Threading the Needle: Maintenance of an Unfolded

Polypeptide by a Cognate Chaperone in Bacterial Type III

SecretionAndrew Perrin

Department of Microbiology

University of Guelph

Bacterial Pathogenesis?

• Bacteria as pathogens

• Implications

Common Themes in Pathogenesis (Salmonella)

1. Infect host

2. Replicate

3. Subvert Host

Design a

TIM

Barrel

I Think I’ll Design a TIM

Barrel

Digression

L.M. Prescott, J.P. Harley, D.A. Klein, Microbiology, 5th Ed. McGraw Hill (2001).

How Do You Circumvent This?

“Standard Secretion”

V.T. Lee and O. Schneewind, Genes Dev. 15, 1725-1752 (2001).

But How Do You Get Proteins into the Host Cytosol?

Two Solutions

Choice 1: Receptor-Mediated Endocytosis

A.A. Salyers and D.D. Whitt, Bacterial Pathogenesis: A Molecular Approach, 2nd Ed. ASM Press (2000).

Choice 2: Inject Directly into Host Cell

Type III Secretion

T.G. Kimbrough and S.I. Miller, Microbes Infect. 4, 75-82 (2002). T. Kubori et al., Science 280, 602-605 (1998).

100 nm

Pathogenesis of Salmonella

A.A. Salyers and D.D. Whitt, Bacterial Pathogenesis: A Molecular Approach, 2nd Ed. ASM Press (2000).

Problem: Epithelial Cells Non-Phagocytic!

A.A. Salyers and D.D. Whitt, Bacterial Pathogenesis: A Molecular Approach, 2nd Ed. ASM Press (2000).

Solution: Make the Cells Phagocytic

T.G. Kimbrough and S.I. Miller, Microbes Infect. 4, 75-82 (2002).

Salmonella Entry

J.E. Galán, Personal Communication.

How?

• Activation of RhoGTPases by SopE (GEF)– Cytoskeletal changes

Y. Fu and J.E. Galán, Nature 401, 293-297 (1999).

Resolution

• Deactivation of RhoGTPases by SptP (GAP)

Y. Fu and J.E. Galán, Nature 401, 293-297 (1999).

Summary

C.E. Stebbins and J.E. Galán, Nature 412, 701-705 (2001).

Structure of SptP

Function of SptP

C.E. Stebbins and J.E. Galán, Mol. Cell 6, 1449-1460 (2000).

But How Do You Get SptP into the Host Cell?

Type III Secretion, Of Course!

74 Å

42 Å

39 Å

30 ÅType III System

How Do You “Thread the Needle”?

Type III Chaperones!• Small (12-20 kDa)

• Acidic pI

• Bind non-covalently

Y. Fu and J.E. Galán, J. Bacteriol. 180, 3393-3399 (1998).

SicP Binding to SptP

C.E. Stebbins and J.E. Galán, Nature 414, 77-81 (2001).

•No secretion signal (mRNA?)

•Prevent aggregation/degradation

Chaperones and Secretion

1. Targeting to secretion system

AND

SicP Maintains an Unfolded SptP

C.E. Stebbins and J.E. Galán, Nature 414, 77-81 (2001).

SicP Maintains an Unfolded SptP

Where Does SicP Bind?

C.E. Stebbins and J.E. Galán, Nature 414, 77-81 (2001).

Domain A

C.E. Stebbins and J.E. Galán, Nature 414, 77-81 (2001).

Where Does SicP Bind?

C.E. Stebbins and J.E. Galán, Nature 414, 77-81 (2001).

Domain B

C.E. Stebbins and J.E. Galán, Nature 414, 77-81 (2001).

Where Does SicP Bind?

C.E. Stebbins and J.E. Galán, Nature 414, 77-81 (2001).

Domain C

C.E. Stebbins and J.E. Galán, Nature 414, 77-81 (2001).

Where Does SicP Bind?

C.E. Stebbins and J.E. Galán, Nature 414, 77-81 (2001).

Domain D

C.E. Stebbins and J.E. Galán, Nature 414, 77-81 (2001).

SptP Binding Buries Hydrophobic Surfaces on SicP

C.E. Stebbins and J.E. Galán, Nature 414, 77-81 (2001).

Why a Dimer/Tetramer of SicP?

• Tetramer may be artifact of crystal

• Dimer supported biochemically– Static-light scattering– Isothermal titration calorimetry– Other crystal studies

So, How Does SptP Get to the Host Cell Cytosol?

C.L. Smith and S.J. Hultgren, Nature 414, 29-30 (2001).

Effector Unfolding/Refolding?

M. Mourez et al., Trends Microbiol. 10, 287-293 (2002).

Salmonella is Smart!

• Permanent pathogens versus occasional

• Constant activation of RhoGTPases:– Oncogenesis– Neurofibramatosis

Controlled Parasitism is Key!

Let’s design a TIM barrel together!

Future Directions

• Need full structure and more of them

• Targeting to Type III system?

• Interactions between effector-chaperone and Type III system

References1. Y.Fu and J.E. Galán, J. Bacteriol. 180, 3393-3399 (1998).2. Y. Luo et al., Nat. Struct. Biol. 8, 1031-1036 (2001).3. C.E. Stebbins and J.E. Galán, Nature 414, 77-81 (2001).4. C.L. Smith and S.J. Hultgren, Nature 414, 29-30 (2001).5. T. Kubori et al., Science 280, 602-605 (1998).6. C.E. Stebbins and J.E. Galán, Mol. Cell 6, 1449-1460 (2000).7. V.T. Lee and O. Schneewind, Genes Dev. 15, 1725-1752 (2001).8. Y. Fu and J.E. Galán, Nature 401, 293-297 (1999).9. T.G. Kimbrough and S.I. Miller, Microbes Infect. 4, 75-82 (2002).10. J. Wesche et al., Biochemistry 37, 15737-15746 (1998).11. K. Scheffzek, M. Reza and A. Wittinghofer, Trends Biochem. 23, 257-262

(1998).12. S.R. Sprang, Science 277, 329-330 (1997).13. Hardt et al., Cell 93, 815-826 (1998).14. A.A Salyers and D.D. Whitt, Bacterial Pathogenesis: A Molecular Approach,

2nd Ed. ASM Press (2000).15. L.M. Prescott, J.P. Harley and D.A. Klein, Microbiology, 5th Ed. McGraw Hill

(2001).16. M. Mourez et al., Trends Microbiol. 10, 287-293 (2002).