RESUMO
BACKGROUND: Plasmodium falciparum and Plasmodium vivax are responsible for most of the global burden of malaria. Although the accentuated pathogenicity of P. falciparum occurs because of sequestration of the mature erythrocytic forms in the microvasculature, this phenomenon has not yet been noted in P. vivax. The increasing number of severe manifestations of P. vivax infections, similar to those observed for severe falciparum malaria, suggests that key pathogenic mechanisms (eg, cytoadherence) might be shared by the 2 parasites. METHODS: Mature P. vivax-infected erythrocytes (Pv-iEs) were isolated from blood samples collected from 34 infected patients. Pv-iEs enriched on Percoll gradients were used in cytoadhesion assays with human lung endothelial cells, Saimiri brain endothelial cells, and placental cryosections. RESULTS: Pv-iEs were able to cytoadhere under static and flow conditions to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparum. Although Pv-iE cytoadhesion levels were 10-fold lower than those observed for P. falciparum-infected erythrocytes, the strength of the interaction was similar. Cytoadhesion of Pv-iEs was in part mediated by VIR proteins, encoded by P. vivax variant genes (vir), given that specific antisera inhibited the Pv-iE-endothelial cell interaction. CONCLUSIONS: These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.
Assuntos
Eritrócitos/parasitologia , Malária Vivax/patologia , Plasmodium vivax/patogenicidade , Animais , Adesão Celular , Células Endoteliais/patologia , Eritrócitos/patologia , Feminino , Humanos , Placenta/patologia , Gravidez , SaimiriRESUMO
BACKGROUND: The naturally-acquired immune response to Plasmodium vivax variant antigens (VIR) was evaluated in individuals exposed to malaria and living in different endemic areas for malaria in the north of Brazil. METHODS: Seven recombinant proteins representing four vir subfamilies (A, B, C, and E) obtained from a single patient from the Amazon Region were expressed in Escherichia coli as soluble glutathione S-transferase fusion proteins. The different recombinant proteins were compared by ELISA with regard to the recognition by IgM, IgG, and IgG subclass of antibodies from 200 individuals with patent infection. RESULTS: The frequency of individuals that presented antibodies anti-VIR (IgM plus IgG) during the infection was 49%. The frequencies of individuals that presented IgM or IgG antibodies anti-VIR were 29.6% or 26.0%, respectively. The prevalence of IgG antibodies against recombinant VIR proteins was significantly lower than the prevalence of antibodies against the recombinant proteins representing two surface antigens of merozoites of P. vivax: AMA-1 and MSP119 (57.0% and 90.5%, respectively). The cellular immune response to VIR antigens was evaluated by in vitro proliferative assays in mononuclear cells of the individuals recently exposed to P. vivax. No significant proliferative response to these antigens was observed when comparing malaria-exposed to non-exposed individuals. CONCLUSION: This study provides evidence that there is a low frequency of individuals responding to each VIR antigens in endemic areas of Brazil. This fact may explain the host susceptibility to new episodes of the disease.
Assuntos
Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Variação Genética , Malária Vivax/epidemiologia , Malária Vivax/imunologia , Plasmodium vivax/genética , Plasmodium vivax/imunologia , Adolescente , Adulto , Animais , Anticorpos Antibacterianos/sangue , Brasil/epidemiologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-IdadeRESUMO
In the present study, we evaluate the naturally acquired antibody response to the Plasmodium vivax apical membrane antigen 1 (PvAMA-1), a leading vaccine candidate against malaria. The gene encoding the PvAMA-1 ectodomain region (amino acids 43-487) was cloned by PCR using genomic DNA from a Brazilian individual with patent P. vivax infection. The predicted amino acid sequence displayed a high degree of identity (97.3%) with a previously published sequence from the P. vivax Salvador strain. A recombinant protein representing the PvAMA-1 ectodomain was expressed in Escherichia coli and refolded. By ELISA, this recombinant protein reacted with 85 and 48.5% of the IgG or IgM antibodies, respectively, from Brazilian individuals with patent P. vivax malaria. IgG1 was the predominant subclass of IgG. The frequency of response increased according to the number of malaria episodes, reaching 100% in individuals in their fourth malaria episode. The high degree of recognition of PvAMA-1 by human antibodies was confirmed using a second recombinant protein expressed in Pichia pastoris (PV66/AMA-1). The observation that recognition of the bacterial recombinant PvAMA-1 was only slightly lower than that of the highly immunogenic 19kDa C-terminal domain of the P. vivax Merozoite Surface Protein-1 was also important. DNA sequencing of the PvAMA-1 variable domain from 20 Brazilian isolates confirmed the limited polymorphism of PvAMA-1 suggested by serological analysis. In conclusion, we provide evidence that PvAMA-1 is highly immunogenic during natural infection in humans and displays limited polymorphism in Brazil. Based on these observations, we conclude that PvAMA-1 merits further immunological studies as a vaccine candidate against P. vivax malaria.
