Mep72, a metzincin protease that is preferentially secreted by biofilms of Pseudomonas aeruginosa

Ian J. Passmore, Kahoko Nishikawa, Kathryn S. Lilley, Steven Bowden, Jade C.S. Chung, Martin Welch

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

In this work, we compared the profile of proteins secreted by planktonic and biofilm cultures of Pseudomonas aeruginosa using two-dimensional difference gel electrophoresis (2D-DiGE). This revealed that a novel metzincin protease, Mep72, was secreted during biofilm growth. Subsequent Western blotting and reverse transcription-PCR (RT-PCR) analyses demonstrated that Mep72 was expressed only during biofilm growth. Mep72 has a tridomain structure comprised of a metzincin protease-like domain and two tandem carbohydrate-binding domains. Unlike the only other metzincin (alkaline protease; AprA) in P. aeruginosa, Mep72 is secreted through the type II pathway and undergoes processing during export. During this processing, the metzincin domain is liberated from the carbohydrate-binding domains. This processing may be self-catalyzed, since purified Mep72 autodegraded in vitro. This autodegradation was retarded in the presence of alginate (an extracellular matrix component of many P. aeruginosa biofilms). The expression of full-length mep72 in Escherichia coli was toxic. However, this toxicity could be alleviated by coexpression of mep72 with the adjacent gene, bamI. Mep72 and BamI were found to form a protein-protein complex in vitro. 2D-DiGE revealed that the electrophoretic mobility of several discrete protein spots was altered in the biofilm secretome of an mep72 mutant, including type III secretion proteins (PopD, PcrV, and ExoS) and a flagellum-associated protein (FliD). Mep72 was found to bind directly to ExoS and PcrV and to affect the processing of these proteins in the biofilm secretome. We conclude that Mep72 is a secreted biofilm-specific regulator that affects the processing of a very specific subset of virulence factors.

Original languageEnglish (US)
Pages (from-to)762-773
Number of pages12
JournalJournal of bacteriology
Volume197
Issue number4
DOIs
StatePublished - Jan 1 2015

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Biofilms
Pseudomonas aeruginosa
Peptide Hydrolases
Proteins
Two-Dimensional Difference Gel Electrophoresis
Carbohydrates
Flagella
Poisons
Virulence Factors
Growth
Reverse Transcription
Extracellular Matrix
Western Blotting
Escherichia coli
Polymerase Chain Reaction
Genes

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Mep72, a metzincin protease that is preferentially secreted by biofilms of Pseudomonas aeruginosa. / Passmore, Ian J.; Nishikawa, Kahoko; Lilley, Kathryn S.; Bowden, Steven; Chung, Jade C.S.; Welch, Martin.

In: Journal of bacteriology, Vol. 197, No. 4, 01.01.2015, p. 762-773.

Research output: Contribution to journalArticle

Passmore, Ian J. ; Nishikawa, Kahoko ; Lilley, Kathryn S. ; Bowden, Steven ; Chung, Jade C.S. ; Welch, Martin. / Mep72, a metzincin protease that is preferentially secreted by biofilms of Pseudomonas aeruginosa. In: Journal of bacteriology. 2015 ; Vol. 197, No. 4. pp. 762-773.
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