RESUMO
Shiga toxin-producing E. coli (STEC) produces Stx1 and/or Stx2, and Subtilase cytotoxin (SubAB). Since these toxins may be present simultaneously during STEC infections, the purpose of this work was to study the co-action of Stx2 and SubAB. Stx2 + SubAB was assayed in vitro on monocultures and cocultures of human glomerular endothelial cells (HGEC) with a human proximal tubular epithelial cell line (HK-2) and in vivo in mice after weaning. The effects in vitro of both toxins, co-incubated and individually, were similar, showing that Stx2 and SubAB contribute similarly to renal cell damage. However, in vivo, co-injection of toxins lethal doses reduced the survival time of mice by 24 h and mice also suffered a strong decrease in the body weight associated with a lowered food intake. Co-injected mice also exhibited more severe histological renal alterations and a worsening in renal function that was not as evident in mice treated with each toxin separately. Furthermore, co-treatment induced numerous erythrocyte morphological alterations and an increase of free hemoglobin. This work shows, for the first time, the in vivo effects of Stx2 and SubAB acting together and provides valuable information about their contribution to the damage caused in STEC infections.
Assuntos
Proteínas de Escherichia coli/toxicidade , Síndrome Hemolítico-Urêmica/etiologia , Toxina Shiga II/toxicidade , Subtilisinas/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Síndrome Hemolítico-Urêmica/patologia , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Glomérulos Renais/citologia , Túbulos Renais Proximais/citologia , Masculino , Camundongos Endogâmicos BALB CRESUMO
Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.
Assuntos
Toxinas Bacterianas/toxicidade , Escherichia coli Enterotoxigênica , Enterotoxinas/toxicidade , Proteínas de Escherichia coli/toxicidade , Animais , Toxinas Bacterianas/farmacocinética , Células CACO-2 , Edema/induzido quimicamente , Embrião não Mamífero/anormalidades , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Enterotoxinas/farmacocinética , Proteínas de Escherichia coli/farmacocinética , Cardiopatias Congênitas/induzido quimicamente , Frequência Cardíaca/efeitos dos fármacos , Humanos , Intestinos/metabolismo , Miocárdio/metabolismo , Saco Vitelino/efeitos dos fármacos , Peixe-Zebra/anormalidades , Peixe-Zebra/metabolismoRESUMO
α-Hemolysin (HlyA) is an extracellular protein toxin secreted by uropathogenic strains of Escherichia coli that inserts into membranes of eukaryotic cells. The main goal of this work was to investigate the involvement of tryptophan (W) residues in the hemolytic activity of HlyA. We investigated the hemolytic activity of six single-point mutant proteins, in which one of the four Ws was replaced by cysteine (C) or leucine (L). We also analyzed the photoinactivation of HlyA with pterin (Ptr), an endogenous photosensitizer, as a method of unspecific oxidation of W and tyrosine (Y) residues. HlyA photoinactivation was analyzed by ultraviolet-visible spectrophotometry, hemolytic activity measurement, fluorescence spectroscopy, and electrophoretic analysis. The results indicate that Ws are important in the hemolytic process. Specifically, the chemical structure of the amino acid at position 578 is important for the acylation of HlyA at residue K563. Furthermore, the exposure of HlyA to ultraviolet radiation, with energy similar to that experienced under sun exposure, in the presence of Ptr induces the inactivation of the toxin, causing chemical changes in, at least, W and Y, the rate of damage to W residues being faster than that observed for Y residues. This work not only deepens our understanding of the structure-function relationship of the toxin but also introduces the possibility of using photoinactivation of HlyA for potential applications such as obtaining innocuous molecules for vaccine production and the elimination of the toxin from contaminated surfaces and drinking water.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/toxicidade , Proteínas Hemolisinas/química , Proteínas Hemolisinas/toxicidade , Luz , Triptofano , Substituição de Aminoácidos , Proteínas de Escherichia coli/genética , Proteínas Hemolisinas/genética , Hemólise/efeitos dos fármacos , Hemólise/efeitos da radiaçãoRESUMO
