Structural analysis of CACHE domain of the McpA chemoreceptor from Leptospira interrogans.

Leptospira is a genus of spirochete bacteria highly motile that includes pathogenic species responsible to cause leptospirosis disease. Chemotaxis and motility are required for Leptospira infectivity, pathogenesis, and invasion of bacteria into the host. In prokaryotes, the most common chemoreceptors are methyl-accepting chemotaxis proteins that have a role play to detect the chemical signals and move to a favorable environment for its survival. Here, we report the first crystal structure of CACHE domain of the methyl-accepting chemotaxis protein (McpA) of L. interrogans. The structural analysis showed that McpA adopts similar α/ß architecture of several other bacteria chemoreceptors. We also found a typical dimerization interface that appears to be functionally crucial for signal transmission and chemotaxis. In addition to McpA structural analyses, we have identified homologous proteins and conservative functional regions using bioinformatics techniques. These results improve our understanding the relationship between chemoreceptor structures and functions of Leptospira species.

The interplay between host haemostatic systems and Leptospira spp. infections.

Hemostasis is a defence mechanism that protects the integrity of the vascular system and is comprised of the coagulation cascade, fibrinolysis, platelet aggregation, and vascular endothelium. Besides the primary function in preserving the vascular integrity, the haemostatic system cooperates with immune and inflammatory processes to eliminate invading pathogens during microbial infections. Under pathological manifestations, hemostasis must therefore interact in a coordinated manner with inflammatory responses and immune reactions. Several pathogens can modulate these host-derived countermeasures by specifically targeting certain haemostatic components for their own benefit. Thus, the ability to modulate host defence systems has to be considered as an essential bacterial virulence mechanism. Complications that bacterial pathogens can induce are therefore often the consequence of evoked host responses. A comprehensive understanding of the molecular mechanisms triggered in infectious processes may help to develop prophylactic methods and novel therapies for the patients suffering from a particular infectious disease. This review aims to provide a critical updated compiling of recent studies on how the pathogenic Leptospira can interact with and manipulate the host haemostatic systems and the consequences for leptospirosis pathogenesis.

Neutrophils engage the kallikrein-kinin system to open up the endothelial barrier in acute inflammation.

Neutrophil recruitment and plasma exudation are key elements in the immune response to injury or infection. Activated neutrophils stimulate opening of the endothelial barrier; however, the underlying mechanisms have remained largely unknown. In this study, we identified a pivotal role of the proinflammatory kallikrein-kinin system and consequent formation of bradykinin in neutrophil-evoked vascular leak. In mouse and hamster models of acute inflammation, inhibitors of bradykinin generation, and signaling markedly reduced plasma exudation in response to chemoattractant activation of neutrophils. The neutrophil-driven leak was likewise suppressed in mice deficient in either the bradykinin B2 receptor or factor XII (initiator of the kallikrein-kinin system). In human endothelial cell monolayers, material secreted from activated neutrophils induced cytoskeletal rearrangement, leading to paracellular gap formation in a bradykinin-dependent manner. As a mechanistic basis, we found that a neutrophil-derived heparin-binding protein (HBP/azurocidin) displaced the bradykinin precursor high-molecular-weight kininogen from endothelial cells, thereby enabling proteolytic processing of kininogen into bradykinin by neutrophil and plasma proteases. These data provide novel insight into the signaling pathway by which neutrophils open up the endothelial barrier and identify the kallikrein-kinin system as a target for therapeutic interventions in acute inflammatory reactions.-Kenne, E., Rasmuson, J., Renné, T., Vieira, M. L., Müller-Esterl, W., Herwald, H., Lindbom, L. Neutrophils engage the kallikrein-kinin system to open up the endothelial barrier in acute inflammation.

Heparin-Binding Protein Release Is Strongly Induced by Leptospira Species and Is a Candidate for an Early Diagnostic Marker of Human Leptospirosis.

BACKGROUND: Leptospirosis, caused by spirochetes of the genus Leptospira, is one of the most widespread zoonoses worldwide. Efficient diagnostic methods for early diagnosis of leptospirosis are still lacking, and acute disease presents with nonspecific symptomatology and is often misdiagnosed. The leptospires pathogenic processes and virulence mechanisms remain virtually unknown. In severe infections, hemostatic impairment is frequently observed, and pathophysiological complications often develop when the host response is modulated by the pathogen. The neutrophil heparin-binding protein (HBP) is an inflammatory mediator and potent inducer of vascular leakage. RESULTS: In this study, we found that leptospires and their secreted products induce the release of HBP from stimulated neutrophils through a controlled degranulation mechanism. We acknowledged 2 leptospiral proteins as able to induce HBP degranulation. These findings have clinical implications, as high levels of HBP were detected in serum from patients with leptospirosis, especially at the early phase of the disease. CONCLUSION: In conclusion, we describe a new mechanism by which the leptospirosis pathophysiological complications may arise, such as vascular leakage and edema formation. We also propose HBP as a new early screening biomarker for human leptospirosis.

