Activation of innate and specific immune responses in hemolytic uremic syndrome (HUS)- patients

La función primaria del sistema inmunológico es preservar al individuo sano frente a infinidad de agentes microbianos patógenos o injuriantes. Sin embargo, en determinadas circunstancias los mecanismos agresores normalmente montados contra un agente invasor, pueden tornarse altamente injuriantes para el propio individuo. Hay importantes evidencias tanto clínicas como experimentales, de que la reacción inflamatoria inducida por los distintos componentes de las bacterias Escherichia coli productoras de toxina Shiga (Stx) (STEC), fundamentalmente la Stx y los lipopolisacáridos (LPS), contribuye decisivamente en la evolución a la forma completa de SUH Así los pacientes al ser diagnosticados de SUH, presentan evidencias de haber sufrido un proceso de activación del sistema inmune innato, o reacción inflamatoria muy aguda y temprana en la evolución de la enfermedad. Algunas de estas evidencias pueden resumirse como: una neutrofilia marcada, leucocitos neutrófilos (PMN) circulantes que se encuentran “agotados o exhaustos”, los monocitos diferenciados hacia un fenotipo inflamatorio (menor expresión de CD14 y aumento de CD16), y se encuentra un significativo descenso en los leucocitos que presentan el receptor para la quimioquina Fractalquina (FKN, CX3CL1)) (CX3CR1): los. monocitos clásicos y células Natural Killer (NK). Estas células tienen un alto potencial citotóxico. La FKN se expresa en endotelio y epitelio renal y ha sido involucrada en los mecanismos patogénicos en distintas nefropatías. Llamativamente, encontramos una correlación significativa entre la severidad del cuadro renal y las alteraciones mencionadas. Por último se discute el papel protector que la respuesta inmune específica podría ejercer, fundamentalmente a través de la producción de anticuerpos neutralizantes de la Stx.The central role of the immune system is the preservation of the health against several pathogenic microbes and injury agents. However, on special conditions defensive mechanisms triggered towards the foreign agent can damage the host. Clinical and experimental evidence indicate that inflammatory reaction triggered by the main components of Shiga toxin (Stx)- producing Escherichia coli (STEC), participate in the evolution to the complete form of HUS. When children are diagnosed of HUS, they present evidence that have suffered a very strong and early inflammatory response. These features include: the presence of a marked neutrophilia, the polymorfonuclear leucocytes (PMN) are “deactivated or exhausted” and the monocytes are differentiated towards an inflammatory phenotype (CD14-reduced and CD16-enhanced membrane expression). In addition, HUS-patients show a marked reduction in the absolute and relative number of leucocytes carrying the receptor (CX3CR1) for the chemokine “Fractalkine” (FKN, CX3 CL1), which are the classic monocytes and Natural Killer cells (NK). All these cells express a high cytotoxic potencial. The chemokine FKN is expressed in endothelial and epithelial renal cells, and is involved in the pathogenic mechanism of different nephropathies. Noteworthy, we found a significant correlation between the severity of the renal damage (as days of anuria) and the alterations described above. Finally, the protective role of specific immune response, mainly through the antibody production with Stx-neutralizing capacity, is discussed.Fil: Palermo, Marina Sandra. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fernandez, Gabriela C.. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Ramos, Maria Victoria. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Bentancor, Leticia. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Fernández Brando, Romina Jimena. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Dran, Graciela I.. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Isturiz, Martín Amadeo. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

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.Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: MaCateer, Sean P.. University of Edinburgh; Reino UnidoFil: Montañez Culma, Leidy Johanna. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Cortés Araya, Yennifer. University of Edinburgh; Reino UnidoFil: Tree, Jai. University of Edinburgh; Reino UnidoFil: Bernal, Alan Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fuentes, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fitzgerald, Stephen. University of Edinburgh; Reino UnidoFil: Pineda, Gonzalo Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Ramos, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gally, David. University of Edinburgh; Reino UnidoFil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

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

A subset of Gram negative bacterial pathogens use a type 3 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-minimum inhibitory (sub-MIC) concentrations, leading to significant reductions in bacterial binding and actin-rich pedestal formation on epithelial cells. Pre-incubation 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 when added to epithelial cells pre-exposed to the ketolide. This killing was dependent on expression of the T3SS. Taken together, this research indicates that the ketolide may traffic back into the bacteria via the T3SS from accumulated levels in epithelial cells. Considering that neither ketolide induces the SOS response, non-toxic members of this class of antibiotic, such as solithromycin, should be considered for future testing and trials in relation to EHEC infections. These antibiotics may also have broader significance for treating other pathogenic bacteria, including intracellular bacteria, that express a T3SS.Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. University of Edinburgh; Reino UnidoFil: Yamaguchi, Nao. University of Edinburgh; Reino UnidoFil: Tahoun, Amin. University of Edinburgh; Reino Unido. Kafrelsheikh University; EgiptoFil: McAteer, Sean P.. University of Edinburgh; Reino UnidoFil: Gillespie, Trudi. University of Edinburgh; Reino UnidoFil: Wang, Dai. University of Edinburgh; Reino Unido. Xiamen University; ChinaFil: Argyle, Sally A.. University of Edinburgh; Reino UnidoFil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gally, David L.. University of Edinburgh; Reino Unid

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A(2) and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2 Delta AB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2 Delta AB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.Fundacion Alberto J. RoemmersFundacion Alberto J. RoemmersConsejo Nacional de Investigaciones Cientificas y Tecnologicas (CONICET)Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET)ANPCyT Agencia Nacional de Promocion Cientifica y Tecnologica, ArgentinaAgencia Nacional de Promoción Científica y Tecnológica (ANPCyT

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

Differential Outcome between BALB/c and C57BL/6 Mice after <i>Escherichia coli</i> O157:H7 Infection Is Associated with a Dissimilar Tolerance Mechanism

Enterohemorrhagic Escherichia coli (EHEC) infections can result in a wide range of clinical presentations despite that EHEC strains belong to the O157:H7 serotype, one of the most pathogenic forms. Although pathogen virulence influences disease outcome, we emphasize the concept of host-pathogen interactions, which involve resistance or tolerance mechanisms in the host that determine total host fitness and bacterial virulence. Taking advantage of the genetic differences between mouse strains, we analyzed the clinical progression in C57BL/6 and BALB/c weaned mice infected with an E. coli O157:H7 strain. We carefully analyzed colonization with several bacterial doses, clinical parameters, intestinal histology, and the integrity of the intestinal barrier, as well as local and systemic levels of antibodies to pathogenic factors. We demonstrated that although both strains had comparable susceptibility to Shiga toxin (Stx) and the intestinal bacterial burden was similar, C57BL/6 showed increased intestinal damage, alteration of the integrity of the intestinal barrier, and impaired renal function that resulted in increased mortality. The increased survival rate in the BALB/c strain was associated with an early specific antibody response as part of a tolerance mechanism.Instituto de Estudios Inmunológicos y Fisiopatológico

Role of bacteriophages in STEC infections: new implications for the design of prophylactic and treatment approaches [v2; ref status: indexed, http://f1000r.es/437]

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 promoter1,2. 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