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.

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.

Inactivation of the antimicrobial peptide LL-37 by pathogenic Leptospira.

Leptospires are aerobic, Gram-negative spirochetes with a high invasive capacity. Pathogenic leptospires secrete proteases that inactivate a variety of host's proteins including molecules of the extracellular matrix and of the human complement system. This strategy, used by several pathogens of medical importance, contributes to bacterial invasion and immune evasion. In the current work we present evidence that Leptospira proteases also target human cathelicidin (LL-37), an antimicrobial peptide that plays an important role in the innate immune response. By using six Leptospira strains, four pathogenic and two saprophytic, we demonstrated that proteases present in the supernatants of pathogenic strains were capable of degrading LL-37 in a time-dependent manner, whereas proteolytic degradation was not observed with the supernatants of the two saprophytic strains. Inactivation of LL-37 was prevented by using the 1,10-phenanthroline inhibitor, thus suggesting the involvement of metalloproteinases in this process. In addition, the antibacterial activity of LL-37 against two Leptospira strains was evaluated. Compared to the saprophytic strain, a greater resistance of the pathogenic strain to the action of the peptide was observed. Our data suggest that the capacity to inactivate the host defense peptide LL-37 may be part of the virulence arsenal of pathogenic Leptospira, and we hypothesize that its inactivation by the bacteria may influence the outcome of the disease.

Inactivation of the antimicrobial peptide LL-37 by pathogenic Leptospira

Leptospires are aerobic, Gram-negative spirochetes with a high invasive capacity. Pathogenic leptospires secrete proteases that inactivate a variety of host's proteins including molecules of the extracellular matrix and of the human complement system. This strategy, used by several pathogens of medical importance, contributes to bacterial invasion and immune evasion. In the current work we present evidence that Leptospira proteases also target human cathelicidin (LL-37), an antimicrobial peptide that plays an important role in the innate immune response. By using six Leptospira strains, four pathogenic and two saprophytic, we demonstrated that proteases present in the supernatants of pathogenic strains were capable of degrading LL-37 in a time-dependent manner, whereas proteolytic degradation was not observed with the supernatants of the two saprophytic strains. Inactivation of LL-37 was prevented by using the 1,10-phenanthroline inhibitor, thus suggesting the involvement of metalloproteinases in this process. In addition, the antibacterial activity of LL-37 against two Leptospira strains was evaluated. Compared to the saprophytic strain, a greater resistance of the pathogenic strain to the action of the peptide was observed. Our data suggest that the capacity to inactivate the host defense peptide LL-37 may be part of the virulence arsenal of pathogenic Leptospira, and we hypothesize that its inactivation by the bacteria may influence the outcome of the disease.

Immunoprotective Activity Induced by Leptospiral Outer Membrane Proteins in Hamster Model of Acute Leptospirosis.

Leptospirosis is a zoonotic disease of worldwide distribution, affecting both humans and animals. The development of an effective vaccine against leptospirosis has long been pursued but without success. Humans are contaminated after direct contact with the urine of infected animals or indirectly by contaminated water or soil. The vaccines available consist of inactivated whole-bacterial cells, and the active immunoprotective antigen is the lipopolysaccharide moiety, which is also the basis for serovar classification. However, these vaccines are short-lasting, and protection is only against serovars contained in the preparation. The search for prevalent antigens, present in pathogenic species of Leptospira, represents the most cost-effective strategy for prevention of leptospirosis. Thus, the identification of these antigens is a priority. In this study, we examined the immunoprotective effect of eight leptospiral recombinant proteins using hamster as the challenge model. Animals received subcutaneously two doses of vaccine containing 50 µg of each recombinant protein adsorbed on alum adjuvant. Two weeks after the booster, animals were challenged with virulent leptospires and monitored for 21 days. All proteins were able to induce a specific immune response, although significant protective effects on survival rate were observed only for the proteins Lsa14, rLIC13259, and rLIC11711. Of these, only rLIC13259 and rLIC11711 were found to be highly prospective in promoting renal clearance. The sterilizing potential of both proteins will be further investigated to elucidate the immunoprotective mechanisms involved in leptospirosis control. These are the first proteins involved with human complement components with the capacity to protect against virulent challenge and to eliminate the bacteria from the host.

Seroprevalence and incidence of Leptospira spp. in domestic dogs in the Southeast region of São Paulo State, Brazil / Soroprevalência e incidência de Leptospira spp. em cães domésticos na região sudeste do estado de São Paulo, Brasil

Leptospirosis is a zoonotic disease caused by Leptospira and domestic dogs can act as host of some serovars. In order to analyze the transmission dynamics in a dog population, with and without immunization, a longitudinal study was carried out with a focus to evaluate antibody response and to identify serovars. Blood samples were collected in three consecutive years (2015 to 2017) from 331, 373 and 347 dogs respectively. The dog seroprevalence in each year was 11%, 7% and 14%, respectively, and the incidence in 2016 was 5% and in 2017, 14%. The most frequent serovars were Cynopteri and Butembo in 2015, Cynopteri, Butembo and Hardjoprajitno in 2016, and Canicola and Butembo in 2017. Dogs can play a role as sentinel animals and hosts of Leptospira serovars. The percentage of seropositive dogs due to vaccination was higher than the previous years without immunization and lower than in previous years for other serovars, which we interpret as evidence for the importance of immunization. These parameters associated with active canine population control are important for prevention and control of leptospirosis not only in dogs but alsoto inhibit the transmission between dogs and humans.(AU)

A leptospirose é uma zoonose causada pelo agente etiológico Leptospira. Cães domésticos atuam como hospedeiro de diversos sorovares deste agente. Com intuito de analisar a dinâmica da leptospirose em uma população canina, com e sem imunização, um estudo longitudinal foi realizado avaliando a resposta sorológica destes animais e identificando seus sorovares. Foram coletadas amostras de 331, 373 e 347 cães em três anos consecutivos (2015 a 2017). As soroprevalências foram de 11%, 7% e 14%, respectivamente, e a incidência em 2016 foi de 5% e em 2017 de 14%. Os sorovares mais frequentes foram Cynopteri e Butembo em 2015, Cynopteri, Butembo e Hardjoprajitno em 2016, e Canicola e Butembo em 2017. Estes cães estão atuando como bio-indicadores da presença de Leptospira na região do estudo, incluindo sorovares zoonóticos, e contribuindo com a sua manutenção no ambiente. A soropositividade para sorovares protegidos pela vacina foi mais alta do que nos anos anteriores à imunização, enquanto para os sorovares não protegidos pela vacina diminuiu, demonstrando a importância da imunização para essa população de cães. Medidas de prevenção e controle para a leptospirose, como imunização e controle populacional canino, são recomendadas no local para inibir a transmissão do agente entre as populações de cães e humanos envolvidas.(AU)

Prevalence of Leptospira serogroups in buffaloes from the Brazilian Amazon.

Although Brazil has one of the largest buffalo populations in the Americas, buffalo leptospirosis is still poorly explored when compared to that in bovines; thus, the aim of this research was to carry out a large serological study for leptospirosis in this species in the Brazilian Amazon. For this, we collected 1,405 serum samples from buffaloes raised in the Amazon delta region, which is considered a major area of buffalo production in Brazil. The test used was a microscopic agglutination test (MAT) adopting 34 Leptospira antigens, some of which have never been tested for buffaloes in Brazil, including autochthonous strains; in total, 20 serogroups were evaluated. From the total of 1,405 serum samples, 894 (63.6%) reacted in the MAT to at least one of the 20 serogroups, and 511 (36.4%) did not react. The serogroups Sejroe, Autumnalis and Pomona were the most prevalent, with titres ranging from 100 to 12,800, and the autochthonous strains used were not significant in relation to the reference serovars. Leptospirosis in buffaloes seems to have a serological profile similar to leptospirosis in cattle, mainly due to the prevalence of the Sejroe serogroup; however, the results of this study suggested that in the Brazilian Amazon, Leptospira strains that are serologically distinct from the autochthonous strains isolated in the southeastern region of Brazil may be circulating in these animals. Other serovars could also be inserted into the panel of antigens used in MAT for serological studies on buffaloes.

New strategies for Leptospira vaccine development based on LPS removal.

