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In vitro identification of novel plasminogen-binding receptors of the pathogen Leptospira interrogans
Monica L Vieira1, Marina V Atzingen, Tatiane R Oliveira
1Centro de Biotecnologia, Instituto Butantan, São Paulo, São Paulo, Brazil.
Background:
Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin.
Methodology/Principal Findings:
We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin.
Conclusions/Significance:
PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.
Insights
Leptospira bacteria bind human plasminogen (PLG) using surface proteins. This interaction, involving lysine residues, may help the bacteria spread within the host by degrading tissue barriers.
Area of Science:
- Microbiology
- Molecular Biology
Background:
- Leptospirosis is a severe disease caused by Leptospira bacteria.
- Leptospira can bind plasminogen (PLG) and degrade extracellular matrix proteins.
Purpose of the Study:
- To identify and characterize Leptospira surface proteins involved in plasminogen binding.
- To understand the mechanism and significance of plasminogen binding in Leptospira pathogenesis.
Main Methods:
- Cloned, expressed, and purified 14 leptospiral recombinant proteins.
- Confirmed surface exposure using immunofluorescence microscopy.
- Assessed plasminogen binding, activation to plasmin, and inhibition by lysine analogs.
Main Results:
- Identified eight plasminogen-binding proteins, including LipL32 and a novel protein rLIC12238.
- Demonstrated that bound plasminogen can be activated to active plasmin.
- Showed plasminogen binding is specific, dose-dependent, saturable, and involves lysine residues.
Conclusions:
- Leptospira surface proteins bind and activate plasminogen.
- This interaction may facilitate bacterial invasion and dissemination by overcoming host tissue barriers.
