Escherichia coli enteroinvasora (EIEC) e Shigella sp causam disenteria bacilar que é caracterizada pela invasão e destruição da mucosa do cólon humano. Amostras de EIEC possuem características bioquímicas, genéticas patogênicas semelhantes às espécies de Shigella, porém a doença causada por EIEC se apresenta numa forma mais branda e autolimitante. As células do epitélio intestinal participam ativamente da imunidade da mucosa, expressando e secretando uma série de mediad ores inflamatórios como citocinas, quimiocinas, moléculas de adesão e óxido nítrico. Para melhor entendimento da patogênese de EIEC, estudamos a resposta inflamatória modulada por este microrganismo em células epiteliais intestinais da linhagem Caco-2, comparando-a com Shigella flexneri. Células Caco-2 foram infectadas com EIEC ou S. flexneri por diferentes intervalos de tempo, para posterior analise da capacidade de invasão e disseminação bacterianas (UFC, PLAQUE ASSA Y), indução de morte celular (FACS), analise relativa de genes envolvidos no reconhecimento bacteriano e na resposta inflamatória (RT-PCR, RPA), dosagem de citocinas e quimiocinas pró-inflamatórias (ELISA) e óxido nítrico (GRIESS). Neste trabalho foi possível observar que: (i) a capacidade de disseminação e (ii) a indução da morte celular em células Caco-2 foi significativamente maior na infecção por S. flexneri do que EIEC; (iii) há diferenças em relação à expressão relativa de genes das células Caco-2 envolvidos no reconhecimento das duas cepas bacterianas. Foi evidenciado o papel essencial dos receptores intracelulares no reconhecimento bacteriano das células Caco-2, sendo a expressão relativa do mRNA do receptor intracelular Nod1 foi maior para EIEC quando comparado com S. flexneri; (iv) há diferenças significativas na cinética de produção de NO pelas células Caco¬2 infectadas, em que EIEC induziu mais precocemente a produção de NO quando comparado com S. flexneri. Estes dados sinalizam que as células epiteliais intestinais reconhecem e respondem de forma diferente frente a essas duas espécies bacterianas, apresentando uma resposta inflamatória mais eficiente no controle da infecção induzida por EIEC. / Escherichia coli enteroinvasive (EIEC) and Shigella sp cause bacillary dysentery which is characterized by the invasion and destruction of the human colon mucosa. Samples of EIEC have characteristics biochemical, genetic and pathogenic similar to those of Shigella species, however the disease caused by EIEC is more lenient. The cells of the intestinal epithelium actively participate in the mucosal immunity by expression and production of several inflammatory mediators such as cytokines, chemokines, adhesion molecules and nitric oxide. For better understanding of the EIEC pathogenesis, we studied the inflammatory response modulated by this microorganism in intestinal epithelial cells Caco-2, comparing it with Shigella flexneri. Caco-2 cells were infected with EIEC or S. flexneri during different intervals of time and analyzed the invasiveness and spread bacteria capacity (CFU, PLAQUE ASSAY), induction of cell death (FACS), analysis of genes involved in the recognition of bacterial and inflammatory response (RT-PCR, RPA), production of pro-inflammatory cytokines and chemokines (ELISA) and nitric oxide (NO) (GRIESS). In this work was possible to observe that: (i) the ability to spread and (ii) the induction of cell death in Caco-2 cells was significantly higher in S. flexneri infection than EIEC, (iii) there are differences regarding the relative expression of genes of Caco-2 cells involved in the recognition of two bacterial strains. It was highlighted the essential role of intracellular receptors in recognition of bacterial by Caco-2 cells, and the expression of mRNA of the intracellular receptor Nod 1 was higher for EIEC when compared with S. flexneri, (iv) there are significant differences in the kinetics of NO production by Caco-2 infected cells, EIEC induced a early NO production when compared with S. flexneri. These data indicate that the intestinal epithelial cells recognize and respond in a different way to these bacterial species and induce an inflammatory response more efficient in control of the infection induced by EIEC.

Neutrophile Granulocyten wirken als einer der ersten Abwehrmechanismen gegen invasive Mikroorganismen im angeborenen Immunsystem von Mammalia. Aktiviert durch inflammatorische Signale verlassen diese Granulocyten das vaskuläre System und migrieren durch das Gewebe zum Infektionsherd. Dort binden sie die Mikroorganismen, phagozytieren und eliminieren diese schließlich mit hoher Effizienz. Humane Neutrophile Elastase (NE) ist Bestandteil der neutrophilen Granula und spielt eine entscheidende Rolle im Abbau von Virulenzfaktoren enteroinvasiver Bakterien, einschließlich der Shigella Virulenzfaktoren IpaB (invasion antigen plasmid B) und IcsA (intracellular spread A). NE gehört zu der Familie der Chymotrypsin-ähnlichen Serinproteasen, die sich durch Sequenz- und Strukurähnlichkeit auszeichnen, jedoch sehr unterschiedliche biologische Funktionen aufweisen. Cathepsin G (CG) ist wie NE eine Chymotrypsin-ähnliche Serinprotease und ebenfalls in neutrophilen Granula lokalisiert. Allerdings zeigt CG keine Aktivität gegenüber Virulenzfaktoren von Shigella. Obwohl die Kristallstrukturen von CG und NE fast identisch sind, konnten einzelne oder mehrere Aminosäuren in der Substratbindungsspalte identifiziert werden, die zwischen den beiden Enzymen differieren. Dies legte die Vermutung nahe, dass die Spezifität von NE gegenüber Virulenzfaktoren in diesen Unterschieden codiert sein könnte. Daher wurden diese Aminosäuren durch die analogen CG Aminosäuren oder durch Alanin ersetzt. Der Vergleich der funktionellen Eigenschaften der NE Mutanten mit wildtyp NE zeigte, dass die Aminosäuren an den Positionen 98 und 216-224 entscheidend für die Substratspezifität von NE sind. Die NE Mutanten N98A, 216-218 und 216-224 waren nicht mehr in der Lage, die Virulenzfaktoren IcsA und IpaB sowie das NE Peptidsubstrat abzubauen. Stattdessen haben diese Mutanten die Fähigkeit erlangt, das CG Peptidsubstrat abzubauen. Zusammenfassend