Secreted autotransporter toxin (Sat) is a 107-kDa serine protease autotransporter of Enterobacteriaceae (SPATE) presenting cytotoxic activity in renal and bladder cells. Further studies have detected the Sat-encoding gene (sat) in enteroaggregative Escherichia coli (EAEC) and in E. coli strains isolated from neonatal septicemia and meningitis. Here, we investigated the role of Sat as a cytotoxin of EAEC. Sat was purified from a strain of E. coli harboring sat (DEC/Sat+, O126:H2) and used to raise antibodies in rabbit. The presence of Sat was detected by ELISA in the supernatant of 93.7% of EAEC strains harboring sat and in none lacking the gene. The effect of Sat during infection was investigated in polarized Caco-2 cells infected with Sat-producing EAEC (CV323/77, O125ab:H21). This strain induced intense cell detachment, which was inhibited by PMSF or Sat antiserum. Also, sat transcription and Sat production were detected during infection. Here we demonstrate that Sat is internalized in polarized cells leading to F-actin disruption which preceded cell detachment. A comparative study of the toxin action in cell lines corresponding to the infection sites in which bacteria carrying the sat gene have been isolated was performed. Cells originating from the gastrointestinal tract (Caco-2), urinary (LLC-PK1) and endothelium (HUVEC) were incubated with purified Sat. The time required for observation of cell damage differed according to the cell line. HUVEC cells were more sensitive to Sat than cells derived from urinary and intestinal tracts. The intense activity of Sat on the endothelial cells suggests that Sat could also be a virulence factor for the bacteria in the bloodstream. In addition, this is the first work demonstrating that Sat induces cytotoxic effect during EAEC infection in vitro. The cell damage observed during infection indicates that Sat may be another toxin with cytotoxic role in the EAEC pathogenesis.
Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Toxinas Bacterianas/toxicidade , Células CACO-2 , Citotoxinas/metabolismo , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/toxicidade , Humanos , Serina Endopeptidases/metabolismo , Sistemas de Secreção Tipo V/metabolismo , Fatores de Virulência/metabolismoRESUMO
Hemolytic uremic syndrome (HUS) is a consequence of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection and is the most frequent cause of acute renal failure (ARF) in children. Subtilase cytotoxin (SubAB) has also been associated with HUS pathogenesis. We previously reported that Stx2 and SubAB cause different effects on co-cultures of human renal microvascular endothelial cells (HGEC) and human proximal tubular epithelial cells (HK-2) relative to HGEC and HK-2 monocultures. In this work we have analyzed the secretion of pro-inflammatory cytokines by co-cultures compared to monocultures exposed or not to Stx2, SubAB, and Stx2+SubAB. Under basal conditions, IL-6, IL-8 and TNF-α secretion was different between monocultures and co-cultures. After toxin treatments, high concentrations of Stx2 and SubAB decreased cytokine secretion by HGEC monocultures, but in contrast, low toxin concentrations increased their release. Toxins did not modulate the cytokine secretion by HK-2 monocultures, but increased their release in the HK-2 co-culture compartment. In addition, HK-2 monocultures were stimulated to release IL-8 after incubation with HGEC conditioned media. Finally, Stx2 and SubAB were detected in HGEC and HK-2 cells from the co-cultures. This work describes, for the first time, the inflammatory responses induced by Stx2 and SubAB, in a crosstalk model of renal endothelial and epithelial cells.
Assuntos
Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Proteínas de Escherichia coli/toxicidade , Túbulos Renais Proximais/efeitos dos fármacos , Microvasos/efeitos dos fármacos , Toxina Shiga II/toxicidade , Subtilisinas/toxicidade , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Sinergismo Farmacológico , Células Endoteliais/imunologia , Células Epiteliais/imunologia , Síndrome Hemolítico-Urêmica , Humanos , Rim/irrigação sanguíneaRESUMO
Alpha hemolysin (HlyA) is the major virulence factor of uropathogenic Escherichia coli (UPEC) strains. Once in circulation, a low concentration of the toxin induces an increase in intracellular calcium that activates calpains - which proteolyse cytoskeleton proteins - and also favours the exposure of phosphatidylserine (PS) in the outer leaflet of erythrocyte membranes. All these events are considered part of eryptosis, as well as the delivery of microvesicles (MVs). Within this context, we studied the delivery of MVs by erythrocytes treated with sublytic concentrations of HlyA and demonstrated that HlyA-treated erythrocytes secrete MVs of diameter â¼200â nm containing HlyA and PS by a mechanism involving an increment of intracellular calcium concentration and purinergic receptor activation. Despite the presence of toxin in their membrane, HlyA-MVs are not hemolytically active and do not induce ATP release in untreated erythrocytes, thus suggesting that the delivery of HlyA-MVs might act as a protective mechanism on the part of erythrocytes that removes the toxin from the membrane to prevent the spread of infection. Although erythrocytes have been found to eliminate denatured hemoglobin and several membrane proteins by shedding MVs, the present work has revealed for the first time that an exogenous protein, such as a toxin, is eliminated by this process. This finding sheds light on the mechanism of action of the toxin and serves to further elucidate the consequences of UPEC infection in patients exhibiting HlyA-related diseases.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Eritrócitos/efeitos dos fármacos , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/toxicidade , Proteínas Hemolisinas/toxicidade , Micropartículas Derivadas de Células/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/metabolismo , Escherichia coli/metabolismo , Infecções por Escherichia coli/fisiopatologia , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Fosfatidilserinas/metabolismoRESUMO
Background: Escherichia coli has been widely used as a host to clone and express heterologous genes. However, there are few vectors available for cloning and expressing extremely toxic genes, which limits further basic and applied research on extremely toxic proteins. Results: In this study, a novel vector pAU10 was constructed in E. coli. pAU10 utilizes the combination of the efficient but highly repressible T7-lacO promoter/operator and the strong rrnBT2 transcriptional terminator upstream of the T7 promoter to strictly control unwanted transcription of the extremely toxic gene; in addition, the trp promoter/operator is oriented opposite to the T7 promoter to control the production of the antisense RNA that may block the translation of leaky mRNA. Without the supplementation of IPTG and L-tryptophan in the culture medium, transcription of the extremely toxic gene by the T7 promoter is highly repressed, and the trp promoter produces the antisense RNA, which strictly prevents unwanted expression of the extremely toxic protein in E. coli. With the supplementation of IPTG and L-tryptophan, the T7 promoter efficiently transcribes the extremely toxic gene, and the trp promoter does not produce the antisense RNA, ensuring efficient expression of the extremely toxic protein in E. coli. Tight regulation and efficiency of expression of an extremely toxic gene cloned in the vector pAU10 were confirmed by cloning and expressing the restriction endonuclease-encoding gene bamHI without its corresponding methylase gene in E. coli JM109(DE3). Conclusion: pAU10 is a good vector used for cloning and expressing extremely toxic genes in E. coli.
Assuntos
Proteínas de Escherichia coli/toxicidade , Escherichia coli/genética , Vetores Genéticos , Triptofano/metabolismo , Desoxirribonuclease BamHI/metabolismo , Western Blotting , Reação em Cadeia da Polimerase , RNA Antissenso , Regiões Promotoras Genéticas , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Proteínas Correpressoras , Genes Bacterianos , Isopropiltiogalactosídeo/metabolismoRESUMO
Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in children. The majority of cases are associated with Shiga toxin (Stx)-producing Escherichia coli (STEC). In Argentina, HUS is endemic and presents the highest incidence rate in the world. STEC strains expressing Stx type 2 (Stx2) are responsible for the most severe cases of this pathology. Subtilase cytotoxin (SubAB) is another STEC virulence factor that may contribute to HUS pathogenesis. To date, neither a licensed vaccine nor effective therapy for HUS is available for humans. Considering that Ouabain (OUA) may prevent the apoptosis process, in this study we evaluated if OUA is able to avoid the damage caused by Stx2 and SubAB on human glomerular endothelial cells (HGEC) and the human proximal tubule epithelial cell (HK-2) line. HGEC and HK-2 were pretreated with OUA and then incubated with the toxins. OUA protected the HGEC viability from Stx2 and SubAB cytotoxic effects, and also prevented the HK-2 viability from Stx2 effects. The protective action of OUA on HGEC and HK-2 was associated with a decrease in apoptosis and an increase in cell proliferation. Our data provide evidence that OUA could be considered as a therapeutic strategy to avoid the renal damage that precedes HUS.
Assuntos
Proteínas de Escherichia coli/toxicidade , Ouabaína/farmacologia , Substâncias Protetoras/farmacologia , Toxina Shiga II/toxicidade , Subtilisinas/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , Rim/citologia , Necrose/induzido quimicamente , Necrose/prevenção & controleRESUMO
Subtilase cytotoxin (SubAB) is a member of the AB5 cytotoxin family and is produced by certain strains of Shiga toxigenic Escherichia coli. The toxin is known to be lethal to mice, but the pathological mechanisms that contribute to Uremic Hemolytic Syndrome (HUS) are poorly understood. In this study we show that intraperitoneal injection of a sublethal dose of SubAB in rats triggers a systemic response, with ascitic fluid accumulation, heart hypertrophy and damage to the liver, colon and kidney. SubAB treated rats presented microalbuminuria 20 days post inoculation. At this time we found disruption of the glomerular filtration barrier and alteration of the protein reabsorption mechanisms of the proximal tubule. In the kidney, SubAB also triggered an epithelial to mesenchymal transition (Wuyts et al., 1996). These findings indicate that apart from direct cytotoxic effects on renal tissues, SubAB causes significant damage to the other organs, with potential consequences for HUS pathogenesis. IMPORTANCE: Uremic Hemolytic Syndrome is an endemic disease in Argentina, with over 400 hundred new cases each year. We have previously described renal effects of Shiga Toxin and its ability to alter renal protein handling. Bearing in mind that Subtilase Cytotoxin is an emerging pathogenic factor, that it is not routinely searched for in patients with HUS, and that to the date its systemic effects have not been fully clarified we decided to study both its systemic effects, and its renal effects to assess whether SubAB could be contributing to pathology seen in children.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli Shiga Toxigênica/metabolismo , Subtilisinas/metabolismo , Albuminúria/induzido quimicamente , Animais , Ascite/induzido quimicamente , Cardiomegalia/induzido quimicamente , Colo/efeitos dos fármacos , Colo/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas de Escherichia coli/toxicidade , Síndrome Hemolítico-Urêmica/etiologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Ratos Sprague-Dawley , Subtilisinas/toxicidadeRESUMO
Postdiarrheal hemolytic uremic syndrome (HUS) affects children under 5 years old and is responsible for the development of acute and chronic renal failure, particularly in Argentina. This pathology is a complication of Shiga toxin (Stx)-producing Escherichia coli infection and renal damage is attributed to Stx types 1 and 2 (Stx1, Stx2) produced by Escherichia coli O157:H7 and many other STEC serotypes. It has been reported the production of Subtilase cytotoxin (SubAB) by non-O157 STEC isolated from cases of childhood diarrhea. Therefore, it is proposed that SubAB may contribute to HUS pathogenesis. The human kidney is the most affected organ because very Stx-sensitive cells express high amounts of biologically active receptor. In this study, we investigated the effects of Stx2 and SubAB on primary cultures of human glomerular endothelial cells (HGEC) and on a human tubular epithelial cell line (HK-2) in monoculture and coculture conditions. We have established the coculture as a human renal proximal tubule model to study water absorption and cytotoxicity in the presence of Stx2 and SubAB. We obtained and characterized cocultures of HGEC and HK-2. Under basal conditions, HGEC monolayers exhibited the lowest electrical resistance (TEER) and the highest water permeability, while the HGEC/HK-2 bilayers showed the highest TEER and the lowest water permeability. In addition, at times as short as 20-30 minutes, Stx2 and SubAB caused the inhibition of water absorption across HK-2 and HGEC monolayers and this effect was not related to a decrease in cell viability. However, toxins did not have inhibitory effects on water movement across HGEC/HK-2 bilayers. After 72 h, Stx2 inhibited the cell viability of HGEC and HK-2 monolayers, but these effects were attenuated in HGEC/HK-2 bilayers. On the other hand, SubAB cytotoxicity shows a tendency to be attenuated by the bilayers. Our data provide evidence about the different effects of these toxins on the bilayers respect to the monolayers. This in vitro model of communication between human renal microvascular endothelial cells and human proximal tubular epithelial cells is a representative model of the human proximal tubule to study the effects of Stx2 and SubAB related to the development of HUS.
Assuntos
Células Endoteliais/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Proteínas de Escherichia coli/toxicidade , Toxina Shiga II/toxicidade , Subtilisinas/toxicidade , Transporte Biológico/efeitos dos fármacos , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Humanos , Glomérulos Renais/citologia , Glomérulos Renais/efeitos dos fármacos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacosRESUMO
The hemolytic uremic syndrome (HUS) associated with diarrhea is a complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection. In Argentina, HUS is endemic and responsible for acute and chronic renal failure in children younger than 5 years old. The human kidney is the most affected organ due to the presence of very Stx-sensitive cells, such as microvascular endothelial cells. Recently, Subtilase cytotoxin (SubAB) was proposed as a new toxin that may contribute to HUS pathogenesis, although its action on human glomerular endothelial cells (HGEC) has not been described yet. In this study, we compared the effects of SubAB with those caused by Stx2 on primary cultures of HGEC isolated from fragments of human pediatric renal cortex. HGEC were characterized as endothelial since they expressed von Willebrand factor (VWF) and platelet/endothelial cell adhesion molecule 1 (PECAM-1). HGEC also expressed the globotriaosylceramide (Gb3) receptor for Stx2. Both, Stx2 and SubAB induced swelling and detachment of HGEC and the consequent decrease in cell viability in a time-dependent manner. Preincubation of HGEC with C-9 -a competitive inhibitor of Gb3 synthesis-protected HGEC from Stx2 but not from SubAB cytotoxic effects. Stx2 increased apoptosis in a time-dependent manner while SubAB increased apoptosis at 4 and 6 h but decreased at 24 h. The apoptosis induced by SubAB relative to Stx2 was higher at 4 and 6 h, but lower at 24 h. Furthermore, necrosis caused by Stx2 was significantly higher than that induced by SubAB at all the time points evaluated. Our data provide evidence for the first time how SubAB could cooperate with the development of endothelial damage characteristic of HUS pathogenesis.
Assuntos
Células Endoteliais/efeitos dos fármacos , Proteínas de Escherichia coli/farmacologia , Glomérulos Renais/efeitos dos fármacos , Toxina Shiga II/farmacologia , Subtilisinas/farmacologia , Antígenos Glicosídicos Associados a Tumores/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Proteínas de Escherichia coli/toxicidade , Humanos , Glomérulos Renais/metabolismo , Necrose/tratamento farmacológico , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Toxina Shiga II/toxicidade , Subtilisinas/toxicidade , Fator de von Willebrand/metabolismoRESUMO
The type I and type II heat-labile enterotoxins (LT-I and LT-II) are strong mucosal adjuvants when they are coadministered with soluble antigens. Nonetheless, data on the parenteral adjuvant activities of LT-II are still limited. Particularly, no previous study has evaluated the adjuvant effects and induced inflammatory reactions of LT-II holotoxins or their B pentameric subunits after delivery via the intradermal (i.d.) route to mice. In the present report, the adjuvant and local skin inflammatory effects of LT-IIa and its B subunit pentamer (LT-IIaB(5)) were determined. When coadministered with ovalbumin (OVA), LT-IIa and, to a lesser extent, LT-IIaB(5) exhibited serum IgG adjuvant effects. In addition, LT-IIa but not LT-IIaB(5) induced T cell-specific anti-OVA responses, particularly in respect to induction of antigen-specific cytotoxic CD8(+) T cell responses. LT-IIa and LT-IIaB(5) induced differential tissue permeability and local inflammatory reactions after i.d. injection. Of particular interest was the reduced or complete lack of local reactions, such as edema and tissue induration, in mice i.d. inoculated with LT-IIa and LT-IIaB(5,) respectively, compared with mice immunized with LT-I. In conclusion, the present results show that LT-IIa and, to a lesser extent, LT-IIaB(5) exert adjuvant effects when they are delivered via the i.d. route. In addition, the low inflammatory effects of LT-IIa and LT-IIaB(5) in comparison to those of LT-I support the usefulness of LT-IIa and LT-IIaB(5) as parenterally delivered vaccine adjuvants.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/imunologia , Enterotoxinas/administração & dosagem , Enterotoxinas/imunologia , Proteínas de Escherichia coli/administração & dosagem , Proteínas de Escherichia coli/imunologia , Pele/efeitos dos fármacos , Pele/imunologia , Adjuvantes Imunológicos/toxicidade , Animais , Toxinas Bacterianas/toxicidade , Linfócitos T CD8-Positivos/imunologia , Enterotoxinas/toxicidade , Proteínas de Escherichia coli/toxicidade , Feminino , Imunoglobulina G/sangue , Inflamação/patologia , Injeções Intradérmicas , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid-encoded toxin (Pet), which exerts cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known whether this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes the redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with the depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, before the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephosphorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication.
Assuntos
Toxinas Bacterianas/toxicidade , Enterotoxinas/toxicidade , Proteínas de Escherichia coli/toxicidade , Escherichia coli/fisiologia , Adesões Focais/efeitos dos fármacos , Serina Endopeptidases/toxicidade , Actinina/metabolismo , Actinas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular , Enterotoxinas/genética , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Citometria de Fluxo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Paxilina/metabolismo , Plasmídeos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Espectrina/metabolismo , Vinculina/metabolismoRESUMO
Shiga toxin (Stx) and hemolysin (Hly) of Escherichia coli O157:H7 produced an increase of reactive oxygen species (ROS) in normal human blood. In vitro assays showed that stimuli of ROS with these toxins oxidized proteins to carbonyls in plasma and raised the degradation of oxidized macromolecules, with the AOPP/carbonyl relationship also increasing. The oxidative stress generated by toxins during the Hemolytic Uremic Syndrome (HUS) produced oxidation of blood proteins with a rise in advanced oxidation protein products (AOPP) in children with HUS. There was a response from the antioxidant system in these patients, evaluated through the determination of the total antioxidant capacity of plasma by the Ferric Reducing Antioxidant Power (FRAP), which reduced the stimuli of ROS during in vitro incubation with Stx or Hly. The application of natural antioxidants was sufficient to reduce in vitro the oxidative stress provoked by both toxins in blood.
