PslD Is a Secreted Protein Required for Biofilm Formation by Pseudomonas aeruginosa

The function of pslD, which is part of the psl operon from Pseudomonasaeruginosa, was investigated in this study. The psl operon isinvolved in exopolysaccharide biosynthesis and biofilm formation.An isogenic marker-free pslD deletion mutant of P. aeruginosaPAO1 which was deficient in the formation of differentiatedbiofilms was generated. Expression of only the pslD gene codingregion restored the wild-type phenotype. A C-terminal, hexahistidinetag fusion enabled the identification of PslD. LacZ and PhoAtranslational fusions with PslD indicated that PslD is a secretedprotein required for biofilm formation, presumably via its rolein exopolysaccharide export.

INTRODUCTION

Top

Introduction

Pseudomonas aeruginosa is an opportunistic human pathogen whichcauses severe infections in immunologically compromised patientsand is the major pathogen in cystic fibrosis patients. An importantvirulence mechanism is the formation of a mucoid biofilm. Secretedalginate is a crucial constituent of the mucoid biofilm matrix(16, 17). However, alginate-negative mutants of P. aeruginosaare also able to form nonmucoid biofilms, showing an architecturedifferent from that of biofilms formed by alginate-overproducingmucoid P. aeruginosa (13, 20). Previous investigations haveshown that the gene cluster PA2231-2245 (designated psl) iscotranscribed and required for nonmucoid biofilm formation (5,7, 12). The putative pslD gene (PA2234) is part of the psl operonand represents an open reading frame comprising 771 bp.

PslD sequence analysis.

Top

Psld sequence analysis.

PslD shows about 72% similarity to hypothetical proteins thatare derived from genomes of various pseudomonads and that havea predicted function in exopolysaccharide export. However, themost similar proteins with experimental and functional assignmentwere Wza_K30 from Escherichia coli strain E69 and Wza_Vv fromVibrio vulnificus, exerting similarities of 33% and 25% to PslD,respectively (4, 21). Only Wza_K30 has been experimentally demonstratedas a multimeric lipoprotein in the outer membrane of E. colistrain E69, which is presumably involved in the export of thegroup 1 capsular polysaccharides (4). A nonpolar mutation inthe gene putatively encoding Wza_Vv in Vibrio vulnificus didnot abolish capsular polysaccharide biosynthesis, but the polysaccharidewas residing in the periplasm and was not exported (21). ThePslD sequence analysis by LipoP 1.0 (3) showed a strong indicationfor a lipoprotein signal peptide recognized by signal peptidaseII, with a cleavage site between amino acid residues 15 and16 (19). Amino acid 16 is a cysteine residue which providesa potential target for lipid modification, enabling anchoringin the outer membrane. No transmembrane domains were predictedboth by the dense alignment surface method (2) and by TMHMM2.0 (9). Signal peptides for lipoproteins are described as containinga positively charged region at the N terminus, a hydrophobicregion, and a lipobox with a four-amino-acid consensus region(1). In PslD, the first two amino acid residues (MK) are considereda positively charged region at the N terminus, the hydrophobicTLLMLAMLA sequence is the predicted hydrophobic region, andthe lipobox sequence is LAAC. A Conserved Domain Database (11)search shows that PslD contains a conserved pfam02563 domain,which is conserved in a family of outer membrane auxiliary proteinsinvolved in polysaccharide export.

Generation of the isogenic pslD knockout mutant of P. aeruginosa PAO1.

Top

Generation of the isogenic...

The isogenic pslD knockout mutant was obtained and verifiedas described by Pham et al. (15). The inserted gentamicin cassettewas removed as previously described (18), resulting in the marker-freeP. aeruginosa PAO1 ${Delta}$ pslD mutant containing a deletion of 170 bpin the chromosomal pslD gene. P. aeruginosa PAO1 ${Delta}$ pslD showeda biofilm-negative phenotype in the abiotic solid surface assay(SSA) for biofilm formation (Table 1) and in flow chamber analysis(Fig. 1).

View this table:
[in this window]
[in a new window]
TABLE 1. SSA analysis and reporter enzyme activity of various P. aeruginosa strains^a

View larger version (125K):
[in this window]
[in a new window]
FIG. 1. Fluorescence microscopy analysis of biofilms formed by P. aeruginosa after 3 days of growth in flow cell chambers. (A) P. aeruginosa PAO1. (B) P. aeruginosa PAO1 ${Delta}$ pslD. (C) P. aeruginosa PAO1 ${Delta}$ pslD(pBBR1-MCS5). (D) P. aeruginosa PAO1 ${Delta}$ pslD(pBBR1-MCS5::pslD).

Biofilm analysis.

Top

Biofilm analysis.

