RESUMO
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.
RESUMO
Leptospirosis is a widespread zoonosis caused by bacteria of the genus Leptospira. Rodents appear to be the most important reservoirs of infection. They contaminate the environment and food and can transmit the pathogen when they are consumed by carnivores. Capybara ( Hydrochaeris hydrochaeris ) are efficient reservoirs of Leptospira, and because they are in close contact with farm animals and are found in semiurban areas, they represent a risk to public health. We isolated five Leptospira strains from capybara kidneys in Sao Paulo State, Brazil, in 2001 and typed them using serologic and molecular techniques. These strains include the Leptospira santarosai serogroup Grippotyphosa serovar Bananal. Pulsed field gel electrophoresis resulted in a unique pattern distinct from the reference strains, and the isolates clustered with greater than 85% similarity. The isolates also presented higher growth rates than other Leptospira serovars, with high minimal inhibitory concentration values for most of the tested antibiotics, with the exception of penicillin and ampicillin. This isolation and characterization of the L. santarosai serogroup Grippotyphosa serovar Bananal from capybara, highlights the importance of wild and sinantropic rodents as carriers of pathogenic leptospires.
Assuntos
Leptospira/classificação , Leptospirose/veterinária , Roedores/microbiologia , Animais , Brasil/epidemiologia , Leptospira/genética , Leptospira/isolamento & purificação , Leptospirose/epidemiologia , Leptospirose/microbiologia , FilogeniaRESUMO
Leptospirosis is a widespread systemic zoonosis, considered as reemerging in certain developing countries. Although the cross agglutinin absorption test is still considered the standard method for Leptospira identification, it presents several disadvantages. The aim of this study was to characterize Leptospira spp. isolated from various hosts by genotyping and broth microdilution susceptibility testing in an attempt to differentiate Leptospira species, serogroups and serovars. Forty-seven isolates were studied. They were previously serotyped, and species confirmation was performed by 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) and pulsed-field gel electrophoresis (PFGE) analysis enabled the distinction of L. interrogans from L. santarosai, L. meyeri and L. borgpetersenii in two main clusters. Among L. interrogans, it was possible to differentiate into two new clusters the serogroup Icterohaemorrhagiae from the serogroups Canicola and Pomona. L. santarosai isolates presented higher genetic variation than the other species in both techniques. Interestingly, the minimum inhibitory concentration (MIC) cluster analysis also provided Leptospira serogroup differentiation. Further studies are necessary regarding serovar Bananal isolates, as they presented the highest MIC values for most of the antimicrobials tested. All studied techniques successfully distinguished Leptospira species and serogroups. Despite being library-dependent methods, these approaches are less labor intensive and more economically viable, particularly SE-AFLP, and can be implemented in most reference laboratories worldwide to enable faster Leptospira typing.
Assuntos
Leptospira/classificação , Leptospira/genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Eletroforese em Gel de Campo Pulsado , Variação Genética , Genótipo , Leptospira/efeitos dos fármacos , Leptospira/imunologia , Leptospira interrogans/classificação , Leptospira interrogans/genética , Leptospira interrogans/imunologia , Testes de Sensibilidade Microbiana , Polimorfismo Genético , RNA Ribossômico 16S , Análise de Sequência de RNA , Sorogrupo , SorotipagemRESUMO
INTRODUCTION: Leptospira interrogans swine infection is a cause of serious economic loss and a potential human health hazard. In Brazil, the most common serovars associated with swine infections are Pomona, Icterohaemorrhagie and Tarassovi. Cross-reactions among serovars and the failure of infected animals to seroconvert may complicate the interpretation of serological tests. Molecular methods with better discriminatory powers are useful tools for swine leptospirosis characterization and diagnosis. METHODOLOGY: This study evaluated nine L. interrogans isolates from the States of Sao Paulo and Minas Gerais during different time periods. Isolates from diseased and apparently healthy swine were characterized by microscopic agglutination tests with polyclonal antibodies and were genotyped by VNTR, PFGE and MLST techniques. Broth microdilution was used to determine the minimal inhibitory concentration of the antimicrobials of veterinary interest. RESULTS: The strains were identified as L. interrogans serogroup Pomona serovar Pomona Genotype A, while MLST grouped all of the isolates in sequence type 37. The PFGE analysis resulted in two pulsotypes with more than 70% similarity, distinguishing serovar Pomona isolates from the serovar Kennewicki reference strain. All of the isolates presented low MIC values to penicillin, ampicillin, ceftiofur and tulathromycin. High MIC values for fluoroquinolones, tiamulin, gentamicin, tetracyclines, neomycin, tilmicosin and sulfas were also observed. CONCLUSIONS: All molecular techniques were concordant in L. interrogans serovar Pomona identification. This serovar may have a different antibiotic susceptibility profile than previously reported for Leptospira isolates.
Assuntos
Testes de Aglutinação , Portador Sadio/veterinária , Leptospira interrogans serovar pomona/classificação , Leptospira interrogans serovar pomona/isolamento & purificação , Leptospirose/veterinária , Tipagem Molecular , Doenças dos Suínos/microbiologia , Animais , Antibacterianos/farmacologia , Brasil , Portador Sadio/microbiologia , Feminino , Leptospira interrogans serovar pomona/efeitos dos fármacos , Leptospira interrogans serovar pomona/genética , Leptospirose/microbiologia , Testes de Sensibilidade Microbiana , SuínosRESUMO
Pathogenic bacteria of the genus Leptospira are the etiological agents of leptospirosis, a disease that affects humans and animals worldwide. Although there are an increasing number of studies on the biology of Leptospira, the mechanisms of pathogenesis are not yet understood. We report in this work that Leptospira interrogans FIOCRUZ L1-130 virulent, M20 culture attenuated and the saprophyte L. biflexa Patoc 1 strains do not bind prothrombin. Leptospiral binding to thrombin was detected with the virulent, followed by culture-attenuated M20, and practically none was observed with the saprophyte strain. The interaction of Leptospira with thrombin mostly occurs via exosite I, with a minor participation of catalytic site, as determined by employing the thrombin inhibitors hirugen, hirudin and argatroban. Leptospira interrogans binding to thrombin inhibits its catalytic activity reducing fibrin clot formation in thrombin-catalyzed reaction of fibrinogen. This inhibition was more efficient with the virulent FIOCRUZ L1-130 than with the M20 culture attenuated, while none was seen with the saprophyte strain, suggesting that this binding might be important for bacterial virulence. This is the first study reporting the binding of pathogenic Leptospira to thrombin promoting a decrease in fibrin clotting that could lead to hemorrhage, helping bacteria dissemination.
Assuntos
Coagulação Sanguínea , Fibrina/metabolismo , Interações Hospedeiro-Patógeno , Leptospira interrogans/fisiologia , Trombina/antagonistas & inibidores , Humanos , Ligação ProteicaRESUMO
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.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Leptospira interrogans/genética , Animais , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Genoma Bacteriano , Humanos , Leptospira interrogans/imunologia , Leptospirose/sangue , Leptospirose/imunologia , Leptospirose/microbiologia , Camundongos Endogâmicos BALB C , Filogenia , Plasminogênio/química , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease of human and veterinary concern. The quest for novel antigens that could mediate host-pathogen interactions is being pursued. Owing to their location, these antigens have the potential to elicit numerous activities, including immune response and adhesion. This study focuses on a hypothetical protein of Leptospira, encoded by the gene LIC11089, and its three derived fragments: the N-terminal, intermediate and C terminus regions. The gene coding for the full-length protein and fragments was cloned and expressed in Escherichia coli BL21(SI) strain by using the expression vector pAE. The recombinant protein and fragments tagged with hexahistidine at the N terminus were purified by metal affinity chromatography. The leptospiral full-length protein, named Lsa32 (leptospiral surface adhesin, 32 kDa), adheres to laminin, with the C terminus region being responsible for this interaction. Lsa32 binds to plasminogen in a dose-dependent fashion, generating plasmin when an activator is provided. Moreover, antibodies present in leptospirosis serum samples were able to recognize Lsa32. Lsa32 is most likely a new surface protein of Leptospira, as revealed by proteinase K susceptibility. Altogether, our data suggest that this multifaceted protein is expressed during infection and may play a role in host-L. interrogans interactions.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Laminina/metabolismo , Leptospira interrogans/genética , Leptospira interrogans/metabolismo , Leptospirose/microbiologia , Plasminogênio/metabolismo , Aderência Bacteriana , Clonagem Molecular , Biologia Computacional , Matriz Extracelular , Expressão Gênica , Genes Bacterianos , Ligação Proteica , Transporte Proteico , Transcrição GênicaRESUMO