Functional and immunological evaluation of two novel proteins of Leptospira spp.

This work shows the production and characterization of two novel putative lipoproteins encoded by the genes LIC10645 and LIC10731 identified in the genome sequences of Leptospira interrogans. In silico conservation analysis indicated that the proteins are well conserved among pathogenic leptospiral serovars and species. Recombinant proteins were obtained in Escherichia coli BL21(DE3) Star pLysS strain, purified by metal-affinity chromatography, and used for characterization and immunological evaluations. Recombinant proteins were capable of eliciting a combination of humoral and cellular immune responses in animal models, and could be recognized by antibodies present in human serum samples. The recombinant proteins Lsa44 and Lsa45 were able to bind laminin, and were named Lsa44 and Lsa45 for leptospiral surface adhesins of 44 and 45 kDa, respectively. The attachment to laminin was dose-responsive with KD values of 108.21 and 250.38 nM for Lsa44 and Lsa45, respectively. Moreover, these proteins interact with plasminogen (PLG) with KD values of 53.56 and 36.80 nM, respectively. PLG bound to the recombinant proteins could be converted to plasmin (PLA) in the presence of an activator. Cellular localization assays suggested that the Lsa44 and Lsa45 were surface-exposed. These are versatile proteins capable of interacting with laminin and PLG/PLA, and hence could mediate bacterial adhesion and contribute to tissue penetration.

Global proteome of the saprophytic strain Leptospira biflexa and comparative analysis with pathogenic strain Leptospira interrogans uncover new pathogenesis mechanisms.

Leptospira is a genus of bacteria that includes free-living saprophytic species found in water or soil, and pathogenic species, which are the etiologic agents of leptospirosis. Besides all the efforts, there are only a few proteins described as virulence factors in the pathogenic strain L. interrogans. This work aims to perform L. biflexa serovar Patoc1 strain Paris global proteome and to compare with the proteome database of pathogenic L. interrogans serovar Copenhageni strain Fiocruz L1-130. We identified a total of 2327 expressed proteins of L. biflexa by mass spectrometry. Using the Get Homologues software with the global proteome of L. biflexa and L. interrogans, we found orthologous proteins classified into conserved, low conserved, and specific proteins. Comparative bioinformatic analyses were performed to understand the biological functions of the proteins, subcellular localization, the presence of signal peptide, structural domains, and motifs using public softwares. These results lead to the selection of 182 low conserved within the saprophyte, and 176 specific proteins of L. interrogans. It is anticipated that these findings will indicate further studies to uncover virulence factors in the pathogenic strain. This work presents for the first time the global proteome of saprophytic strain L. biflexa serovar Patoc, strain Patoc1. SIGNIFICANCE: The comparative analysis established an array of specific proteins in pathogenic strain that will narrow down the identification of immune protective proteins that will help fight leptospirosis.

Interaction of Leptospira interrogans with human proteolytic systems enhances dissemination through endothelial cells and protease levels.

We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date.

OmpL1 is an extracellular matrix- and plasminogen-interacting protein of Leptospira spp.

Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical ß-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with K(D) (equilibrium dissociation constant) values of 2,099.93 ± 871.03 nM and 1,239.23 ± 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a K(D) of 368.63 ± 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection.

Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions.

BACKGROUND: Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. RESULTS: We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (KD) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a KD of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. CONCLUSIONS: We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp.

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coli BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K(D)) of 292 ± 24 nm and 157 ± 35 nm, respectively. Moreover, the Lsa30 is a plasminogen (PLG) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system.

Plasminogen binding proteins and plasmin generation on the surface of Leptospira spp.: the contribution to the bacteria-host interactions.

Leptospirosis is considered a neglected infectious disease of human and veterinary concern. Although extensive investigations on host-pathogen interactions have been pursued by several research groups, mechanisms of infection, invasion and persistence of pathogenic Leptospira spp. remain to be elucidated. We have reported the ability of leptospires to bind human plasminogen (PLG) and to generate enzimatically active plasmin (PLA) on the bacteria surface. PLA-coated Leptospira can degrade immobilized ECM molecules, an activity with implications in host tissue penetration. Moreover, we have identified and characterized several proteins that may act as PLG-binding receptors, each of them competent to generate active plasmin. The PLA activity associated to the outer surface of Leptospira could hamper the host immune attack by conferring the bacteria some benefit during infection. The PLA-coated leptospires obstruct complement C3b and IgG depositions on the bacterial surface, most probably through degradation. The decrease of leptospiral opsonization might be an important aspect of the immune evasion strategy. We believe that the presence of PLA on the leptospiral surface may (i) facilitate host tissue penetration, (ii) help the bacteria to evade the immune system and, as a consequence, (iii) permit Leptospira to reach secondary sites of infection.

