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1.
Medicina (B Aires) ; 78(2): 107-112, 2018.
Artigo em Espanhol | MEDLINE | ID: mdl-29659360

RESUMO

The typical hemolytic uremic syndrome (HUS) is an orphan disease caused by Shiga toxin(Stx) producing Escherichia coli strains and characterized by acute kidney damage, microangiopathic hemolytic anemia and low platelet count. It is endemic in Argentina, the country with the highest incidence of HUS in the world. Stx is essential for its development and therefore, HUS is considered a toxemic non-bacteremic disorder, which could be treated with antibodies. Herein we describe the development of a new treatment capable of neutralizing the toxic effect of Stx and its variants. The treatment consists of F(ab')2 fragments from an equine antiserum whose efficacy and potency against Stx1 and Stx2 were proved in different preclinical models. The product was shown to be safe in animals. Furthermore, the anti-Stx F(ab')2 pharmacokinetic was shown to be similar to that of analogous compounds and a therapeutic window for its administration was determined. Altogether, these preclinical results warrant testing in humans. The phase I clinical trial will be performed at the Hospital Italiano in Buenos Aires to evaluate the safety and pharmacokinetics of the product in healthy adult volunteers. Based on the results of this study, a phase II clinical trial will be planned in pediatric patients diagnosed with infection by Stx-producing E. coli strains.


Assuntos
Drogas em Investigação , Infecções por Escherichia coli/tratamento farmacológico , Síndrome Hemolítico-Urêmica/prevenção & controle , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga II/antagonistas & inibidores , Anticorpos/imunologia , Argentina , Ensaios Clínicos Fase II como Assunto , Escherichia coli/imunologia , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/complicações , Síndrome Hemolítico-Urêmica/imunologia , Humanos , Toxina Shiga I/imunologia , Toxina Shiga II/imunologia
2.
Medicina (B.Aires) ; 78(2): 107-112, abr. 2018. ilus
Artigo em Espanhol | LILACS | ID: biblio-954958

RESUMO

El síndrome urémico hemolítico (SUH) típico es una enfermedad huérfana causada por cepas de Escherichia coli productoras de toxina Shiga (Stx) y caracterizada por daño renal agudo, anemia hemolítica microangiopática y plaquetopenia. Es endémico en Argentina, el país con mayor incidencia de SUH en el mundo. Debido al rol fundamental de la Stx en su patogenia, se puede considerar que, como otras toxemias conocidas, el SUH podría ser tratado con anticuerpos. Este trabajo describe el desarrollo de un nuevo tratamiento capaz de neutralizar el efecto tóxico de distintas variantes de la Stx. El tratamiento consiste en fragmentos F(ab')2 provenientes de un antisuero equino cuya eficacia y potencia contra Stx1 y Stx2 se comprobó en diferentes modelos preclínicos. El producto mostró ser seguro en animales, presentó la farmacocinética descripta para compuestos similares y se pudo establecer una posible ventana terapéutica para su adecuada administración. En conjunto, los resultados preclínicos obtenidos validan la realización de un estudio clínico de primer uso en humanos. En dicho estudio, que se realizará en el Hospital Italiano de Buenos Aires, se analizará la seguridad y la farmacocinética del producto en voluntarios adultos sanos. Estos resultados sentarán las bases para la realización del estudio clínico fase II en pacientes pediátricos con infección por cepas de E. coli productoras de Stx.


The typical hemolytic uremic syndrome (HUS) is an orphan disease caused by Shiga toxin(Stx) -producing Escherichia coli strains and characterized by acute kidney damage, microangiopathic hemolytic anemia and low platelet count. It is endemic in Argentina, the country with the highest incidence of HUS in the world. Stx is essential for its development and therefore, HUS is considered a toxemic non-bacteremic disorder, which could be treated with antibodies. Herein we describe the development of a new treatment capable of neutralizing the toxic effect of Stx and its variants. The treatment consists of F(ab')2 fragments from an equine antiserum whose efficacy and potency against Stx1 and Stx2 were proved in different preclinical models. The product was shown to be safe in animals. Furthermore, the anti-Stx F(ab')2 pharmacokinetic was shown to be similar to that of analogous compounds and a therapeutic window for its administration was determined. Altogether, these preclinical results warrant testing in humans. The phase I clinical trial will be performed at the Hospital Italiano in Buenos Aires to evaluate the safety and pharmacokinetics of the product in healthy adult volunteers. Based on the results of this study, a phase II clinical trial will be planned in pediatric patients diagnosed with infection by Stx-producing E. coli strains.