konnten wir Aminosäuren in NE identifizieren, die sowohl die Spezifität von NE für das Peptidsubstrat als auch für die Virulenzfaktoren von Shigella flexneri determinieren. / Neutrophil granulocytes are one of the first lines of defense of the mammalian innate immune system against invading microorganisms. In response to inflammatory stimuli, neutrophils migrate from the blood stream to infected tissues where they bind, engulf and inactivate microorganisms efficiently. Human neutrophil elastase (NE), a neutrophil granule component, is a key host defense protein that rapidly destroys virulence factors of enteroinvasive pathogens including IpaB (invasion plasmid antigen B) and IcsA (intracellular spread A) from Shigella. NE belongs to the family of chymotrypsin-like serine proteases with sequence and structural similarity but with very different biological functions. Cathepsin G (CG) is another abundant chymotrypsin-like serine protease in neutrophil granules. However, in contrast to NE, CG does not cleave virulence factors of Shigella. The crystallographic structures of NE and CG are very similar but we identified single or multiple residues in the substrate-binding cleft to differ in these two enzymes. We hypothesized that NE specificity for bacterial virulence factors resides within these structural differences. Therefore these specific residues in NE were replaced with the analogous amino acids of CG or with alanine. By comparing the functional properties of these NE mutants to wildtype NE we were able to show that the amino acids at position 98 and 216-224 are crucial for the substrate specificity of NE. The NE mutants N98A, 216-218 and 216-224 did not cleave the virulence factors IcsA and IpaB as well as the NE peptide substrate but cleaved the CG peptide substrate. In summary, we identified residues in NE that determine the specificity of NE for the peptide substrate and for the Shigella flexneri virulence factors.

Shigella spp. está entre as quatro bactérias mais isoladas de fezes diarreicas no Brasil. No mundo cerca de 164,7 milhões de casos de shigelose ocorrem anualmente, sendo a maioria em países em desenvolvimento. O gênero Shigella spp. possui quatro espécies, sendo Shigella sonnei e Shigella flexneri as espécies mais frequentemente isoladas no Brasil e no mundo. O monitoramento de linhagens resistentes de Shigella spp. é essencial, pois este garante uma terapia eficiente quando necessária. Especificamente, a maioria dos estudos realizados com linhagens de S. sonnei no país verificaram apenas a ocorrência dessa e há poucos estudos que investigaram o potencial patogênico e a diversidade genotípica dessa espécie. Os objetivos desse projeto foram analisar o potencial patogênico, o perfil de resistência a antimicrobianos e a diversidade genotípica de linhagens de S. sonnei isoladas durante três décadas no Estado de São Paulo. No total foram caracterizadas 72 linhagens de S. sonnei isoladas de humanos, entre os anos de 1983 a 2014, quanto à presença de 12 genes de virulência por PCR, perfil de suscetibilidade frente a 16 antimicrobianos por disco difusão e tipagem molecular por Pulsed-field gel electrophoresis (PFGE), Enterobacterial repetitve intergenic consensus PCR (ERIC-PCR), Multiple-locus variable-number tandem-repeat analysis (MLVA) e 20 linhagens tipadas por Multi-locus sequence typing (MLST). Todas as linhagens apresentaram os genes de virulência ipaH, iuc e sigA. O gene ipaBCD foi encontrado em 14 (19%) linhagens, os genes ial e virF em 13 (18%) linhagens e o gene sen em sete (10%) linhagens. Os genes set1A, set1B, pic, sat e sepA não foram detectados. As mais altas taxas de resistência foram frente à sulfametoxazol-trimetoprim encontrada em 42 (58,3%) linhagens e frente à tetraciclina encontrada em 30 (41,6%) linhagens. Onze (15,5%) linhagens foram resistentes à ampicilina e piperacilina. Três (4,2%) linhagens foram resistentes à cefotaxima. Três (4,2%) linhagens foram resistentes ao cloranfenicol. Duas (2,8%) linhagens foram resistentes à ampicilina-sulbactam. Duas (2,8%) linhagens foram resistentes ao ácido nalidíxico. Uma (1,4%) linhagem foi resistente à amoxicilina-ácido clavulânico. Cinco (7%) linhagens foram multidroga resistentes (MDR). O dendrograma gerado pelo PFGE agrupou as 72 linhagens de S. sonnei em dois clusters designados PFGE-A e PFGE-B. O cluster PFGE-A agrupou 39 linhagens isoladas entre 1983-2014 com uma similaridade >=73,6% e mais especificamente 35 dessas linhagens apresentaram uma similaridade >=80,3%. O cluster PFGE-B agrupou 33 linhagens de S. sonnei isoladas entre 1984-2014 com uma similaridade >=74,7% e 27 dessas linhagens exibiram uma similaridade >=83,0%. Similarmente, o dendrograma gerado pelo ERIC-PCR agrupou as 72 linhagens de S. sonnei em dois clusters designados ERIC-A e ERIC-B. O cluster ERIC-A agrupou 37 linhagens isoladas entre 1983-2014 que exibiram uma similaridade >=78,8% e mais especificamente 36 dessas linhagens apresentaram uma similaridade >=82,3%. O cluster ERIC-B agrupou 34 linhagens de S. sonnei isoladas entre 1987-2014 que exibiram uma similaridade >=84,0%. Também por MLVA as linhagens foram agrupadas em dois clusters designados MLVA-A e MLVA-B. O cluster MLVA-A agrupou 31 linhagens isoladas entre 1983-2014 com uma similaridade >=40%. O cluster MLVA-B agrupou 41 linhagens isoladas entre 1983-2014 com uma similaridade >=21,6%. Todas as 20 S. sonnei foram tipadas por MLST como ST152. Conclui-se que o potencial patogênico das linhagens estudadas foi destacado pela presença de importantes genes de virulência. A alta porcentagem de resistência para alguns antimicrobianos testados, tais como, sulfametoxazol-trimetoprim e tetraciclina é preocupante e pode levar à falha terapêutica. Os resultados da tipagem molecular sugerem que existam dois subtipos prevalentes nas linhagens de S. sonnei estudadas que se diferenciaram pouco geneticamente e contaminaram humanos durante 31 anos na região metropolitana de Ribeirão Preto no Estado de São Paulo. O resultado do MLST indica que as linhagens estudadas de Shigella sonnei isoladas no Brasil descendem de um precursor comum / Shigella