Subcloning and Cysteine Mutagenesis of the Transmembrane Regions of the Shigella IpaB Virulence Protein

Voiceover

Presenter(s)

Eric Haberman

Abstract or Description

The type III secretion system (T3SS) is a well-known mechanism of pathogenicity for infections caused by bacteria such as Shigella, Salmonella, and E. coli. The atomic structure of the translocon of the T3SS, which creates a pore on the host membrane, is currently unknown. In Shigella, the membrane protein IpaB is needed to assemble the translocon. IpaB is expected to contain two transmembrane (TM) regions, however, it is currently unknown how these two regions of IpaB interact with host membranes. Using Takara In-Fusion methods, the point mutation S333C was introduced in TM1, and the point mutation S401C was introduced in TM2. Additionally, full-length IpaB and a smaller IpaB construct spanning residues 66-425 were subcloned into pET-22b for protein expression. The new plasmids for various IpaB constructs will be used for protein expression of IpaB for biophysical characterization of the transmembrane regions of IpaB in membrane mimetics. Knowledge of the structure of IpaB in membranes will improve our understanding of how the translocon of T3SS is assembled.

Mentor

Dr. Roberto N. De Guzman