Investigation of the Structure and Function of Type III Secretion Needle and Tip Proteins
University of Kansas
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Many Gram-negative pathogens possess type III secretion systems as part of their required virulence factor repertoire. The type III secretion apparatus (TTSA) spans the bacterial inner and outer membranes and resembles a syringe and a needle. It is used to inject proteins into a host cell's membrane and cytoplasm to subvert normal cellular processes. The external portion of the TTSA is a needle that is composed of a single type of protein that is polymerized in a helical fashion to form an elongated tube with a central channel that is 2-3 nm in diameter. We report the first structure of a TTSA needle protein called BsaL from Burkholderia pseudomallei determined by nuclear magnetic resonance (NMR) spectroscopy. The central part of the protein assumes a helix-turn-helix core domain with two well-defined α-helices that are joined by a four-residue linker. Residues that flank this presumably exposed core region are not completely disordered, but adopt a partial helical conformation. The atomic structure of BsaL and its sequence homology with other TTSA needle proteins suggest potentially unique structural dynamics that could be linked with a universal mechanism for control of type III secretion in diverse Gram-negative bacterial pathogens. The pathogenesis of Shigella flexneri requires a functional TTSA to inject host-altering effector proteins directly into the targeted cell. The TTSA exposed needle 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 Yersinia and BipD from Burkholderia) all have a similar overall shape. We have identified key MxiH residues located in its PSNP loop and the contiguous surface that uniquely contribute to the formation of the IpaD-needle interface as determined by NMR chemical shift mapping. Mutation of some of these MxiH residues severely affects the stable maintenance of IpaD at the Shigella surface and thus compromises the invasive phenotype. Other residues could be mutated to give rise to intermediate phenotypes, suggesting they have a role in tip complex stabilization while not being essential for tip complex formation. Initial in vitro fluorescence polarization (FP) studies confirmed that specific amino acid changes adversely affect the MxiH-IpaD interaction. Meanwhile, none of the mutations appeared to have a negative effect on the MxiH-MxiH interactions required for efficient needle assembly. 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. FP studies showed that the bile salt deoxycholate (DOC) binds to IpaD. NMR spectroscopy confirms a DOC-IpaD interaction and suggests that the IpaD conformation changes upon DOC binding. We have identified key IpaD residues that appear to contribute to the formation of the IpaD-DOC interface. DOC appears to bind at the middle of the IpaD coiled-coil where the top of the N-terminal globular domain packs against the coiled-coil. Several IpaD residues in the distal globular domain are also perturbed upon the binding of DOC, suggesting an overall conformational change, which may lead to the recruitment of translocator protein IpaB to the needle tip. Mutation of some of these perturbed residues affects the ability of IpaD to recruit IpaB to the bacterial surface and thus impacts the invasive phenotype of S. flexneri.
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