The role of Lsa23 to mediate the interaction of Leptospira interrogans with the terminal complement components pathway.

Leptospirosis is a severe worldwide zoonotic disease caused by pathogenic Leptospira spp. It has been demonstrated that pathogenic leptospires are resistant to the bactericidal activity of normal human serum while saprophytic strains are susceptible. Pathogenic strains have the ability to bind soluble complement regulators and these activities are thought to contribute to bacterial immune evasion. One strategy used by some pathogens to evade the complement cascade, which is not well explored, is to block the terminal pathway. We have, thus, examined whether leptospires are able to interact with components of the terminal complement pathway. ELISA screening using anti-leptospires serum has shown that the pathogenic, virulent strain L. interrogans L1-130 can bind to immobilized human C8 (1 µg). However, virulent and saprophyte L. biflexa strains showed the ability to interact with C8 and C9, when these components were employed at physiological concentration (50 µg/mL), but the virulent strain seemed more competent. Lsa23, a putative leptospiral adhesin only present in pathogenic strains, interacts with C8 and C9 in a dose-dependent mode, suggesting that this protein could mediate the binding of virulent Leptospira with these components. To our knowledge, this is the first work reporting the binding of Leptospira to C8 and C9 terminal complement components, suggesting that the inhibition of this pathway is part of the strategy used by leptospires to evade the innate immunity.

Characterization of two new putative adhesins of Leptospira interrogans.

We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host -pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.

Leptospira spp.: Novel insights into host-pathogen interactions.

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira spp. It is an important infectious disease that affects humans and animals. The disease causes economic losses as it affects livestock, with decreased milk production and death. Our group is investigating the genome sequences of L. interrogans targeting surface-exposed proteins because, due to their location, these proteins are capable to interact with several host components that could allow establishment of the infection. These interactions may involve adhesion of the bacteria to extracellular matrix (ECM) components and, hence, help bacterial colonization. The bacteria could also react with the host fibrinolytic system and/or with the coagulation cascade components, such as, plasminogen (PLG) and fibrinogen (Fg), respectively. The binding with the first system generates plasmin (PLA), increasing the proteolytic power of the bacteria, while the second interferes with clotting in a thrombin-catalyzed reaction, which may promote hemorrhage foci and increase bacterial dissemination. Interaction with the complement system negative regulators may help bacteria to evade the host immune system, facilitating the invasion. This work compiles the main described leptospiral proteins that could act as adhesins, as PLG and fibrinogen receptors and as complement regulator binding proteins. We present models in which we suggest possible mechanisms of how leptospires might colonize and invade host tissues, causing the disease. Understanding leptospiral pathogenesis will help to identify antigen candidates that would contribute to the development of more effective vaccines and diagnostic tests.

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 µM 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.

Leptospiral extracellular matrix adhesins as mediators of pathogen-host interactions.

Leptospirosis is been considered an important infectious disease that affects humans and animals worldwide. This review summarizes our current knowledge of bacterial attachment to extracellular matrix (ECM) components and discusses the possible role of these interactions for leptospiral pathogenesis. Leptospiral proteins show different binding specificity for ECM molecules: some are exclusive laminin-binding proteins (Lsa24/LfhA/LenA, Lsa27), while others have broader spectrum binding profiles (LigB, Lsa21, LipL53). These proteins may play a primary role in the colonization of host tissues. Moreover, there are multifunctional proteins that exhibit binding activities toward a number of target proteins including plasminogen/plasmin and regulators of the complement system, and as such, might also act in bacterial dissemination and immune evasion processes. Many ECM-interacting proteins are recognized by human leptospirosis serum samples indicating their expression during infection. This compilation of data should enhance our understanding of the molecular mechanisms of leptospiral pathogenesis.

Adhesins of Leptospira interrogans mediate the interaction to fibrinogen and inhibit fibrin clot formation in vitro.

We report in this work that Leptospira strains, virulent L. interrogans serovar Copenhageni, attenuated L. interrogans serovar Copenhageni and saprophytic L. biflexa serovar Patoc are capable of binding fibrinogen (Fg). The interaction of leptospires with Fg inhibits thrombin- induced fibrin clot formation that may affect the haemostatic equilibrium. Additionally, we show that plasminogen (PLG)/plasmin (PLA) generation on the surface of Leptospira causes degradation of human Fg. The data suggest that PLA-coated leptospires were capable to employ their proteolytic activity to decrease one substrate of the coagulation cascade. We also present six leptospiral adhesins and PLG- interacting proteins, rLIC12238, Lsa33, Lsa30, OmpL1, rLIC11360 and rLIC11975, as novel Fg-binding proteins. The recombinant proteins interact with Fg in a dose-dependent and saturable fashion when increasing protein concentration was set to react to a fix human Fg concentration. The calculated dissociation equilibrium constants (K D ) of these reactions ranged from 733.3 ± 276.8 to 128 ± 89.9 nM for rLIC12238 and Lsa33, respectively. The interaction of recombinant proteins with human Fg resulted in inhibition of fibrin clot by thrombin-catalyzed reaction, suggesting that these versatile proteins could mediate Fg interaction in Leptospira. Our data reveal for the first time the inhibition of fibrin clot by Leptospira spp. and presents adhesins that could mediate these interactions. Decreasing fibrin clot would cause an imbalance of the coagulation cascade that may facilitate bleeding and help bacteria dissemination.

Characterization of three novel adhesins of Leptospira interrogans.

We report cloning, expression, purification, and characterization of three predicted leptospiral membrane proteins (LIC11360, LIC11009, and LIC11975). In silico analysis and proteinase K accessibility data suggest that these proteins might be surface exposed. We show that proteins encoded by LIC11360, LIC11009 and LIC11975 genes interact with laminin in a dose-dependent and saturable manner. The proteins are referred to as leptospiral surface adhesions 23, 26, and 36 (Lsa23, Lsa26, and Lsa36), respectively. These proteins also bind plasminogen and generate active plasmin. Attachment of Lsa23 and Lsa36 to fibronectin occurs through the involvement of the 30-kDa and 70-kDa heparin-binding domains of the ligand. Dose-dependent, specific-binding of Lsa23 to the complement regulator C4BP and to a lesser extent, to factor H, suggests that this protein may interfere with the complement cascade pathways. Leptospira spp. may use these interactions as possible mechanisms during the establishment of infection.