spp. is among the four most isolated bacteria from diarrheal faeces in Brazil. In the world about 164.7 million cases of shigellosis occur annually, mostly in developing countries. The genus Shigella spp. comprises four species, being Shigella sonnei and Shigella flexneri the most frequently isolated species in Brazil and worldwide. The monitoring of resistant strains of Shigella spp. is essential and ensures an effective therapy when necessary. Specifically, the majority of the studies with S. sonnei performed in the country verified only the occurrence of this bacterium and there are few studies that investigated the pathogenic potential and genotypic diversity of this species. The aims of this project were to analyze the pathogenic potential, antimicrobial resistance profile and genotypic diversity of S. sonnei strains isolated during three decades in the State of São Paulo. In total, 72 of S. sonnei strains isolated from humans, between the years 1983-2014, were characterized for the presence of 12 virulence genes by PCR, resistance profile against 16 antimicrobials by disk diffusion and molecular typing by Pulsed-field gel electrophoresis (PFGE), Enterobacterial repetitve intergenic consensus PCR (ERIC-PCR), Multiple-locus variable-number tandem-repeat analysis (MLVA) and 20 strains typed by Multi-locus sequence typing (MLST). All the strains contained the ipaH, iuc and sigA genes. The ipaBCD gene was detected in 14 (19%) strains, the ial and virF genes in 13 (18%) strains and the sen gene in seven (10%) strains. The set1A, set1B, pic, sepA and sat genes were not detected. The highest resistance rates were against trimethoprim-sulfamethoxazole found in 42 (58.3%) strains and against tetracycline found in 30 (41.6%) strains. Eleven (15.5%) strains were resistant to ampicillin and piperacillin. Three (4.2%) strains were resistant to cefotaxime. Three (4.2%) strains were resistant to chloramphenicol. Two (2.8%) strains were resistant to ampicillin-sulbactam. Two (2.8%) strains were resistant to nalidixic acid. One (1.4%) strain was resistant to amoxicillin-clavulanic acid. Five (7%) strains were multidrug resistant (MDR). The dendrogram generated by PFGE grouped the 72 S. sonnei strains into two clusters designated PFGE-A and PFGE-B. The PFGE-A cluster comprised, 39 S. sonnei strains isolated between 1983 and 2014 with a similarity above 73.6% and more specifically 35 of those strains exhibited a similarity >= 80.3%. The PFGE-B cluster grouped, 33 S. sonnei strains isolated between 1984 and 2014 with a similarity above 74.7%, and 27 of those strains exhibited a similarity above 83.0.Similarly, the dendrogram generated by ERIC-PCR grouped the 72 S. sonnei strains into two clusters designated ERIC-A and ERIC-B. The ERIC-A cluster comprised, 37 S. sonnei strains isolated between 1983 and 2014 that exhibited a similarity above 78.8% and specifically 36 strains of those exhibited a similarity >= 82.3%. The ERIC-B cluster grouped, 34 S. sonnei strains isolated between 1987 and 2014 that exhibited a similarity above 84.0%. Also, by MLVA strains were grouped into two clusters designated MLVA-A and MLVA-B. The MLVA-A cluster comprised 31 strains isolated between 1983 and 2014 with a similarity >=40%. The MLVA-B cluster comprised 41 strains isolated between 1983 and 2014 with a similarity >=21.6%. All the 20 S. sonnei were typed by MLST as ST152. In conclusion, the possible pathogenic potential of the strains studied was highlighted by the presence of important virulence genes. The high percentage of resistance to some of the antimicrobials tested such as trimethoprim-sulfamethoxazole and tetracycline is worrying and may lead to therapeutic failure. Molecular typing results may suggest that there are two prevalent subtypes of S. sonnei strains studied that differed little genetically and have been contaminating humans over 31 years in the metropolitan region of Ribeirão Preto in the São Paulo State in Brazil. The result of MLST indicates that the Shigella sonnei strains studied isolated in Brazil descended from a common precursor

Escherichia coli enteroinvasora (EIEC) é um dos agentes etiológicos da disenteria bacilar, caracteriza-se pela destruição do epitélio do cólon provocado pela resposta inflamatória induzida após invasão da mucosa por bactérias. Cepas de EIEC são bioquímica, genética e patogênica semelhante a Shigella spp. A patogenicidade de EIEC e Shigella dependem da presença da pInv plasmídeo, que contém os genes necessários para a colonização bacteriana na mucosa intestinal. Recentemente, demonstrou-se que genes plasmidias ospC1, ospG e ospF de S. flexneri estão envolvidos na inibição da resposta inflamatória em células epiteliais intestinais, um fator importante na iniciação da colonização bacteriana e produção de doença. Como a EIEC mostrou doença menos grave, foi analisada as sequências de aminoácido, avaliada a transcrição destes genes plasmídiais e resposta inflamatória modulada por este micro-organismo na célula epitelial intestinal Caco-2. As células Caco-2 foram infectadas em momentos diferentes com 11 sorotipos de EIEC e S. flexneri (M90T). Os dados sobre a capacidade de invasão e sobrevivência de bactérias, expressão de genes de bactérias e da quimiocina IL-8 foram obtidos por CFU, RT-PCR, e ELISA, respectivamente. A significância estatística foi avaliada por ANOVA de dois fatores. Os 11 sorotipos de EIEC estudados apresentaram similaridade de 100% com S.flexneri para OspC1 e OspF,contudo, foram diferentes na homologia do OspG. Quando comparamos as sequências de aminoácido dos 11 sorotipos observamos 100% de similaridade entre eles para OspG, sugerindo o envolvimento destas proteínas na modulação da resposta imune induzida por estes micro-organismos. Os sorotipos de EIEC apresentam diferenças na capacidade de invasão dos enterócitos. Algumas diferenças significativas foram observadas na transcrição dos genes e na produção de IL-8. Os sorotipos de EIEC O29: H-e O167: H-apresentou um baixo transcrição de genes ospC1 e ospF, e um aumento significativo na produção de IL-8 quando comparado com outros sorotipos. Além disso, demonstrou