B-294. Functional Analysis of Invasion Plasmid Antigen B (IpaB) During Type III Secretion by Shigella flexneri

C. M. Terry, N. L. Shelton, W. L. Picking, W. D. Picking;
Univ. of Kansas, Lawrence, KS.

Shigella flexneri, the causative agent of bacillary dysentery or shigellosis, affects more that 150 million people world wide each year. Most of these cases occur in developing countries and cause more than 1.5 million deaths, especially among children. S. flexneri utilizes a type III secretion apparatus (TTSA), common to many Gram negative pathogens, to facilitate invasion of the colonic epithelium. The TTSA resembles a molecular syringe composed of a basal body that spans the inner and outer bacterial membranes and an external needle. Effector proteins are passed through the apparatus where they make contact with or are transferred into the host cell. One such effector protein is the invasion plasmid antigen IpaB. In the bacterial cytoplasm, IpaB is bound to the molecular chaperone IpgC, which also serves as a chaperone for a second effector protein IpaC. This interaction prevents IpaB and IpaC from interacting before they are inserted into the host cell membrane by the TTSA. Under conditions that mimic the human intestinal environment, we have found that IpaB is stably recruited to the tip of the needle after IpaD. Once IpaB is recruited to the tip of the TTSA, IpaC can be secreted and appears to stably associate with the needle tip in an IpaB-dependent manner. In the host membrane, IpaB and IpaC form a pore that allows passage of subsequent effector proteins through the needle and into the host cell cytoplasm. We have therefore also used multiple techniques to determine IpaB’s binding characteristics with IpgC and IpaD at the TTSA needle tip. Tryptophan scanning mutagenesis with fluorescence spectroscopy has shown us that both the N- and C-termini of IpaC are critical for binding to IpgC. Using this approach, we now show that IpaB also has critical residues that appear to be involved in chaperone binding. Additional fluorescence polarization experiments are used to show that IpaB also binds to IpaD. We propose that the IpaB interactions with these binding partners will allow us to determine how it resides at the needle tip as part of the Shigella TTSA.