Detection of plasma anti-lipopolysaccharide (LPS) antibodies against enterohemorrhagic Escherichia coli (EHEC) in asymptomatic kindergarten teachers from Buenos Aires province.

In Argentina, hemolytic uremic syndrome (HUS) caused by EHEC has the highest incidence in the world. EHEC infection has an endemo-epidemic behavior, causing 20-30% of acute bloody diarrhea syndrome in children under 5 years old. In the period 2016-2020, 272 new cases per year were notified to the National Health Surveillance System. Multiple factors are responsible for HUS incidence in Argentina including person-to-person transmission. In order to detect possible EHEC carriers, we carried out a preliminary study of the frequency of kindergarten teachers with anti-LPS antibodies against the most prevalent EHEC serotypes in Argentina. We analyzed 61 kindergarten teachers from 26 institutions from José C. Paz district, located in the suburban area of Buenos Aires province, Argentina. Fifty-one percent of the plasma samples had antibodies against O157, O145, O121 and O103 LPS: 6.4% of the positive samples had IgM isotype (n=2), 61.3% IgG isotype (n=19) and 32.3% IgM and IgG (n=10). Given that antibodies against LPS antigens are usually short-lived specific IgM detection may indicate a recent infection. In addition, the high percentage of positive samples may indicate a frequent exposure to EHEC strains in the cohort studied, as well as the existence of a large non-symptomatic population of adults carrying pathogenic strains that could contribute to the endemic behavior through person-to-person transmission. The improvement of continuous educational programs in kindergarten institutions could be a mandatory measure to reduce HUS cases not only in Argentina but also globally.

Etiological diagnosis of post-diarrheal hemolytic uremic syndrome (HUS): humoral response contribution.

BACKGROUND: Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolysis, thrombocytopenia, and thrombus formation leading to tissue injury. HUS is classified according to its etiology as post-diarrheal or atypical HUS. Differential diagnosis of both entities continues to be a challenge for pediatric physicians. METHODS: The aim was to improve the rapid etiological diagnosis of post-diarrheal HUS cases based on the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection by screening of stx1/stx2 and rfbO157 in cultured stools by multiplex PCR, and the additional detection of anti-lipopolysaccharide (anti-LPS) O157, O145, and O121 antibodies by Glyco-iELISA test. In addition, we studied patients' relatives to detect circulating pathogenic strains that could contribute to HUS diagnosis and/or lead to the implementation of measures to prevent dissemination of familial outbreaks. This study describes the diagnosis of 31 HUS patients admitted to Hospital Municipal de Niños Prof Dr Ramón Exeni during the 2017-2020 period. RESULTS: Stool PCR confirmed the diagnosis of STEC associated with HUS in 38.7% of patients (12/31), while anti-LPS serology did in 88.9% (24/27). In those patients in which both methods were carried out (n = 27), a strong association between the results obtained was found. We found that 30.4% of HUS patients had at least one relative positive for STEC. CONCLUSIONS: We could identify 96.3% (26/27) of HUS cases as secondary to STEC infections when both methods (genotyping and serology) were used. The results demonstrated a high circulation of STEC in HUS families and the prevalence of the STEC O157 serotype (83%) in our pediatric cohort. A higher-resolution version of the Graphical abstract is available as Supplementary information.

Mechanisms involved in the adaptation of Escherichia coli O157:H7 to the host intestinal microenvironment.

Host adaptation of pathogens may increase intra- and interspecies transmission. We showed previously that the passage of a clinically isolated enterohemorrhagic Escherichia coli (EHEC) O157 strain (125/99) through the gastrointestinal tract of mice increases its pathogenicity in the same host. In this work, we aimed to elucidate the underlying mechanism(s) involved in the patho-adaptation of the stool-recovered (125RR) strain. We assessed the global transcription profile by microarray and found almost 100 differentially expressed genes in 125RR strain compared with 125/99 strain. We detected an overexpression of Type Three Secretion System (TTSS) proteins at the mRNA and protein levels and demonstrated increased adhesion to epithelial cell lines for the 125RR strain. Additional key attributes of the 125RR strain were: increased motility on semisolid agar, which correlated with an increased fliC mRNA level; reduced Stx2 production at the mRNA and protein levels; increased survival at pH 2.5, as determined by acid resistance assays. We tested whether the overexpression of the LEE-encoded regulator (ler) in trans in the 125/99 strain could recreate the increased pathogenicity observed in the 125RR strain. As anticipated ler overexpression led to increased expression of TTSS proteins and bacterial adhesion to epithelial cells in vitro but also increased mortality and intestinal colonization in vivo. We conclude that this host-adaptation process required changes in several mechanisms that improved EHEC O157 fitness in the new host. The research highlights some of the bacterial mechanisms required for horizontal transmission of these zoonotic pathogens between their animal and human populations.

