Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

O pEO5, que codifica hemolisina, foi isolado de uma amostra de EPEC do sorotipo O26. Este plasmídio mostrou ser conjugativo e compatível com o pO157, e pelos testes de hibridização observou-se que estes plasmídios não são geneticamente relacionados. Para o estudo comparativo de similaridade foi seqüenciada uma região de 9227 pb de DNA do pEO5 que compreende todo o operon hlyCABD e suas regiões a montante e a jusante. A região do operon hemolitico (7225 pb) e a região promotora do operon foram similares às mesmas regiões do pHly152, que em uma amostra de E. coli isolada de roedor, codifica uma a hemolisina. No entanto, verificou-se a presença de elementos de inserção na região a montante do gene hlyC no pHly152. O pEO5 mostrou ser semelhante a outros plasmídios que também codificam a hemolisina em cepas de EPEC O26 de origem humana e de bovinos. A presença de estruturas semelhantes a transposons em ambas as extremidades do operon a hemolítico do pEO5 indica que esse fator de virulência provavelmente foi adquirido por transferência horizontal de genes. / The conjugative pEO5 encoding haemolysin in strains of EPEC O26 was investigated for its relationship with EHEC haemolysin-encoding of EHEC O26 and O157 strains. pEO5 was found to be compatible with EHEC virulence plasmids and did not hybridize in Southern blots with pO157, indicating that both plasmids were unrelated. A 9227 bp stretch pEO5 DNA encompassing the entire operon hlyCABD was sequenced and compared for similarity to plasmid and chromosomally inherited hly determinants. The a hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of pHly152, in particular, the structural a-hlyCABD and hlyR regions. pEO5 and hly of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural a-hlyCABD. The major difference found between the hly regions of pHly152 and pEO5 is caused by the IS2 upstream of the hlyC in pHly152. The presence of transposonlike structures at both ends of hly sequence indicates that pEO5 was probably acquired by horizontal gene transfer.

A serinoprotease Pic é uma proteína autotransportadora que apresenta papel importante na colonização da mucosa intestinal por E. coli enteroagregativa (EAEC). Uma cepa de E. coli enteropatogênica atípicas (aEPEC) albergando o gene pic foi detectada em um estudo prévio. O presente estudo teve como objetivo caracterizar a proteína Pic produzida por essa cepa de aEPEC (BA589). O gene pic em BA589 está presente em um plasmídeo de alto peso molecular (~98 kb) e o sequenciamento desse gene mostrou identidade de 99% com pic de EAEC 042. Pic da cepa BA589 (Pic589) foi capaz de clivar mucina bovina, hemaglutinar eritrócitos de coelho e clivar moléculas das três vias sistema complemento. O mutante BA589Dpic todas essas atividades in vitro. A cepa selvagem foi capaz de colonizar o intestino de camundongos tratados com estreptomicina, o que não foi observado com o mutante BA589Dpic. Desta forma, a serinoprotease Pic representa um fator de virulência adicional na cepa de aEPEC BA589, relacionada às etapas de adesão, colonização e evasão do sistema imune inato. / The serine protease Pic is an autotransporter protein that plays an important role in the colonization of the intestinal mucosa by enteroaggregative E. coli (EAEC). An atypical enteropathogenic E. coli (aEPEC) strain harboring the pic gene has been detected in a previous study. The present study aimed to characterize the protein Pic produced by that aEPEC strain (BA589). In BA589 pic is located in a high molecular weight plasmid (~98 kb), and sequencing of this gene showed 99% identity with pic from EAEC 042. Pic from BA589 (Pic589) was able to cleave bovine mucin, hemagglutinate rabbit erythrocytes and cleave molecules of the three complement system pathways. The mutant BA589Dpic lost all these capacities. The wild type strain was able to colonize the intestine of streptomycin treated mice, which was not observed with the mutant BA589Dpic. Thus, the serine protease Pic represents an additional virulence factor in aEPEC strain BA589, associated with adherence, colonization and evasion of innate immune system.

Orientador: Marcelo Palma Sircili / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T01:00:20Z (GMT). No. of bitstreams: 1 Culler_HebertFabricio_D.pdf: 4509234 bytes, checksum: 996664e9d72f80df8f43076b618a7f73 (MD5) Previous issue date: 2015 / Resumo: As Escherichia coli enteropatogênicas atípicas são capazes de formar biofilme em superfícies abióticas e bióticas. Diversos mecanismos em E. coli são regulados por Quorum Sensing, incluindo a expressão de fatores de virulência e formação de biofilme. Quorum Sensing é um sistema de sinalização que confere às bactérias a habilidade de responder à moléculas químicas denominadas autoindutores (AI). SdiA e QseC são receptores de quorum sensing encontrados em diversas bactérias, entre elas EPECa. SdiA detecta moléculas autoindutoras do tipo 1 (AI-1) denominadas N-acil homoserina lactonas (AHLs). Entretanto as Escherichia coli não possuem a sintase para estas moléculas e, deste modo, SdiA detecta AHLs produzidas por outras bactérias. O receptor QseC detecta moléculas autoindutoras do tipo 3, além dos hormônios humanos adrenalina e noradrenalina. Neste estudo verificamos a influência da deleção de sdiA e qseC na formação e arquitetura do biofilme, formação de película, anel e também na transcrição de alguns dos genes envolvidos nestes fenótipos (bcsA, csgA, csgD, fliC, fimA e rpoS) em duas amostras de EPECa, sendo uma do sorotipo O55:H7 e outra do sorotipo ONT:H25. Os resultados das análises nas duas amostras de EPECa foram distintos, confirmando a grande heterogeneidade reportada em outros estudos em amostras deste patótipo. A amostra ONT:H25?sdiA formou espesso biofilme em placas de 96 poços e espessa estrutura (em anel) como anéis na parede do tubo de ensaio em relação às amostras selvagem e complementada. Além disso, o mutante sdiA desta amostra foi capaz de formar película na superfície do meio enquanto as amostras selvagem e complementada foram negativas. A amostra O55:H7?sdiA não apresentou diferença significativa na formação (das estruturas analisadas) destas estruturas em relação às amostras selvagem e complementada. Análises de qRT-PCR demonstraram (maiores níveis de transcrição de) um aumento na transcrição de csgA, csgD e fimA na amostra ONT:H25 deletada em sdiA, possivelmente indicando que o aumento na formação de biofilme por esta amostra esteja relacionado ao aumento da expressão das fímbrias tipo 1 e curli. A adição de AHLs à amostra selvagem ONT:H25 diminuiu a formação de biofilme e os níveis transcricionais de csgD e fimA, enquanto na amostra deletada não houve diferença, indicando que sdiA participa da regulação da formação de biofilme em EPECa e que as AHLs aumentam os efeitos repressores deste receptor nos genes relacionados à formação de biofilme. Quanto às amostras deletadas em qseC foi possível verificar uma diminuição da formação de biofilme em relação às amostras selvagens e complementadas, mas não houve diferença na formação de película na interface ar-líquido e também na formação de anel na parede dos tubos. A amostra ONT:H25?qseC foi negativa para expressão de fímbria curli em placas contendo vermelho congo e apresentou aumento da transcrição de bcsA e fimA, enquanto teve a transcrição de csgA, csgD e fliC diminuída em relação à amostra selvagem. A adição de adrenalina aumentou a formação de biofilme e motilidade nas duas amostras mutantes indicando a possível presença de outro receptor em EPECa responsável pela detecção destes hormônios na ausência de QseC. Portanto, conclui-se que estes dois receptores de quorum sensing estão relacionados, direta ou indiretamente, à formação de biofilme em EPECa / Abstract: Atypical enteropathogenic Escherichia coli are capable to form biofilm on biotic and abiotic surfaces. Several E. coli mechanisms are regulated by Quorum Sensing, including expression of virulence factors and biofilm formation. Quorum Sensing is a signaling system that confers bacteria the ability to respond to chemical molecules known as autoinducers. SdiA and QseC are Quorum Sensing receptors found in several bacteria, including aEPEC. SdiA detects type 1 autoinducer molecules (AI-1) known as N-acil homoserin lactones. However, Escherichia coli do not produce this kind of molecules and SdiA detects AHLs produced by other bacteria. QseC receptor detects type 3 autoinducer molecules and the human hormones adrenalin and noradrenaline. In this study the influence of the sdiA and qseC deletion in the biofilm formation and architecture, pellicle and ring-like structure formation and transcription of some genes (bcsA, csgA, csgD, fliC, fimA and rpoS) in two strains of aEPEC (O55:H7 and ONT:H25) was verified. The results of the analysis of the two aEPEC strains were distinct, confirming the heterogeneity reported by other studies in the same patotypes. The strain ONT:H25?sdiA formed a thick biofilm in 96-well plates and thick ring-like structure on the tube wall compared with the wild type and complemented strains. Furthermore, the sdiA mutant strain was capable to form pellicle on both surfaces, while the wild type and complemented strains were negative. The strain O55:H7?sdiA did not show a significant difference on the formation of these structures compared to the wild type and complemented strains. qRT-PCR analysis demonstrated an enhance on the transcription of csgA, csgD and fimA in the deleted sdiA ONT:H25 strain, suggesting that a relative increase of biofilm formation in ONT:H25 strain could be related to the increase of the expression of type 1 and curli fimbriae. The addition of AHLs to the wild type strain ONT:H25 decreased the biofilm formation and the transcriptional levels of csgD and fimA, but not in the mutant strain, indicating that sdiA plays a role in the regulation of biofilm formation in aEPEC. AHLs also increased the repressor efects of this receptor in biofilm related genes. The mutant qseC strains showed a decrease in biofilm formation when compared to wild type and complemented strains, but there was no difference in the pellicle formation in the air-liquid interface, as in the ring-like structure formation in the tube wall. The ONT:H25?qseC strain was negative to curli fimbriae expression in congo red plates and displayed an increase in the transcription of bcsA and fimA genes, while a decrease was verified in the csgA, csgD and fliC genes compared to the wild type strain. The addition of adrenalin increased the biofilm formation and motility in both mutant strains, possibly indicating the presence of other receptor in aEPEC involved in the detection of these hormones in the absence of QseC. In conclusion, these two Quorum Sensing receptors are related with biofilm formation in aEPEC / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

In der vorliegenden Arbeit wurde in einem in vitro-Modell mit porcinen intestinalen Epithelzellen (IPEC-J2) der Einfluss des probiotischen E. coli Nissle 1917 (EcN) auf die Infektion mit atypischen EPEC (aEPEC) untersucht. EcN reduzierte bei Vorinkubation auf IPEC-J2 die aEPEC-Infektion drastisch. Konfokale Laserscanning- und Elektronenmikroskopie zeigten, dass EcN die Adhäsion und Mikrokoloniebildung inhibierte, jedoch nicht die Ausbildung von Attaching and Effacing-Läsionen adhärenter aEPEC. Der inhibierende Effekt von EcN wurde durch dessen sehr gute Adhäsionsfähigkeit an IPEC-J2 vermittelt. Die F1C-Fimbrien wurden als wichtigster Adhäsionsfaktor von EcN identifiziert. Darüber hinaus waren auch H1-Flagellen durch Ausbildung interbakterieller Verbindungen maßgeblich an der Adhäsion des Stammes beteiligt. In gleichem Maß wie die Vorinkubation von EcN reduzierte die Koinkubation seines Kulturüberstandes die aEPEC-Infektion, was auf die Abgabe eines inhibierenden Faktors in den Kulturüberstand schließen lässt. Dieser Faktor wurde auch von anderen pathogenen sowie nicht pathogenen E. coli-Stämmen in Schüttelkultur