que a maior transcrição destes genes por alguns sorotipos de EIEC parecem estar relacionados com a menor indução de IL-8. Estes dados sugerem que as proteínas OspC1 e OspF desempenham um papel na resposta inflamatória. No entanto, não se observou relação na transcrição ospG para a produção de IL-8. Estes resultados sugerem que as proteinas efetoras OspC1 e OspF estão envolvidas na inibição da resposta inflamatória em células epiteliais do intestino favorecendo a invasão da EIEC. / Enteroinvasive E. coli (EIEC) is one of the etiological agents of bacillary dysentery, it is characterized by the destruction of the colonic epithelium caused by the inflammatory response induced upon invasion of the mucosa by bacteria. Strains of EIEC are biochemical, genetic and pathogenic similar to Shigella spp. The pathogenecity of EIEC and Shigella depend on the presence of the plasmid pInv, which contains the genes necessary for bacterial colonization in the intestinal mucosa. Recently, it was demonstrated that the plasmid genes ospC1, ospG and ospF of S. flexneri are involved in inhibition of the inflammatory response in intestinal epithelial cells, an important factor in the initiation of bacterial colonization and production of disease. As EIEC has showed less severe disease, we evaluated the transcription of these plasmid genes and inflammatory response modulated by this microorganism in the intestinal epithelial cell Caco-2. The Caco-2 cells were infected in different times with 11 serotypes of EIEC and S. flexneri M90T strain. The data about sequences of amino acids, invasiveness and survival of bacteria, bacterial genes expression, and chemokine IL-8 were obtained by CFU, RT-PCR, and ELISA, respectively. The statistical significance was evaluated by two-way ANOVA. All EIEC serotypes studied showed 100% similarity with S.flexneri to OspC1 and OSPF, however, were different in the homology of OspG. Compared the amino acid sequences of the 11 serotypes observed 100% similarity between them to OspG, suggesting the involvement of them in modulating of the immune response induced by these microorganisms. There were no differences in the invasion the enterocytes among EIEC serotypes. However, some significant differences were observed in the transcription of those genes and production of IL-8. The EIEC serotypes O29:H- and O167:H- showed a low transcription of genes ospC1 and ospF, and a significant increase in production of IL-8 when compared with other serotypes. Furthermore, it was shown that the high transcription of ospF and ospC1 by some EIEC serotypes are related to low induction of IL-8. These data suggested that the proteins OspC1 and OspF play a role in the inflammatory response. However, we did not observed association between ospG transcription to the production of IL-8. These results lead us to believe that the effector proteins OspF and OspC1 are involved in inhibition of the inflammatory response in intestinal epithelial cells favoring the EIEC invasion.

Escherichia coli enteroinvasora (EIEC) é um dos agentes etiológicos da disenteria bacilar, caracteriza-se pela destruição do epitélio do cólon provocado pela resposta inflamatória induzida após invasão da mucosa por bactérias. Cepas de EIEC são bioquímica, genética e patogênica semelhante a Shigella spp. A patogenicidade de EIEC e Shigella dependem da presença da pInv plasmídeo, que contém os genes necessários para a colonização bacteriana na mucosa intestinal. Recentemente, demonstrou-se que genes plasmidias ospC1, ospG e ospF de S. flexneri estão envolvidos na inibição da resposta inflamatória em células epiteliais intestinais, um fator importante na iniciação da colonização bacteriana e produção de doença. Como a EIEC mostrou doença menos grave, foi analisada as sequências de aminoácido, avaliada a transcrição destes genes plasmídiais e resposta inflamatória modulada por este micro-organismo na célula epitelial intestinal Caco-2. As células Caco-2 foram infectadas em momentos diferentes com 11 sorotipos de EIEC e S. flexneri (M90T). Os dados sobre a capacidade de invasão e sobrevivência de bactérias, expressão de genes de bactérias e da quimiocina IL-8 foram obtidos por CFU, RT-PCR, e ELISA, respectivamente. A significância estatística foi avaliada por ANOVA de dois fatores. Os 11 sorotipos de EIEC estudados apresentaram similaridade de 100% com S.flexneri para OspC1 e OspF,contudo, foram diferentes na homologia do OspG. Quando comparamos as sequências de aminoácido dos 11 sorotipos observamos 100% de similaridade entre eles para OspG, sugerindo o envolvimento destas proteínas na modulação da resposta imune induzida por estes micro-organismos. Os sorotipos de EIEC apresentam diferenças na capacidade de invasão dos enterócitos. Algumas diferenças significativas foram observadas na transcrição dos genes e na produção de IL-8. Os sorotipos de EIEC O29: H-e O167: H-apresentou um baixo transcrição de genes ospC1 e ospF, e um aumento significativo na produção de IL-8 quando comparado com outros sorotipos. Além disso, demonstrou que a maior transcrição destes genes por alguns sorotipos de EIEC parecem estar relacionados com a menor indução de IL-8. Estes dados sugerem que as proteínas OspC1 e OspF desempenham um papel na resposta inflamatória. No entanto, não se observou relação na transcrição ospG para a produção de IL-8. Estes resultados sugerem que as proteinas efetoras OspC1 e OspF estão envolvidas na inibição da resposta inflamatória em células epiteliais do intestino favorecendo a invasão da EIEC. / Enteroinvasive E. coli (EIEC) is one of the etiological agents of bacillary dysentery, it is characterized by the destruction of the colonic epithelium caused by the inflammatory response induced upon invasion of the mucosa by bacteria. Strains of EIEC are biochemical, genetic and pathogenic similar to Shigella spp. The pathogenecity of EIEC and Shigella depend on the presence of the plasmid pInv, which contains the genes necessary for bacterial colonization in the intestinal mucosa. Recently, it was demonstrated that the plasmid genes ospC1, ospG and ospF of S. flexneri are involved in inhibition