title: 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.
authors.affiliation: 1] State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China [2] Biotechnology Research Center, Shandong University, Jinan 250100, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.affiliation: 1] State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China [2] Biotechnology Research Center, Shandong University, Jinan 250100, China.
authors.forename: Hui-Lin
authors.initials: HL
authors.lastname: Zhao
authors.affiliation: 1] State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China [2] Biotechnology Research Center, Shandong University, Jinan 250100, China.
authors.forename: Li-Yuan
authors.initials: LY
authors.lastname: Ran
authors.affiliation: 1] State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China [2] Biotechnology Research Center, Shandong University, Jinan 250100, China.
authors.forename: Chun-Yang
authors.initials: CY
authors.lastname: Li
authors.affiliation: 1] State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China [2] Biotechnology Research Center, Shandong University, Jinan 250100, China.
authors.forename: Xi-Ying
authors.initials: XY
authors.lastname: Zhang
authors.affiliation: 1] State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China [2] Biotechnology Research Center, Shandong University, Jinan 250100, China.
authors.forename: Hai-Nan
authors.initials: HN
authors.lastname: Su
authors.affiliation: 1] State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China [2] Biotechnology Research Center, Shandong University, Jinan 250100, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Shi
authors.affiliation: Biotechnology Research Center, Shandong University, Jinan 250100, China.
authors.forename: Bai-Cheng
authors.initials: BC
authors.lastname: Zhou
authors.affiliation: 1] State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China [2] Biotechnology Research Center, Shandong University, Jinan 250100, China [3] Collaborative Innovation Center of Deep Sea Biology, Shandong University, Jinan 250100, China.
authors.forename: Xiu-Lan
authors.initials: XL
authors.lastname: Chen
authors.affiliation: 1] State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China [2] Biotechnology Research Center, Shandong University, Jinan 250100, China [3] Collaborative Innovation Center of Deep Sea Biology, Shandong University, Jinan 250100, China.
authors.forename: Yu-Zhong
authors.initials: YZ
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D002417:Cattle; D004549:Elastin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014333:Tropoelastin / Q000235:genetics / Q000378:metabolism; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014333:Tropoelastin; D037521:Virulence Factors; D004549:Elastin; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa