Apesar de Escherichia coli O157:H7 ainda ser considerado o principal sorotipo envolvido com surtos de enfermidades veiculadas por alimentos entre as E. coli produtoras de toxina de Shiga (STEC), outros sorogrupos estão ganhando importância, como O26, O45, O103, O111, O121 e O145, que estão sendo denominados de \"Top Six STEC non O157\". As STEC são responsáveis por sintomas que variam de uma simples diarreia até diarreia sanguinolenta, que pode evoluir ainda para síndrome hemolítica urêmica e púrpura trombótica trombocitopênica, podendo ocasionar danos crônicos como falência renal e levar a óbito. Para tanto, apresentam diversos fatores de virulência, entre eles, as toxinas de Shiga (Stx) ou verotoxinas (Vtx). Os veículos destes micro-organismos são diversos alimentos, sendo o principal deles, as carnes moídas. Apesar da importância da carne moída como veículo transmissor de STEC, pouco se conhece sobre a sua presença nesse alimento comercializado na cidade de São Paulo, SP. Sendo assim, o objetivo deste trabalho foi pesquisar a presença de STEC em carne moída comercializada no varejo da cidade de São Paulo e caracterizar tais isolados quanto à presença dos seguintes fatores de virulência: stx1, stx2, eae e ehx. Foram coletadas 248 amostras em diferentes bairros da cidade de São Paulo. Para a detecção de E. coli sorogrupo O157 foi utilizada a metodologia ISO 16654 e para a detecção dos sorogrupos O103, O111, O145 e O26 foi empregada a metodologia descrita pelo Surveillance Group for Diseases and Infections of Animals (NRM 006). Uma amostra de carne moída (0,4%) apresentou o micro-organismo pesquisado. A identificação genotípica e bioquímica caracterizou esse isolado como STEC O157:H7, portador de todos os fatores de virulência pesquisados: stx1, stx2, eae e ehx. Foi constatada, também, a expressão das proteínas stx em células Vero. Esse é o primeiro relato da presença de E. coli O157:H7 produtora de toxina de Shiga em carne moída no Brasil. / Although Escherichia coli O157:H7 is still considered the most important serotype involved in foodborne disease outbreaks among Shiga toxin-producing E. coli (STEC), other serogroups are receiving more attention such as O26, O45, O103, O111, O121 and O145, that are being called the \"Top Six STEC non O157\". STEC are responsible for symptoms ranging from simple diarrhea to bloody diarrhea, which can further evolve to hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, which may cause damage such as chronic renal failure and lead to death. To do so, they have several virulence factors, including the Shiga toxins (Stx) or verotoxins (Vtx). The vehicles of these microorganisms are many foods, most notably, the ground beef. Despite the importance of ground beef as a vehicle for transmitting STEC, little is known about their presence in this kind of food sold in São Paulo, SP. Therefore, the objective of this study was to investigate the presence of STEC in ground beef sold at retail level in Sao Paulo city and characterize the isolates for the presence of the following virulence factors: stx1, stx2, eae and ehx. 248 samples were collected in different districts of Sao Paulo city. For the detection of E. coli O157 serogroup the methodology ISO 16654 was used and for the detection of serogroups O103, O111, O145 and O26 the methodology described by the Surveillance Group for Diseases and Infections of Animals (NRM 006) was used. One sample of ground beef (0.4%) showed the presence of the microorganism studied. The biochemical and genotypical identification characterized this isolate as STEC O157:H7, carrying all of the investigated virulence factors: stx1, stx2, eae and ehx. The expression of stx proteins in Vero cells was also observed. This is the first report on the isolation of E. coli O157:H7 Shiga toxin-producing from ground beef in Brazil.