Assuntos
Humanos , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Drogas em Investigação , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga II/antagonistas & inibidores , Infecções por Escherichia coli/tratamento farmacológico , Síndrome Hemolítico-Urêmica/prevenção & controle , Argentina , Ensaios Clínicos Fase II como Assunto , Toxina Shiga I/imunologia , Toxina Shiga II/imunologia , Escherichia coli/isolamento & purificação , Escherichia coli/imunologia , Infecções por Escherichia coli/complicações , Síndrome Hemolítico-Urêmica/imunologia , Anticorpos/imunologia
3.
Artif Cells Nanomed Biotechnol ; 46(sup1): 841-851, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29447477

RESUMO

Escherichia coli O157:H7 is a pathogen, which can generate Shiga-like toxins (SLTs) and cause hemolytic-uremic syndrome. Foodborne illness outbreaks caused by E. coli O157:H7 have become a global issue. Since SLTs are quite toxic, effective medicines that can reduce the damage caused by SLTs should be explored. SLTs consist of a single A and five B subunits, which can inhibit ribosome activity for protein synthesis and bind with the cell membrane of host cells, respectively. Pigeon ovalbumin (POA), i.e. a glycoprotein, is abundant in pigeon egg white (PEW) proteins. The structure of POA contains Gal-α(1→4)-Gal-ß(1→4)-GlcNAc ligands, which have binding affinity toward the B subunit in SLT type-1 (SLT-1B). POA immobilized gold nanoparticles (POA-Au NPs) can be generated by reacting PEW proteins with aqueous tetrachloroauric acid in one-pot. The generated POA-Au NPs have been demonstrated to have selective trapping-capacity toward SLT-1B previously. Herein, we explore that POA-Au NPs can be used as protective agents to neutralize the toxicity of SLT-1 in SLT-1-infected model cells. The results show that the cells can be completely rescued when a sufficient amount of POA-Au NPs is used to treat the SLT-1-infected cells within 1 h.


Assuntos
Ouro/química , Ouro/farmacologia , Nanopartículas Metálicas/química , Toxina Shiga I/antagonistas & inibidores , Animais , Escherichia coli O157/efeitos dos fármacos , Escherichia coli O157/fisiologia , Células Hep G2 , Humanos , Teste de Materiais , Ovalbumina/química , Toxina Shiga I/toxicidade
4.
Clin Vaccine Immunol ; 22(4): 448-55, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25716230

RESUMO

In the United States, Shiga toxin (Stx)-producing Escherichia coli (STEC) is the most frequent infectious cause of hemorrhagic colitis. Hemolytic uremic syndrome (HUS) is a serious sequela that may develop after STEC infection that can lead to renal failure and death in up to 10% of cases. STEC can produce one or more types of Stx, Stx1 and/or Stx2, and Stx1 and Stx2 are responsible for HUS-mediated kidney damage. We previously generated two monoclonal antibodies (MAbs) that neutralize the toxicity of Stx1 or Stx2. In this study, we evaluated the protective efficacy of human/mouse chimeric versions of those monoclonal antibodies, named cαStx1 and cαStx2. Mice given an otherwise lethal dose of Stx1 were protected from death when injected with cαStx1 either 1 h before or 1 h after toxin injection. Additionally, streptomycin-treated mice fed the mouse-lethal STEC strain B2F1 that produces the Stx2 variant Stx2d were protected when given a dose of 0.1 mg of cαStx2/kg of body weight administered up to 72 h post-oral bacterial challenge. Since many STEC strains produce both Stx1 and Stx2 and since either toxin may lead to the HUS, we also assessed the protective efficacy of the combined MAbs. We found that both antibodies were required to protect mice from the presence of both Stx1 and Stx2. Pharmacokinetic studies indicated that cαStx1 and cαStx2 had serum half-lives (t1/2) of about 50 and 145 h, respectively. We propose that cαStx1 and cαStx2, both of which have been tested for safety in humans, could be used therapeutically for prevention or treatment early in the development of HUS.


