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Characteristics of the biologically active 35-kDa metalloprotease virulence factor from Listeria monocytogenes
A Coffey1, B van den Burg, R Veltman
1Laboratory of Food Microbiology, Wageningen Agricultural University, The Netherlands.
Abstract:
Listeria monocytogenes, a facultative intracellular pathogen, synthesizes an extracellular protease which is responsible for the maturation of phosphatidylcholine phospholipase C (lecithinase), a virulence factor involved in cell-to-cell spread. This work describes the environmental parameters necessary for increased production of mature, 35-kDa active protease in strains of L. monocytogenes, and its detection using polyclonal antibodies raised against Bacillus subtilis neutral protease. High performance liquid affinity chromatography was exploited to isolate the biologically active form of the mature protease, which was then subjected to biochemical characterization using casein as a substrate. The protease is a zinc-dependent metalloprotease which degrades casein over a wide range of temperatures and pH values. It can also degrade actin, the most abundant protein in many eukaryotic cells. The Listeria protease was shown to exhibit a high thermal stability and a relatively narrow substrate specificity. A three-dimensional model built on the basis of the homology with thermolysin was used to understand the structural basis of these characteristics.
Insights
Listeria monocytogenes produces an extracellular protease essential for virulence. This study identifies optimal conditions for its production and characterizes its properties, revealing it as a heat-stable metalloprotease.
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- Listeria monocytogenes is a pathogen that spreads via cell-to-cell transmission.
- Extracellular protease production is crucial for Listeria monocytogenes virulence, specifically for lecithinase maturation.
- Understanding protease production and characteristics is key to developing control strategies.
Purpose of the Study:
- To determine environmental factors promoting the production of active Listeria monocytogenes protease.
- To isolate and biochemically characterize the mature protease.
- To elucidate the structural basis for the protease's stability and specificity.
Main Methods:
- Optimizing environmental parameters for protease production.
- Developing polyclonal antibodies for protease detection.
- Utilizing high-performance liquid affinity chromatography for isolation.
- Biochemical assays using casein and actin as substrates.
- Homology modeling for structural analysis.
Main Results:
- Identified environmental conditions for increased production of the 35-kDa active protease.
- Isolated the active protease and characterized it as a zinc-dependent metalloprotease.
- Demonstrated protease activity against casein across broad pH and temperature ranges.
- Showed the protease degrades actin and possesses high thermal stability with narrow substrate specificity.
- Developed a 3D model based on thermolysin homology.
Conclusions:
- Established optimal conditions for producing a key virulence factor of Listeria monocytogenes.
- Characterized the protease as a stable metalloprotease with implications for virulence.
- Structural insights provide a basis for understanding the protease's functional characteristics.