gebildet und scheint deshalb nicht spezifisch für EcN zu sein. Jedoch ermöglichte erst die gute Adhäsionsfähigkeit von EcN auf der Epithelzelloberfläche die Abgabe ausreichender Mengen des Inhibitors und eine Beeinflussung der aEPEC-Infektion. Die Ergebnisse weisen darauf hin, dass durch EcN die initiale Anheftung von aEPEC an die Wirtszelle unterbunden wird. Der inhibierende Effekt von EcN auf die aEPEC-Infektion war zeitabhängig. Im Gegensatz zur Vorinkubation erhöhten Ko- und Nachinkubation von EcN die Adhäsion von aEPEC und hatten einen geringeren inhibierenden Effekt auf die Mikrokoloniebildung. Dieser gegensätzliche Effekt auf die Adhäsion von aEPEC wird möglicherweise von einem zweiten Faktor hervorgerufen. Dieser scheint nur dann wirksam zu sein, wenn der inhibierende Faktor in zu geringer Konzentration oder erst nach Adhäsion von aEPEC vorliegt. / In this study, the effects of the probiotic E. coli strain Nissle 1917 (EcN) on host cell infection with atypical enteropathogenic E. coli (aEPEC) were investigated in an in vitro porcine intestinal epithelial cell model (IPEC-J2). In pre-incubation experiments, EcN drastically reduced the infection efficiencies of aEPEC. Using confocal laser scanning microscopy and scanning electron microscopy, it was shown that EcN inhibited the attachment and formation of microcolonies, but not the formation of attaching and effacing lesions by adherent aEPEC. The inhibitory effect was mediated by the adherent properties of EcN to epithelial cells. The F1C fimbriae were identified as the most important adhesion factor of EcN in vitro. Furthermore, the H1 flagellae were also shown to be involved in the adhesion of EcN, serving as bridges between bacterial cells. Co-incubation of culture supernatants of EcN reduced the infection efficiencies of aEPEC to the same extent as in pre-incubation with EcN bacteria, indicating the secretion of an inhibitory factor by EcN. This factor was also secreted by other pathogenic and non-pathogenic E. coli strains in shaking culture and therefore does not appear to be specific for EcN. However, the outstanding ability of EcN to adhere to epithelial cells largely contributes to the secretion of sufficient concentrations of this inhibitory factor und to the influence on the aEPEC infection. The results suggest that EcN interferes with the initial adhesion of aEPEC to host cells. The inhibitory effect of EcN was found to be time-dependent. In contrast to pre-incubation experiments, co- and post-incubation of EcN actually increased the adhesion efficiencies of aEPEC and showed only minor effects on microcolony formation. This second effect of EcN on aEPEC adhesion, possibly due to a second factor, appears only to be effective when the putative inhibitory factor is either present at low concentrations or after aEPEC is already adherent to host cells.

A Escherichia coli enteropatogênica (EPEC) é um dos principais agentes etiológicos da diarréia infecciosa tanto em crianças no primeiro ano de vida, como em adultos. As infecções por EPEC são prevalentes nos países em desenvolvimento, principalmente nas populações de baixo nível sócio-econômico, como as encontradas no Brasil. A resposta imune na infecção por EPEC permanece pobremente caracterizada. O uso das novas tecnologias no desenvolvimento de vacinas vem reforçar à importância de se levar em consideração a via natural de infecção do patógeno e utilizá-la como tema de estudo, quando se pretende estudar a resposta imune a um determinado agente infeccioso. O objetivo deste trabalho foi efetuar o estudo da resposta imune em animais inoculados com bactérias vivas ou mortas, por meio de diferentes vias de imunização. As bactérias em estudo foram: a cepa de E. coli O86:H34 e a cepa protótipo de E. coli O127:H6. A cepa de E. coli pertencente ao sorotipo O86:H34 foi isolada de fezes de crianças com diarréia. Foram empregadas as cepas : E2348/69, DH5α e as