RESUMO
Leptospirosis is a neglected disease caused by bacteria of the genus Leptospira and is more prevalent in tropical and subtropical countries. This pathogen infects humans and other animals, responsible for the most widespread zoonosis in the world, estimated to be responsible for 60 000 deaths and 1 million cases per year. To date, commercial vaccines against human leptospirosis are available only in some countries such as Japan, China, Cuba and France. These vaccines prepared with inactivated Leptospira (bacterins) induce a short-term and serovar-specific immune response, with strong adverse side effects. To circumvent these limitations, several research groups are investigating new experimental vaccines in order to ensure that they are safe, efficient, and protect against several pathogenic Leptospira serovars, inducing sterilizing immunity. Most of these protocols use attenuated cultures, preparations after LPS removal, recombinant proteins or DNA from pathogenic Leptospira spp. The aim of this review was to highlight several promising vaccine candidates, considering their immunogenicity, presence in different pathogenic Leptospira serovars, their role in virulence or immune evasion and other factors.
Assuntos
Vacinas Bacterianas , Leptospira , Leptospirose , Leptospirose/prevenção & controle , Leptospirose/imunologia , Humanos , Leptospira/imunologia , Vacinas Bacterianas/imunologia , Animais , Vacinas de Produtos Inativados/imunologiaRESUMO
The complement system plays an important role in the innate and acquired immune response against pathogens. A sophisticated network of activating and regulating proteins allows the distinction between intact and damaged host and non-host surfaces such as bacteria and other parasites. Non-host structures trigger the alternative pathway which may lead to their elimination by phagocytosis or cell lysis. In addition, complement proteins such as C1q, mannose binding lectin (MBL), and ficolins act as pathogen pattern-recognition molecules. Biological functions such as opsonization, activation of B lymphocytes and production of antibodies, degranulation of mast cells and basophils, and cell lysis that are important for elimination of microorganisms are dependent on complement activation. However, several pathogens including spirochetes have developed several specialized mechanisms to evade the complement system, thereby contributing to survival in the host. In this review, we give a brief overview of complement activation and regulation, and discuss in detail the strategies used by spirochetes from the genera Borrelia, Leptospira, and Treponema to overcome complement activation.
Assuntos
Proteínas do Sistema Complemento/imunologia , Evasão da Resposta Imune , Spirochaetales/imunologia , Borrelia/imunologia , Ativação do Complemento , Humanos , Leptospira/imunologia , Lectina de Ligação a Manose/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Treponema/imunologiaRESUMO
In addition to its effector functions, complement is an important regulator of adaptive immune responses. Murine studies suggest that complement modulates helper T-cell differentiation, and Th1 responses in particular are impaired in the absence of functional complement. Here, we have studied humoral responses to toxoid vaccines in eight patients with C3 deficiency, representing more than 25% of all the known patients worldwide. Serum cytokine levels were also studied. The patients developed normal Ig responses to tetanus and diphtheria toxoids, but IgE levels were low. The pattern of antigen-specific IgG subclasses was abnormal, with increased Th1-related IgG3 responses, low IgG2, and almost completely undetectable IgG4. The patients also had increased amounts of Th1-related cytokines IL-12p70 and IL-21, and these showed a positive correlation with IgG3 levels. Our results confirm that complement modulates Th differentiation, but reveal a more nuanced outcome than previously reported. Since IgG4 has been linked to tolerogenic responses, the data also suggest that in the absence of functional complement at least some aspects of systemic tolerance are impaired.
