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Dantas.pdf: 4741082 bytes, checksum: e499e75b7a77189538a9f8be0d6be226 (MD5) Previous issue date: 2016-11-18 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Shigella is a gram-negative and intracellular bacterium responsible for triggering Shigellosis, a diarrheal disease characterized by the presence of mucus and blood. . In an epidemiological study conducted by our group in the city of Manaus-Amazonas with children with severe diarrhea from 2007 to 2009, the genus Shigella was the fifth most isolated pathogen (2.2%). Based on the clinical data we observed a heterogenous symptomatology among patients with shigellosis, since then, our group has been investigating the genotypic and phenotypic characteristics of these isolates. Shigella is a highly virulent bacterium, its mechanisms of pathogenesis is due to the presence of plasmidial and chromosomal genes. Among the genes responsible for its virulence are the Ipas genes that promote the invasion of Shigella in epithelial cells and the death of macrophages, the IpaHs genes, which modulate the host's immune response and the enterotoxins Shet1 and Shet2. In the present study, our main objective was to verify the genetic variability of the main virulence factors in clinical samples of Shigella flexneri. The virulence genes were amplified with specific primers developed in the study, the obtained amplicons were sequenced and analyzed. The Ipa, IpaH, Set1A, Se1B and Sen genes were successfully amplified. A total of 474 sequences referring to the 19 virulence genes of Shigella were sequenced, however, only 12 genes, a total of 154 sequences were analyzed for the presence of polymorphism. In this study 9 point mutations were identified in 5 Shigella virulence genes, being the IpaD, SenA, IpaH3, IpaH2.5 and IpaH6 gene. The study of the identification of specific genetic determinants is essential to understand the mechanism of pathogenesis and the propagation of virulent variants in the population of Shigella. Our work is based on previous studies conducted by our group, since the initial amplification of the virulence genes of these isolates was ineffective. Although few genes are analyzed, nucleotide variations have been found in different isolates, which may or may not interfere in the expression of the virulence phenotype. A new sequencing will be required to confirm the variations found in the study, as well as in vitro and in vivo studies, to verify if these changes may interfere with the virulence phenotype of the Shigella clinical samples. / A Shigella é uma bactéria gram-negativa e intracelular, responsável pelo desencadeamento da Shigelose, uma doença diarreica caracterizada pela presença de muco e sangue. Em um estudo epidemiológico realizado por nosso grupo na cidade de Manaus-Amazonas com crianças que apresentavam diarreia severa nos anos de 2007 a 2009, o gênero Shigella foi o quinto patógeno mais isolado (2,2%). A partir dos dados clínicos observamos uma sintomatologia heterogênia entre os pacientes com shigelose, desde então, nosso grupo vem investigando as características genotípicas e fenotípicas desses isolados. Shigella é uma bactéria altamente virulenta, seus mecanismos de patogenicidade se deve a presença de genes plasmidiais e cromossomais. Entre os genes responsáveis pela sua virulência temos, os genes Ipas que promovem a invasão de Shigella nas células epiteliais e a morte dos macrófagos, os genes IpaHs, que modulam a resposta imunológica do hospedeiro e as enterotoxinas Shet1 e Shet2. No presente estudo, nosso principal objetivo foi verificar a variabilidade genética dos principais fatores de virulência em amostras clínicas de Shigella flexneri. Os genes de virulência foram amplificados com iniciadores específicos desenvolvidos no estudo, os amplicons obtidos foram sequenciados e analisados. Os genes Ipa, IpaH, Set1A, Se1B e Sen foram amplificados com sucesso. Um total de 474 sequências referentes aos 19 genes de virulência de Shigella foi sequenciado, no entanto, apenas 12 genes, num total de 154 sequências foram analisadas quanto à presença de polimorfismo. Neste estudo foram identificadas 9 mutações de ponto em 5 genes de virulência de Shigella, sendo eles, o gene IpaD, SenA, IpaH3, IpaH2.5 e IpaH6. O estudo da identificação de determinantes genéticos específicos é essencial para compreender o mecanismo de patogênese e a propagação de variantes virulentas na população de Shigella. Nosso trabalho se baseia em estudos anteriores realizados por nosso grupo, uma vez que a amplificação inicial dos genes de virulência desses isolados foi ineficaz. Apesar de poucos genes serem analisados, variações de nucleotídeos foram encontradas em isolados diferentes, que podem interferir na expressão do fenótipo de virulência. Um novo sequenciamento será necessário para confirmar as variações encontradas no estudo, assim como estudos in vitro e in vivo, para verificar se essas alterações podem interferir no fenótipo de virulência das amostras clinicas de Shigella.

Shigellae are pathogenic bacteria that cause the disease shigellosis. Two methods for studying secreted effectors encoded by this pathogen’s virulence plasmid are described. First, protein microarrays were used to identify substrates of an E3 ubiquitin ligase called IpaH7.8. Second, a deletion collection containing mutants for every gene on the virulence plasmid was used in two screens: one to identify mutants that elicit atypical levels of Interleukin-8 (IL-8) from U937 cells, and one to identify mutants that bind the dye Congo red abnormally. Although protein microarrays were an ineffective tool, the deletion collection proved valuable. Most mutants were less effective at sequestering Congo red than wild-type S. flexneri, although this ability was enhanced in several mutants. Four mutants, ?ospB, ?orf186, ?mxiH and ?mxiK, elicited higher levels of IL-8 from U937 cells than wild type S. flexneri. These results validate the use of the deletion collection as a tool for studying bacterial pathogenesis.