Leptospirosis, a worldwide zoonotic infection, is an important human and veterinary health problem. We have previously identified a leptospiral multipurpose adhesin, Lsa66, capable of binding extracellular matrix (ECM) components and plasminogen (PLG). In this work, we report the cloning, expression, purification and characterization of three fragments derived from the full-length Lsa66: N-terminal, intermediate and C-terminal regions. We employed Escherichia coli BL21-SI as expression cells. The recombinant fragments tagged with N-terminal His6 were purified by metal-charged chromatography to major protein bands that were recognized by anti-His-tag mAbs. The recombinant fragments were evaluated for their capacity to attach to ECM components and to PLG. The intermediate region bound to laminin, plasma fibronectin and PLG. Laminin also bound to the C-terminal region. Antibodies in leptospirosis-positive serum samples recognized Lsa66, being the immune epitopes located at the N-terminal and intermediate fragments. The data confirm that Lsa66 is expressed during infection and that this protein might have a role in bacterial infection.
Assuntos
Adesinas Bacterianas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Plasminogênio/metabolismo , Mapeamento de Interação de Proteínas , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Cromatografia de Afinidade , Clonagem Molecular , Escherichia coli/genética , Feminino , Expressão Gênica , Camundongos Endogâmicos BALB C , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
This work shows the production and characterization of two novel putative lipoproteins encoded by the genes LIC10645 and LIC10731 identified in the genome sequences of Leptospira interrogans. In silico conservation analysis indicated that the proteins are well conserved among pathogenic leptospiral serovars and species. Recombinant proteins were obtained in Escherichia coli BL21(DE3) Star pLysS strain, purified by metal-affinity chromatography, and used for characterization and immunological evaluations. Recombinant proteins were capable of eliciting a combination of humoral and cellular immune responses in animal models, and could be recognized by antibodies present in human serum samples. The recombinant proteins Lsa44 and Lsa45 were able to bind laminin, and were named Lsa44 and Lsa45 for leptospiral surface adhesins of 44 and 45 kDa, respectively. The attachment to laminin was dose-responsive with KD values of 108.21 and 250.38 nM for Lsa44 and Lsa45, respectively. Moreover, these proteins interact with plasminogen (PLG) with KD values of 53.56 and 36.80 nM, respectively. PLG bound to the recombinant proteins could be converted to plasmin (PLA) in the presence of an activator. Cellular localization assays suggested that the Lsa44 and Lsa45 were surface-exposed. These are versatile proteins capable of interacting with laminin and PLG/PLA, and hence could mediate bacterial adhesion and contribute to tissue penetration.
Assuntos
Adesinas Bacterianas/imunologia , Adesinas Bacterianas/metabolismo , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Laminina/metabolismo , Leptospira interrogans/imunologia , Leptospira interrogans/metabolismo , Adesinas Bacterianas/genética , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Cromatografia de Afinidade , Sequência Conservada , Escherichia coli/genética , Humanos , Cinética , Leptospira interrogans/genética , Leucócitos Mononucleares/imunologia , Lipoproteínas/genética , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Plasminogênio/metabolismo , Ligação Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities.