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.

Leptospira interrogans Bat proteins impair host hemostasis by fibrinogen cleavage and platelet aggregation inhibition.

Leptospirosis is a worldwide spread zoonosis, caused by pathogenic Leptospira. Evidences suggest that compromised hemostasis might be involved in the leptospirosis pathophysiology. In the genome of L. interrogans serovar Copenhageni, we found two genes coding for proteins which comprise von Willebrand factor (VWF) A domains (BatA and BatB). As VWF A domains exhibit multiple binding sites which contributes to human VWF hemostatic functions, we hypothesized that the L. interrogans BatA and BatB proteins could be involved in the hemostatic impairment during leptospirosis. We have cloned, expressed in Escherichia coli, and purified recombinant BatA and BatB. The influence of recombinant BatA and BatB on different in vitro hemostatic assays evaluating the enzymatic activity, platelet aggregation and fibrinogen integrity was investigated. We describe BatB as a new serine protease which is able to cleave thrombin chromogenic substrate, fibrin, fibrinogen, gelatin and casein; while BatA is active only towards fibrinogen. BatA and BatB interfere with the platelet aggregation induced by VWF/ristocetin and thrombin. Our results suggest an important role of the L. interrogans serovar Copenhageni Bat proteins in the hemostasis dysfunction observed during leptospirosis and contribute to the understanding of the leptospirosis pathophysiological mechanisms.

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.

Interaction of spirochetes with the host fibrinolytic system and potential roles in pathogenesis.

The pathogenic spirochetes Borrelia burgdorferi, B. hermsii, B. recurrentis, Treponema denticola and Leptospira spp. are the etiologic agents of Lyme disease, relapsing fever, periodontitis and leptospirosis, respectively. Lyme borreliosis is a multi-systemic disorder and the most prevalent tick-borne disease in the northern hemisphere. Tick-borne relapsing fever is persistent in endemic areas worldwide, representing a significant burden in some African regions. Periodontal disease, a chronic inflammatory disorder that often leads to tooth loss, is caused by several potential pathogens found in the oral cavity including T. denticola. Leptospirosis is considered the most widespread zoonosis, and the predominant human disease in tropical, undeveloped regions. What these diseases have in common is that they are a significant burden to healthcare costs in the absence of prophylactic measures. This review addresses the interaction of these spirochetes with the fibrinolytic system, plasminogen (Plg) binding to the surface of bacteria and the generation of plasmin (Pla) on their surface. The consequences on host-pathogen interactions when the spirochetes are endowed with this proteolytic activity are discussed on the basis of the results reported in the literature. Spirochetes equipped with Pla activity have been shown to degrade extracellular matrix (ECM) components, in addition to digesting fibrin, facilitating bacterial invasion and dissemination. Pla generation triggers the induction of matrix metalloproteases (MMPs) in a cascade of events that enhances the proteolytic capacity of the spirochetes. These activities in concert with the interference exerted by the Plg/Pla on the complement system - helping the bacteria to evade the immune system - should illuminate our understanding of the mechanisms involved in host infection.

Mammalian cell entry (Mce) protein of Leptospira interrogans binds extracellular matrix components, plasminogen and ß2 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--r-esponse 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 αLß2 [(CD11a/CD18)-LFA-1] and αMß2 [(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 αMß2 [(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 αLß2(CD11a/CD18) and αMß2(CD11b/CD18).

Structural and biochemical characterization of Leptospira interrogans Lsa45 reveals a penicillin-binding protein with esterase activity.

Leptospirosis is a bacterial disease that affects humans and animals and is caused by Leptospira. The recommended treatment for leptospirosis is antibiotic therapy, which should be given early in the course of the disease. Despite the use of these antibiotics, their role during the course of the disease is still not completely clear because of the lack of effective clinical trials, particularly for severe cases of the disease. Here, we present the characterization of L. interrogans Lsa45 protein by gel filtration, protein crystallography, SAXS, fluorescence and enzymatic assays. The oligomeric studies revealed that Lsa45 is monomeric in solution. The crystal structure of Lsa45 revealed the presence of two subdomains: a large α/ß subdomain and a small α-helical subdomain. The large subdomain contains the amino acids Ser122, Lys125, and Tyr217, which correspond to the catalytic triad that is essential for ß-lactamase or serine hydrolase activity in similar enzymes. Additionally, we also confirmed the bifunctional promiscuity of Lsa45, in hydrolyzing both the 4-nitrophenyl acetate (p-NPA) and nitrocefin ß-lactam antibiotic. Therefore, this study provides novel insights into the structure and function of enzymes from L. interrogans, which furthers our understanding of this bacterium and the development of new therapies for the prevention and treatment of leptospirosis.

The crystal structure of the leptospiral hypothetical protein LIC12922 reveals homology with the periplasmic chaperone SurA.

Leptospirosis is a world spread zoonosis caused by members of the genus Leptospira. Although leptospires were identified as the causal agent of leptospirosis almost 100 years ago, little is known about their biology, which hinders the development of new treatment and prevention strategies. One of the several aspects of the leptospiral biology not yet elucidated is the process by which outer membrane proteins (OMPs) traverse the periplasm and are inserted into the outer membrane. The crystal structure determination of the conserved hypothetical protein LIC12922 from Leptospira interrogans revealed a two domain protein homologous to the Escherichia coli periplasmic chaperone SurA. The LIC12922 NC-domain is structurally related to the chaperone modules of E. coli SurA and trigger factor, whereas the parvulin domain is devoid of peptidyl prolyl cis-trans isomerase activity. Phylogenetic analyses suggest a relationship between LIC12922 and the chaperones PrsA, PpiD and SurA. Based on our structural and evolutionary analyses, we postulate that LIC12922 is a periplasmic chaperone involved in OMPs biogenesis in Leptospira spp. Since LIC12922 homologs were identified in all spirochetal genomes sequenced to date, this assumption may have implications for the OMPs biogenesis studies not only in leptospires but in the entire Phylum Spirochaetes.

A Novel Leptospira interrogans Protein LIC13086 Inhibits Fibrin Clot Formation and Interacts With Host Components.

Leptospirosis is a neglected zoonosis, caused by pathogenic spirochetes bacteria of the genus Leptospira. The molecular mechanisms of leptospirosis infection are complex, and it is becoming clear that leptospires express several functionally redundant proteins to invade, disseminate, and escape the host's immune response. Here, we describe a novel leptospiral protein encoded by the gene LIC13086 as an outer membrane protein. The recombinant protein LIC13086 can interact with the extracellular matrix component laminin and bind plasminogen, thus possibly participating during the adhesion process and dissemination. Also, by interacting with fibrinogen and plasma fibronectin, the protein LIC13086 probably has an inhibitory effect in the fibrin clot formation during the infection process. The newly characterized protein can also bind molecules of the complement system and the regulator C4BP and, thus, might have a role in the evasion mechanism of Leptospira. Taken together, our results suggest that the protein LIC13086 may have a multifunctional role in leptospiral pathogenesis, participating in host invasion, dissemination, and immune evasion processes.

Virulent Leptospira interrogans Induce Cytotoxic Effects in Human Platelets in vitro Through Direct Interactions.

Leptospirosis is a prevalent zoonotic disease, caused by bacteria of the genus Leptospira. Leptospirosis frequently leads to hemostatic disturbances, and the severe cases are marked by hemorrhages and low platelet number in circulation, which is associated with the patients' poor outcomes. Nevertheless, Leptospira-platelet interactions remain poorly explored. In this study, we performed a series of in vitro experiments evaluating whether leptospires induce human platelet aggregation, activation, and morphological changes. Platelets were incubated with virulent L. interrogans and the platelet outcomes were assessed by aggregometry, flow cytometry, and scanning and transmission electron microscopy. Our results show that leptospires alone do not induce platelet aggregation and activation, and induce platelet cytotoxic effects instead, by clearly inducing platelet disruption and detachment. We show for the first time that virulent leptospires do interact directly with platelets, an event that could trigger pathophysiological effects during the infection. This study might serve as a basis for the development of novel treatments for the disease.

The Leptospira interrogans LIC10774 is a multifunctional surface protein that binds calcium and interacts with host components.

Leptospirosis is a global re-emerging zoonosis, caused by pathogenic bacteria of the genus Leptospira. Humans are infected mainly through contact with contaminated water or soil. The understanding of the molecular mechanisms of leptospirosis through the characterization of unknown outer membrane proteins may contribute to the development of new treatments, diagnostic methods and vaccines. We have identified using bioinformatics analysis a protein that is encoded by the gene LIC10774, predicted to be localized at the leptospiral outer membrane and exhibit beta-roll folding. Surface exposure was confirmed by flow cytometry, ELISA and immunofluorescence-based confocal microscopy. Through circular dichroism spectroscopy and hydrophobic dye binding we have shown that rLIC10774 binds calcium ions, which imposes changes to secondary and tertiary structures. The recombinant protein was capable of binding to several host extracellular matrix and serum components. Therefore, we describe LIC10774 as a calcium-binding protein exposed in the outer surface of pathogenic leptospires with possible multifunctional roles in adhesion to host tissues, evasion of the immune system and participation in dissemination processes during leptospirosis. In addition, we hypothesize that the calcium binding is important for temperature-dependent functional roles during leptospirosis.

The leptospiral antigen Lp49 is a two-domain protein with putative protein binding function.

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 A resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.

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.

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.

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.

Features of two new proteins with OmpA-like domains identified in the genome sequences of Leptospira interrogans.

Leptospirosis is an acute febrile disease caused by pathogenic spirochetes of the genus Leptospira. It is considered an important re-emerging infectious disease that affects humans worldwide. The knowledge about the mechanisms by which pathogenic leptospires invade and colonize the host remains limited since very few virulence factors contributing to the pathogenesis of the disease have been identified. Here, we report the identification and characterization of two new leptospiral proteins with OmpA-like domains. The recombinant proteins, which exhibit extracellular matrix-binding properties, are called Lsa46 - LIC13479 and Lsa77 - LIC10050 (Leptospiral surface adhesins of 46 and 77 kDa, respectively). Attachment of Lsa46 and Lsa77 to laminin was specific, dose dependent and saturable, with KD values of 24.3 ± 17.0 and 53.0 ± 17.5 nM, respectively. Lsa46 and Lsa77 also bind plasma fibronectin, and both adhesins are plasminogen (PLG)-interacting proteins, capable of generating plasmin (PLA) and as such, increase the proteolytic ability of leptospires. The proteins corresponding to Lsa46 and Lsa77 are present in virulent L. interrogans L1-130 and in saprophyte L. biflexa Patoc 1 strains, as detected by immunofluorescence. The adhesins are recognized by human leptospirosis serum samples at the onset and convalescent phases of the disease, suggesting that they are expressed during infection. Taken together, our data could offer valuable information to the understanding of leptospiral pathogenesis.

Adjuvant activity of Mycobacterium bovis BCG expressing CRM197 on the immune response induced by BCG expressing tetanus toxin fragment C.

In order to develop a combined recombinant Mycobacterium bovis BCG (rBCG) vaccine against diphtheria, pertussis and tetanus (DPT), we have constructed different strains of rBCG expressing tetanus toxin fragment C (FC), driven by the up-regulated M. fortuitum beta-lactamase promoter, pBlaF*. Tetanus toxin FC was expressed in comparable levels in native form or in fusion with the beta-lactamase exportation signal sequence; however, in both constructs it was localized to the cytosol. Immunization of mice with rBCG-FC or its combination with rBCG expressing CRM197, induced anti-tetanus toxin antibodies with a Th2 immunoglobulin profile. Administration of a subimmunizing dose of the diphtheria-tetanus toxoid vaccine showed that rBCG-FC primed mice for production of an intense humoral response. Interestingly, the combination of rBCG-FC and rBCG-CRM197 reduced the time required for maturation of the immune response and increased anti-tetanus toxin antibody levels, suggesting adjuvant properties for rBCG-CRM197; this combination induced 75% protection in mice challenged with 100 minimum lethal doses (MLD) of tetanus toxin. Antisera from guinea pigs immunized with this combination were shown to neutralize tetanus toxin and diphtheria toxin. Our results suggest reciprocal adjuvant effects of rBCG-FC and rBCG-CRM197, which may contribute to induction of a more effective immune response against both diseases.