Pathogenic spirochetes from genus Leptospira are etiologic agents of leptospirosis. Cellular vaccines against Leptospira infection often elicit mainly response against the LPS antigen of the serovars present in the formulation. There is no suitable protein candidate capable of replacing whole-cell vaccines, thus requiring new approaches on vaccine development to improve leptospirosis prevention. Our goal was to develop a whole-cell vaccine sorovar-independent based on LPS removal and conservation of protein antigens exposure, to evaluate the protective capacity of monovalent or bivalent vaccines against homologous and heterologous virulent Leptospira in hamster. Leptospire were subjected to heat inactivation, or to LPS extraction with butanol and in some cases further inactivation with formaldehyde. Hamsters were immunized and challenged with homologous or heterologous virulent serovars, blood and organs were collected from the survivors for bacterial quantification, chemokine evaluation, and analysis of sera antibody reactivity and cross-reactivity by Western blot. Immunization with either heated or low LPS vaccines with serovar Copenhageni or Canicola resulted in 100% protection of the animals challenged with homologous virulent bacteria. Notably, different from the whole-cell vaccine, the low LPS vaccines produced with serovar Canicola provided only partial protection in heterologous challenge with the virulent Copenhageni serovar. Immunization with bivalent formulation results in 100% protection of immunized animals challenged with virulent serovar Canicola. All vaccines produced were able to eliminate bacteria from the kidney of challenged animals. All the vaccines raised antibodies capable to recognize antigens of serovars not present in the vaccine formulation. Transcripts of IFNγ, CXCL16, CCL5, CXCL10, CXCR6, and CCR5, increased in all immunized animals. Conclusion: Our results showed that bivalent vaccines with reduced LPS may be an interesting strategy for protection against heterologous virulent serovars. Besides the desirable multivalent protection, the low LPS vaccines are specially promising due to the expected lower reatogenicity.

New strategies for Leptospira vaccine development based on LPS removal

Pathogenic spirochetes from genus Leptospira are etiologic agents of leptospirosis. Cellular vaccines against Leptospira infection often elicit mainly response against the LPS antigen of the serovars present in the formulation. There is no suitable protein candidate capable of replacing whole-cell vaccines, thus requiring new approaches on vaccine development to improve leptospirosis prevention. Our goal was to develop a whole-cell vaccine sorovar-independent based on LPS removal and conservation of protein antigens exposure, to evaluate the protective capacity of monovalent or bivalent vaccines against homologous and heterologous virulent Leptospira in hamster. Leptospire were subjected to heat inactivation, or to LPS extraction with butanol and in some cases further inactivation with formaldehyde. Hamsters were immunized and challenged with homologous or heterologous virulent serovars, blood and organs were collected from the survivors for bacterial quantification, chemokine evaluation, and analysis of sera antibody reactivity and cross-reactivity by Western blot. Immunization with either heated or low LPS vaccines with serovar Copenhageni or Canicola resulted in 100% protection of the animals challenged with homologous virulent bacteria. Notably, different from the whole-cell vaccine, the low LPS vaccines produced with serovar Canicola provided only partial protection in heterologous challenge with the virulent Copenhageni serovar. Immunization with bivalent formulation results in 100% protection of immunized animals challenged with virulent serovar Canicola. All vaccines produced were able to eliminate bacteria from the kidney of challenged animals. All the vaccines raised antibodies capable to recognize antigens of serovars not present in the vaccine formulation. Transcripts of IFN?, CXCL16, CCL5, CXCL10, CXCR6, and CCR5, increased in all immunized animals. Conclusion: Our results showed that bivalent vaccines with reduced LPS may be an interesting strategy for protection against heterologous virulent serovars. Besides the desirable multivalent protection, the low LPS vaccines are specially promising due to the expected lower reatogenicity