Assuntos
Anticorpos Antibacterianos/uso terapêutico , Antitoxinas/uso terapêutico , Infecções por Escherichia coli/prevenção & controle , Intoxicação/prevenção & controle , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga II/antagonistas & inibidores , Animais , Anticorpos Antibacterianos/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/uso terapêutico , Modelos Animais de Doenças , Feminino , Meia-Vida , Masculino , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapêutico , Análise de Sobrevida , Resultado do Tratamento
5.
Infect Immun ; 83(1): 286-91, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25368111

RESUMO

Hemolytic-uremic syndrome (HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), remains untreatable. Production of human monoclonal antibodies against Stx, which are highly effective in preventing Stx sequelae in animal models, is languishing due to cost and logistics. We reported previously that the production and evaluation of a camelid heavy-chain-only VH domain (VHH)-based neutralizing agent (VNA) targeting Stx1 and Stx2 (VNA-Stx) protected mice from Stx1 and Stx2 intoxication. Here we report that a single intramuscular (i.m.) injection of a nonreplicating adenovirus (Ad) vector carrying a secretory transgene of VNA-Stx (Ad/VNA-Stx) protected mice challenged with Stx2 and protected gnotobiotic piglets infected with STEC from fatal systemic intoxication. One i.m. dose of Ad/VNA-Stx prevented fatal central nervous system (CNS) symptoms in 9 of 10 animals when it was given to piglets 24 h after bacterial challenge and in 5 of 9 animals when it was given 48 h after bacterial challenge, just prior to the onset of CNS symptoms. All 6 placebo animals died or were euthanized with severe CNS symptoms. Ad/VNA-Stx treatment had no impact on diarrhea. In conclusion, Ad/VNA-Stx treatment is effective in protecting piglets from fatal Stx2-mediated CNS complications following STEC challenge. With a low production cost and further development, this could presumably be an effective treatment for patients with HUS and/or individuals at high risk of developing HUS due to exposure to STEC.


Assuntos
Adenovírus Humanos/genética , Anticorpos Neutralizantes/uso terapêutico , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli O157/imunologia , Síndrome Hemolítico-Urêmica/tratamento farmacológico , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga II/antagonistas & inibidores , Animais , Anticorpos Neutralizantes/genética , Modelos Animais de Doenças , Portadores de Fármacos/administração & dosagem , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Feminino , Vetores Genéticos , Síndrome Hemolítico-Urêmica/imunologia , Síndrome Hemolítico-Urêmica/microbiologia , Injeções Intramusculares , Camundongos , Toxina Shiga I/imunologia , Toxina Shiga II/imunologia , Análise de Sobrevida , Suínos , Fatores de Tempo
6.
J Appl Microbiol ; 116(5): 1322-33, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24447276

RESUMO

AIMS: To use the phage display technique to develop peptides with the capability to neutralize the cytotoxicity induced by Stx1 and Stx2 toxins produced by Shiga toxin-producing Escherichia coli (STEC). METHODS AND RESULTS: The phage display technique permitted the development of three peptides, named PC7-12, P12-26 and PC7-30, which bind to the globotriaosylceramide (Gb3) receptor for Shiga toxins produced by STEC. Moreover, these peptides were capable of competing efficiently with the Shiga toxins for binding to Gb3. The peptides described herein partially inhibited the Stx-induced cytotoxicity of cell-free filtrates of STEC O157 : H7 and purified Stx toxins in Vero cells. The inhibition of lethality induced by Stx toxins in mice indicated that peptide PC7-30 inhibited the lethality caused by Stx1 (2LD50) in mice. CONCLUSIONS: The phage display technique permitted the development of peptides that inhibited the cytotoxicity induced by Stx toxins in vitro. Peptide PC7-30 inhibited the lethality of Stx1 in vivo; this molecule would be a promising candidate for the development of therapeutic agents for STEC-related diseases in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The selection of Gb3, the common receptor for Stx1 and Stx2, may contribute to the development of efficient neutralizers for both toxins, and our approach would be an interesting alternative for the development of therapeutic molecules for the treatment of diseases caused by STEC strains.


Assuntos
Peptídeos/farmacologia , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga II/antagonistas & inibidores , Animais , Chlorocebus aethiops , Humanos , Camundongos , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Toxina Shiga I/toxicidade , Toxina Shiga II/toxicidade , Escherichia coli Shiga Toxigênica/metabolismo , Triexosilceramidas/metabolismo , Células Vero
7.
Org Lett ; 15(20): 5190-3, 2013 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-24099046

RESUMO

A convenient scaffold based on poly(N-vinyl-2-pyrrolidone-co-vinyl alcohol) is proposed for presenting ligands in multivalent format. This amphiphilic polymer supports synthesis of conjugates in both organic and aqueous media, permits enzymatic processing of the ligand precursor, and, finally, offers a choice of formats for evaluation of biological activity either as a soluble inhibitor or as a capture reagent after deposition on a hydrophobic surface or standard microtiter plates.


