Higuchi Biosciences Center - University of Kansas

Fall 2008 Science Talks
December 5, 2008

B1. Erythrocyte Lipids Recruit IpaC to the Type III Secretion Needle Tip
Chelsea R. Epler, Wendy L. Picking, and William D. Picking.
Dept. of Molecular Biosciences. The University of Kansas, Lawrence, KS 66045

Shigella flexneri, a Gram-negative bacterium, is the causative agent of bacillary dysentery. Shigella uses a type III secretion system (TTSS) to invade and deliver effector proteins into host cells to undermine normal cell function. The type III secretion apparatus (TTSA) consists of a basal body and an extracellular needle. Invasion plasmid antigen D (IpaD) controls secretion at the tip of the needle and acts as an environmental sensor for the recruitment of translocator IpaB to the surface. Contact between the TTSA and the host cell triggers TTSS function, the first step of which is that translocator IpaB and IpaC are secreted and form a pore in the membrane of the host cell to allow other effectors to enter the host cytoplasm. IpaB, the first translocator protein, can be brought to the tip when bile salts are sensed in the environment, while IpaC remains inside the bacterium. The addition of liposomes from erythrocytes has brought IpaC to the surface and allowed direct visualization by immunofluorescence. During this process, IpaB may act as an environmental sensor for the recruitment of IpaC. Currently, how IpaC is brought to the surface is unknown and there are no translocator structures available. Deletion mutants were used to get a better understanding of the protein-protein interaction between IpaB and IpaC. With this information we will be better able to understand the invasion process of Shigella.

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B2. In vivo & In vitro analysis of the DNA binding activity of ChxR: a transcriptional regulator in Chlamydia trachomatis
Lindsey Spedding, John Hickey, and P. Scott Hefty
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045

The mechanisms that regulate the chlamydial developmental cycle are poorly understood; however, transcription appears to play a governing role in chlamydial development. An OmpR subfamily response regulator termed ChxR exhibits expression patterns that indicate an important role during the conversion of reticulate body (RB) to elementary body (EB). We are interested in elucidating the role of ChxR in chlamydial development and the mechanism of transcriptional activation. ChxR is a unique response regulator that is not activated through phosphorylation and prior experiments have demonstrated that unmodified ChxR is transcriptionally active. Usually as a result of phosphorylation, OmpR response regulators form homo-dimers as an integral step in transcriptional activation. We hypothesize that native, unmodified ChxR forms homodimers and are maintained in a structural conformation that corresponds to the transient active state of phosphorylated OmpR response regulators. Using chemical crosslinker we have demonstrated that ChxR forms homodimers through the amino terminal domain in vitro, similar to phosphorylation-induced and transcriptionally active OmpR response regulators. Importantly, we have also detected ChxR dimer formation within Chlamydia infected eukaryotic host cells. Previously, it was demonstrated that ChxR binds to five regions upstream of its own promoter. To facilitate identification of other gene targets of ChxR, we identified the ChxR cis-acting recognition sequence within this promoter. We have located this ChxR recognition sequence within promoter elements of type III secretion operons and tested their interactions with ChxR. The resulting data support the hypothesis that ChxR plays an important role in gene expression during RB to EB transition.

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B3. Interaction of deoxycholate with IpaD, the type III secretion apparatus needle tip protein of Shigella flexneri
Philip R. Adam, Cassandra D. La Mar, Ken Stensrud, Richard S. Givens, Gerald H. Lushington, Wendy L. Picking, and William D. Picking
Depts. of Molecular Biosciences and Chemistry, The University of Kansas, Lawrence, KS 66045

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.

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B4. NMR Analysis of the Influence Bile Salt Binding Has on the Structure of Invasion Plasmid Antigen D (IpaD) from Shigella flexneri
Lingling Zhang, Wendy L. Picking and William D. Picking
Higuchi Biosciences Center, University of Kansas, Lawrence, KS; Department of Molecular Biosciences, University of Kansas, Lawrence, KS

The pathogenesis of Shigella flexneri requires a functional type III secretion apparatus (TTSA) to inject host-altering effector proteins directly into the targeted cell’s membrane and cytoplasm. The TTSA is composed of a basal body and an exposed needle that is an extended polymer of MxiH. Invasion plasmid antigen D (IpaD) resides at the tip of the needle to control Shigella type III secretion. IpaD (36.6 kDa) has a dumbbell shape with two globular domains flanking a central coiled-coil that stabilizes the protein. Known structures of IpaD homologs (LcrV from Yersina and BipD from Burkholderia) all have a similar overall shape. Using NMR (nuclear magnetic resonance) chemical shift mapping, we have demonstrated a MxiH-IpaD interaction and identified key MxiH residues that appear to uniquely contribute to the formation of the IpaD-needle interface. We recently demonstrated that bile salts stimulate recruitment of the translocator protein IpaB to the Shigella surface where it stably resides with IpaD at the TTSA needle tip. This process appears to be initiated by a direct interaction between the bile salt and IpaD. Fluorescence polarization studies showed that the bile salt deoxycholate (DOC) binds to IpaD. NMR spectroscopy confirms a DOC-IpaD interaction and suggests the local IpaD conformation changes upon DOC binding. When DOC is titrated into IpaD, one of the four tryptophans’ side chain resonances is perturbed due to the DOC binding. By mutating the tryptophans individually we have identified the one that appears to be influenced by the DOC binding and this residue is probably influenced as part of a conformational change in IpaD. Mutation of this residue affects the ability of IpaD to control type III secretion and thus impacts the invasive phenotype of S. flexneri. In contrast, this mutation does not appear to have a negative effect on the overall structure of IpaD.

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Higuchi Biosciences Center
University of Kansas
2099 Constant Avenue
Lawrence, KS 66047-2535
785-864-5183
hbc@ku.edu