Assuntos
Diferenciação Celular/imunologia , Complemento C3/deficiência , Tolerância Imunológica , Imunidade Humoral/imunologia , Síndromes de Imunodeficiência/imunologia , Células Th1/imunologia , Criança , Pré-Escolar , Complemento C3/imunologia , Feminino , Doenças da Deficiência Hereditária de Complemento , Humanos , Imunidade Humoral/efeitos dos fármacos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Síndromes de Imunodeficiência/sangue , Síndromes de Imunodeficiência/patologia , Interleucina-12/sangue , Interleucina-12/imunologia , Interleucinas/sangue , Interleucinas/imunologia , Masculino , Toxoide Tetânico/administração & dosagem , Células Th1/metabolismo , Células Th1/patologia , Adulto JovemRESUMO
Enteroaggregative and uropathogenic Escherichia coli, Shigella flexneri 2a, and the hybrid enteroaggregative/Shiga toxin-producing E. coli strain (O104:H4) are important pathogens responsible for intestinal and urinary tract infections, as well as sepsis and hemolytic uremic syndrome. They have in common the production of a serine protease called Pic. Several biological roles for Pic have been described, including protection of E. coli DH5α from complement-mediated killing. Hereby we showed that Pic significantly reduces complement activation by all 3 pathways. Pic cleaves purified C3/C3b and other proteins from the classic and lectin pathways, such as C4 and C2. Cleavage fragments of C3, C4, and C2 were also observed with HB101(pPic1) culture supernatants, and C3 cleavage sites were mapped by fluorescence resonance energy transfer peptides. Experiments using human serum as a source of complement proteins confirmed Pic proteolytic activity on these proteins. Furthermore, Pic works synergistically with the human complement regulators factor I and factor H, promoting inactivation of C3b. In the presence of both regulators, further degradation of C3 α' chain was observed. Therefore, Pic may contribute to immune evasion of E. coli and S. flexneri, favoring invasiveness and increasing the severity of the disorders caused by these pathogens.
Assuntos
Proteínas do Sistema Complemento/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/fisiologia , Evasão da Resposta Imune , Serina Endopeptidases/metabolismo , Fatores de Virulência/metabolismo , Humanos , HidróliseRESUMO
Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn(2+)-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion.
Assuntos
Proteínas de Bactérias/química , Leptospira interrogans/química , Leptospira/química , Fragmentos de Peptídeos/química , Vitronectina/química , Anticorpos Monoclonais/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/imunologia , Sítios de Ligação , Ligação Competitiva , Ativação do Complemento , Proteína de Ligação ao Complemento C4b/química , Proteína de Ligação ao Complemento C4b/imunologia , Complemento C9/química , Complemento C9/imunologia , Fator H do Complemento/química , Fator H do Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/química , Heparina/química , Humanos , Evasão da Resposta Imune , Leptospira/imunologia , Leptospira/patogenicidade , Leptospira interrogans/imunologia , Leptospira interrogans/patogenicidade , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/imunologia , Ligação Proteica , Vitronectina/imunologia , Zinco/químicaRESUMO
Alcoholic liver disease (ALD) is an important worldwide public health issue characterized by liver steatosis, inflammation, necrosis, and apoptosis of hepatocytes with eventual development of fibrosis and cirrhosis. Comparison of murine models with different inflammatory responses for ALD is important for an evaluation of the importance of genetic background in the interpretation of ethanol-induced phenotypes. Here, we investigated the role of inflammation and genetic background for the establishment of ALD using two different mouse strains: C57BL/6 (B6) and A/J. B6 and A/J mice were treated with a high fat diet containing ethanol (HFDE) and compared to the controls for 10 weeks. Hepatomegaly and steatohepatitis were similar in B6 and A/J mice, but only A/J mice were resistant to weight gain. On the other hand, HFDE-fed B6 accumulated more triglycerides (TG) and cholesterol and presented more intense cellular infiltrate in the liver when compared to HFDM-fed mice. Liver inflammatory environment was distinct in these two mouse strains. While HFDE-fed B6 produced more liver IL-12, A/J mice increased the TNF-α production. We concluded that mouse genetic background could dictate the intensity of the HFDE-induced liver injury.