Assuntos
Sondas Moleculares/química , Álcool de Polivinil/análogos & derivados , Povidona/análogos & derivados , Tensoativos/química , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Ligantes , Sondas Moleculares/síntese química , Sondas Moleculares/farmacologia , Estrutura Molecular , Álcool de Polivinil/síntese química , Álcool de Polivinil/química , Povidona/síntese química , Povidona/química , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga I/metabolismo , Relação Estrutura-Atividade , Tensoativos/síntese química , Tensoativos/farmacologia
8.
Infect Immun ; 81(6): 2133-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23545297

RESUMO

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli that occasionally causes fatal systemic complications. We recently developed a tetravalent peptide (PPP-tet) that neutralizes the cytotoxicity of Stx2 using a multivalent peptide library approach. In this study, we used this technique to identify a series of tetravalent peptides that bound to Stx1, another major Stx family member, with high affinity by targeting one receptor-binding site of the B subunit. One peptide, MMA-tet, markedly inhibited Stx1 and Stx2 cytotoxicity with greater potency than PPP-tet. After forming a complex with Stx1 through its specific receptor-binding region, MMA-tet did not affect vesicular transport of the toxin to the endoplasmic reticulum but substantially rescued inhibition of the protein synthesis induced by Stx1. Oral application of MMA-tet protected mice from a fatal dose of an E. coli O157:H7 strain producing both toxins. MMA-tet may be a promising therapeutic agent against the infection.


Assuntos
Peptídeos/farmacologia , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga II/antagonistas & inibidores , Substituição de Aminoácidos , Animais , Sobrevivência Celular , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Ensaio de Imunoadsorção Enzimática , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli O157/patogenicidade , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/uso terapêutico , Subunidades Proteicas , Toxina Shiga I/metabolismo , Toxina Shiga I/toxicidade , Toxina Shiga II/metabolismo , Toxina Shiga II/toxicidade , Organismos Livres de Patógenos Específicos , Células Vero
9.
PLoS One ; 7(2): e31191, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22355345

RESUMO

Shiga-like toxins are ribosome-inactivating proteins (RIP) produced by pathogenic E. coli strains that are responsible for hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A(1) chain of Shiga-like toxin 1 (SLT-1), a representative RIP, first docks onto a conserved peptide SD[D/E]DMGFGLFD located at the C-terminus of all three eukaryotic ribosomal stalk proteins and halts protein synthesis through the depurination of an adenine base in the sarcin-ricin loop of 28S rRNA. Here, we report that the A(1) chain of SLT-1 rapidly binds to and dissociates from the C-terminal peptide with a monomeric dissociation constant of 13 µM. An alanine scan performed on the conserved peptide revealed that the SLT-1 A(1) chain interacts with the anionic tripeptide DDD and the hydrophobic tetrapeptide motif FGLF within its sequence. Based on these 2 peptide motifs, SLT-1 A(1) variants were generated that displayed decreased affinities for the stalk protein C-terminus and also correlated with reduced ribosome-inactivating activities in relation to the wild-type A(1) chain. The toxin-peptide interaction and subsequent toxicity were shown to be mediated by cationic and hydrophobic docking surfaces on the SLT-1 catalytic domain. These docking surfaces are located on the opposite face of the catalytic cleft and suggest that the docking of the A(1) chain to SDDDMGFGLFD may reorient its catalytic domain to face its RNA substrate. More importantly, both the delineated A(1) chain ribosomal docking surfaces and the ribosomal peptide itself represent a target and a scaffold, respectively, for the design of generic inhibitors to block the action of RIPs.