mutantes E2348/69 flic-, E2348/69 Δtir, E2348/69 EscN-, CVD 206 ΔeaeA, UMD 872 ΔEspA, UMD 864 ΔEspB, UMD 870 ΔEspD. No presente estudo os camundongos BALB/c foram inoculados pela via intragástrica com a cepa de E. coli O86:H34 viva ou cepas O86:H34 e O127:H6 mortas por formol, que foram utilizadas nas imunizações pela via intragástrica e pela via intramuscular. Por meio de ELISA foram determinados os níveis de anticorpos específicos dos isotipos IgG, IgA e IgM, assim como o direcionamento da resposta imune para importantes antígenos que participam do mecanismo de patogenicidade da bactéria. De acordo com o perfil de reatividade no Immunoblot foi avaliada a especificidade dos anticorpos presentes nos soros imunes obtidos, frente aos antígenos de \"whole cells\" ou complexo de membrana externa bacteriana, empregados na técnica de \"immunoblotting\". A resposta imune a proteínas, como EspA, EspB, Tir, intimina, flagelos e BFP observada em camundongos, tem um importante papel no esclarecimento da infecção por este patógeno .Pela primeira vez foi realizado um estudo utilizando diferentes vias de imunização por EPEC em camundongos. Este estudo permitiu a análise de antígenos de E. coli reconhecidos pelos anticorpos produzidos pelas inoculações de bactérias vivas ou mortas por meio de vias intragástrica e intramuscular em camundongos, em comparação com aqueles reconhecidos na infecção natural ou experimental humana. Por conseguinte, os dados obtidos poderão auxiliar no esclarecimento desse complexo mecanismo de patogenicidade e orientar na seleção de peptídeos a serem utilizados no preparo de produtos vacinais específicos. / Enteropathogenic Escherichia coli is one of the major ethiologic agent that causes infectious diarrhoea in both infants and adults individuals. EPEC infections are prevalent in developing countries, mainly in low social-economic populations, as those found in Brazil. The immune response of this infection is still insufficiently known. Use of new technologies in the development of vaccines has been reinforced the importance of taking in account the natural route of infeccion of pathogens and use of it in investigation on immune response to be elicited against a certain to infectious agent. The aim of the present investigation was to study the immune response in mice inoculated with dead or alive bacteria, by means of diverse immunization routes. E. coli O86:H34 strain and E. coli O127:H6 prototype were employed for immunization. E. coli strain belonging to O86:H34 serotype was isolated from faeces from infants with diarrhoea. The strains: E2348/69, DH5 α and the mutants strains E2348/69 flic-, E2348/69 Δtir, E2348/69 EscN-, CVD 206 ΔeaeA, UMD 872 ΔEspA, UMD 874 ΔEspB, UMD 870 ΔEspD were employed. BALB/c mice were inoculated by intragastric route with alive E. coli O86:H34 strain or formalin-killed O86:H34 and O127:H6 strains intragastric and intramuscular immunization routes. The specific antibodies of isotypes IgA, IgG and IgM were determinated by means of ELISA and the course of the immune response for important antigens that participate in the patogenicity mechanism of bacteria could be analysed. By means of reactivity profile on immunobloting, the specificity of antibodies present in obtained sera against whole cells or the outer membrane complex of the bacteria were analysed. Immune response to proteins like EspA, EspB, Tir, intimin, flagelin and BFP in immunized mice may have an important meaning for elucidation of infection in this pathogen At the first time a research using different routes of immunization with EPEC strains in mice has been conducted. This study allowed to compare antigens from E. coli recognized in natural or experimental human infection, and consequentently these data may help in the elucidation of this complex mechanism of pathogenicity, and also to orientate the selection of peptides to be used in preparation of specific vaccines.

Orientador: Marcelo Palma Sircili / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T15:41:18Z (GMT). No. of bitstreams: 1 Higa_JulianaSuyama_M.pdf: 2740628 bytes, checksum: 20ced0f2848628f2541d4313632cfc0b (MD5) Previous issue date: 2015 / Resumo: Escherichia coli (E.coli) enteropatogênica (EPEC), é um dos agentes causadores de diarreia em crianças, principalmente em países em desenvolvimento. EPEC pode ser dividida em típica (tEPEC) ou atípica (aEPEC) pela presença ou ausência do plasmídeo EAF, respectivamente e, mais precisamente pela expressão da fímbria Bfp. Uma característica de EPEC é a capacidade de causar uma lesão histopatológica denominada "attaching and effacing" (lesão A/E) no epitélio intestinal e os genes responsáveis pela formação da lesão A/E estão localizados na ilha de patogenicidade LEE (locus of enterocyte effacement). Outra característica de EPEC é a formação de microcolônias que possibilitam a formação de biofilme. Um dos mecanismos de regulação da formação de biofilme envolve a molécula sinalizadora Bis-(3'-5')-monofosfato de guanosina cíclico (c-di-GMP), um mensageiro secundário universal em bactérias que está envolvido na regulação de uma grande variedade de processos celulares. Para exercer sua função, c-di-GMP precisa se ligar e alterar alostericamente a estrutura de uma molécula efetora. Um dos receptores conhecidos para c-di-GMP é YcgR, uma proteína de domínio PilZ que possui um sítio de ligação para c-di-GMP e está envolvida diretamente na regulação do movimento flagelar através da ligação do complexo YcgR-c-di-GMP às proteínas da base do motor flagelar. Existem poucos dados na literatura sobre as funções de YcgR, todos focados apenas no seu papel na regulação da motilidade. Assim, o presente trabalho teve como objetivo avaliar a influência de YcgR na motilidade, formação de biofilme, adesão e formação de lesão A/E em um amostra de aEPEC do sorotipo O55:H7. O gene ycgR foi deletado da amostra selvagem através da técnica de recombinação homóloga proposta por Datsenko e Wanner (2000). A complementação da amostra mutante foi realizada através da clonagem do gene ycgR no plasmídeo de expressão pBAD Myc HisA. Os resultados obtidos indicam que a deleção do gene ycgR reduz a motilidade e aumenta a formação do biofilme inicial na amostra O55:H7. Além disso, a adesão em células epiteliais e a formação da lesão A/E também foram reduzidas em comparação à amostra selvagem. Os resultados fenotípicos corroboram os observados na análise transcricional dos genes eae, ler e espA, que participam da formação da lesão A/E, e dos genes bscA, fimA e csgD, envolvidos na formação do biofilme inicial. Com exceção do gene csgD que apresentou um aumento na transcrição na amostra mutante, todos os outros genes avaliados apresentaram uma menor transcrição em relação à amostra selvagem. Poucos trabalhos na literatura demonstram o papel do mensageiro secundário em amostras de E. coli patogênicas, assim, estes resultados são os primeiros descritos para uma amostra de aEPEC e possibilitam que no futuro novos estudos possam analisar com mais detalhes a participação de c-di-GMP na regulação de fatores de virulência não só de aEPEC mas também de outras E.coli patogênicas / Abstract: Enteropathogenic Escherichia coli (EPEC) is a causative agent of diarrhea in children, especially in developing countries. EPEC can be categorized in 2 subgroups termed typical (tEPEC) or atypical (aEPEC) by the presence or absence of the EAF plasmid respectively, and more precisely by the expression of the Bfp fimbriae. One characteristic of EPEC is the ability to cause a histopathological lesion on epithelial cells called "attaching and effacing" (A/E lesion). The genes responsible for the production of the A/E lesion are encoded in a pathogenicity island named "locus of enterocyte effacement" (LEE). Another feature of EPEC is the formation of microcolonies, which allow biofilm formation. One of the regulation mechanisms of biofilm formation involves the signaling molecule bis- (3'-5') cyclic guanosine monophosphate (c-di-GMP), a ubiquitous second messenger in bacteria that participates in the regulation of a wide variety of cellular processes. To perform its function, c-di-GMP needs to bind and alter allosterically the structure of an effector molecule. One of the known (many?) receptors for c-di-GMP is YcgR, a Pilz domain protein that has a c-di-GMP binding site and is involved directly in the regulation of flagellar movement through the binding of the YcgR-c-di-GMP complex to flagellar motor proteins. There are few published data on the YcgR functions and they focus mainly on the role of the YcgR in motility regulation. The aim of this study was to evaluate the influence of YcgR in motility, biofilm formation, adhesion and A/E lesion formation in an aEPEC strain serotype O55:H7. ycgR gene deletion was performed by homologous recombination as proposed by Datsenko and Wanner (2000). Complementation of O55:H7 mutant strain was achieved by cloning ycgR in pBAD/Myc-HisA plasmid. The results indicate that the deletion of ycgR gene decreases the motility and increases the formation of initial biofilm on O55:H7 strain. Moreover, the adhesion on epithelial cells and the A/E lesion formation were also diminished in comparision to the wild type strain. The phenotypic results are consistent with the transcriptional analysis of eae, ler and espA genes involved in A/E lesion formation, and of bcsA, fimA and csgD genes involved in the initial steps of biofilm formation. With the exception of csgD gene that showed an increased transcription level in the mutant strain, all other analysed genes showed a decrease in transcription when compared to the wild type strain. Few studies demonstrate the role of a second messenger molecule in Escherichia coli pathogenic samples, and therefore, these results are the first report in this regard for an aEPEC strain. This work should encourage further studies in order to analyze in more detail the involvement of c-di GMP in the regulation of virulence factors not only in aEPEC, but also in other pathogenic Escherichia coli pathotypes / Mestrado / Microbiologia / Mestra em Genética e Biologia Molecular

A Escherichia coli enteropatogênica (EPEC) é um dos principais agentes etiológicos da diarréia infecciosa tanto em crianças no primeiro ano de vida, como em adultos. As infecções por EPEC são prevalentes nos países em desenvolvimento, principalmente nas populações de baixo nível sócio-econômico, como as encontradas no Brasil. A resposta imune na infecção por EPEC permanece pobremente caracterizada. O uso das novas tecnologias no desenvolvimento de vacinas vem reforçar à importância de se levar em consideração a via natural de infecção do patógeno e utilizá-la como tema de estudo, quando se pretende estudar a resposta imune a um determinado agente infeccioso. O objetivo deste trabalho foi efetuar o estudo da resposta imune em animais inoculados com bactérias vivas ou mortas, por meio de diferentes vias de imunização. As bactérias em estudo foram: a cepa de E. coli O86:H34 e a cepa protótipo de E. coli O127:H6. A cepa de E. coli pertencente ao sorotipo O86:H34 foi isolada de fezes de crianças com diarréia. Foram empregadas as cepas : E2348/69, DH5α e as mutantes E2348/69 flic-, E2348/69 Δtir, E2348/69 EscN-, CVD 206 ΔeaeA, UMD 872 ΔEspA, UMD 864 ΔEspB, UMD 870 ΔEspD. No presente estudo os camundongos BALB/c foram inoculados pela via intragástrica com a cepa de E. coli O86:H34 viva ou cepas O86:H34 e O127:H6 mortas por formol, que foram utilizadas nas imunizações pela via intragástrica e pela via intramuscular. Por meio de ELISA foram determinados os níveis de anticorpos específicos dos isotipos IgG, IgA e IgM, assim como o direcionamento da resposta imune para importantes antígenos que participam do mecanismo de patogenicidade da bactéria. De acordo com o perfil de reatividade no Immunoblot foi avaliada a especificidade dos anticorpos presentes nos soros imunes obtidos, frente aos antígenos de \"whole cells\" ou complexo de membrana externa bacteriana, empregados na técnica de \"immunoblotting\". A resposta imune a proteínas, como EspA, EspB, Tir, intimina, flagelos e BFP observada em camundongos, tem um importante papel no esclarecimento da infecção por este patógeno .Pela primeira vez foi realizado um estudo utilizando diferentes vias de imunização por EPEC em camundongos. Este estudo permitiu a análise de antígenos de E. coli reconhecidos pelos anticorpos produzidos pelas inoculações de bactérias vivas ou mortas por meio de vias intragástrica e intramuscular em camundongos, em comparação com aqueles reconhecidos na infecção natural ou experimental humana. Por conseguinte, os dados obtidos poderão auxiliar no esclarecimento desse complexo mecanismo de patogenicidade e orientar na seleção de peptídeos a serem utilizados no preparo de produtos vacinais específicos. / Enteropathogenic Escherichia coli is one of the major ethiologic agent that causes infectious diarrhoea in both infants and adults individuals. EPEC infections are prevalent in developing countries, mainly in low social-economic populations, as those found in Brazil. The immune response of this infection is still insufficiently known. Use of new technologies in the development of vaccines has been reinforced the importance of taking in account the natural route of infeccion of pathogens and use of it in investigation on immune response to be elicited against a certain to infectious agent. The aim of