Efficacy of a recombinant Intimin, EspB and Shiga toxin 2B vaccine in calves experimentally challenged with Escherichia coli O157:H7.

Escherichia coli O157:H7 is a zoonotic pathogen of global importance and the serotype of Shiga toxin-producing E.coli (STEC) most frequently associated with Hemolytic Uremic Syndrome (HUS) in humans. The main STEC reservoir is cattle. Vaccination of calves with the carboxy-terminal fraction of Intimin γ (IntC280) and EspB can reduce E.coli O157:H7 fecal shedding after experimental challenge. Shiga toxin (Stx) exerts local immunosuppressive effects in the bovine intestine and Stx2B fused to Brucella lumazine synthase (BLS-Stx2B) induces Stx2-neutralizing antibodies. To determine if an immune response against Stx could improve a vaccine's effect on fecal shedding, groups of calves were immunized with EspB + IntC280, with EspB + IntC280 + BLS-Stx2B, or kept as controls. At 24 days post vaccination calves were challenged with E.coli O157:H7. Shedding of E.coli O157:H7 was assessed in recto-anal mucosal swabs by direct plating and enrichment followed by immunomagnetic separation and multiplex PCR. Calves were euthanized 15 days after the challenge and intestinal segments were obtained to assess mucosal antibodies. Vaccination induced a significant increase of IntC280 and EspB specific antibodies in serum and intestinal mucosa in both vaccinated groups. Antibodies against Stx2B were detected in serum and intestinal mucosa of animals vaccinated with 3 antigens. Sera and intestinal homogenates were able to neutralize Stx2 verocytotoxicity compared to the control and the 2-antigens vaccinated group. Both vaccines reduced E.coli O157:H7 shedding compared to the control group. The addition of Stx2B to the vaccine formulation did not result in a superior level of protection compared to the one conferred by IntC280 and EspB alone. It remains to be determined if the inclusion of Stx2B in the vaccine alters E.coli O157:H7 shedding patterns in the long term and after recurrent low dose exposure as occurring in cattle herds.

Immunization of pregnant cows with Shiga toxin-2 induces high levels of specific colostral antibodies and lactoferrin able to neutralize E. coli O157:H7 pathogenicity.

E. coli O157:H7 is a foodborne pathogen responsible for bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). The objective of the present work was to evaluate the ability of colostral IgG obtained from Stx2-immunized cows to prevent against E. coli O157:H7 infection and Stx2 cytotoxicity. Hyperimmune colostrum (HC) was obtained from cows intramuscularly immunized with inactivated Stx2 or vehicle for controls. Colostral IgG was purified by affinity chromatography. Specific IgG antibodies against Stx2 and bovine lactoferrin (bLF) levels in HC and the corresponding IgG (HC-IgG/bLF) were determined by ELISA. The protective effects of HC-IgG/bLF against Stx2 cytotoxicity and adhesion of E. coli O157:H7 and its Stx2-negative mutant were analyzed in HCT-8 cells. HC-IgG/bLF prevention against E. coli O157:H7 was studied in human colon and rat colon loops. Protection against a lethal dose of E. coli O157:H7 was evaluated in a weaned mice model. HC-IgG/bLF showed high anti-Stx2 titers and high bLF levels that were able to neutralize the cytotoxic effects of Stx2 in vitro and in vivo. Furthermore, HC-IgG/bLF avoided the inhibition of water absorption induced by E. coli O157:H7 in human colon and also the pathogenicity of E. coli O157:H7 and E. coli O157:H7Δstx2 in rat colon loops. Finally, HC-IgG/bLF prevented in a 100% the lethality caused by E. coli O157:H7 in a weaned mice model. Our study suggests that HC-IgG/bLF have protective effects against E. coli O157:H7 infection. These beneficial effects may be due to specific anti-Stx2 neutralizing antibodies in combination with high bLF levels. These results allow us to consider HC-IgG/bLF as a nutraceutical tool which could be used in combination with balanced supportive diets to prevent HUS. However further studies are required before recommendations can be made for therapeutic and clinical applications.

Relevance of Bacteriophage 933W in the Development of Hemolytic Uremic Syndrome (HUS).

