Type III secretion machines: devices for protein delivery into eukaryotic cells

Main features of type III protein secretion systems Associated with phenotypes involving intimate bacteria/host-cell interactions Present in symbiotic and pathogenic gram negative bacteria for animals, plants, and insects Substrates lack typical sec-dependent signal sequence Require activating signals for full function Encoded in extrachromosomal elements or pathogenicity islands Evolved to deliver bacterial proteins into host cells

Stimulation of Ruffling Interaction of Salmonella with Intestinal Epithelial Cells: Type III Secretion at Work IL-8 and other cytokines Stimulation of type III secretion Stimulation of Ruffling and nuclear responses (AP-1 & NFkB) Internalization and cytokine production

Interaction of Salmonella with intestinal epithelial cells

Virulence effector proteins delivered by the Salmonella type III secretion organelle SopE (exchange factor for Cdc42 & Rac1) SptP (GAP for Cdc42 & Rac1 and tyrosine phosphatase) SopB (phosphoinositide phosphatase) SipA (actin nucleator) SipC (actin bundling) AvrA (inhibitor of MAP kinase pathways; putative protease)

Salmonella typhimurium type III secretion organelle: the needle complex PrgI Outer InvG membrane PrgK Inner PrgH membrane Export machinery 100 nM Kubori et al. Science (1998); Kubori et al. PNASc (2000)

Needle complex assembly pathway “Base” “Needle complex” Sukhan et al. J. Bacteriol. (2001); Sukhan et al. J. Bacteriol. (2003)

High resolution view of the Salmonella typhimurium type III secretion organelle (needle complex) and its assembly pathway

Isolated needle complexes and needle complex bases with optimized purification protocols Needle complex Needle complex base

Single Particle Selection

Selection and classification of individual particles orig. filt. align.

Averaging of single-class particles to improve the signal/noise ratio Particle Selection/ Noise Reduction/ Aligning Class-Averages original micrograph many particles (one class) one particle

Three-dimensional reconstitution from averaged projections

Needle complex dimensions

Conformational changes upon needle assembly

PrgJ is recruited as a structural component to the base during needle assembly Selective Needle Disassembly Repurification (CsCl) and Western

The needle complex organelle exhibits varying symmetries 19 fold 20 fold 21 fold 22 fold

Summary The ~15Å resolution structures of the needle complex of Salmonella typhimurium and assembly intermediates were obtained by cryo electron microscopy and single particle analysis The structure shows varying symmetries ranging from19 to 22 fold indicating heterogeneity in the needle complex The needle anchors at the top of the base The base shows the presence of an inner rod composed largely of PrgJ, which anchors at the bottom of the base structure Comparison of the needle complex structure with assembly intermediates showed significant conformational changes in the base substructure upon needle assembly that may provide the bases for substrate switching during type III secretion

Acknowledgments Thomas Marlovits Vinzenz Unger Tomoko Kubori Anand Sukhan Vinzenz Unger Dennis Thomas (Brandeis)