A proteína dispersina, codificada pelo gene aap, é um dos fatores de virulência de Escherichia coli enteroagregativa (EAEC). A detecção de aap tem sido utilizada juntamente com aggR e aatA no diagnóstico molecular de EAEC. Para verificar sua especificidade, esses genes foram pesquisados em 243 amostras de E. coli diarreiogênica (DEC). Todas as amostras foram negativas para os genes aggR e aatA, enquanto 26 amostras foram positivas para aap. Estas amostras haviam sido descritas como E. coli enteropatogênica atípica, entretanto essa classificação não foi confirmada, uma vez que elas não apresentaram o gene eae. Três amostras aderiram em células HEp-2 no padrão agregativo e foram classificadas como EAEC. O restante constituiu um grupo de amostras sem marcadores que caracterizam os patótipos de DEC. O seqüenciamento do gene aap de 6 amostras demonstrou alta homologia entre as seqüências obtidas e a seqüência da amostra protótipo de EAEC 042. Este estudo revelou que o gene aap não é exclusivo de EAEC. / The protein dispersin, encoded by aap, is one of the virulence factors of enteroaggregative Escherichia coli (EAEC). Detection of aap has been employed along with aggR and aatA in the molecular diagnosis of EAEC. In order to verify their specificity, these genes were searched in a collection of 243 diarrheagenic E. coli (DEC) strains. All of them were negative for aggR and aatA, whereas 26 were positive for aap. These strains have been described as atypical enteropathogenic E. coli. However, this classification was not confirmed since they did not harbor the eae gene. Three strains showed the aggregative adherence pattern to HEp-2 cells and were classified as EAEC. The remaining strains were part of a group that did not harbor any specific markers of DEC pathotypes. The DNA sequence analysis of 6 aap-harboring strains showed high homology between the sequence of these strains and the aap sequence of the EAEC prototype strain 042. This study revealed that aap is not exclusive of EAEC.

Escherichia coli isoladas de infecções do trato urinário (ITU) são conhecidas como E. coli uropatogênicas (UPEC). Dentre as E. coli diarreiogênicas, o patótipo denominado E. coli enteroagregativa (EAEC) é definido pela produção do padrão de adesão agregativa em células epiteliais cultivadas. Estudos recentes mostraram que algumas cepas de UPEC albergam propriedades de virulência de EAEC, indicando que cepas de EAEC podem causar ITU. Assim sendo, o objetivo deste estudo foi analisar as relações filogenéticas entre cepas de EAEC que apresentam marcadores genéticos de E. coli extraintestinais (ExPEC) e cepas de UPEC com e sem marcadores genéticos de EAEC. Para tal, foram selecionadas 92 EAEC, 8 UPEC com e 10 sem marcadores de EAEC. As 92 EAEC foram analisadas quanto à presença dos genes considerados como marcadores de cepas de ExPEC (papA/papC, sfa/foc, afa/dra, iutA, kpsMT II), detectando 30 (32,6%) cepas com esse perfil. Estas 30 cepas foram selecionadas para análises de filogrupos e multilocus sequence type (MLST) junto às cepas de UPEC. Foi observado que 17 (54,4%) cepas de EAEC e 3 (16,6%) de UPEC pertenceram ao filogrupo A, 2 (6,45%) EAEC e 1 (5,5%) UPEC ao filogrupo B1, 3 (9,68%) EAEC e 8 (44,4%) UPEC ao filogrupo B2, 6 (19,35%) EAEC e 2 (11,1%) UPEC ao filogrupo D, 1 (3,2%) EAEC e 4 (22,2%) UPEC ao filogrupo E, 1 (3,2%) EAEC ao filogrupo F e 1 EAEC (3,2%) não pôde ser classificada de acordo com esta metodologia. Comparando os dois grupos de UPEC notou-se que dentre as cepas com marcadores de EAEC 3 (37,5%) pertenceram ao filogrupo E, 2 (25%) aos filogrupos A e D e 1 (12,5%) ao filogrupo B1. Dentre