Assuntos
Formação de Anticorpos/imunologia , Antígenos de Protozoários/imunologia , Malária Vivax/imunologia , Proteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Brasil , Clonagem Molecular/métodos , DNA de Protozoário/análise , Doenças Endêmicas , Ensaio de Imunoadsorção Enzimática , Escherichia coli/imunologia , Humanos , Imunoglobulina G/imunologia , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/genética , Proteína 1 de Superfície de Merozoito , Plasmodium vivax/genética , Plasmodium vivax/imunologia , Polimorfismo Genético/genética , Polimorfismo Genético/imunologia , Proteínas de Protozoários/genética , Proteínas Recombinantes/imunologia , Análise de Sequência de Proteína/métodos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
The regulation of gene expression by environmental signals, such as temperature and osmolarity, has been correlated with virulence. In this study, we characterize the protein LipL53 from Leptospira interrogans, previously shown to react with serum sample of individual diagnosed with leptospirosis and to be up-regulated by shift to physiological osmolarity. The recombinant protein was expressed in Escherichia coli system, in insoluble form, recovered by urea solubilization and further refolded by decreasing the denaturing agent concentration during the purification procedure. The secondary structure content of the recombinant LipL53, as assessed by circular dichroism, showed a mixture of beta-strands and alpha-helix. The presence of LipL53 transcript at 28 degrees C was only detected within the virulent strains. However, upon shifted of attenuated cultures of pathogenic strains from 28 degrees C to 37 degrees C and to 39 degrees C, this transcript could also be observed. LipL53 binds laminin, collagen IV, cellular and plasma fibronectin in dose-dependent and saturable manner. Animal challenge studies showed that LipL53, although immunogenic, elicited only partial protection in hamsters. LipL53 is probably surface exposed as seen through immunofluorescence confocal microscopy. Our results suggest that LipL53 is a novel temperature regulated adhesin of L. interrogans that may be relevant in the leptospiral pathogenesis.
Assuntos
Adesinas Bacterianas/metabolismo , Matriz Extracelular/metabolismo , Leptospira interrogans/patogenicidade , Fatores de Virulência/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/imunologia , Animais , Dicroísmo Circular , Cricetinae , Escherichia coli/genética , Feminino , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Humanos , Leptospirose/imunologia , Leptospirose/prevenção & controle , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
BACKGROUND: Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin. METHODOLOGY/PRINCIPAL FINDINGS: We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin. CONCLUSIONS/SIGNIFICANCE: PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.
Assuntos
Leptospira interrogans/metabolismo , Plasminogênio/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Microscopia de Fluorescência , Ligação Proteica , Receptores de Superfície Celular/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. The search for novel antigens that could be relevant in host-pathogen interactions is being pursued. These antigens have the potential to elicit several activities, including adhesion. This study focused on a hypothetical predicted lipoprotein of Leptospira, encoded by the gene LIC12895, thought to mediate attachment to extracellular matrix (ECM) components. The gene was cloned and expressed in Escherichia coli BL21 Star (DE3)pLys by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. The capacity of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC12895, named Lsa27 (leptospiral surface adhesin, 27 kDa), bound strongly to laminin in a dose-dependent and saturable fashion. Moreover, Lsa27 was recognized by antibodies from serum samples of confirmed leptospirosis specimens in both the initial and the convalescent phases of the disease. Lsa27 is most likely a surface protein of Leptospira as revealed in liquid-phase immunofluorescence assays with living organisms. Taken together, these data indicate that this newly identified membrane protein is expressed during natural infection and may play a role in mediating adhesion of L. interrogans to its host.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Laminina/metabolismo , Leptospira interrogans/genética , Leptospira interrogans/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Adesinas Bacterianas/metabolismo , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Aderência Bacteriana/genética , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/imunologia , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , Genes Bacterianos , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Leptospira interrogans/imunologia , Leptospira interrogans/patogenicidade , Leptospirose/imunologia , Leptospirose/metabolismo , Leptospirose/microbiologia , Lipoproteínas/genética , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
Leptospirosis is a zoonosis of multisystem involvement caused by pathogenic strains of the genus Leptospira. In the last few years, intensive studies aimed at the development of a vaccine have provided important knowledge about the nature of the immunological mechanisms of the host. The purpose of this study was to analyze the immune responses to two recombinant proteins, MPL17 and MPL21 (encoded by the genes LIC10765 and LIC13131, respectively) of Leptospira interrogans serovar Copenhageni in individuals during infection. The recombinant proteins were expressed in Escherichia coli as six-His tag fusion proteins and were purified from the soluble bacterial fraction by affinity chromatography with Ni(2+)-charged resin. The recombinant proteins were used to evaluate their ability to bind to immunoglobulin G (IgG) (and IgG subclass) or IgM antibodies in serum samples from patients in the early and convalescent phases of leptospirosis (n = 52) by enzyme-linked immunosorbent assays. The prevalences of total IgG antibodies against MPL17 and MPL21 were 38.5% and 21.2%, respectively. The titers achieved with MPL17 were statistically significantly higher than those obtained by the reference microscopic agglutination test. The specificity of the assay was estimated to be 95.5% for MPL17 and 80.6% for MPL21 when serum samples from individuals with unrelated febrile diseases and control healthy donors were tested. The proteins are conserved among Leptospira strains that cause human and animal diseases. MPL17 and MPL21 are most likely new surface proteins of leptospires, as revealed by liquid-phase immunofluorescence assays with living organisms. Our results demonstrate that these recombinant proteins are highly immunogenic and, when they are used together, might be useful as a means of diagnosing leptospirosis.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias , Proteínas de Bactérias , Leptospira interrogans/imunologia , Leptospirose/diagnóstico , Proteínas Recombinantes de Fusão , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Expressão Gênica , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Leptospira interrogans/genética , Proteínas Recombinantes de Fusão/genética , Sensibilidade e EspecificidadeRESUMO
Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L. interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L. interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira.