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1. Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7

Author

Ángel Adrián Cataldi, Marina S. Palermo, Luisina Martorelli, Daniel A. Vilte, E.C. Mercado, Cristina Ibarra, Sergio Garbaccio, Maria Pilar Mejias, and Adriana Andrea Albanese

Subjects
Bacterial Diseases, Male, 0301 basic medicine, Erythrocytes, Respuesta Inmunológica, Physiology, lcsh:Medicine, Calves, Toxicology, Pathology and Laboratory Medicine, medicine.disease_cause, Biochemistry, Shiga Toxin 2, Antígenos Bacterianos, Immune Physiology, hemic and lymphatic diseases, Chlorocebus aethiops, Medicine and Health Sciences, Type III Secretion Systems, Toxins, Public and Occupational Health, Enzyme-Linked Immunoassays, lcsh:Science, Immune Response, Escherichia coli Infections, Mammals, Bacterial Shedding, Escherichia Coli, Immune System Proteins, Multidisciplinary, Virulence, biology, Escherichia coli Vaccines, Escherichia coli Proteins, Vaccination, Agriculture, Shiga toxin, Ruminants, Vaccination and Immunization, Antibodies, Bacterial, Recombinant Proteins, Infectious Diseases, Toxina Shiga, Vertebrates, Antibody, Research Article, Bacterial Outer Membrane Proteins, Livestock, Bacterial Antigens, Immunology, Toxic Agents, 030106 microbiology, Cattle Diseases, Research and Analysis Methods, Escherichia coli O157, Antibodies, Microbiology, 03 medical and health sciences, Immune system, Antigen, Bovines, Cell Adhesion, medicine, Animals, Antigens, Immunoassays, Adhesins, Bacterial, Vero Cells, Escherichia coli, Intimin, lcsh:R, Vacunación, Organisms, Biology and Life Sciences, Proteins, Virology, Immunity, Humoral, Bacterial adhesin, 030104 developmental biology, Immunoglobulin G, Amniotes, Immunologic Techniques, biology.protein, lcsh:Q, Cattle, Preventive Medicine, Ternero
Abstract

Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo Inst. de Patobiología Fil: Martorelli, Luisina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina Fil: Garbaccio, Sergio Gabriel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina Fil: Vilte, Daniel Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina Fil: Albanese, Adriana Andrea. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Mejias, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires. Laboratorio de Patogénesis e Inmunología de Procesos Infecciosos; Argentina Fil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires. Laboratorio de Patogénesis e Inmunología de Procesos Infecciosos; Argentina Fil: Mercado, Elsa Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina Fil: Ibarra, Cristina E. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina

Published
2017

2. Functional capacity of Shiga-toxin promoter sequences in eukaryotic cells

Author

Romina Jimena Fernandez-Brando, Marcos Fabian Bilen, Cecilia Analia Panek, Gabriela C. Fernández, Leticia V. Bentancor, Pablo Daniel Ghiringhelli, Maria Pilar Mejias, María Victoria Ramos, Martín A. Isturiz, and Marina S. Palermo

Subjects
Cellular differentiation, lcsh:Medicine, Pathogenesis, medicine.disease_cause, HEMOLYTIC UREMIC SYNDROME (HUS), Plasmid, Cricetinae, hemic and lymphatic diseases, Molecular Cell Biology, Chlorocebus aethiops, SHIGA TOXIN, purl.org/becyt/ford/3.4 [https], Promoter Regions, Genetic, lcsh:Science, Multidisciplinary, biology, Cell Differentiation, Shiga toxin, Transfection, Bacterial Pathogens, Host-Pathogen Interaction, Eukaryotic Cells, Medical Microbiology, purl.org/becyt/ford/3 [https], Cellular Types, BACTERIOPHAGE, Research Article, CIENCIAS MÉDICAS Y DE LA SALUD, Protein subunit, Tecnologías que involucran la identificación de ADN, proteínas y enzimas, y cómo influyen en el conjunto de enfermedades y mantenimiento del bienestar, Microbiology, Cell Line, Shiga Toxin, Biotecnología de la Salud, Sequence Homology, Nucleic Acid, Genetics, medicine, Animals, Biology, Vero Cells, Escherichia coli, Gene, Base Sequence, lcsh:R, Promoter, biochemical phenomena, metabolism, and nutrition, Molecular biology, Emerging Infectious Diseases, Genetics of Disease, biology.protein, bacteria, lcsh:Q
Abstract

