RESUMO
Pil-fimbriae is a type IV pili member, which is a remarkably versatile component with a wide variety of functions, including motility, attachment to different surfaces, electrical conductance, DNA acquisition, and secretion of a broad range of structurally distinct protein substrates. Despite the previous functional characterization of Pil, more studies are required to understand the regulation of Pil expression and production, since the exact mechanisms involved in these steps are still unknown. Therefore it is extremely important to have a protein with the correct secondary and tertiary structure that will enable an accurate characterization and a specific antisera generation. For this reason, the aim of this work was to generate potential tools for further investigations to comprehend the mechanisms involved in Pil regulation and its role in pathogenic E. coli infections with the obtaining of a precise native-like recombinant PilS and the corresponding antisera. The pilS gene was successfully cloned into an expression vector, and recombinant PilS (rPilS) was efficiently solubilized and purified by metal affinity chromatography. Protein characterization analyses indicated that rPilS presented native-like secondary and tertiary structures after the refolding process. The generated anti-rPilS sera efficiently recognized recombinant and native proteins from atypical enteropathogenic E. coli strains.
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Shiga toxin (Stx)-producing Escherichia coli (STEC) and its subgroup enterohemorrhagic E. coli are important pathogens involved in diarrhea, which may be complicated by hemorrhagic colitis and hemolytic uremic syndrome, the leading cause of acute renal failure in children. Early diagnosis is essential for clinical management, as an antibiotic treatment in STEC infections is not recommended. Previously obtained antibodies against Stx1 and Stx2 toxins were employed to evaluate the sensitivity and specificity of the latex Agglutination test (LAT), lateral flow assay (LFA), and capture ELISA (cEIA) for STEC detection. The LAT (mAb Stx1 plus mAb stx2) showed 99% sensitivity and 97% specificity. Individually, Stx1 antibodies showed 95.5% and 94% sensitivity and a specificity of 97% and 99% in the cEIA and LFA assay, respectively. Stx2 antibodies showed a sensitivity of 92% in both assays and a specificity of 100% and 98% in the cEIA and LFA assay, respectively. These results allow us to conclude that we have robust tools for the diagnosis of STEC infections.
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We standardized an immunochromatographic test (IC) for heat-labile toxin I (LT-I) detection using LT-I antibodies and a specific platform containing the apparatus for application, assembly and cutting. IC detected as little as 62.5ng/mL of purified LT-I toxin and presented 91% sensitivity, 99.5% specificity and 96.0% accuracy, thereby proving to be an excellent point-of-care test for the diagnosis of enterotoxigenic E. coli infection in low-income countries.
Assuntos
Toxinas Bacterianas/isolamento & purificação , Testes Diagnósticos de Rotina/métodos , Diarreia/diagnóstico , Escherichia coli Enterotoxigênica/isolamento & purificação , Enterotoxinas/isolamento & purificação , Infecções por Escherichia coli/diagnóstico , Proteínas de Escherichia coli/isolamento & purificação , Imunoensaio/métodos , Anticorpos Antibacterianos/imunologia , Toxinas Bacterianas/imunologia , Testes Diagnósticos de Rotina/instrumentação , Diarreia/microbiologia , Escherichia coli Enterotoxigênica/metabolismo , Escherichia coli Enterotoxigênica/patogenicidade , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Temperatura Alta , Humanos , Imunoensaio/instrumentação , Sensibilidade e EspecificidadeRESUMO
Stx1 toxin is one of the AB5 toxins of Shiga toxin-producing Escherichia coli (STEC) responsible for foodborne intoxication during outbreaks. The single-chain variable fragment (scFv) is the most common recombinant antibody format; it consists of both variable chains connected by a peptide linker with conserved specificity and affinity for antigen. The drawbacks of scFv production in bacteria are the heterologous expression, conformation and stability of the molecule, which could change the affinity for the antigen. In this work, we obtained a stable and functional scFv-Stx1 in bacteria, starting from IgG produced by hybridoma cells. After structural modifications, i.e., change in protein orientation, vector and linker, its solubility for expression in bacteria was increased as well as the affinity for its antigen, demonstrated by a scFv dissociation constant (KD) of 2.26 × 10-7 M. Also, it was able to recognize purified Stx1 and cross-reacted with Stx2 toxin by ELISA (Enzyme-Linked Immunosorbent Assay), and detected 88% of Stx1-producing strains using a rapid latex agglutination test. Thus, the scFv fragment obtained in the present work is a bacteria-produced tool for use in a rapid diagnosis test, providing an alternative for STEC diagnosis.
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BACKGROUND: Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes. METHODS AND FINDINGS: In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA. CONCLUSION: In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.
Assuntos
Anticorpos Neutralizantes/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismo , Toxina Shiga II/antagonistas & inibidores , Escherichia coli Shiga Toxigênica/metabolismo , Anticorpos de Cadeia Única/metabolismo , Animais , Linhagem Celular , Humanos , Hibridomas/imunologia , Camundongos , Biblioteca de Peptídeos , Sensibilidade e Especificidade , Escherichia coli Shiga Toxigênica/imunologia , Anticorpos de Cadeia Única/genéticaRESUMO
E. coli O111 strains are responsible for outbreaks of blood diarrhea and hemolytic uremic syndrome throughout the world. Because of their phenotypic variability, the development of a vaccine against these strains which targets an antigen that is common to all of them is quite a challenge. Previous results have indicated, however, that O111 LPS is such a candidate, but its toxicity makes LPS forbidden for human use. To overcome this problem, O111 polysaccharides were conjugated either to cytochrome C or to EtxB (a recombinant B subunit of LT) as carrier proteins. The O111-cytochrome C conjugate was incorporated in silica SBA-15 nanoparticles and administered subcutaneously in rabbits, while the O111-EtxB conjugate was incorporated in Vaxcine(TM), an oil-based delivery system, and administered orally in mice. The results showed that one year post-vaccination, the conjugate incorporated in silica SBA-15 generated antibodies in rabbits able to inhibit the adhesion of all categories of O111 E. coli to epithelial cells. Importantly, mice immunized orally with the O111-EtxB conjugate in Vaxcine(TM) generated systemic and mucosal humoral responses against all categories of O111 E. coli as well as antibodies able to inhibit the toxic effect of LT in vitro. In summary, the results obtained by using 2 different approaches indicate that a vaccine that targets the O111 antigen has the potential to prevent diarrhea induced by O111 E. coli strains regardless their mechanism of virulence. They also suggest that a conjugated vaccine that uses EtxB as a carrier protein has potential to combat diarrhea induced by ETEC.
Assuntos
Anticorpos Antibacterianos/sangue , Portadores de Fármacos/uso terapêutico , Infecções por Escherichia coli/prevenção & controle , Escherichia coli/imunologia , Polissacarídeos Bacterianos/imunologia , Vacinas Conjugadas/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Aderência Bacteriana/imunologia , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Linhagem Celular , Citocromos c/química , Citocromos c/imunologia , Endotoxinas/imunologia , Enterotoxinas/química , Enterotoxinas/imunologia , Escherichia coli/classificação , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/imunologia , Feminino , Síndrome Hemolítico-Urêmica/imunologia , Síndrome Hemolítico-Urêmica/microbiologia , Síndrome Hemolítico-Urêmica/prevenção & controle , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/uso terapêutico , Coelhos , Dióxido de Silício/química , Vacinas Conjugadas/uso terapêuticoRESUMO
BACKGROUND: Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC) are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens. METHODOLOGY: First, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco's modified Eagle's medium (DMEM) or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT) was developed and tested with the same collection of bacterial isolates. PRINCIPAL FINDINGS: EspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world. CONCLUSION: RALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Infecções por Escherichia coli/diagnóstico , Proteínas de Escherichia coli/análise , Escherichia coli/isolamento & purificação , Testes de Fixação do Látex/métodos , Anticorpos Antibacterianos/análise , Biomarcadores/análise , Países em Desenvolvimento , Infecções por Escherichia coli/microbiologia , HumanosRESUMO
INTRODUCTION: Escherichia coli causes gastroenteritis in humans and animals. CASE PRESENTATION: In this study, both Shiga toxin-producing E. coli (STEC) and atypical enteropathogenic E. coli (EPEC) strains were identified in a stool sample from a healthy child, and they were serotyped as Shiga toxin-producing E. coli (STEC) ONTâ:âH19 and atypical enteropathogenic E. coli (EPEC) O37â: H45. CONCLUSION: This is the first report, to our knowledge, of a concomitant presence of diarrhoeagenic E. coli (DEC) strains in an asymptomatic child. None of the microorganisms was able to produce diarrhoea, maybe because they were transient bacteria or because of the good immune status of the child. Attention should be paid to this result and it could be of interest in vaccine prospects.
RESUMO
Enterotoxigenic Escherichia coli (ETEC) produce heat-labile (LT) and/or heat-stable enterotoxins (ST). Despite that, the mechanism of action of both toxins are well known, there is great controversy in the literature concerning the in vitro production and release of LT and, for ST, no major concerns have been discussed. Furthermore, the majority of published papers describe the use of only one or a few ETEC isolates to define the production and release of these toxins, which hinders the detection of ETEC by phenotypic approaches. Thus, the present study was undertaken to obtain a better understanding of ST and LT toxin production and release under laboratory conditions. Accordingly, a collection of 90 LT-, ST-, and ST/LT-producing ETEC isolates was used to determine a protocol for toxin production and release aimed at ETEC detection. For this, we used previously raised anti-LT antibodies and the anti-ST monoclonal and polyclonal antibodies described herein. The presence of bile salts and the use of certain antibiotics improved ETEC toxin production/release. Triton X-100, as chemical treatment, proved to be an alternative method for toxin release. Consequently, a common protocol that can increase the production and release of LT and ST toxins could facilitate and enhance the sensitivity of diagnostic tests for ETEC using the raised and described antibodies in the present work.
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Toxinas Bacterianas/metabolismo , Bioensaio/métodos , Escherichia coli Enterotoxigênica/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Animais , Antibacterianos/farmacologia , Toxinas Bacterianas/imunologia , Ácidos e Sais Biliares/farmacologia , Ciprofloxacina/farmacologia , Escherichia coli Enterotoxigênica/efeitos dos fármacos , Escherichia coli Enterotoxigênica/isolamento & purificação , Enterotoxinas/imunologia , Ensaio de Imunoadsorção Enzimática , Proteínas de Escherichia coli/imunologia , Feminino , Imunoglobulina G/imunologia , Lincomicina/farmacologia , Masculino , Camundongos Endogâmicos BALB C , CoelhosRESUMO
Escherichia coli strains of serogroup O26 comprise two distinct groups of pathogens, characterized as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among the several genes related to type III secretion system-secreted effector proteins, espK was found to be highly specific for EHEC O26:H11 and its stx-negative derivative strains isolated in European countries. E. coli O26 strains isolated in Brazil from infant diarrhea, foods, and the environment have consistently been shown to lack stx genes and are thus considered atypical EPEC. However, no further information related to their genetic background is known. Therefore, in this study, we aimed to discriminate and characterize these Brazilian O26 stx-negative strains by phenotypic, genetic, and biochemical approaches. Among 44 isolates confirmed to be O26 isolates, most displayed flagellar antigen H11 or H32. Out of the 13 nonmotile isolates, 2 tested positive for fliCH11, and 11 were fliCH8 positive. The identification of genetic markers showed that several O26:H11 and all O26:H8 strains tested positive for espK and could therefore be discriminated as EHEC derivatives. The presence of H8 among EHEC O26 and its stx-negative derivative isolates is described for the first time. The interaction of three isolates with polarized Caco-2 cells and with intestinal biopsy specimen fragments ex vivo confirmed the ability of the O26 strains analyzed to cause attaching-and-effacing (A/E) lesions. The O26:H32 strains, isolated mostly from meat, were considered nonvirulent. Knowledge of the virulence content of stx-negative O26 isolates within the same serotype helped to avoid misclassification of isolates, which certainly has important implications for public health surveillance.