Dataset Information

Structural and Biochemical Analysis of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from the Opportunistic Pathogen Pseudomonas aeruginosa PAO1.

ABSTRACT: Biofilms are important for cell communication and growth in most bacteria, and are responsible for a number of human clinical infections and diseases. TpbA (PA3885) is a dual specific tyrosine phosphatase (DUSP) that negatively regulates biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa PAO1 by converting extracellular quorum sensing signals into internal gene cascade reactions that result in reduced biofilm formation. We have determined the three-dimensional crystal structure of wild-type TpbA from P. aeruginosa PAO1 in the phosphate-bound state and a TpbA (C132S) mutant with phosphotyrosine. Comparison between the phosphate-bound structure and the previously reported ligand-free TpbA structure reveals the extent of conformational changes that occur upon substrate binding. The largest changes occur in the functional loops that define the substrate binding site, including the PTP, general acid and ?4-?5 loops. We further show that TpbA efficiently catalyzes the hydrolysis of two phosphotyrosine peptides derived from the periplasmic domain of TpbB (YfiN, PA1120), with a strong preference for dephosphorylating Tyr48 over Tyr62. This work adds to the small repertoire of DUSP structures in both the ligand-free and ligand-bound states, and provides a starting point for further study of the role of TpbA in biofilm formation.

SUBMITTER: Xu K

PROVIDER: S-EPMC4409338 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Structural and Biochemical Analysis of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from the Opportunistic Pathogen Pseudomonas aeruginosa PAO1.

Xu Kun K Li Shanshan S Yang Wen W Li Kan K Bai Yuwei Y Xu Yueyang Y Jin Jin J Wang Yingying Y Bartlam Mark M

PloS one 20150424 4

Biofilms are important for cell communication and growth in most bacteria, and are responsible for a number of human clinical infections and diseases. TpbA (PA3885) is a dual specific tyrosine phosphatase (DUSP) that negatively regulates biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa PAO1 by converting extracellular quorum sensing signals into internal gene cascade reactions that result in reduced biofilm formation. We have determined the three-dimensional crystal structu ...[more]

PMID: 25909591

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Project description:With the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing based on homoserine lactones was found to influence biofilm formation. Here we discern a mechanism by which quorum sensing controls biofilm formation by screening 5850 transposon mutants of P. aeruginosa PA14 for altered biofilm formation. This screen identified the PA3885 mutant, which had 147-fold more biofilm than the wild-type strain. Loss of PA3885 decreased swimming, abolished swarming, and increased attachment, although this did not affect production of rhamnolipids. The PA3885 mutant also had a wrinkly colony phenotype, formed pronounced pellicles, had substantially more aggregation, and had 28-fold more exopolysaccharide production. Expression of PA3885 in trans reduced biofilm formation and abolished aggregation. Whole transcriptome analysis showed that loss of PA3885 activated expression of the pel locus, an operon that encodes for the synthesis of extracellular matrix polysaccharide. Genetic screening identified that loss of PelABDEG and the PA1120 protein (which contains a GGDEF-motif) suppressed the phenotypes of the PA3885 mutant, suggesting that the function of the PA3885 protein is to regulate 3,5-cyclic diguanylic acid (c-di-GMP) concentrations as a phosphatase since c-di-GMP enhances biofilm formation by activating PelD, and c-di-GMP inhibits swarming. Loss of PA3885 protein increased cellular c-di-GMP concentrations; hence, PA3885 protein is a negative regulator of c-di-GMP production. Purified PA3885 protein has phosphatase activity against phosphotyrosine peptides and is translocated to the periplasm. Las-mediated quorum sensing positively regulates expression of the PA3885 gene. These results show that the PA3885 protein responds to AHL signals and likely dephosphorylates PA1120, which leads to reduced c-di-GMP production. This inhibits matrix exopolysaccharide formation, which leads to reduced biofilm formation; hence, we provide a mechanism for quorum sensing control of biofilm formation through the pel locus and suggest PA3885 should be named TpbA for tyrosine phosphatase related to biofilm formation and PA1120 should be TpbB.

Project description:Among various environmental stresses, the depletion of inorganic phosphate, which is an important signaling molecule that modulates virulence production, is notable. In our previous study, we demonstrated that lapA, encoding alkaline phosphatase, plays a key role in Pseudomonas aeruginosa biofilm formation in porcine skin explants, an ex vivo chronic wound model. However, the mechanism by which lapA participates in the virulence and biofilm formation of P. aeruginosa remains unclear. In the present study, the lapA deletion mutant was constructed in P. aeruginosa PAO1, and the phenotypic characterization was assessed. Our data demonstrated that phosphate depletion stress significantly enhanced elastase activity, hemolysis, rhamnolipid production, swarming and swimming motilities, and 3-oxo-C12-homoserine lactone (HSL) production in P. aeruginosa. Moreover, phosphate starvation evidently induced the virulence of P. aeruginosa in Caenorhabditis elegans (C. elegans) through fast-kill and slow-kill infections. Deletion of the lapA gene led to broad phenotypic changes, including reduced elastase activity, swimming motility, C4-HSL, and 3-oxo-C12-HSL production, as well as increased chitinase activity and rhamnolipid production under phosphate depletion stress, which was unrelated to C4-HSL. Deletion of the lapA gene also significantly inhibited PAO1 biofilm formation in porcine skin explants by reducing the expression levels of las and rhl quorum sensing systems and extracellular polymeric substance synthesis. Finally, lapA deletion also reduced the virulence of PAO1 in C. elegans in fast-kill and slow-kill infection assays. Thus, the findings suggest that targeting lapA can help control biofilm formation and reduce virulence in nonhealing-infected wounds, where P. aeruginosa is a persistent problem. IMPORTANCE Our previous study demonstrated that the expression of lapA was induced under phosphate depletion conditions, but its roles in virulence and biofilm formation by Pseudomonas aeruginosa remain largely unknown. This study presents a systematic investigation of the roles of lapA in virulence induction and biofilm formation by constructing a lapA-deficient strain with P. aeruginosa PAO1. The results showed that deletion of the lapA gene evidently reduced elastase activity, swimming motility, C4-HSL, and 3-oxo-C12-HSL production, and increased rhamnolipid production under phosphate depletion stress. Moreover, lapA gene deletion inhibited PAO1 biofilm formation in porcine skin explants by reducing the expression levels of las and rhl quorum sensing systems and extracellular polymeric substance synthesis. Finally, lapA gene deletion also reduced the virulence of PAO1 in Caenorhabditis elegans in fast-kill and slow-kill infection assays. This study provides insights into the roles of lapA in modulating P. aeruginosa virulence and biofilm formation under phosphate depletion stress.

Dataset Information

Structural and Biochemical Analysis of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from the Opportunistic Pathogen Pseudomonas aeruginosa PAO1.

Publications

Structural and Biochemical Analysis of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from the Opportunistic Pathogen Pseudomonas aeruginosa PAO1.

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