1. Regulatory interplay between pap operons in Uropathogenic Escherichia coli
Makrina Totsika, Scott A. Beatson, Nicola Holden, David L. Gally (2008)
Presentation by Zachary Kurtz
with a little help from Dr. David Thanassi

2. Urinary Tract Infections
E. coli
Coagulase
negative
Staphylococci
Other Gram
Negatives
positives
Proteus mirabilis
Community-acquired UTI

3. Infection Model for UTI

4. Infection Model for UTI

5. Bacterial Cell Surface
‘The front line’
Adhesins
required for infection
prevents mictruition
several homologous types
Act as surface antigens for host immune system

6. How do UPEC evade immune response?

7. Phase Variation Control the expression of various adhesins
Population is heterogeneous
Adaptation to changing environments
Limit coexpression
Sequential presentation on single cell
Avoid physical interference
Aide in colonization of microenvironments
urinary tract, multi-organ system

8. Pyelonephritis-associated Pili (Pap)
AKA P fimbriae
Bind to erythrocytes of uroepithelial cells
Symptomatic UTI
Induces mucosal inflammation
Expression controlled by operon
Promoter, operator, structural genes
UPEC contain multiple copies of operon, with sequence variation

9. P pili

10. pap regulatory region The players of the epigenetic switch
Leucine-responsive regulatory protein (Lrp) – global regulator
PapI – regulatory protein
DNA-adenine methylase – Dam
PapB – PapI activator

11. the two expression phase states of pap transcription - OFF
Lrp binds to proximal sites (1,2 & 3)
Blocks GATCproximal methylation from Dam
Inhibits transcription via steric hindrance of RNAP
GATCdistal is methylated.

12. the two expression phase states of pap transcription - ON
PapI interacts with Lrp-pap complex
Lrp binds to distal sites (4,5,6)
GATCproximal site is methylated by Dam
RNA polymerase initiates transcription
PapB is papI activator – positive feedback at low conc.

13. Summary

14. How are the expression of homologous fimbrial operons coordinated at the single-cell level

15. The Answer
Sequence variation in regulatory region and regulators of pap operon
This variation alters pap expression
Study – survey of clinical isolates to measure sequence variation, expression level, binding activity

16. PapI

17. PapI

18. PapI Point mutations altered the capacity to activate expression
Lrp binding sites and nonbinding regions contained variation
Sequence variations affect Lrp, DNA and PapI interactions.
Mutations in clinical isolates are under positive selection by host

19. PapB Highly conserved or frame shift mutations which turn off activity
Frame shift only occurred if intact copy of the operon also present
This turns off some copies of operons but leaves others present
This limits coexpression of different alleles within a clinical isolate

20. Conclusions
Bacteria have multiple copies of homologous pap operons (other pili too)
Sequence variation modulates or changes expression of P fimbriae
Pressures from hosts immune system select for variation
This allows UPEC to hide from host

21. Thank you for coming!