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1. Immunization with BLS-Stx2B chimera totally protects dams from early pregnancy loss induced by Shiga toxin type 2 (Stx2) and confers anti-Stx2 immunity to the offspring

Author

Cristina Ibarra, María Marta Amaral, Marina S. Palermo, Andrea Cecilia Bruballa, Maria Pilar Mejias, Romina Soledad Alvarez, and Flavia Sacerdoti

Subjects
0301 basic medicine, Male, animal diseases, Early Pregnancy Loss, Recombinant Fusion Proteins, Active immunization, Shiga Toxin 2, Microbiology, Foodborne Diseases, Rats, Sprague-Dawley, 03 medical and health sciences, 0302 clinical medicine, Immunity, Multienzyme Complexes, Pregnancy, hemic and lymphatic diseases, medicine, Animals, Escherichia coli Infections, 030219 obstetrics & reproductive medicine, General Veterinary, General Immunology and Microbiology, biology, Shiga-Toxigenic Escherichia coli, Escherichia coli Vaccines, Public Health, Environmental and Occupational Health, Antibody titer, Shiga toxin, biochemical phenomena, metabolism, and nutrition, medicine.disease, Antibodies, Bacterial, Antibodies, Neutralizing, Brucella, Rats, Vaccination, Abortion, Spontaneous, 030104 developmental biology, Infectious Diseases, Immunology, Hemolytic-Uremic Syndrome, biology.protein, bacteria, Molecular Medicine, Female, Antibody, Immunity, Maternally-Acquired
Abstract

Shiga toxin producing Escherichia coli (STEC) are bacterial pathogens involved in food-borne diseases. Shiga toxin (Stx) is the main virulence factor of STEC and is responsible for systemic complications including Hemolytic Uremic Syndrome (HUS). It has been previously demonstrated that Shiga toxin type 2 (Stx2) induces pregnancy loss in rats in early stage of pregnancy. The main purpose of this study was to determine if an active immunization prevents Stx2 mediated pregnancy loss and confers passive protective immunity to the offspring. For that purpose Sprague Dawley female rats were immunized with the chimera based on the enzyme lumazine synthase from Brucella spp. (BLS) and the B subunit of Shiga toxin 2 (Stx2B) named BLS-Stx2B. After immunization females were mated with males. At day 8 of gestation, dams were challenged intraperitoneally with a sublethal and abortifacient dose of Stx2. The immunization induced high anti-Stx2B-specific antibody titers in sera and most important, prevented pregnancy loss. Pups born and breastfeed by immunized dams had high anti-Stx2B-specific antibody titers in sera. Cross-fostering experiments indicated that passive protective immunity against Stx2 was transmitted through lactation. These results indicate that immunization of adult female rats with BLS-Stx2B prevents Stx2-induced pregnancy loss and confers anti Stx2 protective immunity to the offspring.

Published
2016

2. Leukotriene C4 increases the susceptibility of adult mice to Shigatoxin-producing Escherichia coli infection

Author

María Victoria Ramos, Romina Jimena Fernandez-Brando, Gabriel Cabrera, Maria Jimena Abrey-Recalde, Silvia Vanzulli, Marina S. Palermo, Maria Pilar Mejias, and Mónica Vermeulen

Subjects
Microbiology (medical), CIENCIAS MÉDICAS Y DE LA SALUD, Ciencias de la Salud, Inflammation, Hemolytic Uremic Syndrome, medicine.disease_cause, Microbiology, Proinflammatory cytokine, Shiga Toxin, Pathogenesis, chemistry.chemical_compound, purl.org/becyt/ford/3.3 [https], Stec, fluids and secretions, medicine, Animals, Pathogen, Escherichia coli, Escherichia coli Infections, Mice, Inbred BALB C, Shiga-Toxigenic Escherichia coli, biology, Leukotriene C4, Shiga toxin, General Medicine, respiratory system, bacterial infections and mycoses, Survival Analysis, Neutrophilia, Intestines, Enfermedades Infecciosas, Disease Models, Animal, Infectious Diseases, chemistry, Hemolytic-Uremic Syndrome, Immunology, biology.protein, bacteria, lipids (amino acids, peptides, and proteins), purl.org/becyt/ford/3 [https], Disease Susceptibility, medicine.symptom
Abstract

tShiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that causes hemorrhagic colitis.Under some circumstances, Shiga toxin (Stx) produced within the intestinal tract enters the bloodstream,leading to systemic complications that may cause the potentially fatal hemolytic-uremic syndrome(HUS). Despite STEC human infection is characterized by acute inflammation of the colonic mucosa,little is known regarding the role of proinflammatory mediators like cysteine leukotrienes (cysLTs) inthis pathology. Thus, the aim of this work was to analyze whether leukotriene C4 (LTC4) influences STECpathogenesis in mice. We report that exogenous LTC4 pretreatment severely affected the outcome of STECgastrointestinal infection. LTC4-pretreated (LTC4+) and STEC-infected (STEC+) mice showed an increasedintestinal damage by histological studies, and a decreased survival compared to LTC4-non-pretreated(LTC4−) and STEC+ mice. LTC4+/STEC+ mice that died after the infection displayed neutrophilia andhigh urea levels, indicating that the cause of death was related to Stx2-toxicity. Despite the differencesobserved in the survival between LTC4+ and LTC4− mice after STEC infection, both groups showed thesame survival after Stx2-intravenous inoculation. In addition, LTC4 pretreatment increased the perme-ability of mucosal intestinal barrier, as assessed by FITC-dextran absorption experiments. Altogetherthese results suggest that LTC4 detrimental effect on STEC infection is related to the increased passageof pathogenic factors to the bloodstream.Finally, we showed that STEC infection per se increases the endogenous LTC4 levels in the gut, suggestingthat this inflammatory mediator plays a role in the pathogenicity of STEC infection in mice, mainly bydisrupting the mucosal epithelial barrier. Fil: Cabrera, Gabriel. 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: 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: 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: Abrey Recalde, Maria 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: Vanzulli, Silvia. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Vermeulen, Elba Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; 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
2015

3. Protection of mice against Shiga toxin 2 (Stx2)-associated damage by maternal immunization with a Brucella lumazine synthase-Stx2 B subunit chimera

Author

Giselle Ghersi, Elizabeth Miliwebsky, Maria Jimena Abrey-Recalde, Maria Pilar Mejias, Marta Rivas, Fernando Alberto Goldbaum, Roberto Meiss, Marina S. Palermo, Gabriel Cabrera, Vanesa Zylberman, Romina Jimena Fernandez-Brando, and Ariela Baschkier

Subjects
Immunogen, Offspring, Recombinant Fusion Proteins, Immunology, Microbiology, Shiga Toxin 2, Lumazine synthase, Ciencias Biológicas, Mice, Shigella Vaccines, Biología Celular, Microbiología, Immunity, Multienzyme Complexes, hemic and lymphatic diseases, vaccine, Animals, Escherichia coli Infections, Mice, Inbred BALB C, biology, Lethal dose, shiga toxin, Shiga toxin, biochemical phenomena, metabolism, and nutrition, Acquired immune system, protection, Virology, Antibodies, Bacterial, Brucella, Disease Models, Animal, Infectious Diseases, Enterohemorrhagic Escherichia coli, Hemolytic-Uremic Syndrome, Microbial Immunity and Vaccines, biology.protein, hemolytic uremic syndrome, bacteria, Parasitology, Female, Immunization, Antibody, Immunity, Maternally-Acquired, CIENCIAS NATURALES Y EXACTAS
Abstract

Hemolytic-uremic syndrome (HUS) is defined as the triad of anemia, thrombocytopenia, and acute kidney injury. Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC), which causes a prodromal hemorrhagic enteritis, remains the most common etiology of the typical or epidemic form of HUS. Because no licensed vaccine or effective therapy is presently available for human use, we recently developed a novel immunogen based on the B subunit of Shiga toxin 2 (Stx2B) and the enzyme lumazine synthase from Brucella spp. (BLS) (BLS-Stx2B). The aim of this study was to analyze maternal immunization with BLS-Stx2B as a possible approach for transferring anti-Stx2 protection to the offspring. BALB/c female mice were immunized with BLS-Stx2B before mating. Both dams and pups presented comparable titers of anti-Stx2B antibodies in sera and fecal extracts. Moreover, pups were totally protected against a lethal dose of systemic Stx2 injection up to 2 to 3 months postpartum. In addition, pups were resistant to an oral challenge with an Stx2-producing EHEC strain at weaning and did not develop any symptomatology associated with Stx2 toxicity. Fostering experiments demonstrated that anti-Stx2B neutralizing IgG antibodies were transmitted through breast-feeding. Pups that survived the EHEC infection due to maternally transferred immunity prolonged an active and specific immune response that protected them against a subsequent challenge with intravenous Stx2. Our study shows that maternal immunization with BLS-Stx2B was very effective at promoting the transfer of specific antibodies, and suggests that preexposure of adult females to this immunogen could protect their offspring during the early phase of life. Fil: Mejias, Maria Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina Fil: Cabrera, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina Fil: Baschkier, Ariela. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina Fil: Ghersi, Giselle. Inmunova S.a; Argentina Fil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina Fil: Miliwebsky, Elyzabeth. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina Fil: Meiss, Roberto. Academia Nacional de Medicina. Centro de Estudios Oncológicos. Departamento de Patología; Argentina Fil: Goldbaum, Fernando Alberto. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Inmunova S.a; Argentina Fil: Zylberman, Vanesa. Inmunova S.a; Argentina. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina Fil: Rivas, Marta. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina Fil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina

Published
2014

4. Promoter Sequence of Shiga Toxin 2 (Stx2) Is Recognized In Vivo , Leading to Production of Biologically Active Stx2

Author

Marcos Fabian Bilen, Jorge Goldstein, Luciano P. Pedrotti, Maria Cecilia Rodriguez-Galan, Leticia V. Bentancor, Roberto Meiss, Pablo Daniel Ghiringhelli, Natalia S. Baez, Maria Pilar Mejias, Marina S. Palermo, and Alipio Pinto

Subjects
biology, Kidney metabolism, Shiga toxin, Transfection, medicine.disease_cause, Microbiology, QR1-502, Plasmid, In vivo, STX2, hemic and lymphatic diseases, Virology, biology.protein, medicine, bacteria, Cytotoxic T cell, Escherichia coli
Abstract

Shiga toxins (Stx) are the main agent responsible for the development of hemolytic-uremic syndrome (HUS), the most severe and life-threatening systemic complication of infection with enterohemorrhagic Escherichia coli (EHEC) strains. We previously described Stx2 expression by eukaryotic cells after they were transfected in vitro with the stx 2 gene cloned into a prokaryotic plasmid (pStx2). The aim of this study was to evaluate whether mammalian cells were also able to express Stx2 in vivo after pStx2 injection. Mice were inoculated by hydrodynamics-based transfection (HBT) with pStx2. We studied the survival, percentage of polymorphonuclear leukocytes in plasma, plasma urea levels, and histology of the kidneys and the brains of mice. Mice displayed a lethal dose-related response to pStx2. Stx2 mRNA was recovered from the liver, and Stx2 cytotoxic activity was observed in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, and markers of central nervous system (CNS) damage were observed, including increased expression of glial fibrillary acidic protein (GFAP) and fragmentation of NeuN in neurons. Moreover, anti-Stx2B-immunized mice were protected against pStx2 inoculation. Our results show that Stx2 is expressed in vivo from the wild stx 2 gene, reproducing pathogenic damage induced by purified Stx2 or secondary to EHEC infection. IMPORTANCE Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC) infections are a serious public health problem, and Stx is the main pathogenic agent associated with typical hemolytic-uremic syndrome (HUS). In contrast to the detailed information describing the molecular basis for EHEC adherence to epithelial cells, very little is known about how Stx is released from bacteria in the gut, reaching its target tissues, mainly the kidney and central nervous system (CNS). In order to develop an efficient treatment for EHEC infections, it is necessary to understand the mechanisms involved in Stx expression. In this regard, the present study demonstrates that mammals can synthesize biologically active Stx using the natural promoter associated with the Stx-converting bacteriophage genome. These results could impact the comprehension of EHEC HUS, since local eukaryotic cells transduced and/or infected by bacteriophage encoding Stx2 could be an alternative source of Stx production.

Published
2013
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5. Oral administration of Shiga toxin-producing Escherichia coli induces intestinal and systemic specific immune response in mice

Author

Maria Jimena Abrey-Recalde, Maria Pilar Mejias, Leticia V. Bentancor, Ariela Baschkier, Marta Rivas, María Victoria Ramos, Romina Jimena Fernandez-Brando, Cecilia Analia Panek, Elizabeth Miliwebsky, Gabriel Cabrera, and Marina S. Palermo

Subjects
Microbiology (medical), Male, Serum, CIENCIAS MÉDICAS Y DE LA SALUD, T-Lymphocytes, Serum Anti-Stx2 Antibodies, Immunology, Ciencias de la Salud, Administration, Oral, medicine.disease_cause, Microbiology, Mucosal Immune Response, Feces, Peyer's Patches, Immune system, STX2, hemic and lymphatic diseases, medicine, Ehec, Immunology and Allergy, Mesenteric lymph nodes, Animals, Intestinal Mucosa, Escherichia coli, B cell, B-Lymphocytes, Mice, Inbred BALB C, biology, Shiga-Toxigenic Escherichia coli, Escherichia coli Vaccines, General Medicine, biochemical phenomena, metabolism, and nutrition, Acquired immune system, Antibodies, Bacterial, Specific Iga, Immunoglobulin A, Enfermedades Infecciosas, Disease Models, Animal, medicine.anatomical_structure, Lymphatic system, Blood, Immunoglobulin G, Hemolytic-Uremic Syndrome, biology.protein, bacteria, Female, Antibody
Abstract