as cepas sem marcadores de EAEC 1 (10%) pertenceu ao filogrupo A, 1 (10%) ao filogrupo E e 8 (80%) ao filogrupo B2. As análises de MLST através do sequenciamento dos genes recA, fumC, icd, mdh, purA, adk e gyrB permitiram determinar 42 sequence types (ST) distintos, dos quais 22 foram descritos neste estudo. Os mais comuns foram o ST 10 (5 cepas) e ST 95 e ST 746 (ambos com 2 cepas cada). A árvore filogenética gerada confirmou esses dados, mostrando o grupamento das cepas de EAEC com marcadores de ExPEC com as cepas de UPEC com marcadores de EAEC. Em resumo, o presente estudo mostrou que um subgrupo de cepas de EAEC está inserido nos mesmos grupos filogenéticos de cepas de UPEC com marcadores de EAEC apresentando, portanto, correlação filogenética. Houve diferenças de distribuição filogenética entre cepas de UPEC com e sem marcador de EAEC. Concui-se que cepas de EAEC podem apresentar potencial uropatogênico, tanto no curso de uma infecção diarreica, quanto em carreadores assintomáticos. / Escherichia coli isolated from urinary tract infections (UTI) are known as uropathogenic E. coli (UPEC). Among the diarrheagenic E. coli, the enteroaggregative E. coli (EAEC) pathotype is defined by the production of the aggregative adherence on cultured epithelial cells. Recent studies have shown that some UPEC strains harbor virulence properties of EAEC, indicating that EAEC strains can cause UTI. Therefore, the aim of this study was to analyze the phylogenetic relationships among EAEC strains that have genetic markers of extraintestinal E. coli (ExPEC) and UPEC strains, with and without genetic markers of EAEC. For that reason, we selected 92 EAEC, 8 UPEC with and 10 without EAEC markers. The 92 EAEC were analyzed for the presence of genes considered as markers for ExPEC strains (papA/papC, sfa/foc, afa/dra, iutA, kpsMT II), detecting 30 (32.6%) strains with that profile. These 30 strains were selected for phylogroup and multilocus sequence type (MLST) analysis with the UPEC strains. It was observed that 17 (54.4%) EAEC and 3 (16.6%) UPEC belonged to the phylogroup A, 2 (6.45%) EAEC and 1 (5.5%) UPEC to the phylogroup B1, 3 (9.68%) EAEC and 8 (44.4%) UPEC to the phylogroup B2, 6 (19.35%) EAEC and 2 (11.1%) UPEC to the phylogroup D, 1 (3.2%) EAEC and 4 (22.2%) UPEC to the phylogroup E, 1 (3.2%) EAEC to the phylogroup F and 1 (3.2%) EAEC could not be classified according to this methodology. Comparing the two groups of UPEC it was observed that among the UPEC strains with EAEC markers, 3 (37.5%) belonged to the phylogroup E, 2 (25%) to the phylogroups A and D and 1 (12.5%) to the phylogroup B1. Among the UPEC strains without EAEC markers, 1 (10%) belonged to the phylogroup A, 1 (10%) to the phylogroup E and 8 (80%) to the phylogroup B2. The MLST analysis by sequencing of recA, fumC, icd, mdh, purA, adk and gyrB genes allowed to determine 42 distinct sequence types (ST), of whom, 22 were described in this study. The most common were ST 10 (5 strains), and ST 95 and ST 746 (both with two strains each). The phylogenetic tree generated confirmed that data, showing the clustering of EAEC strains (harboring ExPEC markers) with the UPEC strains (harboring EAEC markers). In summary, the current study showed that a subgroup of EAEC strains are clustered in the same phylogenetic groups of UPEC strains with EAEC markers and, thus, present phylogenetic correlation. Also, there were differences in phylogenetic distribution among UPEC strains with and without EAEC markers. In conclusion, EAEC strains may have uropathogenic potential, either in the course of a diarrheal infection or in asymptomatic carriers.

