Probing the effects of lipid phase order on the localization of Shigella type three secretion system translocon proteins in artificial membranes

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Nicholas E. Dickenson


Shigella spp. are gram-negative, non-motile bacteria that can 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. Each year, isolated outbreaks occur in first world countries, but the bigger concerns are related to children of third world countries who suffer an estimated 61% of the 1.1 million Shigella related deaths annually. 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 (TIRFM) 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. 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. These results provides the first insights into lipid phase order effects on T3SS protein localization and they lay the foundation for future studies in which the effect of lipid membrane composition ad phase order on directing interaction with virulent Shigella organisms will be performed.

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