Hemolytic uremic syndrome (HUS) is the major complication of gastrointestinal infections with enterohemorrhagic Escherichia coli (EHEC) and is mediated by the production of Shiga toxins (Stx). Although it has been previously reported that not only HUS patients but healthy children have anti-Stx antibodies, very little is known about how these infections impact on mucosal immune system to generate a specific immune response. This work aimed to evaluate the immune responses elicited after a single oral dose of EHEC in a mouse model of HUS at weaning. We found sequential activation of T and B lymphocytes together with an increased percentage of IgA-bearing B cells in Peyer´s patches and mesenteric lymph nodes. We also found fecal anti-EHEC IgA and serum anti-Stx2 IgG in EHEC-inoculated mice. Besides, these mice were partially protected against an intravenous challenge with Stx2. These data demonstrate that one episode of EHEC infection is enough to induce activation in the gut-associated lymphoid tissue, especially the B cell compartment, and lead to the production of specific IgA in mucosal tissue and the generation of systemic protection against Stx2 in a percentage of intragastrically inoculated mice. These data also support the epidemiologic observation that a second episode of HUS is very rare. 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: Cabrera, Gabriel Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Baschkier, Ariela. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; 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: 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: Miliwebsky, Elizabeth. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina Fil: Abrey Recalde, Maria 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: 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: 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: Rivas, Marta. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; 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

6. Immunization with a chimera consisting of the B subunit of Shiga toxin type 2 and brucella lumazine synthase confers total protection against Shiga toxins in mice

Author

Maria Pilar Mejias, Ariela Baschkier, Cecilia Analia Panek, Giselle Ghersi, Leticia V. Bentancor, Marina S. Palermo, Vanesa Zylberman, Fernando Alberto Goldbaum, and Patricio O. Craig

Subjects
Male, CIENCIAS MÉDICAS Y DE LA SALUD, Immunogen, SHIGA TOXIN 2, Immunology, Inmunología, VACCINE, Hemolytic Uremic Syndrome, medicine.disease_cause, Shiga Toxin 2, Lumazine synthase, Microbiology, Ciencias Biológicas, Chimera (genetics), Mice, Immune system, Biología Celular, Microbiología, Shigella Vaccines, STX2, Multienzyme Complexes, hemic and lymphatic diseases, medicine, Immunology and Allergy, Animals, Escherichia coli, Mice, Inbred BALB C, biology, Shiga toxin, purl.org/becyt/ford/3.1 [https], ANTI-STX2 ANTIBODIES, biochemical phenomena, metabolism, and nutrition, Virology, Antibodies, Bacterial, Antibodies, Neutralizing, Brucella, Recombinant Proteins, Medicina Básica, Disease Models, Animal, Enterohemorrhagic Escherichia coli, Hemolytic-Uremic Syndrome, biology.protein, bacteria, purl.org/becyt/ford/3 [https], Female, Antibody, CIENCIAS NATURALES Y EXACTAS
Abstract

The striking feature of Enterohemorrhagic Escherichia coli (EHEC) infections is the production of Shiga toxins (Stx) implicated in the development of the life-threatening hemolytic uremic syndrome (HUS). Despite the magnitude of the social impact of EHEC infections, no licensed vaccine or effective therapy is presently available for human use. One of the biggest challenges is to develop an effective and safe immunogen to ensure non toxicity but also a strong input to the immune system to induce long-lasting, high affinity antibodies with anti-Stx neutralizing capacity. The enzyme lumazine synthase from Brucella spp. (BLS) is a highly stable dimer of pentamers and a scaffold with enormous plasticity for displaying foreign antigens on it. Taking into account BLS advantages and the potential capacity of the B subunit of Stx2 (Stx2B) to induce antibodies that prevent Stx2 toxicity by blocking its entrance to the host cells, we engineered a new immunogen inserting Stx2B at the amino termini of BLS. The resulting chimera demonstrated a strong capacity to induce long-lasting humoral immune response in mice. The chimera induced antibodies with high neutralizing capacity for Stx2 and its variants. Moreover, immunized mice were completely protected against i.v. Stx2-challenge and weaned mice receiving an oral challenge with EHEC were completely protected by the transference of immune sera. We conclude that this novel immunogen represents a promising candidate for vaccine or antibody development with preventive or therapeutic ends, for use in HUS endemic areas or during future outbreaks caused by pathogenic strains of Stx-producing E. coli. 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: Ghersi, Giselle. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Craig, Patricio Oliver. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; 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: 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: Baschkier, Ariela. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina Fil: Goldbaum, Fernando Alberto. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Zylberman, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Inmunova S.A; 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

7. 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

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