Assuntos
Interações Hidrofóbicas e Hidrofílicas , Oligopeptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga I/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Dados de Sequência Molecular , Biossíntese de Proteínas , Estrutura Terciária de Proteína , RNA Ribossômico/genética , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Técnicas do Sistema de Duplo-Híbrido
10.
Arch Immunol Ther Exp (Warsz) ; 59(4): 239-47, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21644029

RESUMO

Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC), including O157:H7, causes bloody diarrhea and hemorrhagic colitis in humans, occasionally resulting in fatal systemic complications, such as neurological damage and hemolytic-uremic syndrome. Because Stx is a major virulence factor of the infectious disease, a series of Shiga toxin neutralizers with various structural characteristics has been developed as promising therapeutic agents. Most of these agents function to bind to the toxin directly and inhibit the binding to its receptor present on the target cells. Other neutralizers do not inhibit receptor binding but induce aberrant intracellular transport of the toxin, resulting in effective detoxification. Such a novel type of Stx neutralizer provides a new therapeutic strategy against STEC infections. Here, recent progress of the development of Stx neutralizers is reviewed.


Assuntos
Antibacterianos/química , Infecções por Escherichia coli/tratamento farmacológico , Peptídeos/administração & dosagem , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga II/antagonistas & inibidores , Triexosilceramidas/administração & dosagem , Animais , Antibacterianos/uso terapêutico , Sítios de Ligação/efeitos dos fármacos , Técnicas de Química Combinatória/métodos , Desenho de Fármacos , Retículo Endoplasmático/metabolismo , Escherichia coli O157/metabolismo , Globosídeos/metabolismo , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Macrófagos Peritoneais/metabolismo , Camundongos , Peptídeos/síntese química , Polímeros/farmacologia , Polímeros/uso terapêutico , Coelhos , Componente Amiloide P Sérico/metabolismo , Componente Amiloide P Sérico/uso terapêutico , Toxina Shiga I/química , Toxina Shiga II/química , Silanos/síntese química , Silanos/uso terapêutico , Trissacarídeos/síntese química , Trissacarídeos/uso terapêutico , Fatores de Virulência/metabolismo
11.
Bioconjug Chem ; 21(8): 1486-93, 2010 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-20669970

RESUMO

Shiga toxins (Stx) released by Escherichia coli O157:H7 and Shigella dysentriae cause life-threatening conditions that include hemolytic uremic syndrome (HUS), kidney failure, and neurological complications. Cellular entry is mediated by the B-subunit of the AB(5) toxin, which recognizes cell surface glycolipids present in lipid raft-like structures. We developed gold glyconanoparticles that present a multivalent display similar to the cell surface glycolipids to compete for these toxins. These highly soluble glyconanoparticles were nontoxic to the Vero monkey kidney cell line and protected Vero cells from Stx-mediated toxicity in a dose-dependent manner. The inhibition is highly dependent on the structure and density of the glycans; selective inhibition of Stx1 and the more clinically relevant Stx2 was achieved. Interestingly, natural variants of Stx2, Stx2c, and Stx2d possessing minimal amino acid variation in the receptor binding site of the B-subunit or changes in the A-subunit were not neutralized by either the Stx1- or Stx2-specific gold glyconanoparticles. Our results suggest that tailored glyconanoparticles that mimic the natural display of glycans in lipid rafts could serve as potential therapeutics for Stx1 and Stx2. However, a few amino acid changes in emerging Stx2 variants can change receptor specificity, and further research is needed to develop receptor mimics for the emerging variants of Stx2.


Assuntos
Ouro/farmacologia , Nanopartículas Metálicas/química , Polissacarídeos/farmacologia , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga II/antagonistas & inibidores , Animais , Sítios de Ligação , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Escherichia coli O157/química , Ouro/química , Ligantes , Modelos Moleculares , Estrutura Molecular , Polissacarídeos/síntese química , Polissacarídeos/química , Toxina Shiga I/química , Toxina Shiga I/toxicidade , Toxina Shiga II/química , Toxina Shiga II/toxicidade , Shigella dysenteriae/química , Relação Estrutura-Atividade , Propriedades de Superfície , Células Vero
12.
J Food Sci ; 74(8): M461-6, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19799674

RESUMO

To reduce the amounts of verocytotoxin (VT) produced by Escherichia coli O157:H7, various spices were screened for their ability to suppress VT production. Extracts of these spices were prepared with 70% ethyl alcohol. When E. coli O157:H7 cells were grown to the stationary phase at 37 degrees C in Luria-Bertani medium supplemented with 0.02% allspice extract, the production of both VT1 and VT2 was significantly reduced. Neither growth inhibition nor a delay in the lag phase was observed when the cells were cultured in the presence of 0.02% allspice extract. An active component of the allspice extract was purified by HPLC and was identified as eugenol. When we examined the suppressive effect of eugenol on VT production by E. coli O157:H7, the amounts of both intracellular and extracellular VTs were found to decrease with an increase in eugenol concentration. Our results suggest that eugenol is useful for reducing the virulence of E. coli O157:H7.