of the inflammatory response in intestinal epithelial cells, an important factor in the initiation of bacterial colonization and production of disease. As EIEC has showed less severe disease, we evaluated the transcription of these plasmid genes and inflammatory response modulated by this microorganism in the intestinal epithelial cell Caco-2. The Caco-2 cells were infected in different times with 11 serotypes of EIEC and S. flexneri M90T strain. The data about sequences of amino acids, invasiveness and survival of bacteria, bacterial genes expression, and chemokine IL-8 were obtained by CFU, RT-PCR, and ELISA, respectively. The statistical significance was evaluated by two-way ANOVA. All EIEC serotypes studied showed 100% similarity with S.flexneri to OspC1 and OSPF, however, were different in the homology of OspG. Compared the amino acid sequences of the 11 serotypes observed 100% similarity between them to OspG, suggesting the involvement of them in modulating of the immune response induced by these microorganisms. There were no differences in the invasion the enterocytes among EIEC serotypes. However, some significant differences were observed in the transcription of those genes and production of IL-8. The EIEC serotypes O29:H- and O167:H- showed a low transcription of genes ospC1 and ospF, and a significant increase in production of IL-8 when compared with other serotypes. Furthermore, it was shown that the high transcription of ospF and ospC1 by some EIEC serotypes are related to low induction of IL-8. These data suggested that the proteins OspC1 and OspF play a role in the inflammatory response. However, we did not observed association between ospG transcription to the production of IL-8. These results lead us to believe that the effector proteins OspF and OspC1 are involved in inhibition of the inflammatory response in intestinal epithelial cells favoring the EIEC invasion.

Escherichia coli enteroinvasora (EIEC) e Shigella sp causam disenteria bacilar que é caracterizada pela invasão e destruição da mucosa do cólon humano. Amostras de EIEC possuem características bioquímicas, genéticas patogênicas semelhantes às espécies de Shigella, porém a doença causada por EIEC se apresenta numa forma mais branda e autolimitante. As células do epitélio intestinal participam ativamente da imunidade da mucosa, expressando e secretando uma série de mediad ores inflamatórios como citocinas, quimiocinas, moléculas de adesão e óxido nítrico. Para melhor entendimento da patogênese de EIEC, estudamos a resposta inflamatória modulada por este microrganismo em células epiteliais intestinais da linhagem Caco-2, comparando-a com Shigella flexneri. Células Caco-2 foram infectadas com EIEC ou S. flexneri por diferentes intervalos de tempo, para posterior analise da capacidade de invasão e disseminação bacterianas (UFC, PLAQUE ASSA Y), indução de morte celular (FACS), analise relativa de genes envolvidos no reconhecimento bacteriano e na resposta inflamatória (RT-PCR, RPA), dosagem de citocinas e quimiocinas pró-inflamatórias (ELISA) e óxido nítrico (GRIESS). Neste trabalho foi possível observar que: (i) a capacidade de disseminação e (ii) a indução da morte celular em células Caco-2 foi significativamente maior na infecção por S. flexneri do que EIEC; (iii) há diferenças em relação à expressão relativa de genes das células Caco-2 envolvidos no reconhecimento das duas cepas bacterianas. Foi evidenciado o papel essencial dos receptores intracelulares no reconhecimento bacteriano das células Caco-2, sendo a expressão relativa do mRNA do receptor intracelular Nod1 foi maior para EIEC quando comparado com S. flexneri; (iv) há diferenças significativas na cinética de produção de NO pelas células Caco¬2 infectadas, em que EIEC induziu mais precocemente a produção de NO quando comparado com S. flexneri. Estes dados sinalizam que as células epiteliais intestinais reconhecem e respondem de forma diferente frente a essas duas espécies bacterianas, apresentando uma resposta inflamatória mais eficiente no controle da infecção induzida por EIEC. / Escherichia coli enteroinvasive (EIEC) and Shigella sp cause bacillary dysentery which is characterized by the invasion and destruction of the human colon mucosa. Samples of EIEC have characteristics biochemical, genetic and pathogenic similar to those of Shigella species, however the disease caused by EIEC is more lenient. The cells of the intestinal epithelium actively participate in the mucosal immunity by expression and production of several inflammatory mediators such as cytokines, chemokines, adhesion molecules and nitric oxide. For better understanding of the EIEC pathogenesis, we studied the inflammatory response modulated by this microorganism in intestinal epithelial cells Caco-2, comparing it with Shigella flexneri. Caco-2 cells were infected with EIEC or S. flexneri during different intervals of time and analyzed the invasiveness and spread bacteria capacity (CFU, PLAQUE ASSAY), induction of cell death (FACS), analysis of genes involved in the recognition of bacterial and inflammatory response (RT-PCR, RPA), production of pro-inflammatory cytokines and chemokines (ELISA) and nitric oxide (NO) (GRIESS). In this work was possible to observe that: (i) the ability to spread and (ii) the induction of cell death in Caco-2 cells was significantly higher in S. flexneri infection than EIEC, (iii) there are differences regarding the relative expression of genes of Caco-2 cells involved in the recognition of two bacterial strains. It was highlighted the essential role of intracellular receptors in recognition of bacterial by Caco-2 cells, and the expression of mRNA of the intracellular receptor Nod 1 was higher for EIEC when compared with S. flexneri, (iv) there are significant differences in the kinetics of NO production by Caco-2 infected cells, EIEC induced a early NO production when compared with S. flexneri. These data indicate that the intestinal epithelial cells recognize and respond in a different way to these bacterial species and induce an inflammatory response more efficient in control of the infection induced by EIEC.