In vitro evidence for immune evasion activity by human plasmin associated to pathogenic Leptospira interrogans.

Leptospirosis is a widespread re-emerging zoonosis of human and veterinary concern. It has been shown that virulent leptospires protect themselves against the host's innate immune system, a strategy that allows the bacteria to reach immunologically safe environments. Although extensive studies on host-pathogen interactions have been performed, little is known on how leptospires deal with host immune attack. In a previous work, we demonstrated the ability of leptospires to bind human plasminogen (PLG), that after treatment with activators, conferred plasmin (PLA) activity on the bacteria surface. In this study, we show that the PLA activity associated to the outer surface of Leptospira could interfere with the host immune attack by conferring some evasion advantage during infection. We demonstrate that PLA-coated leptospires interfere with complement C3b and IgG depositions on the bacterial surface, probably through the degradation of these components, thus diminishing opsonization process. Similar decrease on the deposition was observed when normal and immune sera from patients diagnosed with leptospirosis were employed as a source of IgG. We believe that decreasing opsonization by PLA generation might be an important aspect of the leptospiral immune escape strategy and survival. To our knowledge, this is the first proteolytic activity of plasmin associated-Leptospira related to anti-opsonic properties reported to date.

Plasminogen acquisition and activation at the surface of leptospira species lead to fibronectin degradation.

Pathogenic Leptospira species are the etiological agents of leptospirosis, a widespread disease of human and veterinary concern. In this study, we report that Leptospira species are capable of binding plasminogen (PLG) in vitro. The binding to the leptospiral surface was demonstrated by indirect immunofluorescence confocal microscopy with living bacteria. The PLG binding to the bacteria seems to occur via lysine residues because the ligation is inhibited by addition of the lysine analog 6-aminocaproic acid. Exogenously provided urokinase-type PLG activator (uPA) converts surface-bound PLG into enzymatically active plasmin, as evaluated by the reaction with the chromogenic plasmin substrate d-Val-Leu-Lys 4-nitroanilide dihydrochloridein. The PLG activation system on the surface of Leptospira is PLG dose dependent and does not cause injury to the organism, as cellular growth in culture was not impaired. The generation of active plasmin within Leptospira was observed with several nonvirulent high-passage strains and with the nonpathogenic saprophytic organism Leptospira biflexa. Statistically significant higher activation of plasmin was detected with a low-passage infectious strain of Leptospira. Plasmin-coated virulent Leptospira interrogans bacteria were capable of degrading purified extracellular matrix fibronectin. The breakdown of fibronectin was not observed with untreated bacteria. Our data provide for the first time in vitro evidence for the generation of active plasmin on the surface of Leptospira, a step that may contribute to leptospiral invasiveness.

Adjuvanted leptospiral vaccines: Challenges and future development of new leptospirosis vaccines.

Leptospirosis is a neglected infectious disease of global importance. Vaccination is the most viable strategy for the control of leptospirosis, but in spite of efforts for the development of an effective vaccine against the disease, few advances have been made, and to date, bacterin is the only option for prevention of leptospirosis. Bacterins are formulations based on inactivated leptospires that present a series of drawbacks, such as serovar-dependence and short-term immunity. Therefore, bacterins are not widely used in humans, and only Cuba, France and China have these vaccines licensed for at-risk populations. The development of recombinant DNA technology emerges as an alternative to solve the problem. Recombinant protein-based vaccines or DNA vaccines seem to be an attractive strategy, but the use of adjuvants is critical for achievement of a protective immune response. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells. In the last years, several components have been tested as adjuvants, such as aluminum salts, oil based-emulsion adjuvants, bacteria-derived components and liposomes. This review highlights the use of adjuvants in the multiple vaccine approaches that have been used for leptospirosis and their most important immunological aspects. Immune response data generated by these strategies can contribute to the understanding of the immune mechanisms involved in protection against leptospirosis, and consequently, the development of effective vaccines against this disease. This is the first review on leptospiral vaccines focusing on adjuvant aspects.

Leptospira interrogans outer membrane protein LipL21 is a potent inhibitor of neutrophil myeloperoxidase.