Assuntos
Antioxidantes/metabolismo , Proteínas Sanguíneas/metabolismo , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/toxicidade , Proteínas Hemolisinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Toxina Shiga/toxicidade , Antioxidantes/farmacologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Criança , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/isolamento & purificação , Frutas/química , Proteínas Hemolisinas/isolamento & purificação , Síndrome Hemolítico-Urêmica/sangue , Síndrome Hemolítico-Urêmica/metabolismo , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Oxirredução , Extratos Vegetais/farmacologia , Prosopis/química , Carbonilação Proteica/efeitos dos fármacos , Espécies Reativas de Oxigênio/sangue , Toxina Shiga/isolamento & purificação , Ziziphus/químicaRESUMO
Heat-labile toxins (LT) encompass at least 16 natural polymorphic toxin variants expressed by wild-type enterotoxigenic Escherichia coli (ETEC) strains isolated from human beings, but only one specific form, produced by the reference ETEC H10407 strain (LT1), has been intensively studied either as a virulence-associated factor or as a mucosal/transcutaneous adjuvant. In the present study, we carried out a biological/immunological characterization of a natural LT variant (LT2) with four polymorphic sites at the A subunit (S190L, G196D, K213E, and S224T) and one at the B subunit (T75A). The results indicated that purified LT2, in comparison with LT1, displayed similar in vitro toxic activities (adenosine 3',5'-cyclic monophosphate accumulation) on mammalian cells and in vivo immunogenicity following delivery via the oral route. Nonetheless, the LT2 variant showed increased adjuvant action to ovalbumin when delivered to mice via the transcutaneous route while antibodies raised in mice immunized with LT2 displayed enhanced affinity and neutralization activity to LT1 and LT2. Taken together, the results indicate that the two most frequent LT polymorphic forms expressed by wild ETEC strains share similar biological features, but differ with regard to their immunological properties.
Assuntos
Toxinas Bacterianas/imunologia , Toxinas Bacterianas/toxicidade , Escherichia coli Enterotoxigênica/fisiologia , Enterotoxinas/imunologia , Enterotoxinas/toxicidade , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/toxicidade , Polimorfismo Genético , Administração Cutânea , Administração Oral , Substituição de Aminoácidos/genética , Animais , Anticorpos Antibacterianos/imunologia , Antitoxinas/imunologia , Toxinas Bacterianas/genética , Linhagem Celular , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Camundongos , Mutação de Sentido Incorreto , Testes de NeutralizaçãoRESUMO
It has been shown that the autotransporter plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli (EAEC) produces cytotoxic and enterotoxic effects. Both effects can be explained by the proteolytic activity of Pet on its intracellular target alpha-fodrin (alphaII spectrin). In addition, Pet cytotoxicity and enterotoxicity depend on Pet serine protease activity, and on its internalization into epithelial cells. However, the mechanisms of Pet uptake by epithelial cells are unknown. Here, we show that Pet interacts with the plasma membrane of epithelial cells, and afterwards is detected inside the cells. Furthermore, Pet was internalized via clathrin-mediated endocytosis, since its internalization was inhibited by monodansylcadaverine and sucrose, but not by filipin or methyl-beta-cyclodextrin, which are drugs that interfere with protein entry via a clathrin-independent pathway. Additionally, Pet was immunoprecipitated by anti-clathrin antibodies, but not by anti-caveolin antibodies. Moreover, small interfering RNA (siRNA), designed to knock out clathrin gene expression in HEp-2 cells, prevented Pet internalization, and thereby the Pet-induced cytotoxic effect. However, the use of siRNA to knock out caveolin expression had no effect on Pet internalization, and the cytotoxic effect was clearly observed. Together, these data indicate that Pet secreted by EAEC binds to the cell surface via an unknown receptor, to be taken up by clathrin-mediated endocytosis, and exert its toxic effect in the cytoplasm.
Assuntos
Toxinas Bacterianas/toxicidade , Clatrina/farmacologia , Endocitose/efeitos dos fármacos , Enterotoxinas/toxicidade , Células Epiteliais/metabolismo , Proteínas de Escherichia coli/toxicidade , Escherichia coli/patogenicidade , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Endocitose/fisiologia , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Serina EndopeptidasesRESUMO
Bacterial diarrheal diseases remain a major cause of morbidity and mortality in developing countries. Diffusely adhering Escherichia coli (DAEC) is a newly proposed category of diarrheagenic E. coli based on epidemiological studies. Sat, a new virulence factor of some uropathogeic Escherichia coli, was described with a vacuolating cytotoxic action in bladder and kidney tissues. In the present study, we analyzed the Sat effects, produced by a DAEC strain in rabbit ileal intestinal tissue and cultured epithelial cells. We observed enterotoxic activity in rabbit ileum tissues by Ussing chamber assays, a pronounced fluid accumulation in rabbit ileum loops with villous necrosis observed in the histopathologic examination, and morphological changes in monolayer cultures of Y1 adrenal cells. Our results suggest that DAEC strains may be involved in diarrhea.