Escherichia coli Enteropatogênicas são um importante patógeno causador de diarréia. As EPEC podem ser classificadas em típica e atípica, baseado na presença do plasmídeo EAF. As amostras de EPEC apresentam em seu genoma uma ilha de patogenicidade denominada região LEE, na qual estão contidos os genes relacionados a formação da lesão (A/E). A regulação gênica da região LEE é multifatorial, sendo o principal regulador o gene ler. Até o momento não existem trabalhos sobre a participação de Hfq em EPEC, assim sendo, o presente estudo analisa o papel de Hfq na regulação dos fatores de virulência de EPEC típica (O127:H6) e atípica (O55:H7). A mutagênese do gene hfq foi obtida através do sistema l Red de recombinação alélica. As amostras mutantes apresentaram uma diminuição na capacidade aderir e formar a lesão A/E. Analise transcricional dos mutantes revelou uma significativa diminuição na transcrição do gene espA e do gene eae. Foi possível evidenciar uma diminuição da motilidade das amostras mutantes. A analise in silico revelou a possibilidade do dobramento natural do mRNA ler, ocultando o sitio de ligação do ribossomo. Aqui demonstramos a necessidade Hfq para a transcrição dos genes responsáveis pela lesão A/E. responsible for the A/E lesion. / Enteropathogenic Escherichia coli are an important pathogen responsible for causing diarrhea. EPEC can be classified as typical and atypical, based on the presence of the EAF plasmid. EPEC strains have in their genome a pathogenicity island known as LEE region, where it harbours genes related to the formation of a lesion A/E. LEE regulation is multifactorial, being the ler gene its main regulator. Until now there are no studies on the role of Hfq in EPEC, thus, the present study analyzes the function of Hfq on the regulation of virulence factors in typical EPEC (O127:H6) and atypical (O55:H7) strains. Hfq gene mutagenesis was obtained utilizing the allelic recombination l Red system. The mutant strains demonstrated a decrease in the capability of mutant strains to adhere and form A/E lesion. Transcriptional analysis showed a decrease on the espA gene and eae gene transcription. It was possible to notice a decrease in motility of the mutant strains. In silico analysis revealed the possibility of a natural folding of ler mRNA, concealing the ribosome binding site. With this study we could demonstrate the need of Hfq for the transcription of genes responsible for the A/E lesion.

A proteína verde fluorescente (GFPuv), pelo fato de ter como característica a emissão de luz verde fluorescente brilhante quando exposta à luz ultravioleta, tem sido utilizada como marcador em diversos campos de pesquisa. Células transformadas de Escherichia coli DH5-a expressando a GFPuv foram submetidas aos tratamentos: (i) permeação seletiva (congelamento/ descongelamento/ sonicação) associada à extração por partição em três fases (TPP) e posterior purificação em coluna cromatográfica de interação hidrofóbica (HIC), e (ii) extração direta das células por TPP e posterior purificação em coluna HIC. Este trabalho teve por objetivo a extração da GFPuv com a aplicação das metodologias citadas para a extração da proteína, avaliando a influência da permeação seletiva das células de Escherichia coli no processo de extração da GFPuv. Na análise dos resultados observou-se que apesar da permeação seletiva ser uma metodologia mais laboriosa, mostrou-se mais eficiente pela obtenção de aproximadamente 9 vezes mais GFPuv em relação à extração direta das células por TPP. / The green fluorescent protein (GFPuv), for the fact of having as characteristic the emission of brilliant green fluorescent light when exposed to the ultraviolet light, it has been used as marker in several research fields. Transformed cells of Escherichia coli DH5-a expressing GFPuv was subjected to: (i) selective permeation (freezing / thawing/ sonication) associated to the extraction for partition in three phases (TPP) and subsequent purification in hydrophobic interaction chromatography column (HIC), and (ii) direct extraction of the cells for TPP and subsequent purification in HIC column. This work had for objective the extraction of GFPuv with the application of the methodologies mentioned for the extraction of the protein, evaluating the influence of the selective permeation in the cells of Escherichia coli in the process of GFPuv extraction. In the analysis of the results was observed that in spite of the selective permeation to be a more laborious methodology, it was shown more efficient for the obtaining of approximately 9 more times GFPuv in relation to the direct extraction of the cells for TPP.

