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

2. 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

3. 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

4. Interaction of Salmonella with intestinal epithelial cells

5. 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)

6. 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)

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

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

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

10. Single Particle
Selection

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

12. 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

13. Three-dimensional reconstitution from averaged projections

16. Needle complex dimensions

17. Conformational changes upon needle assembly

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

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

20. 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

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