Hemolytic uremic syndrome (HUS), principally caused by shiga toxins (Stxs), is associated with Shiga toxin-producing Escherichia coli (STEC) infections. We previously reported Stx2 expression by host cells in vitro and in vivo. As the genes encoding the two Stx subunits are located in bacteriophage genomes, the aim of the current study was to evaluate the role of bacteriophage induction in HUS development in absence of an E. coli O157:H7 genomic background. Mice were inoculated with a non-pathogenic E. coli strain carrying the lysogenic bacteriophage 933W (C600Φ933W), and bacteriophage excision was induced by an antibiotic. The mice died 72 h after inoculation, having developed pathogenic damage typical of STEC infection. As well as renal and intestinal damage, markers of central nervous system (CNS) injury were observed, including aberrant immunolocalization of neuronal nuclei (NeuN) and increased expression of glial fibrillary acidic protein (GFAP). These results show that bacteriophage 933W without an E. coli O157:H7 background is capable of inducing the pathogenic damage associated with STEC infection. In addition, a novel mouse model was developed to evaluate therapeutic approaches focused on the bacteriophage as a new target.

Soluble CD40 Ligand and Oxidative Response Are Reciprocally Stimulated during Shiga Toxin-Associated Hemolytic Uremic Syndrome.

Shiga toxin (Stx), produced by Escherichia coli, is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS), which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L), which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS) on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC) by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions.

Microbiological and serological control of Escherichia coli O157: H7 in kindergarten staff in Buenos Aires city and suburban areas.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening hemolytic-uremic syndrome (HUS). Despite the magnitude of the social and economic problems caused by HUS, no licensed vaccine or effective therapy is currently available for human use. Prevention of STEC infections continues being the most important measure to reduce HUS incidence. This is especially true for Argentina where HUS incidence among children is extremely high and shows an endemic pattern. The aim of this work was to investigate serologically adult staff of kindergartens in Buenos Aires city and suburban areas in order to detect possible carriers, and to educate personnel about good practices to reduce HUS transmission. We also assessed the microbiological quality of water and meal samples from the same kindergartens. We tested 67 healthy adults, 13 water supplies and 6 meals belonging to 6 public kindergartens. We analysed hand swabs for isolation of STEC and serum samples for the presence of antibodies against Stx and lipopolysaccharide (LPS) of O157 serogroup. We identified 46 Stx2-positive individuals, but only 7 for O157 LPS. No presence of STEC pathogens was detected in hands of staff, water or meal samples.

Microbiological and serological control of Escherichia coli O157: H7 in kindergarten staff in Buenos Aires city and suburban areas

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening hemolytic-uremic syndrome (HUS). Despite the magnitude of the social and economic problems caused by HUS, no licensed vaccine or effective therapy is currently available for human use. Prevention of STEC infections continues being the most important measure to reduce HUS incidence. This is especially true for Argentina where HUS incidence among children is extremely high and shows an endemic pattern. The aim of this work was to investigate serologically adult staff of kindergartens in Buenos Aires city and suburban areas in order to detect possible carriers, and to educate personnel about good practices to reduce HUS transmission. We also assessed the microbiological quality of water and meal samples from the same kindergartens. We tested 67 healthy adults, 13 water supplies and 6 meals belonging to 6 public kindergartens. We analysed hand swabs for isolation of STEC and serum samples for the presence of antibodies against Stx and lipopolysaccharide (LPS) of O157 serogroup. We identified 46 Stx2-positive individuals, but only 7 for O157 LPS. No presence of STEC pathogens was detected in hands of staff, water or meal samples.

Las infecciones bacterianas con Escherichia coli productor de toxina Shiga (Stx) (STEC) están implicadas en el desarrollo del síndrome urémico hemolítico (SUH). A pesar de la magnitud del problema social y económico causado por el SUH, actualmente no existe un tratamiento específico o una vacuna eficaz para uso humano. Por lo tanto, la prevención de las infecciones por STEC es la tarea central para reducir la incidencia del SUH. Esto es especialmente cierto para Argentina en donde el SUH muestra un comportamiento endémico y presenta una incidencia extremadamente alta entre los niños. En efecto, la mediana de casos notificados en menores de 5 años para el periodo 2010-2015 fue 306, mientras que la tasa de notificación fue 8.5 casos cada 100 000 menores/año (http://www.msal.gob.ar/images/stories/boletines/boletin_integrado_vigilancia_N335-SE45.pdf). El objetivo de este trabajo fue analizar serológicamente al personal adulto de jardines de infantes de la ciudad de Buenos Aires y el área suburbana con el fin de detectar portadores, y brindarles formación sobre las buenas prácticas para reducir la transmisión de infecciones con STEC y así evitar el SUH. También se evaluó la calidad microbiológica de las muestras de agua y de la comida elaborada en los mismos jardines. Hemos estudiado 67 adultos, a través del hisopado de manos para la búsqueda de STEC y suero para la presencia de anticuerpos contra Stx y el lipopolisacárido (LPS) de serogrupo O157. También se analizaron 13 suministros de agua y 6 muestras de comida pertenecientes a 6 jardines de infantes públicos. Se identificaron 46 individuos positivos para Stx2, pero solo 7 para LPS-O157. No se detectó presencia de patógenos STEC en las muestras de las manos del personal, ni en los reservorios de agua o muestras de comida.