Assuntos
Toxinas Bacterianas/toxicidade , Diarreia/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/toxicidade , Escherichia coli/patogenicidade , Íleo/patologia , Animais , Aderência Bacteriana , Linhagem Celular , Diarreia/patologia , Modelos Animais de Doenças , Escherichia coli/metabolismo , Infecções por Escherichia coli/patologia , Camundongos , Coelhos , Fatores de Virulência/toxicidadeRESUMO
EspC (Escherichia coli secreted protein C) of enteropathogenic E. coli (EPEC) shows the three classical domains of the autotransporter proteins and has a conserved serine protease motif belonging to the SPATE (serine protease autotransporters of Enterobacteriaceae) subfamily. EspC and its homolog Pet in enteroaggregative E. coli (EAEC) bear the same sequence within the serine protease motif, and both proteins produce enterotoxic effects, suggesting that like Pet, EspC could be internalized to reach and cleave the calmodulin-binding domain of fodrin, causing actin cytoskeleton disruption. Even though both proteins cause cytoskeleton damage by virtue of their serine protease motifs, the following evidence supports the hypothesis that the mechanisms are different. (i) To obtain similar cytotoxic and cytoskeletal effects, a threefold-higher EspC concentration and a twofold-higher exposure time are needed. (ii) EspC internalization into epithelial cells takes more time (6 h) than Pet internalization (30 min), and the distributions of the two proteins inside the cells are also different. (iii) Both proteins have affinity for fodrin and cleave it, but the cleavage sites are different; EspC produces two cleavages, while Pet produces just one. (iv) EspC does not cause fodrin redistribution within epithelial cells. (v) An EspC serine protease motif mutant, but not a Pet serine protease mutant, competes with EspC by blocking cytoskeletal damage. All these data suggest that the protein conformational structure is very important for the activity of the catalytic site, influencing its interaction with the target protein and its internalization. The differences between these proteins may explain the reduced ability of EspC to cause cytopathic effects. However, these differences may confer a specialized role on EspC in the pathogenesis of EPEC, which is different from that of Pet in EAEC pathogenesis.
Assuntos
Toxinas Bacterianas/toxicidade , Enterotoxinas/toxicidade , Células Epiteliais/patologia , Proteínas de Escherichia coli/toxicidade , Escherichia coli/patogenicidade , Serina Endopeptidases/toxicidade , Motivos de Aminoácidos , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Enterotoxinas/química , Enterotoxinas/genética , Células Epiteliais/microbiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Humanos , Proteínas dos Microfilamentos/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Serina Endopeptidases/genéticaRESUMO
Twenty-four strains of cell-detaching Escherichia coli (CDEC) isolated from stool specimens in different cities in Brazil were examined for virulence properties. Aerobactin production and multiple antibiotic resistance were observed in most of the isolates. In hybridization studies, the alphahly, pap, and cnf sequences, common properties of this category of E. coli, were found in a minority of isolates. Half of the CDEC isolates had enteroaggregative DNA sequences (pet, astA, aggA), six strains carried the shet1 gene, nine strains carried the daaC sequence, and one strain carried the stp gene. Thirteen strains induced fluid accumulation in the rabbit intestinal loop assay. Supernatant filtrate of one of those strains, which did not hybridize with any of the toxin probes tested, induced destructive lesions in the rabbit ileal loop and enterotoxic activity in the Ussing chamber. A 12-kDa protein purified by 60% ammonium sulfate precipitation of the supernatant filtrate demonstrated a toxigenic effect that was inhibited by the anti-12-kDa protein antiserum.