Leptospirosis is a widespread zoonotic and neglected infectious disease of human and veterinary concern that is caused by pathogenic Leptospira species. After entrance in the host, pathogenic leptospires evade the host natural defense mechanisms in order to propagate and disseminate to multiple organs. Myeloperoxidase is an enzyme stored in neutrophils azurophilic granules, and is released upon neutrophil activation to produce mainly hypochlorous acid, a strong oxidant and potent antimicrobial agent. In the present investigation, we studied the modulation of myeloperoxidase activity by L. interrogans serovar Copenhageni. We show that leptospires and their culture supernatants are able to inhibit both peroxidase and chlorination activities of myeloperoxidase, without interfering with neutrophil degranulation. By leptospiral outer membrane protein extraction and fractionation, we identified the proteins LipL21 and LipL45 as myeloperoxidase inhibitors, constituting new Leptospira virulence factors. Accordingly, we propose a function for the protein LipL21, one of the most expressed leptospiral outer membrane proteins. Our results show a novel innate immune evasion mechanism by which leptospires interfere with the host response in order to cope with the host oxidative stress and efficiently achieve dissemination and colonization.

A novel leptospiral protein increases ICAM-1 and E-selectin expression in human umbilical vein endothelial cells.

It has been reported previously that activation of vascular endothelium by outer membrane proteins of the spirochetes Borrelia sp. and Treponema sp. resulted in enhanced expression of endothelial cell adhesion molecules. To investigate the role of leptospiral proteins in this process, a predicted lipoprotein encoded by the gene LIC10365 was selected, which belongs to a paralogous family that presents a domain of unknown function, DUF1565. The LIC10365 gene was cloned and the protein expressed in Escherichia coli C43 (DE3) strain using the vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and was used to assess its ability to activate cultured human umbilical vein endothelial cells. The rLIC10365 activated endothelium in such a manner that E-selectin and intercellular adhesion molecule 1 (ICAM-1) became upregulated in a dose-dependent fashion. The LIC10365-encoded protein was identified in vivo in the renal tubules of animal during experimental infection with Leptospira interrogans. Collectively, these results implicate the LIC10365-coding protein of L. interrogans as a potential effector molecule in the promotion of a host inflammatory response. This is the first report of a leptospiral protein capable of up-regulating the expression of endothelial cell adhesion molecules ICAM-1 and E-selectin.

Leptospira Infection Interferes with the Prothrombinase Complex Assembly during Experimental Leptospirosis.

Leptospirosis is a worldwide zoonotic and neglected infectious disease of human and veterinary concern, caused by pathogenic Leptospira species. Although bleeding is a common symptom of severe leptospirosis, the cause of hemorrhage is not completely understood. In severe infections, modulation of hemostasis by pathogens is an important virulence mechanism, and hemostatic impairments such as coagulation/fibrinolysis dysfunction are frequently observed. Here, we analyze the coagulation status of experimentally infected hamsters in an attempt to determine coagulation interferences and the origin of leptospirosis hemorrhagic symptomatology. Hamsters were experimentally infected with L. interrogans. The lungs, kidneys, and livers were collected for culture, histopathology, and coagulation assays. L. interrogans infection disturbs normal coagulation in the organs of animals. Our results suggest the presence of a thrombin-like factor or FX activator, which is able to activate FII in the leptospirosis organ extracts. The activity of those factors is accelerated in the prothrombinase complex. Additionally, we show for the first time that live leptospires act as a surface for the prothrombinase complex assembly. Our results contribute to the understanding of leptospirosis pathophysiological mechanisms and may open new routes for the discovery of novel treatments in the severe manifestations of the disease.

Modulation of Hemostatic and Inflammatory Responses by Leptospira Spp.

Leptospirosis is a worldwide spread zoonotic and neglected infectious disease of human and veterinary concern that is caused by pathogenic Leptospira species. In severe infections, hemostatic impairments such as coagulation/fibrinolysis dysfunction are frequently observed. These complications often occur when the host response is controlled and/or modulated by the bacterial pathogen. In the present investigation, we aimed to analyze the modulation of the hemostatic and inflammatory host responses by the bacterial pathogen Leptospira. The effects of leptospires and their secreted products on stimulation of human intrinsic and extrinsic pathways of coagulation were investigated by means of altered clotting times, assembly and activation of contact system and induction of tissue factor. We show that both extrinsic and intrinsic coagulation cascades are modulated in response to Leptospira or leptospiral secreted proteins. We further find that the pro-inflammatory mediator bradykinin is released following contact activation at the bacterial surface and that pro-coagulant microvesicles are shed from monocytes in response to infection. Also, we show that human leptospirosis patients present higher levels of circulating pro-coagulant microvesicles than healthy individuals. Here we show that both pathways of the coagulation system are modulated by leptospires, possibly leading to altered hemostatic and inflammatory responses during the disease. Our results contribute to the understanding of the leptospirosis pathophysiological mechanisms and may open new routes for the discovery of novel treatments for the severe manifestations of the disease.

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.

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.