Dataset Information

Cell-wall remodeling by the zinc-protease AmpDh3 from Pseudomonas aeruginosa.

ABSTRACT: Bacterial cell wall is a polymer of considerable complexity that is in constant equilibrium between synthesis and recycling. AmpDh3 is a periplasmic zinc protease of Pseudomonas aeruginosa , which is intimately involved in cell-wall remodeling. We document the hydrolytic reactions that this enzyme performs on the cell wall. The process removes the peptide stems from the peptidoglycan, the major constituent of the cell wall. We document that the majority of the reactions of this enzyme takes place on the polymeric insoluble portion of the cell wall, as opposed to the fraction that is released from it. We show that AmpDh3 is tetrameric both in crystals and in solution. Based on the X-ray structures of the enzyme in complex with two synthetic cell-wall-based ligands, we present for the first time a model for a multivalent anchoring of AmpDh3 onto the cell wall, which lends itself to its processive remodeling.

SUBMITTER: Lee M

PROVIDER: S-EPMC3842012 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Cell-wall remodeling by the zinc-protease AmpDh3 from Pseudomonas aeruginosa.

Lee Mijoon M Artola-Recolons Cecilia C Carrasco-López César C Martínez-Caballero Siseth S Hesek Dusan D Spink Edward E Lastochkin Elena E Zhang Weilie W Hellman Lance M LM Boggess Bill B Hermoso Juan A JA Mobashery Shahriar S

Journal of the American Chemical Society 20130815 34

Bacterial cell wall is a polymer of considerable complexity that is in constant equilibrium between synthesis and recycling. AmpDh3 is a periplasmic zinc protease of Pseudomonas aeruginosa , which is intimately involved in cell-wall remodeling. We document the hydrolytic reactions that this enzyme performs on the cell wall. The process removes the peptide stems from the peptidoglycan, the major constituent of the cell wall. We document that the majority of the reactions of this enzyme takes plac ...[more]

PMID: 23931161

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Project description:Pseudomonas aeruginosa induces pathways indicative of low zinc availability in the cystic fibrosis (CF) lung environment. To learn more about P. aeruginosa zinc access in CF, we grew P. aeruginosa strain PAO1 directly in expectorated CF sputum. The P. aeruginosa Zur transcriptional repressor controls the response to low intracellular zinc, and we used the NanoString methodology to monitor levels of Zur-regulated transcripts, including those encoding a zincophore system, a zinc importer, and paralogs of zinc containing proteins that do not require zinc for activity. Zur-controlled transcripts were induced in sputum-grown P. aeruginosa compared to those grown in control cultures but not if the sputum was amended with zinc. Amendment of sputum with ferrous iron did not reduce expression of Zur-regulated genes. A reporter fusion to a Zur-regulated promoter had variable activity in P. aeruginosa grown in sputum from different donors, and this variation inversely correlated with sputum zinc concentrations. Recombinant human calprotectin (CP), a divalent-metal binding protein released by neutrophils, was sufficient to induce a zinc starvation response in P. aeruginosa grown in laboratory medium or zinc-amended CF sputum, indicating that CP is functional in the sputum environment. Zinc metalloproteases comprise a large fraction of secreted zinc-binding P. aeruginosa proteins. Here, we show that recombinant CP inhibited both LasB-mediated casein degradation and LasA-mediated lysis of Staphylococcus aureus, which was reversible with added zinc. These studies reveal the potential for CP-mediated zinc chelation to posttranslationally inhibit zinc metalloprotease activity and thereby affect the protease-dependent physiology and/or virulence of P. aeruginosa in the CF lung environment. IMPORTANCE The factors that contribute to worse outcomes in individuals with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa infections are not well understood. Therefore, there is a need to understand environmental factors within the CF airway that contribute to P. aeruginosa colonization and infection. We demonstrate that growing bacteria in CF sputum induces a zinc starvation response that inversely correlates with sputum zinc levels. Additionally, both calprotectin and a chemical zinc chelator inhibit the proteolytic activities of LasA and LasB proteases, suggesting that extracellular zinc chelators can influence proteolytic activity and thus P. aeruginosa virulence and nutrient acquisition in vivo.

Dataset Information

Cell-wall remodeling by the zinc-protease AmpDh3 from Pseudomonas aeruginosa.

Publications

Cell-wall remodeling by the zinc-protease AmpDh3 from Pseudomonas aeruginosa.

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