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

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

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.

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.

Development of camelid single chain antibodies against Shiga toxin type 2 (Stx2) with therapeutic potential against Hemolytic Uremic Syndrome (HUS).

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening Hemolytic Uremic Syndrome (HUS). Despite the magnitude of the social and economic problems caused by STEC infections, no licensed vaccine or effective therapy is presently available for human use. Single chain antibodies (VHH) produced by camelids exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis and therapy. In the present work, the properties of a recently developed immunogen, which induces high affinity and protective antibodies against Stx type 2 (Stx2), were exploited to develop VHHs with therapeutic potential against HUS. We identified a family of VHHs against the B subunit of Stx2 (Stx2B) that neutralize Stx2 in vitro at subnanomolar concentrations. One VHH was selected and was engineered into a trivalent molecule (two copies of anti-Stx2B VHH and one anti-seroalbumin VHH). The resulting molecule presented extended in vivo half-life and high therapeutic activity, as demonstrated in three different mouse models of Stx2-toxicity: a single i.v. lethal dose of Stx2, several i.v. incremental doses of Stx2 and intragastrical STEC infection. This simple antitoxin agent should offer new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcome.

Type III Secretion-Dependent Sensitivity of Escherichia coli O157 to Specific Ketolides.

A subset of Gram-negative bacterial pathogens uses a type III secretion system (T3SS) to open up a conduit into eukaryotic cells in order to inject effector proteins. These modulate pathways to enhance bacterial colonization. In this study, we screened established bioactive compounds for any that could repress T3SS expression in enterohemorrhagic Escherichia coli (EHEC) O157. The ketolides telithromycin and, subsequently, solithromycin both demonstrated repressive effects on expression of the bacterial T3SS at sub-MICs, leading to significant reductions in bacterial binding and actin-rich pedestal formation on epithelial cells. Preincubation of epithelial cells with solithromycin resulted in significantly less attachment of E. coli O157. Moreover, bacteria expressing the T3SS were more susceptible to solithromycin, and there was significant preferential killing of E. coli O157 bacteria when they were added to epithelial cells that had been preexposed to the ketolide. This killing was dependent on expression of the T3SS. Taken together, this research indicates that the ketolide that has accumulated in epithelial cells may traffic back into the bacteria via the T3SS. Considering that neither ketolide induces the SOS response, nontoxic members of this class of antibiotics, such as solithromycin, should be considered for future testing and trials evaluating their use for treatment of EHEC infections. These antibiotics may also have broader significance for treating infections caused by other pathogenic bacteria, including intracellular bacteria, that express a T3SS.

Leukotriene C4 increases the susceptibility of adult mice to Shiga toxin-producing Escherichia coli infection.

Shiga 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) in this pathology. Thus, the aim of this work was to analyze whether leukotriene C4 (LTC4) influences STEC pathogenesis in mice. We report that exogenous LTC4 pretreatment severely affected the outcome of STEC gastrointestinal infection. LTC4-pretreated (LTC4+) and STEC-infected (STEC+) mice showed an increased intestinal 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 and high urea levels, indicating that the cause of death was related to Stx2-toxicity. Despite the differences observed in the survival between LTC4+ and LTC4- mice after STEC infection, both groups showed the same survival after Stx2-intravenous inoculation. In addition, LTC4 pretreatment increased the permeability of mucosal intestinal barrier, as assessed by FITC-dextran absorption experiments. Altogether these results suggest that LTC4 detrimental effect on STEC infection is related to the increased passage of pathogenic factors to the bloodstream. Finally, we showed that STEC infection per se increases the endogenous LTC4 levels in the gut, suggesting that this inflammatory mediator plays a role in the pathogenicity of STEC infection in mice, mainly by disrupting the mucosal epithelial barrier.

Retinoid levels influence enterohemorrhagic Escherichia coli infection and Shiga toxin 2 susceptibility in mice.

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that produces Shiga toxin (Stx) and causes hemorrhagic colitis. Under some circumstances, Stx produced within the intestinal tract enters the bloodstream, leading to systemic complications that may cause the potentially fatal hemolytic-uremic syndrome. Although retinoids like vitamin A (VA) and retinoic acid (RA) are beneficial to gut integrity and the immune system, the effect of VA supplementation on gastrointestinal infections of different etiologies has been controversial. Thus, the aim of this work was to study the influence of different VA status on the outcome of an EHEC intestinal infection in mice. We report that VA deficiency worsened the intestinal damage during EHEC infection but simultaneously improved survival. Since death is associated mainly with Stx toxicity, Stx was intravenously inoculated to analyze whether retinoid levels affect Stx susceptibility. Interestingly, while VA-deficient (VA-D) mice were resistant to a lethal dose of Stx2, RA-supplemented mice were more susceptible to it. Given that peripheral blood polymorphonuclear cells (PMNs) are known to potentiate Stx2 toxicity, we studied the influence of retinoid levels on the absolute number and function of PMNs. We found that VA-D mice had decreased PMN numbers and a diminished capacity to produce reactive oxygen species, while RA supplementation had the opposite effect. These results are in line with the well-known function of retinoids in maintaining the homeostasis of the gut but support the idea that they have a proinflammatory effect by acting, in part, on the PMN population.

Role of bacteriophages in STEC infections: new implications for the design of prophylactic and treatment approaches.

Shiga toxin (Stx) is considered the main virulence factor in Shiga toxin-producing Escherichia coli (STEC) infections. Previously we reported the expression of biologically active Stx by eukaryotic cells in vitro and in vivo following transfection with plasmids encoding Stx under control of the native bacterial promoter (1,2). Since stx genes are present in the genome of lysogenic bacteriophages, here we evaluated the relevance of bacteriophages during STEC infection. We used the non-pathogenic E. coli C600 strain carrying a lysogenic 933W mutant bacteriophage in which the stx operon was replaced by a gene encoding the green fluorescent protein (GFP). Tracking GFP expression using an In Vivo Imaging System (IVIS), we detected fluorescence in liver, kidney, and intestine of mice infected with the recombinant E. coli strain after treatment with ciprofloxacin, which induces the lytic replication and release of bacteriophages. In addition, we showed that chitosan, a linear polysaccharide composed of d-glucosamine residues and with a number of commercial and biomedical uses, had strong anti-bacteriophage effects, as demonstrated at in vitro and in vivo conditions. These findings bring promising perspectives for the prevention and treatment of haemolytic uremic syndrome (HUS) cases.

Promoter sequence of Shiga toxin 2 (Stx2) is recognized in vivo, leading to production of biologically active Stx2.

UNLABELLED: 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 stx2 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 stx2 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.

Advances in pathogenesis and therapy of hemolytic uremic syndrome caused by Shiga toxin-2.

Shiga toxin (Stx) producing Escherichia coli (STEC) is responsible to bloody diarrhea (hemorrhagic colitis) and the hemolytic uremic syndrome (HUS). STEC strains carry inducible lambda phages integrated into their genomes that encode Stx 1 and/or 2, with several allelic variants each one. O157:H7 is the serotype that was documented in the vast majority of HUS cases although non-O157 serotypes have been increasingly reported to account for HUS cases. However, the outbreak that occurred in central Europe during late spring of 2011 showed that the pathogen was E. coli O104:H4. More than 4,000 persons were infected mainly in Germany, and it produced more than 900 cases of HUS resulting in 54 deaths. E. coli O104:H4 is a hybrid organism that combines some of the virulence genes of STEC and enteroaggregative E. coli specially production of Stx2 and the adherence mechanisms to intestinal epithelium. The differences in the epidemiology and presentation of E. coli pathogen meant a challenge for public health and scientific research to increase the knowledge of HUS-pathophysiology and to improve available therapies to treat HUS.

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.

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. Despite the magnitude of the social impact of EHEC infections, no licensed vaccine or effective therapy is available for human use. One of the biggest challenges is to develop an effective and safe immunogen to ensure nontoxicity, as well as a strong input to the immune system to induce long-lasting, high-affinity Abs 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 on which to display foreign Ags. Taking into account the advantages of BLS and the potential capacity of the B subunit of Stx2 to induce Abs that prevent Stx2 toxicity by blocking its entrance into the host cells, we engineered a new immunogen by inserting the B subunit of Stx2 at the amino termini of BLS. The resulting chimera demonstrated a strong capacity to induce a long-lasting humoral immune response in mice. The chimera induced Abs 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 Ab development with preventive or therapeutic ends, for use in hemolytic uremic syndrome-endemic areas or during future outbreaks caused by pathogenic strains of Stx-producing E. coli.

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

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.