Shigella provoque la dysenterie bacillaire en envahissant les muqueuses du colon. Cette maladie diarrhéique est responsable d’un million de décès par an essentiellement dans les pays en voie développement. Les gènes nécessaires àl’entrée dans les cellules hôtes sont regroupés sur un fragment d’ADN plasmidique de 30-kb. Celui-ci contient deux types de gènes, les gènes ipa(B, C et D) et ipgcodant pour des protéines responsables de l’entrée de la bactérie dans les cellules, et les gènes mxi/spacodant pour un système de sécrétion appelétype III (SST3) nécessaire àla sécrétion des facteurs de virulence. Les gènes mxi/spaetipa/ipgsont exprimés à37°C et les protéines Ipa/Ipgrestent dans le cytoplasme jusqu’àce que le SST3 soit activéau contact de la cellule hôte. Ce contact induit l’internalisation de la bactérie par macropinocytose, suivie de sa dissémination intra-et intercellulaire. Des observations en microscopie électronique (ME) montrent que le SST3 est composéde trois parties: i) une aiguille dont la longueur est régulée à50 nm par la protéine Spa32, ii) un corps basal qui traverse les membranes interneet externe ainsi que le peptidoglycane, et iii) un bulbe cytoplasmique. Le SST3 est le dispositif principal de virulence et permet l’injection de facteurs de virulences du cytoplasme bactérien vers celui de la cellule cible. Shigelladoit sécréter ces protéines de manière ordonnée. Très peu de travaux ont abordécette question. L’objectif principal de ce travail de thèse a étéd’étudier les mécanismes moléculaires impliqués dans la hiérarchie de sécrétion.Nous avons principalement investiguéle rôle de 3 protéines: Spa32, Spa40 et MxiC dans la sécrétion. Nous avons montré, par des études génétiques, que contrairement aux études publiées sur les protéines homologues, Spa32 n’agit pas comme un «molecularruler»pour réguler la taille de l’aiguille. Nous avons montréque cette régulation nécessite l’interaction de Spa32 via ses résidus 206-246 au domaine C-terminal de Spa40 (Spa40C) (Botteaux et al. 2008a). Ayant identifiécette interaction avec Spa40, l’étape suivante de notre travail a portésur la caractérisation de la fonction du gène spa40par des méthodes génétique, biochimique et structurale (ME). Nos résultats montrent que Spa40 joue un rôle important dans l’assemblage du SST3. Des plus, nous avons mis en évidence de nouvelles interactions impliquant Spa40C et des composants du SST3 (Botteaux et al. in preparation).Parallèlement àces travaux, nous avons montréque l’inactivation du gène mxiCaboutit àune dérégulation spécifique de la sécrétion des effecteurs sans altérer celle des translocateurs IpaB et IpaC. Cette augmentation de sécrétion est due àune augmentation de transcription des gènes tardifs, conséquence de la sécrétion précoce de l’anti-activateur transcriptionnel, OspD1 (Botteaux et al. 2008b). De plus, nous avons montréque MxiC est un substrat de l’appareil de sécrétion et que cette sécrétion est associée àsa fonction. Finalement, la mise en évidence d’une interaction entre MxiC et Spa47, l’ATPase du SST3 nous permet de proposer un modèle régulant la hiérarchie de sécrétion des effecteurs.Dans une autre partie de notre travail, nous avons identifié, par des expériences de ME et d’immunomarquage, que l’invasineIpaD est localisée au sommet de l’aiguille du SST3 oùelle lui sert de bouchon. Enfin, de manière très intéressante, nous avons montréque des anticorps anti-IpaD neutralisent l’entrée de Shigelladans les cellules (Sani, Botteaux et al. 2007, dépôt de brevet). IpaD, étant conservée dans les isolats invasifs de Shigella, représente donc un réel candidat vaccinal pouvant pallier la diversitédes sérotypesbactériens.En conclusion, nos travaux représentent une contribution importante àla compréhension des mécanismes de virulence bactériens et dépassent le cadre de Shigellapuisque les systèmes de sécrétion sont hautement conservés parmi plusieurs pathogènes. L’identification de drogues pouvant interférer avec ces systèmes de sécrétion représente une voie d’avenir pour le développement de nouveaux agents anti-infectieux. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

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Die zellulären Rho GTPasen kontrollieren und regulieren zentrale elementare Zellvorgänge wie Phagozytose, Migration und epitheliale Integrität. Aufgrund ihrer zentralen Stellung, interagiert eine Vielzahl von bakteriellen Cytotoxinen und Modulinen mit den Rho GTPasen und wirken so als Pathogenitätsfaktoren. Die zur W-xxx-E Familie gehörenden Effektoren IpgB1 und IpgB2 von Shigella und Map von E. coli (Pathotypen EHEC und EPEC) werden über ein Typ 3 Sekretionssystem (T3SS) in Wirtszellen injiziert und wirken als Rac1, RhoA bzw. Cdc42 GEF Mimetikum. In der vorliegenden Arbeit wurden die Effektor Funktionen von IpgB1 IpgB2 und Map mit Hilfe des Yersinia (Ysc)-T3SS untersucht, was zur Etablierung der „Yersinia-Toolbox“ führte. Damit können heterologe Effektoren isoliert im physiologischen Kontext der Erreger-Zell-Interaktion zellbiologisch untersucht werden unter Vermeidung von simultaner Injektion redundanter oder unbekannter Effektoren. Zur Etablierung der Yersinia-Toolbox wurden zunächst die Gene für die Rho GTPasen