Abstract
BackgroundLeptospirosis 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 FindingsWe 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/SignificancePLG-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.
Highlights
Leptospirosis is an emerging infectious disease with worldwide distribution and a zoonosis that is of human and veterinary concern
The minima at 208 and 222 nm, and the maximum at 192 nm in the Circular dichroism (CD) spectrum showed the high a-helical secondary structure content of the recombinant proteins LipL40, MPL36 and rLIC12922, while rLIC12238 showed a predominant signal of b-strands, with minimum ellipticity around 215 nm
The interaction of the human plasminogen system has been suggested to be a feature that significantly contributes to the virulence of many bacterial pathogens by facilitating the initial anchoring to the endothelium [20]
Summary
Leptospirosis is an emerging infectious disease with worldwide distribution and a zoonosis that is of human and veterinary concern. Pathogenic Leptospira are the etiological agents of leptospirosis, a disease with greater incidence in tropical and subtropical regions [1,2,3,4,5]. It is well documented that the interaction of pathogens with the extracellular matrix (ECM) may play a primary role in the colonization of host tissues, as long-lasting infections may occur if microorganisms reach the sub-epithelial tissues [10]. The proteolytic activity achieved by subversion of host proteases by pathogens, such as plasmin, has been demonstrated to be important in various bacterial infections [20]. 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
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