RESUMO
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.
Assuntos
Adesinas Bacterianas/genética , Proteínas de Bactérias/genética , Genoma Bacteriano , Interações Hospedeiro-Patógeno , Leptospira interrogans/genética , Aderência Bacteriana/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Humanos , Leptospira interrogans/química , Leptospira interrogans/patogenicidade , Leptospirose/microbiologia , Ligação Proteica , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
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.
RESUMO
Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the ß-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.
Assuntos
Engenharia Genética/métodos , Leptospira/fisiologia , RNA Guia de Cinetoplastídeos/genética , Streptococcus pyogenes/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Inativação Gênica , RNARESUMO
Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the ß-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.
RESUMO
Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigA(C)) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigA(C), either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigA(C) or LigA(C) coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Flagelina/administração & dosagem , Leptospira interrogans/imunologia , Leptospirose/prevenção & controle , Hidróxido de Alumínio/administração & dosagem , Animais , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Imunoglobulina G/sangue , Rim/microbiologia , Leptospira interrogans/genética , Leptospirose/imunologia , Masculino , Mesocricetus , Análise de Sobrevida , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Leptospirosis is a re-emergent zoonosis, caused by pathogenic spirochetes from the genus Lepstospira. To date, there is no protein described to be involved in leptospiral hemorrhagic manifestations, although several proteases have been reported for other bacterial infections. In this study we identified 12 putative metalloproteases from the genome of Leptospira interrogans, and characterized for the first time a putative metalloprotease, here named Leptallo I, as a potential Zn(2+) dependent glycylglycine protease belonging to the M23 metalloendopeptidase family. The native protein was detected in extracts from several pathogenic Leptospira species and further shown to be secreted to the culture medium. We expressed the recombinant protein and its C-terminal fragment containing the metalloprotease domain, and both presented regular secondary structures. The sera of humans with leptospirosis were able to recognize rLeptallo I, indicating that the native protein is expressed and presented to the immune system during infection. The recombinant proteins displayed a significant, though relatively low, elastinolytic activity, and the challenge of hamsters immunized with rLeptallo I conferred 33% protection, suggesting a significant importance of this protein in the pathogenesis. The elastinolytic activity may be important for leptospires-host interaction, because elastin constitutes a significant proportion of total lung and blood vessel proteins.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Elastina/metabolismo , Leptospira interrogans/patogenicidade , Leptospirose/prevenção & controle , Metaloproteases/metabolismo , Elastase Pancreática/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cricetinae , Elastina/genética , Humanos , Leptospira interrogans/genética , Leptospira interrogans/metabolismo , Leptospirose/imunologia , Leptospirose/microbiologia , Masculino , Mesocricetus , Metaloproteases/química , Metaloproteases/genética , Metaloproteases/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Elastase Pancreática/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNARESUMO
ABSTRACT It was compared the antibody response of sows immunized with two experimental vaccines produced with L.interrogans, serovar Canicola, strain LO-4, isolated in Brazil.One of the vaccines was the usual bacterin (whole culture inactivated with phenol and adjuvanted with aluminum hydroxide -WC-AlOH3) and the other one was a subunit vaccine produced with a lipopolysaccharide (LPS) fraction extracted from the bacteria outer envelop and with the lipid A, also extracted from the leptospira wall as adjuvant (LPS-MPLA). Experiment was as follows: group 1 (n = 11), not immunized control, group 2, (n = 11): two immunization with 30 days interval of LPS-MPLA vaccine and group 3 (n = 11): two immunization with 30 days interval of WC-AlOH3 vaccine All three groups were simultaneously immunized, independently of pregnancy stage. Both agglutinin and neutralizing post vaccination antibodies levels were measured respectively by the microscopic sera agglutination with live antigens test (MAT) and the in vitro leptospira growth inhibition test (GIT). Sera collections were performed each 30 days during four months after the first vaccination. Non vaccinated control group animals presented no agglutinating antibodies against Canicola serovar during the whole experiment. At 32 and 68 post vaccination days the agglutinating antibodies levels of group 2 (LPS-MPLA) were significantly higher than the observed in group 3 (WC AlOH3), respectively, p = 0.013 and p = 0.031. The differences observed in the growth inhibition antibodies titers of the two vaccines tested were not significant (p > 0.05). Despite the peak of post-vaccination agglutinins have been registered at 68 days after first immunization, higher levels growth inhibition antibodies were detected at 30 days of first vaccination. Subunit vaccine presented the same immunogenic capacity for the production of neutralizing antibodies as the whole culture one.
RESUMO Foi comparada a resposta imune de fêmeas suínas adultas imunizadas contra a leptospirose, com vacinas monovalentes produzidas com L.interrogans, sorovar Canicola estirpe LO4, isolada no Brasil. A vacina foi empregada em duas formas: cultura de bactérias totais inativada e acrescida do adjuvante de hidróxido de alumínio (WC-AlOH3) e a do tipo de subunidade constituída apenas por uma fração de lipopolisacarídios (LPS) extraídos do envelope externo da bactéria tendo como adjuvante o monofosforil lipídio A, também extraído da parede da leptospira (LPS-MPLA). O delineamento experimental incluiu: grupo 1 (n = 11): controle não imunizado; grupo 2 (n = 11): imunizado com duas aplicações em intervalo de 30 dias da vacina LPS MFLA; Grupo 3 (n = 11): imunizado com duas aplicações em intervalo de 30 dias da vacina WC-AlOH3. Todos os grupos foram imunizados simultaneamente sem ser considerado o estágio de gestação dos animais. Os níveis de anticorpos pós-vacinais, aglutinantes e neutralizantes foram avaliados, respectivamente, pelos testes de soroaglutinação microscópica com antígenos vivos (SAM) e o de inibição do crescimento de leptospiras in vitro (ICLIV). O monitoramento sorológico foi efetuado a cada 30 dias durante quatro meses após aplicação da primeira dose da vacina. Os animais do grupo controle, não vacinados, não apresentaram anticorpos aglutinantes para o sorovar Canicola durante todo o período experimental. Aos 32 e 68 dias da primo-vacinação, os níveis de anticorpos aglutinantes do grupo 2 (LPS-MPLA) foram significativamente superiores aos observados no grupo 3 (WC AlOH3), respectivamente p = 0,013 e p = 0,031. As diferenças observadas nos níveis de anticorpos inibidores do crescimento de leptospiras in vitro, induzidos pelas duas vacinas, não foram significativas (p > 0,05). A despeito do pico de anticorpos aglutinantes pós-vacinais ter sido registrado aos 68 dias da primeira imunização, os níveis mais elevados de anticorpos inibidores do crescimento de leptospiras já foram observados aos 30 dias da primo-vacinação. A vacina de subunidade apresentou a mesma capacidade de indução de anticorpos neutralizantes que a vacina de bactérias totais.
RESUMO
It was compared the antibody response of sows immunized with two experimental vaccines produced with L.interrogans, serovar Canicola, strain LO-4, isolated in Brazil.One of the vaccines was the usual bacterin (whole culture inactivated with phenol and adjuvanted with aluminum hydroxide -WC-AlOH3) and the other one was a subunit vaccine produced with a lipopolysaccharide (LPS) fraction extracted from the bacteria outer envelop and with the lipid A, also extracted from the leptospira wall as adjuvant (LPS-MPLA). Experiment was as follows: group 1 (n = 11), not immunized control, group 2, (n = 11): two immunization with 30 days interval of LPS-MPLA vaccine and group 3 (n = 11): two immunization with 30 days interval of WC-AlOH3 vaccine All three groups were simultaneously immunized, independently of pregnancy stage. Both agglutinin and neutralizing post vaccination antibodies levels were measured respectively by the microscopic sera agglutination with live antigens test (MAT) and the in vitro leptospira growth inhibition test (GIT). Sera collections were performed each 30 days during four months after the first vaccination. Non vaccinated control group animals presented no agglutinating antibodies against Canicola serovar during the whole experiment. At 32 and 68 post vaccination days the agglutinating antibodies levels of group 2 (LPS-MPLA) were significantly higher than the observed in group 3 (WC AlOH3), respectively, p = 0.013 and p = 0.031. The differences observed in the growth inhibition antibodies titers of the two vaccines tested were not significant (p > 0.05). Despite the peak of post-vaccination agglutinins have been registered at 68 days after first immunization, higher levels growth inhibition antibodies were detected at 30 days of first vaccination. Subunit vaccine presented the same immunogenic capacity for the production of neutralizing antibodies as the whole culture one.