modulierenden Shigella Effektoren IpgB1 und IpgB2 sowie der E. coli (EHEC)-Effektor Map mit unterschiedlich langen Gensequenzen der N-terminalen Bereiche des Yersinia-Effektorproteins YopE fusioniert (Hybridproteine: YopEi-X:i = 18, 53 bzw. 138 Aminosäurereste, X = IpgB1, IpgB2 bzw. Map). In der vorliegenden Arbeit wird gezeigt, dass die Hybridproteine YopE53-X und YopE138-X (X=IpgB1, IpgB2, Map) in den Kulturüberstand sezerniert bzw. in Zielzellen injiziert wurden. In einem weiteren Schritt konnte die zellbiologische Aktivität der heterologen Proteine fluoreszenzmikroskopisch durch Aktinzytoskelettumlagerungen gezeigt werden. So wurden „Membrane Ruffles“ (Rac1-Aktivierung) durch YopE138-IpgB1, Stressfasern (RhoA-Aktivierung) durch E138-IpgB2 und „Mikrospikes“ (Cdc42-Aktivierung) durch YopE138-Map nachgewiesen. Invasionstudien zeigten, dass YopEi-IpgB1 (i = 53, 138) die Yersinia-Invasion induzierte, wohingegen YopEi-IpgB2 die Invasionsrate der Stämme WA (pT3SS, pEi-IpgB2) (i=53, 138) verglichen mit dem Stamm WA (pT3SS) reduziert war. Durch Kombination verschiedener Yersinia-Toolbox-Stämme konnte im Co-Infektionsmodell mit HeLa-Zellen gezeigt werden, dass (1) die YopE138-IpgB1 vermittelte Invasion durch YopE138-IpgB2 signifikant inhibiert werden kann, was auf eine antagonistische Wirkung zwischen IpgB1 und IpgB2 schließen lässt, dass (2) YopT ebenfalls die IpgB1 vermittelte Invasionsrate reduziert (inhibitorische Wirkung auf Rac1), und dass (3) YopE als GAP für RhoG/Rac1 (bevorzugt RhoG) praktisch nicht die IpgB1-vermittelte Invasion hemmt. Durch Klonierung der YopE138-IpgB1 und YopE138-IpgB2 kodierenden Fusionsgene in zwei kompatible Plasmidvektoren konnten die Hybridproteine simultan transloziert werden und die Co-Infektionsergebnisse bestätigt werden. In der Literatur ist beschrieben, dass die Ysc-Translokationspore YopB/YopD Rho-abhängig Membranporen-bedingte Zellschädigungen verursacht (LDH-Freisetzung, PI-Kernfärbung). Mit der Yersinia-Toolbox konnte mit dem Stamm WA (pT3SS) Zytoplasmamembranschädigung / Zytotoxizität nachgewiesen werden, nicht aber mit den Stämmen WA (pE138-X) X = IpgB1, IpgB2 oder Map. Co-Infektionen jedoch zeigen, dass vermehrt LDH bei der Infektion mit WA (pT3SS) + WA (pT3SS, pE138-IpgB1) detektiert wurde, wohingegen dieser Effekt von YopE138-IpgB2 in einer Co-Infektion von WA (pT3SS) + WA (pT3SS, pE138-IpgB2) inhibiert wurde. Auch hier wurde der Antagonismus zwischen IpgB1 und IpgB2 erneut sichtbar. Diese Befunde widersprechen publizierten Daten, die eine RhoA-Aktivierung/Aktinpolymerisierung mit verstärkter Porenbildung in einen Zusammenhang bringen. Rho GTPasen sind beteiligt an der Erhaltung der polarisierten Eipthelzellschichtintegrität über Adhäsionskomplexbildung. Mittels Infektion von polarisierten MDCK-Zellschichten mit verschiedenen Yersinia-Stämmen und Messung des transepithelialen elektrischen Widerstandes/Resistenz (TER) konnte gezeigt werden, dass die Ysc-T3SS vermittelte Injektion von YopE138-IpgB1 (Rac1-Aktivierung) oder YopE138-Map (Cdc42-Aktivierung) zur Abnahme der TER und damit Schädigung der Zellschichtintegrität führt, wogegen bei YopE138-IpgB2-Injektion der TER-Wert unverändert blieb. Um bakterielle Rho GTPasen-modulierende Effektorproteine detailliert untersuchen zu können und um die Rolle von Rho GTPasen im Mausinfektionsmodell mit Yersinia enterocolitica und Salmonellen zu bestimmen, wurden Mäuse mit deletierten Genen für RhoA, Rac1 bzw. Cdc42 in Makrophagen hergestellt. / Phagocytosis, migration and regulation of epithelial integrity are central cellular aspects that are controlled by the cellular Rho GTPases. In this regard, Rac1, RhoA and Cdc42 have important regulatory roles mediating various cytoskeletal rearrangements in many cell types including epithelial cells as well as professional phagocytes. Because of the central role of the Rho GTPases in cellular integrity and function, bacterial cytotoxins and modulins targeting these cellular switches are very efficient pathogenicity factors. Recently, the T3SS effectors, IpgB1, IpgB2 of Shigella and Map of E. coli (pathotype EHEC/EPEC) were assembled in one protein family sharing the common motif W-xxx-E. Members of this protein family are described to act as GEF mimics for the cellular Rho GTPases. In this study the effector functions of IpgB1, IpgB2 and Map were analyzed with the Yersinia (Ysc)-T3SS which led to the development of the “Yersinia-Toolbox”. Yersinia enterocolitica is very suitable to be used as “T3SS-Toolbox” because (1) a plasmid solely carrying the DNA fragment encoding the Ysc-T3SS without T3SS-effectors is available, (2) in difference to Salmonella and E. coli (EPEC/EHECH) the Ysc-T3SS-effector genes of Yersinia are not localized on the chromosome and (3) heterologous proteins fused to the Ysc-T3SS-effector YopE are secreted and translocated into cells. This allows the analysis of single heterologous effectors without simultanous injection of other (unknown/redundant) T3SS-effectors in a physiological context during the interaction of Yersinia with cells. To develop the Yersinia-Toolbox, the genes of the GTPase modulating effectors IpgB1, IpgB2 of Shigella and Map of E. coli (EHEC) were fused to different long sections of