Shiga toxins (Stx) are the main virulence factors in enterohemorrhagic Escherichia coli (EHEC) infections, causing diarrhea and hemolytic uremic syndrome (HUS). The genes encoding for Shiga toxin-2 (Stx2) are located in a bacteriophage. The toxin is formed by a single A subunit and five B subunits, each of which has its own promoter sequence. We have previously reported the expression of the B subunit within the eukaryotic environment, probably driven by their own promoter. The aim of this work was to evaluate the ability of the eukaryotic machinery to recognize stx2 sequences as eukaryotic-like promoters. Vero cells were transfected with a plasmid encoding Stx2 under its own promoter. The cytotoxic effect on these cells was similar to that observed upon incubation with purified Stx2. In addition, we showed that Stx2 expression in Stx2-insensitive BHK eukaryotic cells induced drastic morphological and cytoskeletal changes. In order to directly evaluate the capacity of the wild promoter sequences of the A and B subunits to drive protein expression in mammalian cells, GFP was cloned under eukaryotic-like putative promoter sequences. GFP expression was observed in 293T cells transfected with these constructions. These results show a novel and alternative way to synthesize Stx2 that could contribute to the global understanding of EHEC infections with immediate impact on the development of treatments or vaccines against HUS Fil: Bentancor, Leticia Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Bilen, Marcos Fabian. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mejias, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Panek, Cecilia Analía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Ramos, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Fernández, Gabriela Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Isturiz, Martín Amadeo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Ghiringhelli, Pablo Daniel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

Published
2013

3. Antibody response to Shiga toxins in Argentinean children with enteropathic hemolytic uremic syndrome at acute and long-term follow-up periods

Author

Claudia Exeni, Ramón Exeni, Romina Jimena Fernandez-Brando, Marina S. Palermo, María del Carmen Laso, María Victoria Ramos, María Pilar Mejías, Martín A. Isturiz, Andrea Exeni, and Leticia V. Bentancor

Subjects
Male, Bacterial Diseases, Time Factors, lcsh:Medicine, Shiga Toxins, medicine.disease_cause, urologic and male genital diseases, Serology, fluids and secretions, STX2, hemic and lymphatic diseases, Gastrointestinal Infections, Child, lcsh:Science, Immune Response, Escherichia Coli, education.field_of_study, Multidisciplinary, biology, Pediatric Nephrology, Shiga toxin, Diarrhea, Treatment Outcome, Infectious Diseases, Nephrology, Child, Preschool, Medicine, Female, medicine.symptom, Antibody, Research Article, Population, Argentina, Enzyme-Linked Immunosorbent Assay, Antibodies, medicine, Humans, Serologic Tests, Immunoassays, education, Immunity to Infections, lcsh:R, Immunity, Pathogenic bacteria, Virology, Case-Control Studies, Hemolytic-Uremic Syndrome, Humoral Immunity, Immunology, Immunologic Techniques, biology.protein, Etiology, Clinical Immunology, lcsh:Q, Follow-Up Studies
Abstract

Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is associated with a broad spectrum of clinical manifestations that include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Systemic Stx toxemia is considered to be central to the genesis of HUS. Distinct methods have been used to evaluate anti-Stx response for immunodiagnostic or epidemiological analysis of HUS cases. The development of enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay to detect the presence of specific antibodies to Stx has introduced important advantages for serodiagnosis of HUS. However, application of these methods for seroepidemiological studies in Argentina has been limited. The aim of this work was to develop an ELISA to detect antibodies against the B subunit of Stx2, and a WB to evaluate antibodies against both subunits of Stx2 and Stx1, in order to analyze the pertinence and effectiveness of these techniques in the Argentinean population. We studied 72 normal healthy children (NHC) and 105 HUS patients of the urban pediatric population from the surrounding area of Buenos Aires city. Using the WB method we detected 67% of plasma from NHC reactive for Stx2, but only 8% for Stx1. These results are in agreement with the broad circulation of Stx2-expressing STEC in Argentina and the endemic behavior of HUS in this country. Moreover, the simultaneous evaluation by the two methods allowed us to differentiate acute HUS patients from NHC with a great specificity and accuracy, in order to confirm the HUS etiology when pathogenic bacteria were not isolated from stools.

