No / Enteroaggregative Escherichia coli (EAEC), increasingly recognized as an important cause of infant and travelers' diarrhoea, exhibits an aggregative, stacked-brick pattern of adherence to epithelial cells. Adherence is mediated by aggregative adherence fimbriae (AAFs), which are encoded on the pAA virulence plasmid. We recently described a highly prevalent pAA plasmid-borne gene, aap, which encodes a protein (nicknamed dispersin) that is secreted to the bacterial cell surface. Dispersin-null mutants display a unique hyper-aggregating phenotype, accompanied by collapse of AAF pili onto the bacterial cell surface. To study the mechanism of this effect, we solved the structure of dispersin from EAEC strain 042 using solution NMR, revealing a stable beta-sandwich with a conserved net positive surface charge of +3 to +4 among 23 dispersin alleles. Experimental data suggest that dispersin binds non-covalently to lipopolysaccharide on the surface of the bacterium. We also show that the AAF organelles contribute positive charge to the bacterial surface, suggesting that dispersin's role in fimbrial function is to overcome electrostatic attraction between AAF and the bacterial surface

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Escherichia coli enteroagregativa (EAEC) à um patÃtipo de E. coli que tem sido cada vez mais identificado como agente etiolÃgico das doenÃas diarrÃicas. Esse trabalho teve como objetivos determinar a prevalÃncia de EAEC e investigar a importÃncia de alguns genes associados à virulÃncia no grau de severidade das doenÃas diarrÃicas causadas pelo microorganismo, alÃm de avaliar o impacto dessas infecÃÃes na inflamaÃÃo intestinal e no estado nutricional de crianÃas carentes de Fortaleza, CearÃ. CrianÃas na faixa etÃria entre 2 e 36 meses, com histÃrico ou nÃo de diarrÃia nos Ãltimos 14 dias, tiveram suas medidas antropomÃtricas avaliadas e suas amostras fecais coletadas. O diagnÃstico de EAEC foi realizado por reaÃÃo de polimerase em cadeia (PCR) dos genes aaiC (cromossomal) e aatA (plasmidial). Amostras positivas foram pesquisadas quanto à presenÃa dos genes de virulÃncia aggR (regulador transcricional), aap (dispersina), pic (enterotoxina), pet (enterotoxina) e astA (enterotoxina). A sequÃncia nucleotÃdica do gene aggR foi analisada atravÃs de seqÃenciamento. AlÃquotas fecais foram submetidas à quantificaÃÃo de lactoferrina (LFF) e citocinas (IL-4, IL-10, TNF-α e IFN-γ) atravÃs de reaÃÃo imunoenzimÃtica (ELISA). Esse estudo analisou 83 crianÃas com diarrÃia (casos) e 83 crianÃas sem diarrÃia (controles). EAEC foi encontrada na mesma proporÃÃo entre ambos os grupos (41,0%). CrianÃas com diarrÃia apresentaram reduÃÃo significativa na espessura da prega cutÃnea, Ãndice de massa corporal e escores-z peso-por-idade (WAZ) e peso-por-altura (WHZ), mas a presenÃa da bactÃria nÃo foi associada com alteraÃÃes nos Ãndices antropomÃtricos analisados. Entre as amostras positivas para EAEC, nÃo houve diferenÃa quanto à presenÃa isolada dos fatores de virulÃncia pesquisados nas crianÃas que desenvolveram ou nÃo diarrÃia. Avaliando as freqÃÃncias desses genes em combinaÃÃo, cepas de EAEC expressando os genes aggR, aap, pic, pet e astA foram isoladas em freqÃÃncia superior significativa de crianÃas doentes quando comparadas com cepas de EAEC expressando somente aggR, aap, pic e astA (excluindo o gene pet). A anÃlise da seqÃÃncia codificadora do gene aggR apresentou 27 polimorfismos em um Ãnico nucleotÃdeo (SNPs), distribuÃdos entre cinco amostras caso e trÃs controles. Mais de 80,0% das crianÃas estudadas apresentaram inflamaÃÃo intestinal caracterizada por elevados nÃveis de LFF, independente da presenÃa de diarrÃia e EAEC. Todas as crianÃas com diarrÃia associada com EAEC apresentaram altas concentraÃÃes de LFF. NÃveis basais de citocinas fecais foram observados entre crianÃas de ambos os grupos. A variabilidade na presenÃa dos fatores de virulÃncia pesquisados ratifica a heterogeneidade das cepas de EAEC. A combinaÃÃo de genes codificadores de fatores de virulÃncia mostrou que a presenÃa do pet està associada com a doenÃa causada por EAEC. A infecÃÃo pela bactÃria nÃo causou impacto significativo nos Ãndices antropomÃtricos analisados. As elevadas concentraÃÃes observadas de LFF sugerem a existÃncia de fatores adicionais desencadeadores do processo inflamatÃrio. / Enteroaggregative Escherichia coli (EAEC) is a pathotype of diarrheagenic E. coli, which has increasingly been identified as an etiological agent of diarrheal disease. This purpose of this study is to determine the prevalence of EAEC and examine the importance of some virulence-related genes in the severity of the diarrheal disease caused by this microorganism, and further evaluate the impact of these infections on intestinal inflammation and the nutritional status of poor children from Fortaleza, Ceara. Children aged 2 to 36 months, with and without an occurrence of diarrhea in the previous 14 days, had their anthropometric data evaluated and their stools collected. Diagnosis of EAEC was done by polymerase chain reaction (PCR) of the aaiC (chromosomal) and aatA (plasmidial) genes. Positive samples were further analyzed for the presence of the virulence genes aggR (transcription regulator), aap (dispersin), pic (enterotoxin), pet (enterotoxin) and astA (enterotoxin). The nucleotide sequence of aggR gene was also analyzed by sequencing. Aliquots of stool samples were quantified for lactoferrin (LFF) and cytokines (IL-4, IL-10, TNF-α e IFN-γ) by enzyme-linked immunosorbent assay (ELISA). This study analyzed 83 children with diarrhea (cases) and 83 children without diarrhea (controls). EAEC was found in the same proportion in both groups (41.0%). Children with diarrhea presented with significantly reduced skin thickness, body mass index and weight-for-age (WAZ) and weight-for-height (WHZ) z-scores. However, the presence of the bacteria was not associated with changes in the analyzed anthropometric measures. Among the positive samples for EAEC, there was no difference in the presence of the isolated virulence genes in the children with or without diarrhea. Observing the frequencies of these genes in combination, EAEC strains carrying the aggR, aap, pic, pet and astA genes together were isolated in significantly higher frequencies from sick children when compared to EAEC strains expressing only aggR, aap, pic, and astA (excluding pet). Nucleotide sequence analysis of the aggR gene presented 27 polymorphisms of a single nucleotide (SNPs), distributed among five case samples and three control samples. More than 80.0% of studied children had intestinal inflammation characterized by elevated levels of LFF, regardless of the presence of illness and EAEC. All children with diarrhea associated with EAEC presented with high concentrations of LFF. Basal levels of fecal cytokines were observed among children from both groups. Variability in the presence of the evaluated virulence factors confirms the heterogeneity of EAEC strains. The combination of the virulence related genes expressed showed that pet has an association with illness caused by EAEC. The infection by this bacterium did not cause significant impact on the anthropometric index analyzed. The high concentrations of LFF observed suggest that there may be additional factors triggering the inflammatory process.

yes / Background The roles of diffusely-adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) in disease are not well understood, in part because of the limitations of diagnostic tests for each of these categories of diarrhoea-causing E. coli. A HEp-2 adherence assay is the Gold Standard for detecting both EAEC and DAEC but DNA probes with limited sensitivity are also employed. Results We demonstrate that the daaC probe, conventionally used to detect DAEC, cross-reacts with a subset of strains belonging to the EAEC category. The cross hybridization is due to 84% identity, at the nucleotide level, between the daaC locus and the aggregative adherence fimbriae II cluster gene, aafC, present in some EAEC strains. Because aaf-positive EAEC show a better association with diarrhoea than other EAEC, this specific cross-hybridization may have contributed to an over-estimation of the association of daaC with disease in some studies. We have developed a discriminatory PCR-RFLP protocol to delineate EAEC strains detected by the daaC probe in molecular epidemiological studies. Conclusions A PCR-RFLP protocol described herein can be used to identify aaf-positive EAEC and daaC-positive DAEC and to delineate these two types of diarrhoeagenic E. coli, which both react with the daaC probe. This should help to improve current understanding and future investigations of DAEC and EAEC epidemiology. / Food Standards Agency

During acute and chronic infectious diseases hosts develop complex immune responses to cope with bacterial persistence. Depending on a variety of host and microbe factors, outcomes range from peaceful co-existence to detrimental disease. Mechanisms underlying immunity to bacterial stimuli span several spatiotemporal magnitudes and the summation of these hierarchical interactions plays a decisive role in pathogenic versus tolerogenic fate for the host. This dissertation integrates diverse data from immunoinformatics analyses, experimental validation and mathematical modeling to investigate a series of hypotheses driven by computational modeling to study mucosal immunity. Two contrasting microbes, enteroaggregative Escherichia coli and Helicobacter pylori, are used to perturb gut immunity in order to discover host-centric targets for modulating the host immune system. These findings have the potential to be broadly applicable to other infectious and immune-mediated diseases and could assist in the development of antibiotic-free and host-targeted treatments that modulate tolerance to prevent disease. / Ph. D.

Yes / Background. The roles of diffusely-adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) in disease are not well understood, in part because of the limitations of diagnostic tests for each of these categories of diarrhoea-causing E. coli. A HEp-2 adherence assay is the Gold Standard for detecting both EAEC and DAEC but DNA probes with limited sensitivity are also employed. Results. We demonstrate that the daaC probe, conventionally used to detect DAEC, cross-reacts with a subset of strains belonging to the EAEC category. The cross hybridization is due to 84% identity, at the nucleotide level, between the daaC locus and the aggregative adherence fimbriae II cluster gene, aafC, present in some EAEC strains. Because aaf-positive EAEC show a better association with diarrhoea than other EAEC, this specific cross-hybridization may have contributed to an over-estimation of the association of daaC with disease in some studies. We have developed a discriminatory PCR-RFLP protocol to delineate EAEC strains detected by the daaC probe in molecular epidemiological studies. Conclusions. A PCR-RFLP protocol described herein can be used to identify aaf-positive EAEC and daaC-positive DAEC and to delineate these two types of diarrhoeagenic E. coli, which both react with the daaC probe. This should help to improve current understanding and future investigations of DAEC and EAEC epidemiology.

As serino-proteases autotransportadoras de Enterobacteriaceae (SPATE) constituem uma família de proteases secretadas pelo sistema de secreção do tipo V, cujos genes foram estudados em Escherichia coli intestinal e extra-intestinal. Sat é uma SPATE citotóxica de 107 kDa, cujo gene foi identificado pela primeira vez em UPEC isolada da urina de um paciente com pielonefrite. A maioria dos estudos envolvendo Sat foram realizados em células renais e da bexiga, embora seu gene seja encontrado em DAEC, EAEC e, mais recentemente, em amostras bacterianas isoladas de septicemia neonatal e meningite. Os objetivos deste trabalho foram: i) purificar Sat; ii) determinar a ação da Sat purificada em diferentes tipos celulares e iii) caracterizar o papel de Sat na infecção in vitro por EAEC. Desta foram, a presença de Sat nos sobrenadantes do cultivo das cepas EAEC CV323 e DEC/Sat, isoladas de diarreia, foi confirmada por LC-MS/MS. Sat foi purificada da cultura de DEC/Sat+ e utilizada para a obtenção de anticorpos anti-Sat em coelho. O efeito citotóxico de Sat purificada foi investigado em células derivadas do endotélio (HUVEC) e do sistema urinário (Y1, LLC-PK1 e HEK-293) e gastrointestinal (Caco-2). Os parâmetros citotóxicos analisados foram: o descolamento celular e alterações na morfologia, permeabilidade e metabolismo mitocondrial das células. Para investigar o papel de Sat na infecção por EAEC, células Y-1 foram infectadas com EAEC CV323 e DEC/Sat+ na presença ou ausência de PMSF (inibidor de serino-protease) e anticorpos anti-Sat. Os parâmetros de citotoxicidade analisados nas culturas infectadas foram descolamento celular e alteração na morfologia. Os resultados demonstraram que: i) Sat é secretada por EAEC CV323 e DEC/Sat+ e, em ambas as cepas, há duas mutações em resíduos de aminoácidos que não interferiram na atividade enzimática; ii) as células do endotélio são mais sensíveis à Sat do que as células derivadas do trato urinário, sendo a linhagem gastrointestinal a mais resistente; iii) Sat secretada por EAEC CV323 durante a infecção induziu intenso dano celular, o qual, em presença de anticorpos anti-Sat e PMSF foi reduzido em cerca de 80 a 90%, respectivamente. Este é o primeiro trabalho que demonstra a expressão de Sat pela EAEC e a ação da toxina em células endoteliais sugerindo que o papel de Sat possa ser mais amplo na patogenia do que o proposto até o momento. / The serine protease autotransporters of Enterobacteriaceae (SPATEs) constitute a family of proteases secreted by the type V secretion system whose genes have been studied in intestinal and extra intestinal Escherichia coli. Sat is a 107 kDa cytotoxic SPATE and its gene was first identified in UPEC isolated from the urine of a patient with pyelonephritis. Most studies involving Sat were performed in renal and bladder cells, although the gene encoding Sat is encountered in other strains of E. coli such as DAEC, EAEC and more recently, in bacterial samples isolated from neonatal septicemia and meningitis. The objectives of this work were: i) purify Sat; ii) to determine the action of Sat in different types of cells and iii) to characterize in vitro the role of Sat in EAEC infection. Accordingly, the presence of Sat in the culture supernatant of EAEC CV323 and DEC/Sat+ derived from diarrhea was confirmed by LCMS/MS. Sat was purified from the culture of DEC/Sat+ and utilized to produce rabbit antibodies anti-Sat. The cytotoxic effect of Sat was investigated in cells derived from the endothelium (HUVEC) and the urinary (Y1, LLC-PK1, HEK-293) and gastrointestinal (Caco-2) systems. The cytotoxic parameters analyzed were cellular detachment and alterations in the morphology, permeability and mitochondrial metabolism of the cells. To investigate the role of Sat in EAEC infection, Y-1 cells were incubated with EAEC CV323 and DEC/Sat+ in the presence or absence of PMSF (a serine protease inhibitor) and rabbit antibodies anti-Sat. The parameters analyzed were cellular detachment and alteration in the morphology of the cells. The results demonstrated that: i) Sat is secreted by EAEC CV323 and DEC/Sat + and in both strains there are two mutations in amino acid residues that did not interfere with enzymatic activity; ii) endothelium cells are more sensitive to the Sat effect than the cells derived from urinary tract system, being the gastrointestinal cell lineage the most resistant one; iii) Sat secreted by EAEC CV323 during infection induced intense cellular damage which in the presence of anti-Sat antibodies and PMSF was reduced in about 80 to 90%, respectively. This is the first work demonstrating the expression of Sat by EAEC and the action of the toxin on endothelial cells suggesting that the role of Sat may be broader in pathogenesis than has been proposed so far.

Orientador: Rodrigo Tavanelli Hernandes / Resumo: A Escherichia coli enteroagregativa (EAEC) é um importante agente causador de diarreia aguda e persistente em crianças e adultos em todo o mundo. EAEC é definida como isolados de E. coli que produzem o padrão de aderência agregativa (AA) em células epiteliais (HeLa e/ou HEp-2) cultivadas in vitro. O patotipo EAEC pode ser dividido em típico e atípico com base na presença do gene aggR, que codifica um ativador transcricional, presente apenas no primeiro grupo. O objetivo deste estudo foi caracterizar uma coleção de isolados de EAEC obtidos de pacientes com diarreia durante um período de 7 anos de vigilância epidemiológica (2010-2016). Um total de 220 isolados de EAEC (194 típicas e 26 atípicas) foi classificado nos distintos grupos filogenéticos de E. coli, e caracterizados quanto aos sorotipos (O:H), padrão de aderência produzidos em células HeLa, sensibilidade aos antimicrobianos, e a presença de 25 genes responsáveis por codificarem fatores de virulência no patotipo EAEC. A maioria dos isolados de EAEC foi classificada nos grupos filogenéticos A (44,1%; 97/220) e B1 (21,4%; 47/220). Em relação ao padrão de aderência, observamos que 92,7% (204/220) produziram o padrão AA. Além disso, foram identificados nove isolados (4,1%; 9/220) que produziram a aderência em cadeia (CLA), dos quais seis produziram concomitantemente o padrão AA, além de isolados de EAEC que produzem um padrão de aderência indefinido (1,4%; 3/220) e isolados não aderentes (3,6%; 8/220). Foi identificado some... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Enteroaggregative Escherichia coli (EAEC) is an important agent that causes acute and persistent diarrhea in children and adults worldwide. EAEC is defined as E. coli isolates that produce the aggregative adherence pattern (AA) on epithelial cells (HeLa and/or HEp-2) cultured in vitro. The EAEC pathotype can be divided in typical and atypical based on the presence of the aggR gene, which encodes a transcriptional activator, in the former group. The aim of this study was to characterize a collection of EAEC isolates obtained from diarrheal patients over a 7-year period of surveillance (2010-2016). A total of 220 EAEC isolates (194 typical and 26 atypical) were evaluated regarding the phylogenetic classification, serotypes, adherence pattern produced on HeLa cells, susceptibility to antimicrobial drugs and the presence of 25 virulence factor-encoding genes. The majority of the EAEC isolates were assigned to phylogroups A (44.1%; 97/220) and B1 (21.4%; 47/220). Regarding the adherence pattern, was observed that 92.7% (204/220) produced AA. Moreover, we identified nine isolates (4.1%; 9/220) that produced the chain-like adherence (CLA), with six of them producing concomitantly the AA pattern, besides EAEC isolates producing an undefined adherence (1.4%; 3/220) and isolates non-adherent (3.6%; 8/220). Only 0.9% (2/220) of the EAEC isolates studied presented the multidrug resistance phenotype. The genes encoding for the major pilin subunit of all five previously described aggregati... (Complete abstract click electronic access below) / Doutor

PhD (Microbiology) / Department of Microbiology / Diarrhoea is still a major problem worldwide. Enteric pathogens such as Enteroaggregative E. coli (EAEC), Enteropathogenic E. coli (EPEC) and Enterotoxigenic E. coli (ETEC) have been reported to cause diarrhoea in children under the age of 5 years. The incidences of these pathogens are due to factors such as poor water quality, sanitation and hygiene practices. Infections with these pathogens result in diarrhoea and have been reported to result in severe disease outcomes more especially in children under 2 years of age. EPEC infections have been well studied using in vitro analyses, with studies highlighting the adherence traits, proteins and virulence genes involved in pathogenesis and inflammatory responses. EPEC is characterized by localized adherence with microcolony formation at the site of infection. In vivo studies have reported on human EPEC infection. However, the current animal models have not been able to replicate clinical outcomes (such as diarrhoea and weigh loss) of EPEC infection similar to humans. Therefore, there is still a need for a suitable small animal model that mimic clinical outcomes of human EPEC infections in vivo. Children living in poor environmental conditions are more susceptible to diarrhoeal pathogens. Furthermore, the incidences of children being exposed to co-infections (more than one pathogen at the same time) is relatively high. The EAEC/EPEC (A/P) and EPEC/ETEC (P/T) co-infections have been increasingly detected in children with and without diarrhoea. It has been suggested that patients infected with these co-infections might result in severe disease outcome than those infected with single pathogens. Pathogens are constantly evolving and the microbe-microbe interaction in the host can result in these pathogens competing for the same niche and thus result in increased virulence. Interaction of co-infections can lead to increased inflammatory responses thus affecting the infected host. The first objective of this study was to develop an EPEC murine model using weaned C57BL/6 mice that have been pretreated with antibiotic cocktail. Mice were orally infected with wild-type (WT) typical EPEC, bfp- and escN mutant strains. The WT had transient weight loss and wet stools with mucous; and the bfp- infected mice also had transient weight loss and bloody stool appearance. Increase in inflammatory biomarkers MPO, LCN-2, CRP, IL-6 and SAA were observed in the WT and bfp- infected mice. The mice infected with escN mutant did not exhibit any weight changes and the stools were similar to the uninfected mice. Furthermore, no inflammatory biomarkers were observed in mice infected with the escN mutant. Metabolic perturbations were observed in WT EPEC infected mice at day 3 post infection with the TCA cycle metabolites (reduced succinate, citrate, fumarate, cis-aconitate) being excreted at lower quantities indicating that the energy production in the infected mice was greatly affected. The second objective of this study was to determine the interaction between the P/T coinfections using in vitro and in vivo analyses. In vitro, human colorectal tumour 8 (HCT-8) cells were infected with single strains of ETEC, EPEC and both the pathogens and incubated for 3 hours. After infection the cells were analysed for bacterial adherence using real-time PCR. The single strains adhered at the same rate similar to the P/T coinfected cells. IL-8, as a marker of inflammatory response, was measured using ELISA. The results indicated that the P/T co-infected cells had a significant increase in IL-8 response higher than the single infections. The P/T co-infections were further analysed in vivo using the EPEC murine model developed in this study. Interestingly, mice infected with P/T co-infections developed severe diarrhoea accompanied with significant increased weight loss and some mice died during the 3-day infection period. The inflammatory responses MPO, LCN-2 and SAA were higher in the co-infected mice indicating a synergistic effect. The bfp and eltA virulence genes were significantly increased in the P/T co-infections. The third objective of this study was to determine the interaction between A/P coinfections using in vitro and in vivo analyses. HeLa cells and HCT-8 cells were infected with EAEC, EPEC and both the pathogens at the same time in order to determine adherence and inflammatory responses. EAEC adherence was higher than EPEC and A/P co-infections adherence. A/P co-infections did not have increased IL-8 response in HCT-8 cells when compared to EAEC alone. The virulence genes involved in EPEC adherence and Type 3 Secretion System (bfp, eae, tir, ler, per, espB and espA) were significantly reduced in A/P co-infected cells. An interesting adherence trait was observed between the A/P co-infections in HeLA cells, EAEC was found to adhere around EPEC altering the localized adherence pattern. The A/P co-infections were further analysed using the EPEC murine model developed in this study. The A/P infected mice had diminished weight changes and EAEC shedding was enhanced when EPEC was present. Faecal inflammatory biomarkers MPO and LCN-2 in A/P infected mice did not have any additive effect. The findings of this study contributed significantly to the knowledge of human EPEC infection in weaned C57BL/6 mice, highlighting clinical outcomes, inflammatory responses and metabolic perturbations. Furthermore, this study also highlighted the interaction of P/T and A/P co-infections using in vitro and in vivo analyses in order to determine the disease severity and outcomes. It was observed in this study that coinfections can result in either synergistic or antagonistic effects. Further studies are therefore, required in order to understand the underlying mechanisms that are involved during co-infections and this can further assist in the development of therapeutic interventions. / NRF