Assuntos
Inflamação/sangue , Inflamação/imunologia , Hepatopatias Alcoólicas/imunologia , Animais , Colesterol/sangue , Citocinas/sangue , Modelos Animais de Doenças , Interleucina-12/sangue , Interleucina-6/sangue , Leucócitos , Metabolismo dos Lipídeos , Fígado/imunologia , Fígado/patologia , Hepatopatias Alcoólicas/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Triglicerídeos/sangue , Fator de Necrose Tumoral alfa/sangueRESUMO
Leptospirosis is an infectious disease of public health importance. To successfully colonize the host, pathogens have evolved multiple strategies to escape the complement system. Here we demonstrate that the culture supernatant of pathogenic but not saprophytic Leptospira inhibit the three complement pathways. We showed that the proteolytic activity in the supernatants of pathogenic strains targets the central complement molecule C3 and specific proteins from each pathway, such as factor B, C2, and C4b. The proteases cleaved α and ß chains of C3 and work in synergy with host regulators to inactivate C3b. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. A recombinant leptospiral metalloprotease from the thermolysin family cleaved C3 in serum and could be one of the proteases responsible for the supernatant activity. We conclude that pathogenic leptospiral proteases can deactivate immune effector molecules and represent potential targets to the development of new therapies in leptospirosis.
Assuntos
Proteínas de Bactérias/metabolismo , Complemento C3/metabolismo , Leptospira/imunologia , Leptospirose/microbiologia , Peptídeo Hidrolases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Via Clássica do Complemento , Humanos , Evasão da Resposta Imune , Leptospira/química , Leptospira/enzimologia , Leptospira/patogenicidade , Leptospirose/imunologia , Modelos Biológicos , Peptídeo Hidrolases/imunologia , Peptídeo Hidrolases/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Termolisina/química , Termolisina/metabolismoRESUMO
Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system. Patients with FH deficiency have a higher risk for development of infections and kidney diseases because of the uncontrolled activation and subsequent depletion of the central regulatory component C3 of the complement system. In this study, we investigated the consequences of the Arg(127)His mutation in FH (FH(R127H)) previously described in an FH-deficient patient, on the secretion of this protein by skin fibroblasts in vitro. We observed that, although the patient cells stimulated with IFN-γ were able to synthesize FH(R127H), the mutant protein was largely retained within the endoplasmic reticulum (ER), whereas normal human fibroblasts stimulated with IFN-γ secrete FH without retention in the ER. Moreover, the retention of FH(R127H) provoked enlargement of ER cisterns after treatment with IFN-γ. A similar ER retention was observed in Cos-7 cells expressing the mutant FH(R127H) protein. Despite this deficiency in secretion, we show that the FH(R127H) mutant is capable of functioning as a cofactor in the Factor I-mediated cleavage of C3. We then evaluated whether a treatment could increase the secretion of FH, and observed that the patient's fibroblasts treated with the chemical chaperones 4-phenylbutiric acid or curcumin increased the secretion rate of FH. We propose that these chemical chaperones could be used as alternative therapeutic agents to increase FH plasma levels in FH-deficient patients caused by secretion delay of this regulatory protein.