Published
2011

4. Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7.

Author

Luisina Martorelli, Sergio Garbaccio, Daniel A Vilte, Adriana A Albanese, María P Mejías, Marina S Palermo, Elsa C Mercado, Cristina E Ibarra, and Angel A Cataldi

Subjects
Medicine, Science
Abstract

Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo.

Published
2017
Full Text
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5. Functional capacity of Shiga-toxin promoter sequences in eukaryotic cells.

Author

Leticia V Bentancor, Marcos F Bilen, María P Mejías, Romina J Fernández-Brando, Cecilia A Panek, Maria V Ramos, Gabriela C Fernández, Martín Isturiz, Pablo D Ghiringhelli, and Marina S Palermo

Subjects
Medicine, Science
Abstract

Shiga toxins (Stx) are the main virulence factors in enterohemorrhagic Escherichia coli (EHEC) infections, causing diarrhea and hemolytic uremic syndrome (HUS). The genes encoding for Shiga toxin-2 (Stx2) are located in a bacteriophage. The toxin is formed by a single A subunit and five B subunits, each of which has its own promoter sequence. We have previously reported the expression of the B subunit within the eukaryotic environment, probably driven by their own promoter. The aim of this work was to evaluate the ability of the eukaryotic machinery to recognize stx2 sequences as eukaryotic-like promoters. Vero cells were transfected with a plasmid encoding Stx2 under its own promoter. The cytotoxic effect on these cells was similar to that observed upon incubation with purified Stx2. In addition, we showed that Stx2 expression in Stx2-insensitive BHK eukaryotic cells induced drastic morphological and cytoskeletal changes. In order to directly evaluate the capacity of the wild promoter sequences of the A and B subunits to drive protein expression in mammalian cells, GFP was cloned under eukaryotic-like putative promoter sequences. GFP expression was observed in 293T cells transfected with these constructions. These results show a novel and alternative way to synthesize Stx2 that could contribute to the global understanding of EHEC infections with immediate impact on the development of treatments or vaccines against HUS.

Published
2013
Full Text
View/download PDF

6. Antibody response to Shiga toxins in Argentinean children with enteropathic hemolytic uremic syndrome at acute and long-term follow-up periods.

Author

Romina J Fernández-Brando, Leticia V Bentancor, María Pilar Mejías, María Victoria Ramos, Andrea Exeni, Claudia Exeni, María del Carmen Laso, Ramón Exeni, Martín A Isturiz, and Marina S Palermo

Subjects
Medicine, Science
Abstract

Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is associated with a broad spectrum of clinical manifestations that include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Systemic Stx toxemia is considered to be central to the genesis of HUS. Distinct methods have been used to evaluate anti-Stx response for immunodiagnostic or epidemiological analysis of HUS cases. The development of enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay to detect the presence of specific antibodies to Stx has introduced important advantages for serodiagnosis of HUS. However, application of these methods for seroepidemiological studies in Argentina has been limited. The aim of this work was to develop an ELISA to detect antibodies against the B subunit of Stx2, and a WB to evaluate antibodies against both subunits of Stx2 and Stx1, in order to analyze the pertinence and effectiveness of these techniques in the Argentinean population. We studied 72 normal healthy children (NHC) and 105 HUS patients of the urban pediatric population from the surrounding area of Buenos Aires city. Using the WB method we detected 67% of plasma from NHC reactive for Stx2, but only 8% for Stx1. These results are in agreement with the broad circulation of Stx2-expressing STEC in Argentina and the endemic behavior of HUS in this country. Moreover, the simultaneous evaluation by the two methods allowed us to differentiate acute HUS patients from NHC with a great specificity and accuracy, in order to confirm the HUS etiology when pathogenic bacteria were not isolated from stools.

Published
2011
Full Text
View/download PDF

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