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Protein D2 porin of the Pseudomonas aeruginosa outer membrane bears the protease activity
E Yoshihara1, N Gotoh, T Nishino
1Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan.
Abstract:
We report here our discovery that protein D2 of the outer membrane of Pseudomonas aeruginosa is a novel porin bearing protease activity. Homogeneously purified protein D2 hydrolyzed several synthetic peptides according to the Michaelis-Menten kinetics. A specific serine protease inhibitor, diisopropyl fluorophosphate (DFP), inactivated the protease activity and [3H]DFP covalently labeled protein D2. We tested the effect of two monoclonal antibodies raised against protein D2 on the protease activity. One antibody lowered the protease activity to about 20%, while the other enhanced it to about 300% of that without antibody. In addition, the fractions derived from the outer membrane of the protein D2-deficient mutants showed negligible protease activity, whereas similarly fractionated outer membrane proteins of the protein D2-positive parent strain showed strong protease activity.
Insights
Pseudomonas aeruginosa outer membrane protein D2 is a novel porin with protease activity. This discovery reveals a new target for antibacterial strategies against this opportunistic pathogen.
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections.
- Outer membrane proteins play crucial roles in bacterial physiology and pathogenesis.
- The function of protein D2 in P. aeruginosa was previously unknown.
Purpose of the Study:
- To investigate the biochemical properties and function of protein D2 from Pseudomonas aeruginosa.
- To determine if protein D2 possesses enzymatic activity.
- To characterize the role of protein D2 in the outer membrane.
Main Methods:
- Purification of homogeneous protein D2.
- Enzymatic assays using synthetic peptides and Michaelis-Menten kinetics.
- Inhibition studies with diisopropyl fluorophosphate (DFP).
- Covalent labeling with [3H]DFP.
- Monoclonal antibody neutralization assays.
- Analysis of protein D2-deficient mutants.
Main Results:
- Protein D2 exhibits serine protease activity, hydrolyzing synthetic peptides.
- Protease activity is inhibited by DFP, which covalently binds to protein D2.
- Monoclonal antibodies modulate protein D2 protease activity, with one inhibiting and another enhancing it.
- Protein D2-deficient mutants show significantly reduced outer membrane protease activity compared to the parent strain.
Conclusions:
- Protein D2 is a novel outer membrane porin with intrinsic serine protease activity.
- This protease activity is essential for the overall protease function of the P. aeruginosa outer membrane.
- Protein D2 represents a potential new target for therapeutic intervention against P. aeruginosa infections.