Assuntos
Substituição de Aminoácidos/imunologia , Fator H do Complemento/deficiência , Fator H do Complemento/metabolismo , Curcumina/farmacologia , Fibroblastos/metabolismo , Chaperonas Moleculares/fisiologia , Fenilbutiratos/farmacologia , Substituição de Aminoácidos/efeitos dos fármacos , Animais , Arginina/genética , Células COS , Células Cultivadas , Criança , Chlorocebus aethiops , Fator H do Complemento/genética , Curcumina/uso terapêutico , Fibroblastos/efeitos dos fármacos , Histidina/genética , Humanos , Chaperonas Moleculares/uso terapêutico , Fenilbutiratos/uso terapêuticoRESUMO
Leptospira, the causative agent of leptospirosis, interacts with several host molecules, including extracellular matrix components, coagulation cascade proteins, and human complement regulators. Here we demonstrate that acquisition of factor H (FH) on the Leptospira surface is crucial for bacterial survival in the serum and that these spirochetes, besides interacting with FH, FH related-1, and C4b binding protein (C4BP), also acquire FH like-1 from human serum. We also demonstrate that binding to these complement regulators is mediated by leptospiral immunoglobulin-like (Lig) proteins, previously shown to interact with fibronectin, laminin, collagen, elastin, tropoelastin, and fibrinogen. Factor H binds to Lig proteins via short consensus repeat domains 5 and 20. Competition assays suggest that FH and C4BP have distinct binding sites on Lig proteins. Moreover, FH and C4BP bound to immobilized Ligs display cofactor activity, mediating C3b and C4b degradation by factor I. In conclusion, Lig proteins are multifunctional molecules, contributing to leptospiral adhesion and immune evasion.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas Inativadoras do Complemento C3b/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Leptospira/patogenicidade , Leptospirose/imunologia , Aderência Bacteriana , Proteínas de Bactérias/genética , Sítios de Ligação , Clonagem Molecular , Complemento C3b/metabolismo , Proteína de Ligação ao Complemento C4b/metabolismo , Fator H do Complemento/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Evasão da Resposta Imune , Imunoglobulinas/química , Leptospira/genética , Leptospira/metabolismo , Leptospirose/microbiologia , PlasmídeosRESUMO
Sphingomyelin is a major constituent of eukaryotic cell membranes, and if degraded by bacteria sphingomyelinases may contribute to the pathogenesis of infection. Among Leptospira spp., there are five sphingomyelinases exclusively expressed by pathogenic leptospires, in which Sph2 is expressed during natural infections, cytotoxic, and implicated in the leptospirosis hemorrhagic complications. Considering this and the lack of information about associations between Sph2 and leptospirosis severity, we use a combination of immunoinformatics approaches to identify its B-cell epitopes, evaluate their reactivity against samples from leptospirosis patients, and investigate the role of antibodies anti-Sph2 in protection against severe leptospirosis. Two B-cell epitopes, Sph2(176-191) and Sph2(446-459), were predicted in Sph2 from L. interrogans serovar Lai, presenting different levels of identity when compared with other pathogenic leptospires. These epitopes were recognized by about 40% of studied patients with a prevalence of IgG antibodies against both Sph2(176-191) and Sph2(446-459). Remarkably, just individuals with low reactivity to Sph2(176-191) presented clinical complications, while high responders had only mild symptoms. Therefore, we identified two B-cell linear epitopes, recognized by antibodies of patients with leptospirosis, that could be further explored in the development of multi-epitope vaccines against leptospirosis.
RESUMO
PURPOSE: To investigate the influence of the Factor H (CFH) Tyr4°²His polymorphism on the plasma levels of the alternative pathway proteins CFH, C3, Factor B (FB), Factor D (FD), and Factor I (FI) and the inflammatory marker C-reactive protein (CRP) in 119 patients with age-related macular degeneration (AMD) and 152 unrelated control individuals. METHODS: Patients with AMD and the control group were separated according to CFH polymorphism, age, and gender. Plasma complement proteins and CRP concentrations were determined with enzyme-linked immunosorbent assay, immunodiffusion, or nephelometry. RESULTS: Significant differences in the concentrations of FD and FI were observed between the patients with AMD and the control individuals. We observed significantly reduced FD plasma levels in patients with AMD. We also identified a significant decrease in CFH plasma levels in female patients with AMD in relation to female controls. Plasma FI levels were significantly increased in patients with AMD compared to the control group. Regarding gender, a significant increase in FI plasma levels was observed in male patients. Finally, we found no significant correlation between the CFH Tyr(402)His polymorphism and the CFH, C3, FB, FD, FI, and CRP plasma levels. CONCLUSIONS: Patients with AMD present altered levels of FD and FI in a manner independent of this CFH polymorphism, and gender apparently contributes to the plasma levels of these two proteins in patients with AMD and control individuals.
