RESUMO
Pathogenic spirochetes from genus Leptospira are etiologic agents of leptospirosis. Cellular vaccines against Leptospira infection often elicit mainly response against the LPS antigen of the serovars present in the formulation. There is no suitable protein candidate capable of replacing whole-cell vaccines, thus requiring new approaches on vaccine development to improve leptospirosis prevention. Our goal was to develop a whole-cell vaccine sorovar-independent based on LPS removal and conservation of protein antigens exposure, to evaluate the protective capacity of monovalent or bivalent vaccines against homologous and heterologous virulent Leptospira in hamster. Leptospire were subjected to heat inactivation, or to LPS extraction with butanol and in some cases further inactivation with formaldehyde. Hamsters were immunized and challenged with homologous or heterologous virulent serovars, blood and organs were collected from the survivors for bacterial quantification, chemokine evaluation, and analysis of sera antibody reactivity and cross-reactivity by Western blot. Immunization with either heated or low LPS vaccines with serovar Copenhageni or Canicola resulted in 100% protection of the animals challenged with homologous virulent bacteria. Notably, different from the whole-cell vaccine, the low LPS vaccines produced with serovar Canicola provided only partial protection in heterologous challenge with the virulent Copenhageni serovar. Immunization with bivalent formulation results in 100% protection of immunized animals challenged with virulent serovar Canicola. All vaccines produced were able to eliminate bacteria from the kidney of challenged animals. All the vaccines raised antibodies capable to recognize antigens of serovars not present in the vaccine formulation. Transcripts of IFNγ, CXCL16, CCL5, CXCL10, CXCR6, and CCR5, increased in all immunized animals. Conclusion: Our results showed that bivalent vaccines with reduced LPS may be an interesting strategy for protection against heterologous virulent serovars. Besides the desirable multivalent protection, the low LPS vaccines are specially promising due to the expected lower reatogenicity.
Assuntos
Vacinas Bacterianas , Leptospira/imunologia , Leptospirose/imunologia , Lipopolissacarídeos/química , Vacinação , Animais , Anticorpos Antibacterianos/imunologia , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Cricetinae , Leptospira/química , Leptospirose/prevenção & controleRESUMO
Acute kidney injury (AKI) from leptospirosis is frequently nonoliguric with hypo- or normokalemia. Higher serum potassium levels are observed in non-survivor patients and may have been caused by more severe AKI, metabolic disarrangement, or rhabdomyolysis. An association between the creatine phosphokinase (CPK) level and maximum serum creatinine level has been observed in these patients, which suggests that rhabdomyolysis contributes to severe AKI and hyperkalemia. LipL32 and Lp25 are conserved proteins in pathogenic strains of Leptospira spp., but these proteins have no known function. This study evaluated the effect of these proteins on renal function in guinea pigs. Lp25 is an outer membrane protein that appears responsible for the development of oliguric AKI associated with hyperkalemia induced by rhabdomyolysis (e.g., elevated CPK, uric acid and serum phosphate). This study is the first characterization of a leptospiral outer membrane protein that is associated with severe manifestations of leptospirosis. Therapeutic methods to attenuate this protein and inhibit rhabdomyolysis-induced AKI could protect animals and patients from severe forms of this disease and decrease mortality.
Assuntos
Injúria Renal Aguda/patologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Leptospirose/complicações , Lipoproteínas/metabolismo , Rabdomiólise/patologia , Injúria Renal Aguda/microbiologia , Animais , Creatina Quinase/sangue , Creatinina/sangue , Modelos Animais de Doenças , Cobaias , Leptospira , Músculos/patologia , Potássio/sangue , Rabdomiólise/microbiologiaRESUMO
Cattle are considered a reservoir of Shiga toxin-producing Escherichia coli (STEC). There is no information about the presence of antibodies against Shiga toxins in Brazilian bovine serum. Using ELISA, all sera tested showed antibodies against the two main STEC virulence factors; Stx1 and Stx2. Neutralizing antibodies against Stx1 and/or Stx2 were detected in all but one serum. In conclusion, our results indicated that these animals had been exposed to STEC producing both toxins.