Foi comparada a resposta imune de fêmeas suínas adultas imunizadas contra a leptospirose, com vacinas monovalentes produzidas com L.interrogans, sorovar Canicola estirpe LO4, isolada no Brasil. A vacina foi empregada em duas formas: cultura de bactérias totais inativada e acrescida do adjuvante de hidróxido de alumínio (WC-AlOH3) e a do tipo de subunidade constituída apenas por uma fração de lipopolisacarídios (LPS) extraídos do envelope externo da bactéria tendo como adjuvante o monofosforil lipídio A, também extraído da parede da leptospira (LPS-MPLA). O delineamento experimental incluiu: grupo 1 (n = 11): controle não imunizado; grupo 2 (n = 11): imunizado com duas aplicações em intervalo de 30 dias da vacina LPS MFLA; Grupo 3 (n = 11): imunizado com duas aplicações em intervalo de 30 dias da vacina WC-AlOH3. Todos os grupos foram imunizados simultaneamente sem ser considerado o estágio de gestação dos animais. Os níveis de anticorpos pós-vacinais, aglutinantes e neutralizantes foram avaliados, respectivamente, pelos testes de soroaglutinação microscópica com antígenos vivos (SAM) e o de inibição do crescimento de leptospiras in vitro (ICLIV). O monitoramento sorológico foi efetuado a cada 30 dias durante quatro meses após aplicação da primeira dose da vacina. Os animais do grupo controle, não vacinados, não apresentaram anticorpos aglutinantes para o sorovar Canicola durante todo o período experimental. Aos 32 e 68 dias da primo-vacinação, os níveis de anticorpos aglutinantes do grupo 2 (LPS-MPLA) foram significativamente superiores aos observados no grupo 3 (WC AlOH3), respectivamente p = 0,013 e p = 0,031. As diferenças observadas nos níveis de anticorpos inibidores do crescimento de leptospiras in vitro, induzidos pelas duas vacinas, não foram significativas (p > 0,05). A despeito do pico de anticorpos aglutinantes pós-vacinais ter sido registrado aos 68 dias da primeira imunização, os níveis mais elevados de anticorpos inibidores do crescimento de leptospiras já foram observados aos 30 dias da primo-vacinação. A vacina de subunidade apresentou a mesma capacidade de indução de anticorpos neutralizantes que a vacina de bactérias totais.
Assuntos
Animais , Suínos/imunologia , Vacinas de Produtos Inativados , Leptospira/crescimento & desenvolvimento , Leptospirose/veterinária , AnticorposRESUMO
O presente estudo determinou a prevalência de anticorpos anti-Leptospira spp. em ovinos do Município de Monte Negro, RO. Foram examinados soros de 141 ovinos de raça, idade e sexo variados provenientes de 15 fazendas, pela técnica de Soroaglutinação Microscópica. Doze (80,0%) propriedades apresentaram pelo menos um animal reagente. Títulos de anticorpos iguais ou superiores a 100 foram detectados em 47 (33,3%) animais, e os sorovares mais frequentes foram Patoc (29,7%), Autumnalis (14,8%), Pyrogenes (10,6%), Australis (4,2%), Bratislava (4,2%), Hardjo (4,2%), Icterohaemorrhagiae (4,2%), Castellonis (2,1%) e Hebdomadis (2,1%). Em 11 (23,4%) soros não foi possível a determinação do provável sorovar envolvido na reação. Alerta-se também para a possibilidade de infecção no homem, tendo em vista as características regionais de fronteira agrícola amazônica.
The present study determined the prevalence of anti-Leptospira spp.antibodies in 141 ovines from 15 farms of the Monte Negro Municipality, Rondonia State, Brazil, by the microscopic agglutination test. Twelve (80.0%) farms presented at least 1 reactive animal. Antibodies titers of ? 100 were detected in 47 (33.3%) animals, the most frequent serovars being Patoc (29.7%), Autumnalis (14.8%), Pyrogenes (10.6%), Australis (4.2%), Bratislava (4.2%), Hardjo (4.2%), Icterohaemorrhagiae (4.2%), Castellonis (2.1%) and Hebdomadis (2.1%). In 11 (23.4%) sera it was not possible to determine the most frequent serovar involved. The results raise a warning as to the possibility of infection in the human being by Leptospira in light of the regional characteristics of the Amazon agricultural frontier.
Assuntos
Animais , Ovinos/imunologia , Leptospirose/sangue , Leptospirose/epidemiologia , Brasil/epidemiologia , Testes de Hemaglutinação/veterinária , Ecossistema AmazônicoRESUMO
The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.