Identification of a novel protein in the genome sequences of Leptospira interrogans with the ability to interact with host's components

Background Leptospirosis is an infectious disease that affects humans and animals worldwide. The etiological agents of this disease are the pathogenic species of the genus Leptospira. The mechanisms involved in the leptospiral pathogenesis are not full understood. The elucidation of novel mediators of host-pathogen interaction is important in the detection of virulence factors involved in the pathogenesis of leptospirosis. Objective This work focused on identification and characterization of a hypothetical protein of Leptospira encoded by the gene LIC10920. Methods The protein of unknown function was predicted to be surface exposed. Therefore, the LIC10920 gene was cloned and the protein expressed in Escherichia coli BL21 (DE3) Star pLysS strain. The recombinant protein was purified by metal affinity chromatography and evaluated with leptospirosis human serum samples. The interaction with host components was also performed. Results The recombinant protein was recognized by antibodies present in leptopsirosis human serum, suggesting its expression during infection. Immunofluorescence and intact bacteria assays indicated that the bacterial protein is surface-exposed. The recombinant protein interacted with human laminin, in a dose-dependent and saturable manner and was named Lsa24.9, for Leptospiral surface adhesin, followed by its molecular mass. Lsa24.9 also binds plasminogen (PLG) in a dose-dependent and saturable fashion, fulfilling receptor ligand interaction. Moreover, Lsa24.9 has the ability to acquire PLG from normal human serum, exhibiting similar profile as observed with the human purified component. PLG bound Lsa24.9 was able of generating plasmin, which could increase the proteolytic power of the bacteria. Conclusions This novel leptospiral protein may function as an adhesin at the colonization steps and may help the invasion process by plasmin generation at the bacterial cell surface.

Identification of a novel protein in the genome sequences of Leptospira interrogans with the ability to interact with host's components

Background Leptospirosis is an infectious disease that affects humans and animals worldwide. The etiological agents of this disease are the pathogenic species of the genus Leptospira. The mechanisms involved in the leptospiral pathogenesis are not full understood. The elucidation of novel mediators of host-pathogen interaction is important in the detection of virulence factors involved in the pathogenesis of leptospirosis. Objective This work focused on identification and characterization of a hypothetical protein of Leptospira encoded by the gene LIC10920. Methods The protein of unknown function was predicted to be surface exposed. Therefore, the LIC10920 gene was cloned and the protein expressed in Escherichia coli BL21 (DE3) Star pLysS strain. The recombinant protein was purified by metal affinity chromatography and evaluated with leptospirosis human serum samples. The interaction with host components was also performed. Results The recombinant protein was recognized by antibodies present in leptopsirosis human serum, suggesting its expression during infection. Immunofluorescence and intact bacteria assays indicated that the bacterial protein is surface-exposed. The recombinant protein interacted with human laminin, in a dose-dependent and saturable manner and was named Lsa24.9, for Leptospiral surface adhesin, followed by its molecular mass. Lsa24.9 also binds plasminogen (PLG) in a dose-dependent and saturable fashion, fulfilling receptor ligand interaction. Moreover, Lsa24.9 has the ability to acquire PLG from normal human serum, exhibiting similar profile as observed with the human purified component. PLG bound Lsa24.9 was able of generating plasmin, which could increase the proteolytic power of the bacteria. Conclusions This novel leptospiral protein may function as an adhesin at the colonization steps and may help the invasion process by plasmin generation at the bacterial cell surface.

Decrease in antithrombin III and prothrombin serum levels contribute to coagulation disorders during leptospirosis

Pathogenic bacteria of the genus Leptospira are the causative agent of leptospirosis, an emergent infectious disease that affects humans and animals worldwide. Severe forms of the disease in humans include jaundice, multiple organ failure and intense haemorrhage. Up to now, mechanisms associated with the haemorrhage foci are poorly understood. We report in this work that, despite the low levels of antithrombin III in convalescent human serum samples, virulent, culture-attenuated and saprophyte strains of Leptospira are unable to bind and/or degrade this thrombin inhibitor, suggesting an indirect mechanism of pathogenesis. Lower levels of prothrombin were found in serum samples at the onset and convalescent phase of the disease when compared to normal human sera. The concomitant decreased levels of antithrombin III and prothrombin suggest a process of stimulated coagulation, which is corroborated by the increase of prothrombin fragment F1+2 in the serum samples. Data obtained with hamsters experimentally infected with virulent Leptospira interrogans serovars Kennewicki and Canicola strongly point out that haemorrhage is correlated with decreased levels of thrombin inhibitors and prothrombin. Activated coagulation might lead to an overconsumption of coagulation factors ultimately leading to bleeding and organ failure

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

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

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 procoagulant 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

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 invadeand 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 andLsa77 - LIC10050 (Leptospiral surface adhesins of 46 and 77 kDa, respectively). Attachmentof Lsa46 and Lsa77 to laminin was specific, dose dependent and saturable, with KD valuesof 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 generatingplasmin (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 saprophyteL. biflexa Patoc 1 strains, as detected by immunofluorescence. The adhesins are recognizedby human leptospiros is serum samples at the on set 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...

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 microscopicagglutination 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...