Assuntos
Fator H do Complemento/genética , Via Alternativa do Complemento/genética , Degeneração Macular/genética , Idoso , Idoso de 80 Anos ou mais , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Complemento C3/metabolismo , Fator B do Complemento/metabolismo , Fator D do Complemento/metabolismo , Fator H do Complemento/metabolismo , Fator I do Complemento/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Humanos , Degeneração Macular/sangue , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores SexuaisRESUMO
Extracellular proteolytic enzymes are produced by a variety of pathogenic microorganisms, and contribute to host colonization by modulating virulence. Here, we present a first characterization of leptolysin, a Leptospira metalloprotease of the pappalysin family identified in a previous exoproteomic study. Comparative molecular analysis of leptolysin with two other pappalysins from prokaryotes, ulilysin and mirolysin, reveals similarities regarding calcium, zinc, and arginine -binding sites conservation within the catalytic domain, but also discloses peculiarities. Variations observed in the primary and tertiary structures may reflect differences in primary specificities. Purified recombinant leptolysin of L. interrogans was obtained as a ~50 kDa protein. The protease exhibited maximal activity at pH 8.0 and 37°C, and hydrolytic activity was observed in the presence of different salts with maximum efficiency in NaCl. Substrate specificity was assessed using a small number of FRET peptides, and showed a marked preference for arginine residues at the P1 position. L. interrogans leptolysin proteolytic activity on proteinaceous substrates such as proteoglycans and plasma fibronectin was also evaluated. All proteins tested were efficiently degraded over time, confirming the protease´s broad-spectrum activity in vitro. In addition, leptolysin induced morphological alterations on HK-2 cells, which may be partially attributed to extracellular matrix (ECM) degradation. Hemorrhagic foci were observed in the dorsal skin of mice intradermally injected with leptolysin, as a plausible consequence of ECM disarray and vascular endothelium glycocalyx damage. Assuming that leptospiral proteases play an important role in all stages of the infectious process, characterizing their functional properties, substrates and mechanisms of action is of great importance for therapeutic purposes.
Assuntos
Leptospira , Metaloproteases , Animais , Arginina/metabolismo , Leptospira/química , Leptospira/metabolismo , Leptospirose , Metaloproteases/metabolismo , Metaloproteases/farmacologia , Camundongos , Peptídeo Hidrolases/metabolismoRESUMO
Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system. FH deficiency is a rare condition that causes unregulated C3 consumption, leading to an increased susceptibility to infections and glomerulopathies. Our previous studies have demonstrated a FH deficient patient carrying a c.452G > A, p.R127H FH mutation which leads to a misfolded protein and its retention in the endoplasmic reticulum. In his cultured fibroblasts, FH-delayed secretion was partially rescued when treated with curcumin, and once secreted, exhibited normal regulatory function. Here, we report a childhood-onset systemic lupus erythematosus (cSLE) in this FH deficient patient and the results of experimental treatment with curcumin aiming to rescue FH secretion and regulatory activity.
RESUMO
Innate immunity contributes effectively to the development of Alcohol-Associated liver disease (ALD). Particularly, human studies and murine models of ALD have shown that Complement activation plays an important role during the initial and later stages of ALD. The Complement System may contribute to the pathogenesis of this disease since it has been shown that ethanol-derived metabolic products activate the Complement cascade on liver membranes, leading to hepatocellular damage. However, studies evaluating the plasma levels of Complement proteins in ALD patients present contradictory results in some cases, and do not establish a well-marked role for each Complement component. The impairment of leukocyte chemoattractant activity observed in these patients may contribute to the susceptibility to bacterial infections in the latter stages of the disease. On the other hand, murine models of ALD have provided more detailed insights into the mechanisms that link the Complement System to the pathogenesis of the disease. It has been observed that Classical pathway can be activated via C1q binding to apoptotic cells in the liver and contributes to the development of hepatic inflammation. C3 contributes to the accumulation of triglycerides in the liver and in adipose tissue, while C5 seems to be involved with inflammation and liver injury after chronic ethanol consumption. In this review, we present a compendium of studies evaluating the role of Complement in human and murine models of ALD. We also discuss potential therapies to human ALD, highlighting the use of Complement inhibitors.
