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.

A review on host-leptospira interactions: what we know and future expectations

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira spp. It is considered a neglected infectious disease of human and veterinary concern. Our group has been investigating proteins annotated as hypothetical, predicted to be located on the leptospiral surface. Because of their location, these proteins may have the ability to interact with various host components, which could allow establishment of the infection. These proteins act as adherence factors by binding to host receptor molecules, such as the extracellular matrix (ECM) components laminin and glycosaminoglycans to help bacterial colonization. Leptospira also interacts with the host fibrinolytic system, which has been demonstrated to be a powerful tool for invasion mechanisms. The interaction with fibrinogen and thrombin has been shown to reduce fibrin clot formation. Additionally, the degradation of coagulation cascade components by secreted proteases or by acquired surface plasmin could also play a role in reducing clot formation, hence facilitating dissemination during infection. Interaction with host complement system regulators also plays a role in helping bacteria to evade the immune system, facilitating invasion. Interaction of Leptospira to cell receptors, such as cadherins, can contribute to investigate molecules that participate in virulence. To achieve a better understanding of the host-pathogen interaction, leptospiral mutagenesis tools have been developed and explored. This work presents several proteins that mediate binding to components of the ECM, plasma, components of the complement system and cells, to gather research achievements that can be helpful in better understanding the mechanisms of leptospiral-host interactions and discuss genetic manipulation for Leptospira spp. aimed at protein function validation.

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.

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.

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.

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

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.

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

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.

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.

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.

Automatized image processing of bovine blastocysts produced in vitro for quantitative variable determination.

There is currently no objective, real-time and non-invasive method for evaluating the quality of mammalian embryos. In this study, we processed images of in vitro produced bovine blastocysts to obtain a deeper comprehension of the embryonic morphological aspects that are related to the standard evaluation of blastocysts. Information was extracted from 482 digital images of blastocysts. The resulting imaging data were individually evaluated by three experienced embryologists who graded their quality. To avoid evaluation bias, each image was related to the modal value of the evaluations. Automated image processing produced 36 quantitative variables for each image. The images, the modal and individual quality grades, and the variables extracted could potentially be used in the development of artificial intelligence techniques (e.g., evolutionary algorithms and artificial neural networks), multivariate modelling and the study of defined structures of the whole blastocyst.

A Method Based on Artificial Intelligence To Fully Automatize The Evaluation of Bovine Blastocyst Images.

Morphological analysis is the standard method of assessing embryo quality; however, its inherent subjectivity tends to generate discrepancies among evaluators. Using genetic algorithms and artificial neural networks (ANNs), we developed a new method for embryo analysis that is more robust and reliable than standard methods. Bovine blastocysts produced in vitro were classified as grade 1 (excellent or good), 2 (fair), or 3 (poor) by three experienced embryologists according to the International Embryo Technology Society (IETS) standard. The images (n = 482) were subjected to automatic feature extraction, and the results were used as input for a supervised learning process. One part of the dataset (15%) was used for a blind test posterior to the fitting, for which the system had an accuracy of 76.4%. Interestingly, when the same embryologists evaluated a sub-sample (10%) of the dataset, there was only 54.0% agreement with the standard (mode for grades). However, when using the ANN to assess this sub-sample, there was 87.5% agreement with the modal values obtained by the evaluators. The presented methodology is covered by National Institute of Industrial Property (INPI) and World Intellectual Property Organization (WIPO) patents and is currently undergoing a commercial evaluation of its feasibility.