Transcript Presentation slides - Yale School of Medicine

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
Interaction of Salmonella with Intestinal
Epithelial Cells: Type III Secretion at Work
IL-8 and other
cytokines
Stimulation of
type III secretion
Stimulation of
Internalization and
Ruffling
cytokine production
and nuclear responses
(AP-1 & NFkB)
Interaction of Salmonella with intestinal epithelial cells
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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
membrane
Inner
membrane
100 nM
InvG
PrgK
PrgH
Export machinery
Kubori et al. Science (1998); Kubori et al. PNASc (2000)
Needle complex assembly pathway
InvG
InvH
PrgI
PrgJ?
IagB?
InvJ
PrgK
PrgH
Intermediate?
InvA
InvC
SpaP
SpaQ
SpaR
Export machinery
SpaS (ATPase + inner membrane
components)
sec-dependent
“Base”
Structure competent for secretion of
translocases and effector proteins
“Needle complex”
sec-independent
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
original
micrograph
Class-Averages
one particle
many particles
(one class)
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
Tomoko Kubori
Anand Sukhan
Vinzenz Unger
Dennis Thomas
(Brandeis)