Evaluation of two novel leptospiral proteins for their interaction with human host components

Pathogenic species of the genus Leptospira are the etiological agents of leptospirosis, the most widespread zoonosis. Mechanisms involved in leptospiral pathogenesis are not well understood. By data mining the genome sequences of Leptospira interrogans we have identified two proteins predicted to be surface exposed, LIC10821 and LIC10064. Immunofluorescence and proteinase K assays confirmed that the proteins are exposed. Reactivity of the recombinant proteins with human sera has shown that rLIC10821, but not rLIC10064, is recognized by antibodies in confirmed leptospirosis serum samples, suggesting its expression during infection. The rLIC10821 was able to bind laminin, in a dose-dependent fashion, and was called Lsa37 (leptospiral surface adhesin of 37 kDa). Studies with human plasma components demonstrated that rLIC10821 interacts with plasminogen (PLG) and fibrinogen (Fg). The binding of Lsa37 with PLG generates plasmin when PLG activator was added. Fibrin clotting reduction was observed in a thrombin-catalyzed reaction, when Fg was incubated with Lsa37, suggesting that this protein may interfere in the coagulation cascade during the disease. Although LIC10064 protein is more abundant than the corresponding Lsa37, binding activity with all the components tested was not detected. Thus, Lsa37 is a novel versatile adhesin that may mediate Leptospira-host interactions

Mammalian cell entry (Mce) protein of Leptospira interrogans binds extracellular matrix components, plasminogen and 2 integrin

A severe re-emergingzoonosis, leptospirosis, is caused by pathogenic spirochetes of the genus Leptospira. Several studies have identified leptospiral surface proteins with the ability to bind ECM and plasma components, which could mediate adhesion and invasion through the hosts. It has been shown that Mce of pathogenic Leptospira spp. is an RGD (Arg-Gly-Asp)-motif-dependent virulence factor, responsible for infection of cells and animals. In the present article, we decided to further study the repertoire of the Mce activities in leptospiral biological properties. We report that the recombinant Mce is a broad-spectrum ECM-binding protein, capable of interacting with laminin, cellular and plasma fibronectin and collagen IV. Dose-response interaction was observed for all the components, fulfilling ligand-receptor requirements. Mce is a PLG binding protein capable to recruit this component from NHS, generating PLA in the presence of PLG activator. Binding of Mce was also observed with the leukocyte cell receptors L2 [(CD11a/CD18)-LFA-1] and M2 [(CD11b/CD18)-Mac-1], suggesting the involvement of this protein in the host immune response. Indeed, virulent Leptospira L1-130 was capable of binding both integrins, whereas culture-attenuated M-20 strain only bind to M2 [(CD11b/CD18)-Mac-1]. To the best of our knowledge, this is the first work to describe that Mce surface protein could mediate the attachment of Leptospira interrogans to human cell receptors L2(CD11a/CD18) and M2(CD11b/CD18)

Leptospira interrogans Lsa23 protein recruits plasminogen, factor H and C4BP from normal human serum and mediates C3b and C4b degradation

It has been reported that pathogenic Leptospira are resistant to normal human serum (NHS) due to their ability to evade the complement immune system by interacting with factor H (FH) and C4b-binding protein (C4BP) regulators. Moreover, plasmin generation on the leptospiral surface diminishes C3b and IgG deposition, decreasing opsonophagocytosis by immune competent cells. We have previously reported that Lsa23 (LIC11360) is a multipurpose protein capable of binding purified extracellular matrix molecules, FH, C4BP and plasminogen (PLG)/plasmin in the presence of PLG activators. In this work, we provide further evidence that Lsa23 is located at the bacterial surface by using immunofluorescence microscopy. We show that Lsa23 has the ability to acquire FH, C4BP and PLG from NHS, and use these interactions to evade innate immunity. The binding with the complement regulators FH and C4BP preserves factor I (FI) activity, leading to C3b and C4b degradation products, respectively. C3b and C4b alpha-chain cleavage was also observed when Lsa23 bound to PLG generating plasmin, an effect blocked by the protease inhibitor aprotinin. Lsa23 also inhibited lytic activity by NHS mediated by both classical and alternative complement pathways. Thus, Lsa23 has the ability to block both pathways of the complement system, and may help pathogenic Leptospira to escape complement-mediated clearance in human hosts. Indeed, NHS treated with Lsa23 confers a partial serum resistance phenotype to Leptospira biflexa, whereas blocking this protein with anti-Lsa23 renders pathogenic L. interrogans more susceptible to complement-mediated killing. Thus, Lsa23 is a multifunctional protein involved in many pathways, featuring C4b cleavage by plasmin, knowledge that may help in the development of preventive approaches to intervene with human complement escape by this versatile pathogen

The recombinant LIC10508 is a plasma fibronectin, plasminogen, fibrinogen and C4BP-binding protein of Leptospira interrogans

Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. In this study, we report that the recombinant proteins LIC10507, LIC10508 and LIC10509 are recognized by confirmed leptospirosis serum samples at both phases of the disease. The recombinant rLIC10508 and rLIC10507 are plasminogen (PLG)-binding proteins, capable of generating plasmin in the presence of a PLG activator. The proteins bind to PLG in a dose-dependent and saturable manner, fulfilling host-ligand interaction. Furthermore, rLIC10508 interacts with fibrinogen (Fg), plasma fibronectin and C4b binding protein (C4BP). The binding of rLIC10508 to Fg decreases the fibrin clotting in a thrombin-catalyzed reaction. The incubation with 4 mu M of protein promoted 40% inhibition upon clotting formation. C4BP bound to rLIC10508 retained its cofactor activity for factor I promoting the cleavage of C4b protein, which may reduce the membrane attack complex formation. Although these proteins have high amino acid sequence similarity, rLIC10508 is the most talented of the three, a behavior that might be explained by its unique putative 3D structure, whereas structures of rLIC10507 and rLIC10509 are very similar. Plasmin generation (rLIC10507 and rLIC10508), together with decreasing fibrin clot formation (rLIC10508) and impairment of the complement system (rLIC10508) may help the bacteria to overcome host defense, facilitating the infection process