336) 716-9928. E-mail: dwozniak{at}wfubmc.edu Back

{triangledown} Published ahead of print on 13 July 2007. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Journal of Bacteriology, November 2007, p. 8353-8356, Vol. 189, No. 22
0021-9193/07/$08.00+0     doi:10.1128/JB.00620-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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