In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

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.FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico)Fundacao Butantan, BrazilFAPESP (Brazil

The Terminal Immunoglobulin-Like Repeats of LigA and LigB of Leptospira Enhance Their Binding to Gelatin Binding Domain of Fibronectin and Host Cells

Leptospira spp. are pathogenic spirochetes that cause the zoonotic disease leptospirosis. Leptospiral immunoglobulin (Ig)-like protein B (LigB) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin, fibrinogen, laminin, elastin, tropoelastin and collagen. A high-affinity Fn-binding region of LigB has been localized to LigBCen2, which contains the partial 11th and full 12th Ig-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, the gelatin binding domain of fibronectin was shown to interact with LigBCen2R (KD = 1.91±0.40 µM). Not only LigBCen2R but also other Ig-like domains of Lig proteins including LigAVar7'-8, LigAVar10, LigAVar11, LigAVar12, LigAVar13, LigBCen7'-8, and LigBCen9 bind to GBD. Interestingly, a large gain in affinity was achieved through an avidity effect, with the terminal domains, 13th (LigA) or 12th (LigB) Ig-like repeat of Lig protein (LigAVar7'-13 and LigBCen7'-12) enhancing binding affinity approximately 51 and 28 fold, respectively, compared to recombinant proteins without this terminal repeat. In addition, the inhibited effect on MDCKs cells can also be promoted by Lig proteins with terminal domains, but these two domains are not required for gelatin binding domain binding and cell adhesion. Interestingly, Lig proteins with the terminal domains could form compact structures with a round shape mediated by multidomain interaction. This is the first report about the interaction of gelatin binding domain of Fn and Lig proteins and provides an example of Lig-gelatin binding domain binding mediating bacterial-host interaction

Association of markers of endothelial activation and dysfunction with occurrence and outcome of pulmonary hemorrhage in dogs with leptospirosis

BACKGROUND: Endothelial dysfunction might contribute to the development of leptospiral pulmonary hemorrhage syndrome (LPHS). HYPOTHESIS: Serum concentrations of markers of endothelial activation and dysfunction are higher in dogs with leptospirosis and correlate with the occurrence of LPHS and a higher case fatality rate. ANIMALS: Clinically healthy dogs (n = 31; 10/31 dogs confirmed healthy based on no detected abnormalities on blood work), dogs with leptospirosis with LPHS (n = 17) and without LPHS (n = 15), dogs with acute kidney injury not due to leptospirosis (AKI‐nL, n = 34). METHODS: Observational study. Serum concentrations of soluble intercellular adhesion molecule 1 (sICAM‐1), vascular endothelial growth factor (VEGF), and angiopoietin‐2 (Ang‐2) at admission were compared between groups. Correlations with outcome and the accuracy to predict LPHS were examined. RESULTS: Soluble intercellular adhesion molecule (sICAM‐1), VEGF, and Ang‐2 concentrations were higher in dogs with AKI‐nL (sICAM‐1 34.7 ng/mL, interquartile range [IQR] = 24.4‐75.5; VEGF 43.1 pg/mL, IQR = 12.3‐79.2; Ang‐2 8.5 ng/mL, IQR = 6.2‐12.3), leptospirosis without LPHS (sICAM‐1 45.1 ng/mL, IQR = 30.6‐59.0; VEGF 32.4 pg/mL, IQR = 12.5‐62.6; Ang‐2 9.6 ng/mL, IQR = 6.9‐19.3), and LPHS (sICAM‐1 69.7 ng/mL, IQR = 42.1‐89.1; VEGF 51.8 pg/mL, IQR = 26.3‐96.7; Ang‐2 8.0 ng/mL, IQR = 5.6‐12.2) compared to controls (P < .001). In dogs with leptospirosis, VEGF and sICAM‐1 were higher in nonsurvivors (sICAM‐1 89.4 ng/mL, IQR = 76.5‐101.0; VEGF 117.0 pg/mL, IQR = 90.3‐232.4) than survivors (P = .004) and sICAM‐1 predicted the development of LPHS. CONCLUSIONS: Soluble intercellular adhesion molecule 1, VEGF, and Ang‐2 do not discriminate leptospirosis from AKI‐nL. In dogs with leptospirosis, sICAM‐1 and VEGF predict outcome and sICAM‐1 might identify dogs at risk for LPHS