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

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

Infection Model for UTI

Infection Model for UTI

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

How do UPEC evade immune response?

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

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

P pili

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

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.

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.

Summary

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

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

PapI

PapI

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

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

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

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