Variabilidade genética dos principais fatores de virulência em amostras Clínicas de Shigella flexneri

Abstract

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.