Análise investigativa dos fatores de virulência de cepas selvagens de shigella SPP. in vivo e seu potencial inflamatório

Abstract

Introduction: Shigellosis is a bacillary dysentery caused by Shigella, a gramnegative intracellular human pathogen. One of most common animal models to evaluate Shigella’s immune response is mice pulmonary infection due easy manipulation and similar gut responses. At this study, we have hypothesized how clinical strains isolated from Shigellosis patients had differential immune response expression when compared to standards strains (M90T). Our main proposition was analyze four clinical Shigella strains with distinct virulence genes and M90T immune responses at murine invasion after 24 and 48h infection. Methods: Shigella Clinical Strains were selected through presence of specific virulence genes by PCR. Primers related to immune system were designed. Clinical strains and M90T Shigella were submitted to expression at Fluidigm (Bioamark Platform). Results were analyzed in statistical software R. Results: We grouped analyzed mRNA in five gene sets: Pro Inflammatory (CXCL15, IL-1β, IL-6, IFN-β, TNF-α), Anti Inflammatory (TGF-β1, TGF- β2 e TGF-β3), Innate Response (NOD 1, NOD 2, TLR4 e TLR9), Adaptive Response (Th1-like, IFN-γ, IL-17A e IL-17B) and Macrophage (NOS, H2 -Aβ1, H2-Eβ, H2-K1, MHC-like 2). As main results, all clinical strains displayed a moderate mRNA expression in 24h infection which gets higher in 48h, while M90T standard had opposite regulation with lower mRNA rates in 48h infection, suggesting major differences between well characterized standards and clinical strains. Clinical strain #5 did not alter mRNA expression in 24 and 48h at adaptive immune response genes, suggesting low invasion rates and host control at initial steps of infection. Clinical strain #11 demonstrates high macrophage activity by superior expression levels of IFN-γ and NOS. Strains #14 and #27 suggest potential of Th1 protection through high MHC-like II and Th1 mRNA expression when compared to all other strains and standard control. Presence of IL-17 high expression in strain #27 demonstrates a possible relation with Th17 cells. The immunological potential of strain #27 may have relationship with Shigella enterotoxins (shET1A, shET1B, and shET2). Enterotoxins presence was confirmed by PCR and results shows this complete set is absent in all other strains studied. Conclusion: The differences between clinical strains and standards were evident at this study. Clinical strain 27 appears as a great candidate to future studies and may provide new perspectives about differences among invasion and immune response seen in the clinical practice.