Assuntos
Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Suscetibilidade a Doenças , Hepatopatias Alcoólicas/etiologia , Hepatopatias Alcoólicas/metabolismo , Animais , Biomarcadores , Ativação do Complemento/genética , Ativação do Complemento/imunologia , Gerenciamento Clínico , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Humanos , Imunomodulação , Hepatopatias Alcoólicas/patologia , Hepatopatias Alcoólicas/terapia , Terapia de Alvo MolecularRESUMO
In the past 20 years, infections caused by coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2 have posed a threat to public health since they may cause severe acute respiratory syndrome (SARS) in humans. The Complement System is activated during viral infection, being a central protagonist of innate and acquired immunity. Here, we report some interactions between these three coronaviruses and the Complement System, highlighting the central role of C3 with the severity of these infections. Although it can be protective, its role during coronavirus infections seems to be contradictory. For example, during SARS-CoV-2 infection, Complement System can control the viral infection in asymptomatic or mild cases; however, it can also intensify local and systemic damage in some of severe COVID-19 patients, due to its potent proinflammatory effect. In this last condition, the activation of the Complement System also amplifies the cytokine storm and the pathogenicity of coronavirus infection. Experimental treatment with Complement inhibitors has been an enthusiastic field of intense investigation in search of a promising additional therapy in severe COVID-19 patients.
Assuntos
COVID-19/imunologia , Proteínas do Sistema Complemento/imunologia , SARS-CoV-2/imunologia , Animais , COVID-19/complicações , COVID-19/patologia , Ativação do Complemento/efeitos dos fármacos , Complemento C3/imunologia , Inativadores do Complemento/farmacologia , Inativadores do Complemento/uso terapêutico , Infecções por Coronavirus/complicações , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Síndrome da Liberação de Citocina/complicações , Síndrome da Liberação de Citocina/tratamento farmacológico , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/patologia , Humanos , Inflamação/complicações , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/complicações , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/patologia , Tratamento Farmacológico da COVID-19RESUMO
We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Leptospira/imunologia , Leptospirose/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Western Blotting , Clonagem Molecular , Proteína de Ligação ao Complemento C4b , Ensaio de Imunoadsorção Enzimática , Genes Bacterianos/genética , Genes Bacterianos/fisiologia , Antígenos de Histocompatibilidade , Humanos , Immunoblotting , Leptospira/genética , Leptospira/fisiologia , Leptospira interrogans/imunologia , Microscopia Imunoeletrônica , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase , Proteínas RecombinantesRESUMO
Leptospires are highly invasive spirochetes equipped with efficient strategies for dissemination in the host. The Leptospira genus currently comprises 64 species divided into two major clades: the saprophytes composed of nonpathogenic, free-living organisms, and the pathogens encompassing all the species that cause mild or severe infections in humans and animals. While saprophytes are highly susceptible to the lytic action of the complement system, pathogenic (virulent) strains have evolved virulence strategies that allow efficient colonization of a variety of hosts and target organs, including mechanisms to circumvent hosts' innate and acquired immune responses. Pathogenic Leptospira avoid complement-mediated killing by recruiting host complement regulatory proteins and by targeting complement proteins using own and host-expressed proteases. This review outlines the role of complement in eradicating saprophytic Leptospira and the stratagems adopted by pathogenic Leptospira to maneuver the host complement system for their benefit.
