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Mechanistic insights into elastin degradation by pseudolysin, the major virulence factor of the opportunistic pathogen Pseudomonas aeruginosa.

ABSTRACT: Pseudolysin is the most abundant protease secreted by Pseudomonas aeruginosa and is the major extracellular virulence factor of this opportunistic human pathogen. Pseudolysin destroys human tissues by solubilizing elastin. However, the mechanisms by which pseudolysin binds to and degrades elastin remain elusive. In this study, we investigated the mechanism of action of pseudolysin on elastin binding and degradation by biochemical assay, microscopy and site-directed mutagenesis. Pseudolysin bound to bovine elastin fibers and preferred to attack peptide bonds with hydrophobic residues at the P1 and P1' positions in the hydrophobic domains of elastin. The time-course degradation processes of both bovine elastin fibers and cross-linked human tropoelastin by pseudolysin were further investigated by microscopy. Altogether, the results indicate that elastin degradation by pseudolysin began with the hydrophobic domains on the fiber surface, followed by the progressive disassembly of macroscopic elastin fibers into primary structural elements. Moreover, our site-directed mutational results indicate that five hydrophobic residues in the S1-S1' sub-sites played key roles in the binding of pseudolysin to elastin. This study sheds lights on the pathogenesis of P. aeruginosa infection.

SUBMITTER: Yang J

PROVIDER: S-EPMC4407726 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Mechanistic insights into elastin degradation by pseudolysin, the major virulence factor of the opportunistic pathogen Pseudomonas aeruginosa.

Yang Jie J Zhao Hui-Lin HL Ran Li-Yuan LY Li Chun-Yang CY Zhang Xi-Ying XY Su Hai-Nan HN Shi Mei M Zhou Bai-Cheng BC Chen Xiu-Lan XL Zhang Yu-Zhong YZ

Scientific reports 20150423

Pseudolysin is the most abundant protease secreted by Pseudomonas aeruginosa and is the major extracellular virulence factor of this opportunistic human pathogen. Pseudolysin destroys human tissues by solubilizing elastin. However, the mechanisms by which pseudolysin binds to and degrades elastin remain elusive. In this study, we investigated the mechanism of action of pseudolysin on elastin binding and degradation by biochemical assay, microscopy and site-directed mutagenesis. Pseudolysin bound ...[more]

PMID: 25905792

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Project description:It is generally thought that mucosal fluids protect underlying epithelial surfaces against opportunistic infection via their antimicrobial activity. However, our published data show that human tear fluid can protect against the major opportunistic pathogen Pseudomonas aeruginosa independently of bacteriostatic activity. Here, we explored the mechanisms for tear protection, focusing on impacts of tear fluid on bacterial virulence factor expression. Results showed that tear fluid suppressed twitching motility, a type of surface-associated movement conferred by pili. Previously, we showed that twitching is critical for P. aeruginosa traversal of corneal epithelia, exit from epithelial cells after internalization, and corneal virulence. Inhibition of twitching by tear fluid was dose-dependent with dilutions to 6.25% retaining activity. Purified lactoferrin, lysozyme, and contrived tears containing these, and many other, tear components lacked the activity. Systematic protein fractionation, mass spectrometry, and immunoprecipitation identified the glycoprotein DMBT1 (Deleted in Malignant Brain Tumors 1) in tear fluid as required. DMBT1 purified from human saliva also inhibited twitching, as well as P. aeruginosa traversal of human corneal epithelial cells in vitro, and reduced disease pathology in a murine model of corneal infection. DMBT1 did not affect PilA expression, nor bacterial intracellular cyclicAMP levels, and suppressed twitching motility of P. aeruginosa chemotaxis mutants (chpB, pilK), and an adenylate cyclase mutant (cyaB). However, dot-immunoblot assays showed purified DMBT1 binding of pili extracted from PAO1 suggesting that twitching inhibition may involve a direct interaction with pili. The latter could affect extension or retraction of pili, their interactions with biotic or abiotic surfaces, or cause their aggregation. Together, the data suggest that DMBT1 inhibition of twitching motility contributes to the mechanisms by which mucosal fluids protect against P. aeruginosa infection. This study also advances our understanding of how mucosal fluids protect against infection, and suggests directions for novel biocompatible strategies to protect our surface epithelia against a major opportunistic pathogen.

Dataset Information

Mechanistic insights into elastin degradation by pseudolysin, the major virulence factor of the opportunistic pathogen Pseudomonas aeruginosa.

Publications

Mechanistic insights into elastin degradation by pseudolysin, the major virulence factor of the opportunistic pathogen Pseudomonas aeruginosa.

Similar Datasets

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