the N-Terminus of the Yersinia-Ysc-T3SS-effector YopE (hybrid proteins: YopEi-X: i = 18, 53 or 138 amino acid residues, X = IpgB1, IpgB2 or Map). This study demonstrates the secretion to the culture supernatent and the injection into target cells of the hybrid proteins YopE53-X and YopE138-X (X = IpgB1, IpgB2 and Map). Furthermore, cell biologic activity was detected for the YopE-X hybrid proteins by fluorescence microscopy as membrane ruffles (Rac1 activation), stress fibres (RhoA activation) and micro spikes (Cdc42 activation) occurred after injection of YopE138-IpgB1,.YopE138-IpgB2 and YopE138-Map, in respective. Invasion studies showed that YopEi-IpgB1 (i = 53, 138) induced invasion of Yersinia, whereas YopEi-IpgB2 reduced invasion of the strains WA (pT3SS, pEi-IpgB2) (i = 53, 138) compared to the strain WA (pT3SS). Combination of different Yersinia-Toolbox strains in the co-infection model with HeLa cells showed that (1) YopE138-IpgB2 reduced the YopE138-IpgB1 induced invasion suggesting an antagonism between IpgB1 and IpgB2, (2) YopT also reduced the YopE138-IpgB1 induced invasion (inhibitory function on Rac1) and (3) that YopE as GAP for RhoG/Rac1 (predominantly RhoG) did not inhibit the YopE138-IpgB1 induced invasion. Because of the construction of two different compatible plasmids carrying the genes for either YopE138-IpgB1 or YopE138-IpgB2, simultanous translocation of the hybrid proteins of one single strain was possible. These studies confirmed the results of the co-infection studies. It has been reported that the Ysc translocation pore YopB/YopD induces Rho dependent membrane pores in cells which leads to cellular damage (LDH release, PI-staining of the nucleus). In this study cellular damage / cytotoxicity was detected after an infection of HeLa cells with the Yersinia-Toolbox strain WA (pT3SS). In contrast to that no cytotoxicity was detected after an infection of HeLa cells with the Yersinia-Toolbox strains WA (pT3SS, pE138-X) X = IpgB1, IpgB2 and Map. Additionally, co-infections with the strains WA (pT3SS) and WA (pT3SS, pE138-IpgB1) resulted in an increased LDH release whereas a co-infection with the strains WA (pT3SS) and WA (pT3SS, pE138-IpgB2) led to the decrease of LDH release compared to single infections with WA (pT3SS), again suggesting an antagonism between IpgB1 and IpgB2. These results are contrary to published data, which suggest a correlation between RhoA activation dependent actin polymerisation and pore formation. The cellular Rho GTPases are involved in the maintenance of epithelial integrity of polarized cells. Infections of polarized MDCK cell layers with different Yersinia-Toolbox strains resulted in a decrease of the transepithelial electric resistance (TER) indicating a damage of the epithelial integrity after injection of YopE138-IpgB1 or YopE138-Map. The TER value was not altered after injection of YopE138-IpgB2 indicating an intact epithelial integrity. To study bacterial Rho GTPase modulating proteins in more detail and to get a deeper insight to the role of Rho GTPases in the murine infection model with Yersinia enterocolitica and Salmonella, mice with gene deletions for RhoA, Rac1 or Cdc42 in macrophages were constructed.

Shigella flexneri is a highly adapted pathogen that invades the host cytosol and causes bacillary dysentery. Shigella has evolved powerful countermeasures to disarm host defense mechanisms; amongst them a family of twelve bacterial E3 ubiquitin ligases (IpaH) that are structurally unrelated to eukaryotic enzymes. IpaH ligases are injected into the host cytosol via the bacterial type III secretion system (T3SS) to manipulate the host cell and counteract anti-bacterial defense pathways. My work demonstrated that IFN-induced guanylate-binding proteins (GBPs) are novel targets for IpaH9.8. GBPs inhibit actin-dependent motility and cell-to-cell spread of bacteria unless they are ubiquitylated by IpaH9.8 and consequently degraded by the proteasome. IpaH9.8 targets GBP1, GBP2, and GBP4, thereby causing a transient poly-ubiquitin coat comprising K48 and K27-linked chains around S. flexneri, which leads to the proteasome-dependent destruction of existing GBP coats and the re-establishment of bacterial motility and cell-to-cell spread. So far, ubiquitylation of bacteria has mostly been associated with anti-bacterial autophagy or immune signaling. However, the ubiquitin coat assembled around intracellular Shigella by IpaH effectors, in particular IpaH9.8, serves a pro-bacterial function, the first observed so far. In addition, I characterized IpaH1.4 and IpaH2.5 for their ability to prevent NF-κB activation by targeting LUBAC. I found that IpaH1.4 specifically binds the LUBAC component HOIP and mediates its proteasomal degradation, thus abolishing linear ubiquitylation of bacteria and consecutive NF-κB activation via NEMO and autophagy induction via optineurin. Lastly, I identified novel potential ubiquitylation targets for IpaH effectors in human cells using a mass spectrometry-based approach. The resulting IpaH interactome presents the groundwork for further investigations and will help to identify potentially unknown cellular defense mechanisms that are antagonized by Shigella flexneri.