Assuntos
Proteínas do Sistema Complemento/imunologia , Evasão da Resposta Imune , Leptospira , Leptospirose/imunologia , Animais , Humanos , Leptospira/imunologia , Leptospira/patogenicidade , Leptospirose/patologiaRESUMO
Like many other pathogens of medical importance, pathogenic Leptospira employ diverse strategies to circumvent Complement System activation. Under physiological conditions, this central humoral arm of innate immunity is tightly controlled by negative Complement regulatory proteins. However, upon infection, pathogenic microorganisms interfere with normal Complement host defense mechanisms by recruiting or mimicking Complement regulators and by secreting endogenous proteases or acquiring host's proteases that inactivate key Complement components. In this chapter, we describe in detail some of the most frequently used assays to evaluate Leptospira Complement resistance.
Assuntos
Bioensaio/métodos , Proteínas do Sistema Complemento/imunologia , Ativação do Complemento/imunologia , Humanos , Imunidade Humoral/imunologia , Imunidade Inata/imunologia , Leptospira/imunologia , Leptospirose/imunologia , Leptospirose/microbiologia , Ligação Proteica/imunologiaRESUMO
The Complement System (CS) plays an important role in the immune response against leptospirosis and can be activated by the Alternative and Lectin Pathways (Innate Immunity) and by the Classical Pathway (Acquired Immunity). Here we analyzed a broad range of nonpathogenic and pathogenic Leptospira strains considering their interaction with each CS pathway. We determined bacterial survival rate and CS protein deposition in the presence of purified proteins, specific component depleted sera and NHS treated with the chelating agents EDTA (inhibits all three activation pathways) or EGTA (inhibits the Classical and Lectin Pathways). We suggest that the Lectin and the Alternative Pathways have an important role to eliminate saprophytic leptospires since i) approximately 50% survival of both saprophytic strains was observed in the presence of MBL-deficient serum; ii) approximately 50% survival of Leptospira biflexa Patoc I was observed in the presence of NHS - EGTA and iii) C1q-depleted serum caused significant bacterial lysis. In all serovars investigated the deposition of C5-C9 proteins on saprophytic Leptospira strains was more pronounced when compared to pathogenic species confirming previous studies in the literature. No difference on C3 deposition was observed between nonpathogenic and pathogenic strains. In conclusion, Leptospira strains interact to different degrees with CS proteins, especially those necessary to form MAC, indicating that some strains and specific ligands could favor the binding of certain CS proteins.
Assuntos
Ativação do Complemento , Leptospira/imunologia , Leptospirose/imunologia , Proteínas do Sistema Complemento/imunologia , Humanos , Evasão da Resposta Imune , Leptospira/patogenicidade , Viabilidade Microbiana/imunologiaRESUMO
Properdin (P) is a positive regulatory protein that stabilizes the C3 convertase and C5 convertase of the complement alternative pathway (AP). Several studies have suggested that properdin can bind directly to the surface of certain pathogens regardless of the presence of C3bBb. Saprophytic Leptospira are susceptible to complement-mediated killing, but the interaction of properdin with Leptospira spp. has not been evaluated so far. In this work, we demonstrate that properdin present in normal human serum, purified properdin, as well as properdin oligomers P2, P3, and P4, interact with Leptospira. Properdin can bind directly to the bacterial surface even in the absence of C3b. In line with our previous findings, AP activation was shown to be important for killing non-pathogenic L. biflexa, and properdin plays a key role in this process since this microorganism survives in P-depleted human serum and the addition of purified properdin to P-depleted human serum decreases the number of viable leptospires. A panel of pathogenic L.interrogans recombinant proteins was used to identify putative properdin targets. Lsa30, an outer membrane protein from L. interrogans, binds to unfractionated properdin and to a lesser extent to P2-P4 properdin oligomers. In conclusion, properdin plays an important role in limiting bacterial proliferation of non-pathogenic Leptospira species. Once bound to the leptospiral surface, this positive complement regulatory protein of the AP contributes to the formation of the C3 convertase on the leptospire surface even in the absence of prior addition of C3b.