Assuntos
Anticorpos Antibacterianos/sangue , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Toxinas Shiga/imunologia , Escherichia coli Shiga Toxigênica/imunologia , Animais , Brasil/epidemiologia , Bovinos , Reservatórios de Doenças/veterinária , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , SorotipagemRESUMO
BACKGROUND: Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains. METHODS AND FINDINGS: Recombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains. CONCLUSION: The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.
Assuntos
Toxinas Bacterianas/imunologia , Escherichia coli Enterotoxigênica/metabolismo , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Anticorpos de Cadeia Única/biossíntese , Sequência de Aminoácidos , Escherichia coli Enterotoxigênica/isolamento & purificação , Dados de Sequência Molecular , Proteínas Recombinantes/isolamento & purificação , Anticorpos de Cadeia Única/químicaRESUMO
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.
Assuntos
Fator H do Complemento/metabolismo , Leptospira/enzimologia , Fator Tu de Elongação de Peptídeos/metabolismo , Plasminogênio/metabolismo , Animais , Coagulação Sanguínea , Fibrinolisina/metabolismo , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Imunidade Inata/imunologia , Leptospira/metabolismo , Leptospira/fisiologia , Lisina/metabolismo , Fator Tu de Elongação de Peptídeos/química , Ligação Proteica , Transporte ProteicoRESUMO
Leptospira interrogans causes leptospirosis, one of the most common zoonotic diseases in the world. This pathogenic spirochete is able to bind to extracellular matrix, to express virulent factors and to cause host death. Until now, there is no effective human vaccine for the disease. Shotgun phage display genomic libraries of L. interrogans were constructed and used for in vivo biopanning in hamsters and screened for ligands able to bind to LLC-PK1 epithelial cells. In both panning procedures, clones coding for the putative lipoprotein LIC12976 were identified and, in order to confirm its adhesin activity, a recombinant protein was produced in Escherichia coli and showed to interact with A31 fibroblasts, LLC-PK1 and Vero epithelial cells in vitro. Moreover, rLIC12976 was shown to bind to laminin, indicating an adhesin function. This protein was also detected in extracts of L. interrogans from different serovars and it was found to be conserved among pathogenic leptospires. Further, the protein was tested as vaccine candidate and immunization of hamsters with LIC12976 did not confer protection against a lethal challenge with the homologous L. interrogans serovar Copenhageni. Nevertheless, LIC12976 seems to act as an adhesin, and may be important for the host-pathogen interaction, so that its study can contribute to the understanding of the virulence mechanisms in pathogenic leptospires.
Assuntos
Adesinas Bacterianas/genética , Interações Hospedeiro-Patógeno , Leptospira interrogans/patogenicidade , Leptospirose/microbiologia , Lipoproteínas/genética , Adesinas Bacterianas/fisiologia , Animais , Chlorocebus aethiops , Cricetinae , Humanos , Laminina/metabolismo , Leptospira interrogans/genética , Lipoproteínas/fisiologia , Camundongos , Biblioteca de Peptídeos , Células VeroRESUMO
Atypical enteropathogenic Escherichia coli (aEPEC) are heterogeneous in terms of serotypes, adherence patterns and the presence of non-locus of enterocyte effacement virulence factors. In this study, the low-molecular mass proteomes of four representative aEPEC, comprising three different adhesion phenotypes (localized-like, aggregative and diffuse) and one non-adherent isolate, were analyzed and compared by 2D gel electrophoresis and LC-MS/MS. By mass spectrometry, a total of 59 proteins were identified according to their annotated function, with most of them being involved in metabolism, protection, and transport; some of them still classified as hypothetical proteins. Thus, in this comparative proteomic analysis of low-molecular mass extracted proteins from different aEPEC isolates, the proteins identified are mainly involved in key metabolic pathways. Also, the majority of the hypothetical and filamentous proteins identified in the isolates studied are products of genes originally identified in the genome of enterohemorrhagic E. coli.