the present investigation was to study the immune response in mice inoculated with dead or alive bacteria, by means of diverse immunization routes. E. coli O86:H34 strain and E. coli O127:H6 prototype were employed for immunization. E. coli strain belonging to O86:H34 serotype was isolated from faeces from infants with diarrhoea. The strains: E2348/69, DH5 α and the mutants strains E2348/69 flic-, E2348/69 Δtir, E2348/69 EscN-, CVD 206 ΔeaeA, UMD 872 ΔEspA, UMD 874 ΔEspB, UMD 870 ΔEspD were employed. BALB/c mice were inoculated by intragastric route with alive E. coli O86:H34 strain or formalin-killed O86:H34 and O127:H6 strains intragastric and intramuscular immunization routes. The specific antibodies of isotypes IgA, IgG and IgM were determinated by means of ELISA and the course of the immune response for important antigens that participate in the patogenicity mechanism of bacteria could be analysed. By means of reactivity profile on immunobloting, the specificity of antibodies present in obtained sera against whole cells or the outer membrane complex of the bacteria were analysed. Immune response to proteins like EspA, EspB, Tir, intimin, flagelin and BFP in immunized mice may have an important meaning for elucidation of infection in this pathogen At the first time a research using different routes of immunization with EPEC strains in mice has been conducted. This study allowed to compare antigens from E. coli recognized in natural or experimental human infection, and consequentently these data may help in the elucidation of this complex mechanism of pathogenicity, and also to orientate the selection of peptides to be used in preparation of specific vaccines.

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

Intimina é uma adesina de natureza protéica das bactérias diarreiogênicas Escherichia coli enteropatogenica (EPEC) e enterohemorrágica (EHEC), capazes de induzir a lesão \'attaching e effacing\' em enterócitos. Os principais subtipos de intiminas de EPEC e EHEC prevalentes no Brasil são α, β e γ. Nosso objetivo foi investigar a transferência de anticorpos maternos anti-intiminas aos recém-nascidos de mães saudáveis de São Paulo, Brasil. Foram pesquisados anticorpos SIgA no colostro e IgG no soro de 50 mulheres saudáveis e no soro de cordão umbilical de seus recém-nascidos, por ELISA utilizando como antígeno proteínas recombinantes purificadas das regiões conservadas e variáveis de intiminas α, β e γ. As concentrações de anticorpos no colostro foram superiores quando comparadas com as concentrações do soro para todos os tipos de intiminas. Não se observaram diferenças estatísticas entre as concentrações de anticorpos reativos com as diferentes intiminas nas amostras de colostro. As concentrações de anticorpos reativos com a região conservada da intimina foram significativamente mais elevadas em comparação com as regiões variáveis no soro dos grupos de mães e de recém-nascidos. Houve alta correlação entre todos os anticorpos anti-intiminas nas amostras de colostro. Comparando-se as concentrações de anticorpos séricos, os coeficientes foram maiores entre anti-α e anti-β que entre os outros pares. Nossos resultados confirmam a transferência de anticorpos maternos para o recém-nascido pela placenta e pelo aleitamento materno e reforça o efeito protetor da amamentação contra infecção por EPEC. / Intimin is a proteic adhesin of enteropatogenic (EPEC) and enterohemorragic (EHEC) Escherichia coli, capable of inducing attachment and effacement lesion in enterocytes. The main subtypes of intimins of EPEC and EHEC prevalent in Brazil are α, β and γ. Our aim is to investigate the transference of maternal anti-intimin antibodies to the newborns of healthy mothers from Sao Paulo, Brazil. IgG and SIgA antibodies were determined in sera and colostrum from 50 healthy women and cord sera from their newborns, by ELISA using as antigens purified recombinant proteins, conserved and variable regions of α, β and γ intimins. The IgA antibody concentrations of colostrum are higher than IgG antibodies in serum for all intimins and there were no statistical differences between them in colostrum samples. The concentrations of antibodies reactive with the conserved region of intimin are significantly higher compared to the variable regions in the sera groups, mothers and newborns. There were high correlation coefficients between all the anti-intimins antibodies in colostrum samples. In the comparison of the seric antibody concentrations, the coefficients were higher between anti-α and anti-β than all the other pairs. Our results confirm the transference of maternal antibodies to the newborns by placenta and breastfeeding and reinforce the high protection effect of breastfeeding against EPEC infection.