The conserved mechanism of IcsA polar targeting among proteobacteria, characterization of the omptin family, and the roles and regulation of IcsP in Shigella flexneri
Shigella flexneri is a gram-negative intracellular pathogen that is a global health problem, causing severe bacillary dysentery. In the past 30 years, research has enabled us to have a better understanding of the molecular basis of Shigella pathogenesis. In this study, we further expand our knowledge of key determinants at molecular and cellular levels. This work is composed of four separate investigations; Firstly, we examined whether the elusive mechanism of protein targeting found in Shigella is conserved in three closely related gamma-proteobacterial families by using GFP fusions and microscopy. Our results indicate that the mechanism of protein localization to the bacterial pole was conserved in the species investigated. Secondly, we further characterized omptin proteins found in three pathogenic organisms: Shigella (IcsP), E. coli (OmpT), and Salmonella (PgtE). We addressed whether the LPS environment and/or the inherent amino acid differences in the surface loops and LPS binding motif affect the cleavage specificity of these proteases by introducing a known substrate into each of these strains and comparing cleavage patterns by western blot analysis. Our results indicate that the cleavage specificity of these omptins are likely due to the inherent amino acid differences in the surface loops and/or LPS binding region. Thirdly, we examined whether IcsP, an outer membrane protease in Shigella, promotes resistance to a cationic antimicrobial peptide, LL-37 by using minimum inhibitory concentration assays. Our results suggest that IcsP does not promote resistance to LL-37. Finally, we examined whether icsP was regulated by RyhB, a small regulatory RNA, by western blot analysis and beta-galactosidase assays. Our results suggest that RyhB does not down-regulate the expression of icsP in S. flexneri; Taken together, this study has improved our understanding of Shigella pathogenesis and omptin family proteases. Ultimately, the relevance of this study has potential use for the development of prevention and intervention strategies against specific bacterial infections and disease