Assuntos
Anti-Infecciosos/farmacologia , Escherichia coli O157/efeitos dos fármacos , Eugenol/farmacologia , Aditivos Alimentares/farmacologia , Extratos Vegetais/farmacologia , Toxinas Shiga/biossíntese , Especiarias , Anti-Infecciosos/análise , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Contagem de Colônia Microbiana , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/metabolismo , Eugenol/análise , Eugenol/isolamento & purificação , Fezes/microbiologia , Aditivos Alimentares/análise , Aditivos Alimentares/isolamento & purificação , Humanos , Testes de Fixação do Látex , Pimenta/química , Extratos Vegetais/química , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga I/biossíntese , Toxina Shiga II/antagonistas & inibidores , Toxina Shiga II/biossíntese , Toxinas Shiga/antagonistas & inibidores
13.
Proc Natl Acad Sci U S A ; 105(44): 16837-42, 2008 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-18955695

RESUMO

We demonstrate that interactions between multimeric receptors and multivalent ligands are dramatically enhanced by recruiting a complementary templating receptor such as an endogenous multimeric protein but only when individual ligands are attached to a polymer as preorganized, covalent, heterobifunctional pairs. This effect cannot be replicated by a multivalent ligand if the same recognition elements are independently arrayed on the scaffold. Application of this principle offers an approach to create high-avidity inhibitors for multimeric receptors. Judicious selection of the ligand that engages the templating protein allows appropriate effector function to be incorporated in the polymeric construct, thereby providing an opportunity for therapeutic applications. The power of this approach is exemplified by the design of exceptionally potent Escherichia coli Shiga toxin antagonists that protect transgenic mice that constitutively express a human pentraxin, serum amyloid P component.


Assuntos
Antibacterianos/química , Escherichia coli O157/efeitos dos fármacos , Toxina Shiga I/antagonistas & inibidores , Animais , Antibacterianos/síntese química , Antibacterianos/farmacologia , Escherichia coli O157/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Polímeros/síntese química , Polímeros/química , Polímeros/farmacologia , Componente Amiloide P Sérico/metabolismo , Toxina Shiga I/química
14.
Chembiochem ; 9(7): 1100-9, 2008 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-18398881

RESUMO

Compared to monovalent carbohydrates, multivalent carbohydrate ligands exhibit significantly enhanced binding affinities to their interacting proteins. Here, we report globotriose (P(k) ligand)-functionalized gold nanoparticle (AuNP) probes for the investigation of multivalent interactions with the B(5) subunit of Shiga-like toxin I (B-Slt). Six P(k)-ligand-encapsulated AuNPs (P(k)-AuNPs) of varying particle size and linker length were synthesized and evaluated for their potential as multivalent affinity probes by using a surface plasmon resonance competition assay. The affinity of these probes for the interacting proteins was greatly affected by nanoparticle size, linker length, and ligand density on nanoparticle surface. For example, the 20-nm 20-P(k)-l-AuNP, which had a relatively long linker showed a >10(8)-fold increase in affinity compared with the mono P(k) ligand. This intrinsic high-affinity AuNP probe specifically captured the recombinant B-Slt from Escherichia coli lysate, and the resulting purity of the B-Slt was >95 %. We also developed a robust P(k)-AuNP-based detection method for Slt-I by combining the technique with silver enhancement.


Assuntos
Técnicas Biossensoriais/instrumentação , Ouro/química , Nanopartículas Metálicas/análise , Nanopartículas Metálicas/química , Toxina Shiga I/análise , Toxina Shiga I/metabolismo , Trissacarídeos/química , Bactérias/citologia , Glicoconjugados/química , Ligantes , Ligação Proteica , Toxina Shiga I/antagonistas & inibidores , Solubilidade , Ressonância de Plasmônio de Superfície , Triexosilceramidas/síntese química , Trissacarídeos/metabolismo , Água/química
15.
J Mol Biol ; 378(2): 375-86, 2008 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-18358491