Inactivation of the antimicrobial peptide LL-37 by pathogenic Leptospira

Leptospires are aerobic, Gram-negative spirochetes with a high invasive capacity. Pathogenic leptospires secrete proteases that inactivate a variety of host's proteins including molecules of the extracellular matrix and of the human complement system. This strategy, used by several pathogens of medical importance, contributes to bacterial invasion and immune evasion. In the current work we present evidence that Leptospira proteases also target human cathelicidin (LL-37), an antimicrobial peptide that plays an important role in the innate immune response. By using six Leptospira strains, four pathogenic and two saprophytic, we demonstrated that proteases present in the supernatants of pathogenic strains were capable of degrading LL-37 in a time-dependent manner, whereas proteolytic degradation was not observed with the supernatants of the two saprophytic strains. Inactivation of LL-37 was prevented by using the 1,10-phenanthroline inhibitor, thus suggesting the involvement of metalloproteinases in this process. In addition, the antibacterial activity of LL-37 against two Leptospira strains was evaluated. Compared to the saprophytic strain, a greater resistance of the pathogenic strain to the action of the peptide was observed. Our data suggest that the capacity to inactivate the host defense peptide LL-37 may be part of the virulence arsenal of pathogenic Leptospira, and we hypothesize that its inactivation by the bacteria may influence the outcome of the disease.

Immunoprotective activity induced by leptospiral outer membrane proteins in hamster model of acute leptospirosis

Leptospirosis is a zoonotic disease of worldwide distribution, affecting both humans and animals. The development of an effective vaccine against leptospirosis has long been pursued but without success. Humans are contaminated after direct contact with the urine of infected animals or indirectly by contaminated water or soil. The vaccines available consist of inactivated whole-bacterial cells, and the active immunoprotective antigen is the lipopolysaccharide moiety, which is also the basis for serovar classification. However, these vaccines are short-lasting, and protection is only against serovars contained in the preparation. The search for prevalent antigens, present in pathogenic species of Leptospira, represents the most cost-effective strategy for prevention of leptospirosis. Thus, the identification of these antigens is a priority. In this study, we examined the immunoprotective effect of eight leptospiral recombinant proteins using hamster as the challenge model. Animals received subcutaneously two doses of vaccine containing 50 μg of each recombinant protein adsorbed on alum adjuvant. Two weeks after the booster, animals were challenged with virulent leptospires and monitored for 21 days. All proteins were able to induce a specific immune response, although significant protective effects on survival rate were observed only for the proteins Lsa14, rLIC13259, and rLIC11711. Of these, only rLIC13259 and rLIC11711 were found to be highly prospective in promoting renal clearance. The sterilizing potential of both proteins will be further investigated to elucidate the immunoprotective mechanisms involved in leptospirosis control. These are the first proteins involved with human complement components with the capacity to protect against virulent challenge and to eliminate the bacteria from the host.

New strategies for Leptospira vaccine development based on LPS removal

Pathogenic spirochetes from genus Leptospira are etiologic agents of leptospirosis. Cellular vaccines against Leptospira infection often elicit mainly response against the LPS antigen of the serovars present in the formulation. There is no suitable protein candidate capable of replacing whole-cell vaccines, thus requiring new approaches on vaccine development to improve leptospirosis prevention. Our goal was to develop a whole-cell vaccine sorovar-independent based on LPS removal and conservation of protein antigens exposure, to evaluate the protective capacity of monovalent or bivalent vaccines against homologous and heterologous virulent Leptospira in hamster. Leptospire were subjected to heat inactivation, or to LPS extraction with butanol and in some cases further inactivation with formaldehyde. Hamsters were immunized and challenged with homologous or heterologous virulent serovars, blood and organs were collected from the survivors for bacterial quantification, chemokine evaluation, and analysis of sera antibody reactivity and cross-reactivity by Western blot. Immunization with either heated or low LPS vaccines with serovar Copenhageni or Canicola resulted in 100% protection of the animals challenged with homologous virulent bacteria. Notably, different from the whole-cell vaccine, the low LPS vaccines produced with serovar Canicola provided only partial protection in heterologous challenge with the virulent Copenhageni serovar. Immunization with bivalent formulation results in 100% protection of immunized animals challenged with virulent serovar Canicola. All vaccines produced were able to eliminate bacteria from the kidney of challenged animals. All the vaccines raised antibodies capable to recognize antigens of serovars not present in the vaccine formulation. Transcripts of IFN?, CXCL16, CCL5, CXCL10, CXCR6, and CCR5, increased in all immunized animals. Conclusion: Our results showed that bivalent vaccines with reduced LPS may be an interesting strategy for protection against heterologous virulent serovars. Besides the desirable multivalent protection, the low LPS vaccines are specially promising due to the expected lower reatogenicity

The interaction of two novel putative proteins of Leptospira interrogans with E-cadherin, plasminogen and complement components with potential role in bacterial infection.

Leptospirosis is a worldwide zoonosis caused by pathogenic species of Leptospira. Leptospires are able to adhere to exposed extracellular matrix in injured tissues and, once in the bloodstream, can survive the attack of the immune system and spread to colonize target organs. In this work, we report that two novel putative proteins, coded by the genes LIC11711 and LIC12587 of L. interrogans serovar Copenhageni are conserved among pathogenic strains, and probably exposed in the bacterial surface. Soluble recombinant proteins were expressed in Escherichia coli, purified and characterized. Both recombinant proteins bound to laminin and E-cadherin, suggesting an initial adhesion function in host epithelial cells. The recombinant protein LIC11711 (rLIC11711) was able to capture plasminogen (PLG) from normal human serum and convert to enzymatically active plasmin (PLA), in the presence of PLG activator. rLIC12587 (recombinant protein LIC12587) displayed a dose dependent and saturable interaction with components C7, C8, and C9 of the complement system, reducing the bactericidal effect of the complement. Binding to C9 may have consequences such as C9 polymerization inhibition, interfering with the membrane attack complex formation. Blocking LIC11711 and LIC12587 on bacterial cells by the respective antiserum reduced leptospiral cell viability when exposed to normal human serum (NHS). Both recombinant proteins could be recognized by serum samples of confirmed leptospirosis, but not of unrelated diseases, suggesting that the native proteins are immunogenic and expressed during leptospirosis. Taken together, our data suggest that these proteins may have a role in leptospiral pathogenesis, participating in immune evasion strategies.

Leptospira spp., rotavirus, norovirus, and hepatitis E virus surveillance in a wild invasive golden-headed lion tamarin (Leontopithecus chrysomelas; Kuhl, 1820) population from an urban park in Niterói, Rio de Janeiro, Brazil.

The world currently faces severe biodiversity losses caused by anthropogenic activities such as deforestation, pollution, the introduction of exotic species, habitat fragmentation, and climate changes. Disease ecology in altered environments is still poorly understood. The golden-headed lion tamarin (GHLT, Leontopithecus chrysomelas) is an endangered species that became invasive in an urban park in Niterói, Rio de Janeiro, Brazil. The initially few invasive GHLT individuals became hundreds, adapted to living in proximity to humans and domestic animals. These GHLTs were captured as part of a conservation project; some animals were translocated to Bahia and some were kept in captivity. This study tested 593 GHLT for Leptospira serology; 100 and 95 GHLT for polymerase chain reaction (PCR) toLeptospira and hepatitis E virus genotype 3 (HEV-3), respectively, and 101 familiar groups for PCR to viruses (rotavirus A, norovirus GI and GII, and HEV-3). One animal had antibodies for Leptospira serovar Shermani and another for serovar Hebdomadis. One saprophyticLeptospira was found by the 16S PCR and sequencing. Viruses were not detected in samples tested. Findings suggest that the epidemiological importance of such pathogens in this GHLT population is either low or nonexistent. These data are important to understand the local disease ecology, as well as monitoring a translocation project, and to contribute data for species conservation.

Characterization of a novel protein of Leptospira interrogans exhibiting plasminogen, vitronectin and complement binding properties.

Leptospirosis is a severe zoonosis caused by pathogenic species of the genus Leptospira. This work focuses on a hypothetical protein of unknown function, encoded by the gene LIC13259, and predicted to be a surface protein, widely distributed among pathogenic leptospiral strain. The gene was amplified from L. interrogans serovar Copenhageni, strain Fiocruz L1-130, cloned and the protein expressed using Escherichia coli as a host system. Immunofluorescence assay showed that the protein is surface-exposed. The recombinant protein LIC13259 (rLIC13259) has the ability to interact with the extracellular matrix (ECM) laminin, in a dose-dependent manner but saturation was not reach. The rLIC13259 protein is a plasminogen (PLG)-binding protein, generating plasmin, in the presence of urokinase PLG-activator uPA. The recombinant protein is able to mediate the binding to human purified terminal complement route vitronectin, C7, C8 and C9, and to recruit and interact with these components from normal human serum (NHS). These interactions are dose-dependent on NHS increased concentration. The binding of rLIC13259 to C8 and vitronectin was slight and pronounced inhibited in the presence of increasing heparin concentration, respectively, suggesting that the interaction with vitronectin occurs via heparin domain. Most interesting, the interaction of rLIC13259 with C9 protein was capable of preventing C9 polymerization, suggesting that the membrane attack complex (MAC) formation was inhibited. Thus, we tentatively assign the coding sequence (CDS) LIC13259, previously annotated as unknown function, as a novel protein that may play an important role in the host's invasion and immune evasion processes, contributing to the establishment of the leptospiral infection.

The interaction of two novel putative proteins of Leptospira interrogans with E-cadherin, plasminogen and complement components with potential role in bacterial infection

Leptospirosis is a worldwide zoonosis caused by pathogenic species of Leptospira. Leptospires are able to adhere to exposed extracellular matrix in injured tissues and, once in the bloodstream, can survive the attack of the immune system and spread to colonize target organs. In this work, we report that two novel putative proteins, coded by the genes LIC11711 and LIC12587 of L. interrogans serovar Copenhageni are conserved among pathogenic strains, and probably exposed in the bacterial surface. Soluble recombinant proteins were expressed in Escherichia coli, purified and characterized. Both recombinant proteins bound to laminin and E-cadherin, suggesting an initial adhesion function in host epithelial cells. The recombinant protein LIC11711 (rLIC11711) was able to capture plasminogen (PLG) from normal human serum and convert to enzymatically active plasmin (PLA), in the presence of PLG activator. rLIC12587 (recombinant protein LIC12587) displayed a dose dependent and saturable interaction with components C7, C8, and C9 of the complement system, reducing the bactericidal effect of the complement. Binding to C9 may have consequences such as C9 polymerization inhibition, interfering with the membrane attack complex formation. Blocking LIC11711 and LIC12587 on bacterial cells by the respective antiserum reduced leptospiral cell viability when exposed to normal human serum (NHS). Both recombinant proteins could be recognized by serum samples of confirmed leptospirosis, but not of unrelated diseases, suggesting that the native proteins are immunogenic and expressed during leptospirosis. Taken together, our data suggest that these proteins may have a role in leptospiral pathogenesis, participating in immune evasion strategies.

Characterization of a novel protein of Leptospira interrogans exhibiting plasminogen, vitronectin and complement binding properties

Leptospirosis is a severe zoonosis caused by pathogenic species of the genus Leptospira. This work focuses on a hypothetical protein of unknown function, encoded by the gene LIC13259, and predicted to be a surface protein, widely distributed among pathogenic leptospiral strain. The gene was amplified from L. interrogans serovar Copenhageni, strain Fiocruz L1-130, cloned and the protein expressed using Escherichia coli as a host system. Immunofluorescence assay showed that the protein is surface-exposed. The recombinant protein LIC13259 (rLIC13259) has the ability to interact with the extracellular matrix (ECM) laminin, in a dose-dependent manner but saturation was not reach. The rLIC13259 protein is a plasminogen (PLG)-binding protein, generating plasmin, in the presence of urokinase PLG-activator uPA. The recombinant protein is able to mediate the binding to human purified terminal complement route vitronectin, C7, C8 and C9, and to recruit and interact with these components from normal human serum (NHS). These interactions are dose-dependent on NHS increased concentration. The binding of rLIC13259 to C8 and vitronectin was slight and pronounced inhibited in the presence of increasing heparin concentration, respectively, suggesting that the interaction with vitronectin occurs via heparin domain. Most interesting, the interaction of rLIC13259 with C9 protein was capable of preventing C9 polymerization, suggesting that the membrane attack complex (MAC) formation was inhibited. Thus, we tentatively assign the coding sequence (CDS) LIC13259, previously annotated as unknown function, as a novel protein that may play an important role in the host’s invasion and immune evasion processes, contributing to the establishment of the leptospiral infection.