A proteína dispersina, codificada pelo gene aap, é um dos fatores de virulência de Escherichia coli enteroagregativa (EAEC). A detecção de aap tem sido utilizada juntamente com aggR e aatA no diagnóstico molecular de EAEC. Para verificar sua especificidade, esses genes foram pesquisados em 243 amostras de E. coli diarreiogênica (DEC). Todas as amostras foram negativas para os genes aggR e aatA, enquanto 26 amostras foram positivas para aap. Estas amostras haviam sido descritas como E. coli enteropatogênica atípica, entretanto essa classificação não foi confirmada, uma vez que elas não apresentaram o gene eae. Três amostras aderiram em células HEp-2 no padrão agregativo e foram classificadas como EAEC. O restante constituiu um grupo de amostras sem marcadores que caracterizam os patótipos de DEC. O seqüenciamento do gene aap de 6 amostras demonstrou alta homologia entre as seqüências obtidas e a seqüência da amostra protótipo de EAEC 042. Este estudo revelou que o gene aap não é exclusivo de EAEC. / The protein dispersin, encoded by aap, is one of the virulence factors of enteroaggregative Escherichia coli (EAEC). Detection of aap has been employed along with aggR and aatA in the molecular diagnosis of EAEC. In order to verify their specificity, these genes were searched in a collection of 243 diarrheagenic E. coli (DEC) strains. All of them were negative for aggR and aatA, whereas 26 were positive for aap. These strains have been described as atypical enteropathogenic E. coli. However, this classification was not confirmed since they did not harbor the eae gene. Three strains showed the aggregative adherence pattern to HEp-2 cells and were classified as EAEC. The remaining strains were part of a group that did not harbor any specific markers of DEC pathotypes. The DNA sequence analysis of 6 aap-harboring strains showed high homology between the sequence of these strains and the aap sequence of the EAEC prototype strain 042. This study revealed that aap is not exclusive of EAEC.

aEPEC é capaz de causar lesão A/E, provocada por proteínas codificadas na região LEE. Foi realizada a análise estrutural e funcional da região LEE de amostras de aEPEC que expressam os padrões ALL, AA e AD, e amostra não aderente (NA). O padrão de adesão característico e capacidade de causar a lesão A/E foram investigados em células epiteliais. As amostras mantiveram o padrão de adesão independentemente da origem da linhagem celular. A lesão A/E foi detectada em algumas linhagens celulares após o contato com as amostras ALL e AD. A presença da região LEE foi detectada intacta e ensaios de PCR em tempo real, microarray e imunodetecção, mostrando a funcionalidade da mesma em todas as amostras. Um plasmídio que expressa a proteína EspFu foi introduzido em todas as 4 amostras, demonstrando não influenciar nos padrões de adesão e nem na capacidade de causar a lesão A/E nas amostras ALL, AA e AD. Mas, a amostra NA expressou o padrão ALL e foi capaz de causar a lesão A/E. Assim, EspFu desempenhou papel na adesão celular além do estabelecimento da lesão A/E in vitro. / aEPEC is capable to cause A/E lesion, triggered by proteins encoded by LEE region. We analyzed structurally and functionally the LEE region of aEPEC strains displaying LAL, AA, DA, and one nonadherent (NA) strain. The adherence characteristics and ability to cause A/E were investigated in epithelial cells. The displayed adherence patterns were independent of the cell line origin. A/E lesion was detected in some cellular lines after contact only with ALL- and AD-strains. LEE region presence was detected intact and real time PCR, microarray and immunodetection, in all samples tested. An EspFu-expressing plasmid was introduced in all strains, demonstrating no influence of this protein neither in the adherence patterns nor in the capacity to cause A/E of the LAL-, AA- and DA-strains. But, NA-strain expressed the LAL pattern and was able to cause A/E. Therefore, EspFu was shown to play a role in cell adhesion in addition to the establishment of the A/E lesion in vitro.

