Mep72, a Metzincin Protease That Is Preferentially Secreted by Biofilms of Pseudomonas aeruginosa

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.

Characterization of a thaumarchaeal symbiont that drives incomplete nitrification in the tropical spongeIanthella basta

“…Ca. N. ianthellae possesses a putatively exported metalloprotease of the M72 family (Drapeau, 1980;Passmore et al, 2015) that was also detected in the metaproteome. This large protease (2027 AA) contains hyaline repeat domains suggestive of an additional adhesive property.…”

Which Microbial Factors Really are Important in Pseudomonas Aeruginosa Infections?

“…This was especially true of 'pleiotropic' mutants affecting multiple virulence phenotypes. For example, the cell-cell signaling mechanism, quorum sensing (QS) has been shown to exert decisive control over the synthesis of many secreted virulence factors in P. aeruginosa [4,6]. Consistent with this, QS mutants display drastically reduced virulence in vivo [7,8].…”

“…Moreover, in the right conditions, the manifold secretory systems (six at the last count) encoded by P. aeruginosa churn out vast quantities of these tissue-damaging exoenzymes, which in addition to the proteases, also include phospholipases, exotoxins and endotoxins (Figure 1). A vivid snapshot of the complexity and diversity of the secretome can be garnered by inspection of any of the manifold 2D-PAGE analyses of P. aeruginosa exoproteins that have been carried out over the years [4,5]. The importance of these virulence factors in infection was inferred from that fact that mutants defective in their synthesis, regulation or secretion often displayed reduced pathogenicity in for example, rodent pulmonary models of infection.…”

A Formidable Foe Is Sabotaging Your Results: What You Should Know about Biofilms and Wound Healing

“…They are a key component of the keratinization process in mammalian epidermis and function to maintain the permeability barrier and evaporative water loss functions of the skin [60, 61]. When bacterial infection is in a biofilm form it induces bacterial expression of proteases [62-64]. These bacterial derived proteases can also activate host matrix metalloproteases [63, 65-67] and stimulate neutrophil respiratory burst.…”