The interaction of two novel putative proteins of Leptospira interrogans with E-cadherin, plasminogen and complement components with potential role in bacterial infection

Leptospirosis is a worldwide zoonosis caused by pathogenic species of Leptospira. Leptospires are able to adhere to exposed extracellular matrix in injured tissues and, once in the bloodstream, can survive the attack of the immune system and spread to colonize target organs. In this work, we report that two novel putative proteins, coded by the genes LIC11711 and LIC12587 of L. interrogans serovar Copenhageni are conserved among pathogenic strains, and probably exposed in the bacterial surface. Soluble recombinant proteins were expressed in Escherichia coli, purified and characterized. Both recombinant proteins bound to laminin and E-cadherin, suggesting an initial adhesion function in host epithelial cells. The recombinant protein LIC11711 (rLIC11711) was able to capture plasminogen (PLG) from normal human serum and convert to enzymatically active plasmin (PLA), in the presence of PLG activator. rLIC12587 (recombinant protein LIC12587) displayed a dose dependent and saturable interaction with components C7, C8, and C9 of the complement system, reducing the bactericidal effect of the complement. Binding to C9 may have consequences such as C9 polymerization inhibition, interfering with the membrane attack complex formation. Blocking LIC11711 and LIC12587 on bacterial cells by the respective antiserum reduced leptospiral cell viability when exposed to normal human serum (NHS). Both recombinant proteins could be recognized by serum samples of confirmed leptospirosis, but not of unrelated diseases, suggesting that the native proteins are immunogenic and expressed during leptospirosis. Taken together, our data suggest that these proteins may have a role in leptospiral pathogenesis, participating in immune evasion strategies.

Characterization of a novel protein of Leptospira interrogans exhibiting plasminogen, vitronectin and complement binding properties

Leptospirosis is a severe zoonosis caused by pathogenic species of the genus Leptospira. This work focuses on a hypothetical protein of unknown function, encoded by the gene LIC13259, and predicted to be a surface protein, widely distributed among pathogenic leptospiral strain. The gene was amplified from L. interrogans serovar Copenhageni, strain Fiocruz L1-130, cloned and the protein expressed using Escherichia coli as a host system. Immunofluorescence assay showed that the protein is surface-exposed. The recombinant protein LIC13259 (rLIC13259) has the ability to interact with the extracellular matrix (ECM) laminin, in a dose-dependent manner but saturation was not reach. The rLIC13259 protein is a plasminogen (PLG)-binding protein, generating plasmin, in the presence of urokinase PLG-activator uPA. The recombinant protein is able to mediate the binding to human purified terminal complement route vitronectin, C7, C8 and C9, and to recruit and interact with these components from normal human serum (NHS). These interactions are dose-dependent on NHS increased concentration. The binding of rLIC13259 to C8 and vitronectin was slight and pronounced inhibited in the presence of increasing heparin concentration, respectively, suggesting that the interaction with vitronectin occurs via heparin domain. Most interesting, the interaction of rLIC13259 with C9 protein was capable of preventing C9 polymerization, suggesting that the membrane attack complex (MAC) formation was inhibited. Thus, we tentatively assign the coding sequence (CDS) LIC13259, previously annotated as unknown function, as a novel protein that may play an important role in the host’s invasion and immune evasion processes, contributing to the establishment of the leptospiral infection.

Evaluation of Lsa46 and Lsa77 Leptospiral Proteins for Their Immunoprotective Activities in Hamster Model of Leptospirosis.

Leptospirosis is a neglected tropical disease caused by pathogenic Leptospira spp. The lack of an effective vaccine favors the increase of the disease. Currently, surface-exposed proteins are the main targets for the search of vaccine candidates. In this study, we examined whether the surface Lsa46 and Lsa77 proteins, previously identified as laminin and plasminogen binding proteins, have the capacity of inducing protection and sterilizing immunity against challenge with virulent Leptospira in hamster model. Animals were subcutaneously immunized with Lsa46, Lsa77, or a combination of both in Alum adjuvant and challenged intraperitoneally with L. interrogans serovar Kennewicki strain Pomona Fromm. Hamster immunization with Lsa46 or Lsa77 or both promoted a strong IgG response. Th2- and Th1-biased immune responses were observed when Lsa46 and Lsa77 were individually administered, respectively, as detected by the IgG1/IgG2/3 ratio. Immunized hamsters with the combined proteins induced a Th1-biased immune response. Although the immunization with Lsa46 and Lsa77 stimulated protective immunity with reduction of bacterial burden, when compared to animals individually immunized with the proteins, the data was not statistically significant. Thus, although promising, more studies are needed before the role of these proteins in stimulating sterilizing immunity in mammals is conclusively determined.