Em caso de envenenamento ofídico, o tratamento no Brasil hoje é realizado pela administração de soros geralmente produzidos por equinos, que apresentam eficácia limitada: são úteis para os efeitos sistêmicos, mas não inibem efetivamente a evolução dos danos locais, podem causar reações adversas e apresentam alto custo de produção. De acordo com a Organização Mundial da Saúde (OMS), trata-se de uma doença negligenciada pelas autoridades científicas mundiais. O presente projeto, em parceria com o Instituto de Pesquisas em Patologias Tropicais da Fundação Oswaldo Cruz - Rondônia, propõe a produção por Escherichia coli de fragmentos de anticorpos de cadeia pesada de camelídeos, denominados VHH, contra as toxinas do veneno de Bothrops jararacussu, utilizando biorreator. Neste trabalho há interesse em produzir VHH, através da otimização do crescimento desta E. coli. A cinética do crescimento bacteriano foi realizada em shaker orbital sob diferentes condições, variando tamanho do frasco, rotação do shaker, composição do meio de cultura e concentração de substrato; e em biorreatores, alternando meios de cultura e modo de operação do reator (descontínuo e descontínuo alimentado), alterando a vazão de alimentação (linear e exponencial) O processo cinético é fortemente limitado pela formação de acetato, por condições auxotróficas da célula e pela transferência de oxigênio. Nos ensaios em frascos agitados, uma melhor condição de crescimento foi obtida utilizando frascos de 1 L, sob rotação a 270 rpm e 5,0 g/L de glicose. Nos ensaios em reator, quando operados em batelada obtiveram-se cerca 5,5 g/L de células finais, contra 9,3 g/L de células em batelada alimentada com vazão constante. Um maior crescimento foi ainda obtido em um reator de 2 L em regime de batelada alimentada exponencialmente. O biorreator varia a agitação do meio e mantém um nível pré-definido de oxigênio dissolvido, evitando a limitação de oxigênio e controlando a oferta de glicose para o crescimento celular. Neste processo, atingimos 25,6 g/L de células e 0,35 g/L de proteína total após purificação, utilizando meio M9 suplementado. / Nowadays in Brazil, the treatment for snakebite poisoning is carried out by the administration of horse sera, which have limited effectiveness: they are useful for systemic effects, but do not effectively inhibit local damage, cause adverse reactions, and present high production costs. According to the WHO, this disease is neglected by the world`s scientific authorities. This project, in partnership with the Institute for Research in Tropical Diseases of Oswaldo Cruz Foundation - Rondonia, proposes the production of heavy chain antibody fragments from camelids, called VHH, using Escherichia coli, to be used against the toxins from the Bothrops jararacussu poison, using a bioreactor. This work is interested in producing VHH through the use of E. coli. The kinetics of bacterial growth were performed in orbital shaker under different conditions, varying vial size, shaker rotation, composition of the culture medium and substrate concentration; and bioreactors, alternating culture media and operation mode of reactor (batch and fed-batch), changing feeding rate (linear and exponential). The kinetic process is statically bound to acetate formation, auxotrophic conditions of the cell and oxygen transfer. In assays in shaken flasks, an output of 270 rpm and 5.0 g/L of glucose. In reactor runs, when operated in a batch 5.5 g/L of final cells were obtained, against 9.3 g/L of final cells in fed-batch with constant flowrate. A larger value was obtained in an exponentially fed-batch reactor of 2 L. The bioreactor varies the agitation of oxygen and controls glucose addition for cell growth. In this process, 25.6 g/L cells and 0.35 g/L total protein after purification were reached, using supplemented M9 medium.