RESUMO

Shiga-like toxin 1 (SLT-1) is a type II ribosome-inactivating protein; its A(1) domain blocks protein synthesis in eukaryotic cells by catalyzing the depurination of a single adenine base in 28 S rRNA. The molecular mechanism leading to this site-specific depurination event is thought to involve interactions with eukaryotic ribosomal proteins. Here, we present evidence that the A(1) chain of SLT-1 binds to the ribosomal proteins P0, P1, and P2. These proteins were identified from a HeLa cell lysate by tandem mass spectrometry, and subsequently confirmed to bind to SLT-1 A(1) chain by yeast-two-hybrid and pull-down experiments using candidate full-length proteins. Moreover, the removal of the last 17 amino acids of either protein P1 or P2 abolishes the interaction with the A(1) chain, whereas P0, lacking this common C terminus, still binds to the A(1) domain. In vitro pull-down experiments using fusion protein-tagged C-terminal peptides corresponding to the common 7, 11, and 17 terminal residues of P1 and P2 confirmed that the A(1) chain of SLT-1 as well as the A chain of ricin bind to this shared C-terminal peptide motif. More importantly, a synthetic peptide corresponding to the 17 amino acid C terminus of P1 and P2 was shown to inhibit the ribosome-inactivating function of the A(1) chain of SLT-1 in an in vitro transcription and translation-coupled assay. These results suggest a role for the ribosomal stalk in aiding the A(1) chain of SLT-1 and other type II ribosome-inactivating proteins in localizing its catalytic domain near the site of depurination in the 28 S rRNA.


Assuntos
Domínio Catalítico , Fosfoproteínas/metabolismo , Proteínas Ribossômicas/metabolismo , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga I/metabolismo , Sequência de Aminoácidos , Extratos Celulares/química , Sequência Conservada , Células HeLa , Humanos , Dados de Sequência Molecular , Fosfoproteínas/análise , Fosfoproteínas/genética , Proteínas Ribossômicas/análise , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ricina/metabolismo , Espectrometria de Massas em Tandem , Técnicas do Sistema de Duplo-Híbrido
17.
Biol Pharm Bull ; 30(9): 1697-701, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17827723

RESUMO

Shiga toxin (Stx) exerts toxic activity by binding to glycosphingolipids, mainly globotriaosyl (Gb(3)) ceramide, on the surface of target cells. The inhibition of toxin-receptor binding is a promising therapeutic approach to prevent Stx-mediated diseases. In this study, we synthesized monovalent Stx-ligands of phosphatidylethanolamine dipalmitoyl-Gb(3) (Gb(3)-PEDP) and galabiosyl (Gb(2))-PEDP and we examined their neutralizing activity against Stx-1 and Stx-2 in vitro. Both Gb(3)-PEDP and Gb(2)-PEDP strongly neutralized the cytotoxicity of Stx-1 and Stx-2. It is likely that the mechanism of neutralization involved formation of liposomes and consequently clustering of sugar units. We propose monovalent Gb(3)-/Gb(2)-derivatives conjugated with phosphatidyl residue as a novel class of Stx-neutralizing agent.


Assuntos
Globosídeos/farmacologia , Fosfolipídeos/química , Toxina Shiga/antagonistas & inibidores , Triexosilceramidas/farmacologia , Sequência de Carboidratos , Escherichia coli/química , Escherichia coli/metabolismo , Globosídeos/síntese química , Células HeLa , Humanos , Lipossomos/química , Dados de Sequência Molecular , Toxina Shiga/toxicidade , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga I/toxicidade , Toxina Shiga II/antagonistas & inibidores , Toxina Shiga II/toxicidade , Triexosilceramidas/síntese química
18.
Microbiol Immunol ; 51(6): 581-92, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17579269

RESUMO

Shiga toxin (Stx) is one of the most critical factors in the development of hemolytic uremic syndrome and other systemic complications following enterohemorrhagic Escherichia coli (EHEC) infection. Substances neutralizing Stx by interfering with toxin-receptor binding have been explored as therapeutic candidates for EHEC infection. In this study, we examined globotriaosyl (Gb3), galabiosyl (Gb2) and galacto-trehalose, each of which was synthetically conjugated with a polyacrylamide backbone, for Stxneutralizing activity. Galacto-trehalose was designed as a Gb2 mimicking, unnatural Stx-ligand that was expected to show tolerance to enzymatic degradation in vivo. Galacto-trehalose copolymer showed neutralizing activity against Stx-1 but not Stx-2 in a HeLa cell cytotoxicity assay. It was thought that galactotrehalose copolymer could be a lead compound for the treatment of Stx-mediated diseases, although it requires modification to show neutralizing activity to Stx-2. The Gb3 copolymer with high sugar unit density showed stronger neutralizing activity against Stx-2 than those with lower density. However, the density-dependency of the neutralizing activity was less obvious against Stx-1. Intravenous administration of the Gb3 copolymer prevented death in mice lethally infected with Stx-1- and Stx-2-producing E. coli O157:H7. Thus, we demonstrated that the artificial Gb3 copolymer could neutralize Stx-1 and the more clinically relevant Stx-2 in vitro and effectively inhibit Stx toxicity in vivo.


Assuntos
Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli O157/metabolismo , Galactose/farmacologia , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga II/antagonistas & inibidores , Trealose/farmacologia , Animais , Sequência de Carboidratos , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Organismos Livres de Patógenos Específicos
19.
Mol Immunol ; 44(9): 2487-91, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17134756

RESUMO

Verotoxins (VTs), or shiga-like toxins, are produced by enterohemorrhagic Escherichia coli (EHEC), which cause hemorrhagic colitis and hemolytic uremic syndrome. VTs are the major virulence factors in EHEC infection due to their cytotoxicity to various types of cells. Here, we present a novel type of VT neutralizer based on pentavalent single-domain antibodies, or pentabodies. Two single-domain antibodies (sdAbs) specific for the receptor binding sites of the B subunit of VT1 (VT1B) were isolated from a naïve llama phage display library. These two sdAbs were pentamerized to generate pentameric VT neutralizers, VTI-1 and VTI-3. Both VT neutralizers bound wild type VT1B specifically with superior functional affinity. In vitro neutralization assays showed that VTI-1 and VTI-3 were able to neutralize 90% and 40%, respectively, of the cytotoxicity caused by VT1. This effort provides the basis of a novel type of VT neutralizer that can potentially be produced at a relatively low cost.


Assuntos
Anticorpos/química , Anticorpos/imunologia , Toxina Shiga I/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/isolamento & purificação , Afinidade de Anticorpos/imunologia , Sítios de Ligação , Camelídeos Americanos/imunologia , Chlorocebus aethiops , Citotoxicidade Imunológica , Cinética , Dados de Sequência Molecular , Proteínas Mutantes/imunologia , Testes de Neutralização , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores de Superfície Celular/imunologia , Toxina Shiga I/antagonistas & inibidores , Células Vero
20.
Infect Immun ; 74(12): 6992-8, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17030576

RESUMO

The 13C4 monoclonal antibody (MAb) recognizes the B subunit of Stx1 (StxB1) and neutralizes the cytotoxic and lethal activities of Stx1. However, this MAb does not bind to the B polypeptide of Stx2, despite the 73% amino acid sequence similarity between StxB1 and StxB2. When we compared the amino acid sequences of StxB1 and StxB2, we noted three regions of dissimilarity (amino acids 1 to 6, 25 to 32, and 54 to 61) located near each other on the crystal structure of StxB1. To identify the 13C4 epitope, we generated seven Stx1/Stx2 B chimeric polypeptides that contained one, two, or three of the dissimilar StxB1 regions. The 13C4 MAb reacted strongly with StxB1 and the triple-chimeric B subunit but not with the other chimeras. Mice immunized with the triple-chimeric B subunit survived a lethal challenge with Stx1 but not Stx2, substantiating the identified regions as the 13C4 MAb epitope and suggesting that the incorporation of this epitope into StxB2 altered sites necessary for anti-Stx2-neutralizing Ab production. Next, single amino acid substitutions were made in StxB1 to mimic Stx1d, a variant not recognized by the 13C4 MAb. The 13C4 MAb reacted strongly to StxB1 with the T1A or G25A mutations but not with the N55T change. Finally, we found that the 13C4 MAb blocked the binding of Stx1 to its receptor, globotriaosyl ceramide. Taken together, these results indicate that the 13C4 MAb prevents the interaction of Stx1 with its receptor by binding three nonlinear regions of the molecule that span receptor recognition sites on StxB1, one of which includes the essential residue 55N.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/química , Toxina Shiga I/antagonistas & inibidores , Toxina Shiga I/imunologia , Triexosilceramidas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Cristalografia por Raios X , Mapeamento de Epitopos , Epitopos/imunologia , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Toxina Shiga I/química , Toxina Shiga II/antagonistas & inibidores , Toxina Shiga II/química , Toxina Shiga II/imunologia
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