B-295. Interaction of Deoxycholate with IpaD, the Type III Secretion Apparatus Needle Tip Protein of Shigella flexneri

P. R. Adam, C. D. LaMar, K. Stensrud, R. S. Givens, G. H. Lushington, W. L. Picking, W. D. Picking;
Univ. of Kansas, Lawrence, KS.

Shigella flexneri causes a severe form of dysentery (shigellosis) that is of global public health significance. Shigella uses a type III secretion apparatus (TTSA) to deliver effector proteins into target host cells to subvert cellular functions and promote cellular invasion. The external portion of the Shigella TTSA is a needle that has a “tip complex” at its outermost point. The tip complex is composed of invasion plasmid antigen D (IpaD) which is required for cellular invasion. Because previous studies showed that bile salts incubated could enhance Shigella’s invasive phenotype without inducing type III secretion, we explored the influence that deoxycholic acid (DOC) has on the nature and composition of the TTSA tip complex. DOC was subsequently found to promote the stable recruitment of IpaB to the IpaD-containing TTSA tip complex. IpaC was not observed on the Shigella surface under these conditions. We now show that IpaD is able to directly bind deoxycholate using a fluorescent derivative of DOC (FITC-DOC) an in vitro fluorescence polarization analysis. FITC-DOC was also used in Forster resonance energy transfer experiments to estimate the site on IpaD at which this bile salt derivative binds and this measurement is in agreement with molecular docking analyses. The accuracy of the in silico analysis of DOC docking was further explored by introducing mutations into ipaD which were found to have altered invasion properties using a gentamycin-protection assay with Henle 407 cells. Likewise, the specificity of the interaction was tested using nonfluorescent competitors for FITC-DOC binding to IpaD in vitro. The findings presented here strongly argue that IpaD is capable of sensing environmental small molecules to influence type III secretion and it is thus a potential target for structure-based drug design.