For biofilm analysis, P. aeruginosa strains were grown in mineralmedium continuous-culture flow cells (channel dimensions, 1by 1 by 120 mm) at room temperature as previously described(14). Channels were inoculated with 0.5 ml of early-stationary-phasecultures containing approximately 2 x 10⁹ cells ml^–1 andincubated without flow for 4 h at room temperature. Flow wasthen started with a mean flow of 0.3 ml min^–1, correspondingto a laminar flow with a Reynolds number of 5. Biofilms werestained and visualized using the LIVE/DEAD BacLight bacterialviability kit (Molecular Probes Inc., Eugene, Oreg.). The wild-typebiofilm was homogenous and showed large microcolonies. A strongprevalence of living cells was observed. The PAO1 ${Delta}$ pslD mutantwas unable to form such complex biofilms and was characterizedby small clusters of cells not developing into the typical microcolonies(Fig. 1). This finding is consistent with the previously identifiedstrong induction of the transcription of the psl operon insidemicrocolonies, which suggested an important function of thepsl operon in microcolony formation (14). For quantitative analysisof biofilm formation, the abiotic SSA was applied as describedpreviously (14).

Complementation of the P. aeruginosa PAO1 ${Delta}$ pslD mutant.

Top

Complementation of the p....

For complementation of the P. aeruginosa PAO1 ${Delta}$ pslD mutant, thecoding region of the pslD gene was cloned into the broad-host-rangevector pBBR1-MCS5 (8), resulting in plasmid pBBR1-MCS5::pslD.The pslD gene was inserted downstream of the lac promoter andafter conjugational transfer, the biofilm formation wild-typephenotype was restored (Table 1 and Fig. 1). Flow cell chamberanalysis showed that the ${Delta}$ pslD deletion mutant harboring pBBR1-MCS5::pslDshowed a fully differentiated and mature biofilm, but LIVE/DEADstaining revealed a larger fraction of dead cells for the maturebiofilm than for the wild-type biofilm. Furthermore, the biofilmshowed additional smaller cell clusters and appeared to be slightlymore heterogenous than the wild-type biofilm. A C-terminal,hexahistidine-tagged PslD protein encoded by plasmid pBBR1-MCS5::HTpslD,constructed as described above, also mediated restoration ofthe biofilm-forming phenotype (Table 1 and Fig. 1).

Detection of PslD.

Top

Detection of psld.

The C-terminal, hexahistidine-tagged PslD protein, with an apparentmolecular mass of 29 kDa, was detected in whole-cell lysatesof P. aeruginosa PAO1 ${Delta}$ pslD harboring plasmid pBBR1-MCS5::HTpslD.Proteins were separated by sodium dodecyl sulfate-polyacrylamidegel electrophoresis and subjected to immunoblot analysis, usingthe SuperSignal West HisProbe kit (Pierce Biotechnology Inc.)according to the manufacturer's protocol (Fig. 2).

View larger version (62K):
[in this window]
[in a new window]
FIG. 2. Immunoblotting of whole-cell lysates of P. aeruginosa strains harboring various plasmids. Antihexahistidine antibodies were used to detect C-terminal, hexahistidine-tagged PslD. Lane 1, molecular mass standard of hexahistidine-tagged proteins; lane 2, P. aeruginosa PAO1; lane 3, P. aeruginosa PAO1 ${Delta}$ pslD(pBBR1-MCS5); and lane 4, P. aeruginosa PAO1 ${Delta}$ pslD(pBBR1-MCS5::HTpslD). The arrow indicates the position of C-terminal, hexahistidine-tagged PslD.

Subcellular localization of PslD.

Top

Subcellular localization of...

The primary structure analysis of PslD suggested a periplasmicand/or outer membrane localization. Translational fusions ofthe reporter enzymes LacZ and PhoA to the C terminus of PslDwere obtained using the respective vectors pJE608 and pJE609as previously described (6, 18). Only the PslD-PhoA fusion wasfunctional and restored biofilm formation in P. aeruginosa PAO1 ${Delta}$ pslD(Table 1). Alkaline phosphatase and ß-galactosidaseenzymatic assays were performed according to the methods ofMiller (12a) and Manoil (10), respectively. Reporter enzymeassays showed both high specific alkaline phosphatase activityand ß-galactosidase activity in P. aeruginosa PAO1 ${Delta}$ pslDmutants harboring the respective plasmids (Table 1). The functionalPslD-PhoA fusion provides evidence for secretion and localizationin the periplasm and/or outer membrane. Immunological detectionof PslD-His₆ in whole-cell lysates provided evidence that theprotein is not released and remains attached to the cells. Sinceno hydrophobic domains are present in PslD and no transmembranehelices are predicted, PslD might not be attached to the cytoplasmicmembrane. Overall, these data indicate that the pslD gene encodesa protein which is localized in the periplasm/outer membranebelonging to the outer membrane auxiliary protein family andwhich is required for the export of a biofilm-relevant exopolysaccharide.

ACKNOWLEDGMENTS

This study was supported by the Massey University Research Fundand the Massey University Postdoctoral Fellowship.

We thank Zoe Jordens for assistance in biofilm experiments.

FOOTNOTES

* Corresponding author. Mailing address: Institute of Molecular Sciences, Massey University, Private Bag 11222, Palmerston North, New Zealand. Phone: 64 6 350 5515 7890. Fax: 64 6 350 5688. E-mail: B.Rehm{at}massey.ac.nz.

REFERENCES

Top