Probing the effects of lipid phase order on the localization of Shigella type three secretion system translocon proteins in artificial membranes
Class
Article
Department
Chemistry and Biochemistry
Faculty Mentor
Nick Dickenson
Presentation Type
Poster Presentation
Abstract
Shigella spp. are Gram-negative, non-motile bacteria that infect humans and higher order primates via a type three secretion system (TTSS). The normal infection includes severe dehydration through bacillary dysentery and can lead to permanent damage of the colonic epithelium. Many aspects of Shigella virulence, including the maturation of the T3SS apparatus, have been studied extensively, however, the events surrounding the initial host-pathogen interaction and its role in cellular uptake and infection remain unclear. Here, we have developed a series of techniques allowing us to directly visualize the interactions between key Shigella T3SS tip proteins and effectors with defined artificial phospholipid membranes. A custom total internal reflection fluorescence microscope was equipped with an atomic force microscope piezoelectric scanning head and stage. This instrument has allowed us to identify favorable conditions for generating artificial phospholipid membranes using the Langmuir Blodgett technique. By varying the pressures and composition of the lipid films, we have been able to mimic the mixed phase order conditions that likely exist within eukaryotic cell membranes. The use of lipophilic dyes and selective fluorescent labeling of the Shigella T3SS translocon proteins, believed to be directly involved in host cell contact (IpaB and IpaC), has allowed us to monitor the lipid phase order of our artificial membranes and protein localization/co-localization within the same films, respectively. Consequently, the use of such fluorophores and dyes, when carefully selected, allows us the ability to conduct Förster resonance energy transfer (FRET) studies, and quantify the protein-lipid interactions. The incorporation of cholesterol and sphingolipids into the membranes moves us one step closer to truly mimicking a cellular environment and helps determine what role it plays in protein localization and ultimately Shigella infection.
Start Date
4-9-2015 1:30 PM
Probing the effects of lipid phase order on the localization of Shigella type three secretion system translocon proteins in artificial membranes
Shigella spp. are Gram-negative, non-motile bacteria that infect humans and higher order primates via a type three secretion system (TTSS). The normal infection includes severe dehydration through bacillary dysentery and can lead to permanent damage of the colonic epithelium. Many aspects of Shigella virulence, including the maturation of the T3SS apparatus, have been studied extensively, however, the events surrounding the initial host-pathogen interaction and its role in cellular uptake and infection remain unclear. Here, we have developed a series of techniques allowing us to directly visualize the interactions between key Shigella T3SS tip proteins and effectors with defined artificial phospholipid membranes. A custom total internal reflection fluorescence microscope was equipped with an atomic force microscope piezoelectric scanning head and stage. This instrument has allowed us to identify favorable conditions for generating artificial phospholipid membranes using the Langmuir Blodgett technique. By varying the pressures and composition of the lipid films, we have been able to mimic the mixed phase order conditions that likely exist within eukaryotic cell membranes. The use of lipophilic dyes and selective fluorescent labeling of the Shigella T3SS translocon proteins, believed to be directly involved in host cell contact (IpaB and IpaC), has allowed us to monitor the lipid phase order of our artificial membranes and protein localization/co-localization within the same films, respectively. Consequently, the use of such fluorophores and dyes, when carefully selected, allows us the ability to conduct Förster resonance energy transfer (FRET) studies, and quantify the protein-lipid interactions. The incorporation of cholesterol and sphingolipids into the membranes moves us one step closer to truly mimicking a cellular environment and helps determine what role it plays in protein localization and ultimately Shigella infection.