Enterohaemorrhagic Escherichia coli O157:H7 causes haemorrhagic diarrhoea and potentially fatal renal failure in humans. Ruminants are considered to be the primary reservoir for human infection. Vaccines that reduce shedding in cattle are only partially protective and their underlying protective mechanisms are unknown. Studies investigating the response of cattle to colonisation generally focus on humoral immunity, leaving the role of cellular immunity unclear. To inform future vaccine development, the cellular immune response of cattle during EHEC O157:H7 colonisation was examined. Calves were challenged with either a phage type (PT) 21/28 strain possessing the Shiga toxin (Stx) 2a and Stx2c genes or a PT32 strain possessing the Stx2c gene only. T-helper cell associated transcripts at the terminal rectum were analysed by reverse transcriptase quantitative PCR (RT-qPCR). Induction of interferon (IFN)γ and T-bet was observed, with peak expression of both genes at 7 days in PT32 challenged calves, whilst up regulation was delayed, peaking at 21 days in PT21/28 challenged calves. Cells isolated from gastro-intestinal lymph nodes demonstrated antigen-specific proliferation and IFNγ release in response to type III secreted proteins (T3SPs); however responsiveness was suppressed in cells isolated from PT32 challenged calves. Lymph node cells showed increased expression of the proliferation marker Ki67 in CD4+ T cells from PT21/28, NK cells from PT32 and CD8+ and γδ T cells from both PT21/28 and PT32 challenged calves following ex vivo stimulation with T3SPs. Epitope mapping of rectal lymph node CD4+ T cell responses to 16 EHEC O157:H7 proteins, identified 20 CD4+ T cell epitopes specific to E. coli. The highly conserved N-terminal region of Intimin, including the signal peptide, was consistently recognised by mucosal CD4+ T cell populations from multiple animals of different major histocompatibility complex (MHC) class II haplotypes. Studies investigating the impact of secreted bacterial proteins on bovine peripheral blood mononuclear cells (PBMC) identified the ability of these proteins to cleave the surface molecule CD8 and that this phenotype was dependent on the ler virulence regulator but not the type III secretory system (T3SS) machinery. This effect was also observed in murine and ovine, but not human lymphocytes. Preliminary in vitro experiments suggest that this activity may reduce the efficiency of CD8+ T cell killing. This study demonstrates that cattle mount cellular immune responses during colonisation with EHEC O157:H7, the temporality of which is strain dependent, with further evidence of strain-specific immunomodulation.

Microorganisms are exposed to constantly changing environments, and consequently have evolved mechanisms to rapidly adapt their physiology upon stress imposition. These adaptive responses are coordinated through the rewiring of gene expression via complex networks that control the transcriptional program and the activity of post-transcriptional regulators. Although transcription factors primarily determine which genes are expressed, post-transcriptional regulation has a major role in fine-tuning the dynamics of gene expression. Post-transcriptional control is exerted by RNA-binding proteins and small regulatory RNAs (sRNAs) that bind to mRNA targets and modulate their synthesis, degradation and translation efficiency. In Escherichia coli, sRNAs associated with an RNA chaperone, Hfq, are key post-transcriptional regulators, yet the functions of most of these sRNAs are still unknown. The first step in understanding the roles of sRNAs in regulating gene expression is to identify their targets. To generate transcriptome-wide maps of Hfq-mediated sRNA-mRNA binding, we applied CLASH (cross-linking, ligation and sequencing of hybrids), a method that combines in vivo capture of RNA-RNA interactions, high-throughput sequencing and computational analyses, in E. coli. We uncovered thousands of dynamic growth-stage dependent association of Hfq to sRNAs and mRNAs. The latter confirmed known sRNA-target pairs and identified additional targets for known sRNAs, as well as novel sRNAs in various genomic features along with their targets. These data significantly expand our knowledge of the sRNA-target interaction networks in E.coli. In particular, the Hfq CLASH data indicated 3'-UTRs of mRNAs as major reservoirs of sRNAs, and the utilization of these may be more common than anticipated. Our findings also provide mechanistic insights that ensue from the identification of tens of sRNA-sRNA interactions that point to extensive sponging activity among regulatory RNAs: many sRNAs appear to be able to interact and repress the functions of other base-pairing sRNAs. We validated and highlighted the biological significance of some of the CLASH results by characterizing a 3'-UTR derived sRNA, MdoR (mal-dependent OMP repressor). This sRNA emerges by processing of the last transcript of malEFG polycistron, encoding components of maltose transport system. We found MdoR directly downregulates several major porins, whilst derepressing the maltose-specific porin LamB via destabilization of its inhibitor, MicA, likely by a sponging mechanism. Physiologically, MdoR contributes to the remodelling of envelope composition and links nutrient sensing to envelope stress responses during maltose assimilation. MdoR is a clear example of how cells integrate circuitry through multiple networks as part of their adaptive responses and how the CLASH methodology can help expand our understanding of sRNA-based regulation.