Assuntos
Escherichia coli Enteropatogênica/genética , Proteínas de Escherichia coli/genética , Proteoma/genética , Fatores de Virulência/genética , Aderência Bacteriana , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Escherichia coli Enteropatogênica/classificação , Escherichia coli Enteropatogênica/isolamento & purificação , Escherichia coli Enteropatogênica/ultraestrutura , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Humanos , Microscopia Eletrônica de Transmissão , Peso Molecular , Proteoma/metabolismo , Proteômica , Espectrometria de Massas em Tandem , Fatores de Virulência/metabolismoRESUMO
Leptospirosis is one of the most widespread zoonosis in the world. The development of a recombinant leptospira vaccine remains a challenge. In this study, we cloned the Leptospira interrogans open reading frame (ORF) coding the external membrane protein LipL32, an immunodominant antigen found in all pathogenic leptospira, downstream of the highly immunogenic cholera toxin B subunit (CTB) ORF. Expression and assembly of the CTB-LipL32 fusion protein into oligomeric structures of pentameric size were observed in soluble fractions by Western blot analysis. The CTB-LipL32 protein demonstrated strong affinity for monosialotetrahexosylgaglioside (GM1-ganglioside) in an enzyme-linked immunosorbent assay (ELISA), suggesting that the antigenic sites for binding and proper folding of the pentameric CTB structure were conserved. Furthermore, antisera against LipL32 also recognized the CTB-LipL32 fusion protein, suggesting that LipL32 also conserved its antigenic sites, a fact confirmed by an ELISA assay showing soluble CTB-LipL32 recognition by sera from convalescent patients. In addition, soluble CTB-LipL32 generated higher specific titers in mice immunized without external adjuvant than co-administration of CTB with LipL32. The data presented here provide support for CTB-LipL32 as a promising antigen for use in the control and study of leptospirosis.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Toxina da Cólera/imunologia , Leptospira interrogans/imunologia , Lipoproteínas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/genética , Western Blotting , Toxina da Cólera/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Gangliosídeo G(M1)/metabolismo , Leptospira interrogans/genética , Lipoproteínas/genética , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichiacoli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). Both pathogens are still important causes of diarrhea inchildren and adults in many developing and industrialized countries. Considering the fact that antibodies areimportant tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int388-667). In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv).Anti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into pGEM-T Easy vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. E. coli BL21(DE3)pLys strainwas transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv,expressed as inclusion bodies (insoluble fraction), was denatured, purified and submitted to refolding. The proteinyield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA andimmunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin,resulting in an absorbance of 0.75 at 492 nm.
Assuntos
Anticorpos Monoclonais , Escherichia coli Enteropatogênica/imunologia , Escherichia coli Enteropatogênica/patogenicidade , Hibridomas/imunologia , Imunofluorescência/métodosRESUMO
Leptospirosis is one of the most widespread zoonosis in the world. The development of a recombinant leptospira vaccine remains a challenge. In this study, we cloned the Leptospira interrogans open reading frame (ORF) coding the external membrane protein LipL32, an immunodominant antigen found in all pathogenic leptospira, downstream of the highly immunogenic cholera toxin B subunit (CTB) ORF. Expression and assembly of the CTB-LipL32 fusion protein into oligomeric structures of pentameric size were observed in soluble fractions by Western blot analysis. The CTB-LipL32 protein demonstrated strong affinity for monosialotetrahexosylgaglioside (GM1-ganglioside) in an enzyme-linked immunosorbent assay (ELISA), suggesting that the antigenic sites for binding and proper folding of the pentameric CTB structure were conserved. Furthermore, antisera against LipL32 also recognized the CTB-LipL32 fusion protein, suggesting that LipL32 also conserved its antigenic sites, a fact confirmed by an ELISA assay showing soluble CTB-LipL32 recognition by sera from convalescent patients. In addition, soluble CTB-LipL32 generated higher specific titers in mice immunized without external adjuvant than co-administration of CTB with LipL32. The data presented here provide support for CTB-LipL32 as a promising antigen for use in the control and study of leptospirosis.