1. Does the Chaperone SpcU Mask the Membrane Localization Domain of the Pseudomonas aeruginosa Type III Toxin ExoU? Presented by: Donald Rowen Work mainly of Master Student – Suresh Kampalli

2. Pseudomonas aeruginosa: Gram-negative rod Opportunistic pathogen Respiratory infections, particularly in Cystic Fibrosis patients Skin infections: Wound, burn Urinary tract infection Corneal infection Nosocomial infections

3. Virulence factors –Capsule – biofilm formation –Antibiotic resistance –Secretion of the exotoxins/effectors by Type III secretion system. Four known exotoxins: ExoS, ExoT, ExoU, ExoY Pathogenicity of P. aeruginosa.

4. Type III secretion Mammalian Cells Pseudomonas Secretion and Translocation

5. Type III secretion machinery ExoU ExoT ExoY ExoS Exotoxin Chaperone

6. ExoU Potent cytotoxin - causes cell death if it gets injected into host cells. Phospholipase – has patatin-like phospholipase domain Has a membrane localization domain (MLD) on C-terminus – targets protein once inside host cell Secretion signals and perhaps chaperone binding on N-terminus Patatin 107-357 1 687 MLD C-Terminus S142 I Secretion Chaperone binding?

7. Role of SpcU in ExoU secretion Specific chaperone for ExoU secretion –Gene located just downstream of exoU that is required for ExoU secretion –Encodes small protein similar to some chaperones –Co-purifies with full length ExoU – But did not co-purify with a mutant from of ExoU lacking residues 3-123 in N-terminus Roles of chaperones is still debated: –It may protect and keep effector in secretion competent state (unfolded) –It may help in delivery of effector to secretion apparatus –New theory - mask “membrane localization domain” of toxins – prevent aggregation and degradation

8. ExoU – Good Test Protein Good protein for testing of the MLD masking theory. Most chaperone binding sites and MLD are on N-terminus after secretion signal MLD of ExoU on C-terminus Patatin 107-357 1 687 MLD C-Terminus S142 I Secretion Chaperone binding?

9. First Objective of This Study 1.Determine residues of ExoU required for SpcU interaction –Testing whether residues on both the N- terminus and C-terminus (MLD) are required for their association –Determining the effect of N- and C-terminal deletions of ExoU on interaction with SpcU in yeast two-hybrid system

10. Detected Signs of Association of SpcU with full-length ExoU in Yeast-Two Hybrid System GAL UAS Reporter genePromoter GAL BD ExoU GAL AD SpcU Transcription of B-gal gene ExoU-BD + AD vector

11. Truncations of ExoU made to Map residues required for SpcU interaction 679 654 605 552 1 C-terminus deletions 369 687 552 C-terminus only 39 57 98 121 687 1 N-terminus deletions S142A 155 369 687 552 Internal deletions 1

12. Preliminary Results with N-terminal deletions 369 552 39 57 98 121 687 1 S142A Blue color Detected B-gal Act +++ ++ - + +* 33.4 13.7 0.8 0.5 0.4 5.7 ND

13. Preliminary Results with C-terminal and Internal deletions 687 1 S142A Blue color Detected B-gal Act +++ - + +* 33.4 0.1 ND 0.1 8.2 ND 679 654 605 551 155369 552

14. Second Objective of This Study 2.Test the importance of SpcU interaction on stability and aggregation of ExoU. –Uncovered MLD hypothesized to promote aggregation and degradation of toxin –Plan to measure levels of the ExoU in bacterial cells in absence and presence of SpcU –Determine whether ExoU with MLD forms aggregates in absence of SpcU.

15. Preliminary Experiment Tagged full length ExoU (70 kD) with HA epitope on C- terminus Express in wild type and exsA mutant (low levels of SpcU) of P. aeruginosa Detect levels of ExoU-HA in cell lysates with anti-HA antibody in Western blots 70 Kd PA0103 wt PAO103 exsA Low levels of SpcU

16. Constructing plasmids expresssing mutant versions of ExoU tagged with HA Will express ExoU in bacterial cells with or without SpcU Will determine levels and location of ExoU in two cell fractions –Pellet fraction (100,000 x g) = aggregate/membrane associated –Supernatant fraction = soluble cytosolic protein + SpcU-SpcU PelletSupernatantPelletSupernatant ExoU - full length-+++++/- ExoU  MLD -+++- Ongoing Work

17. ExoU Mutants to be Tested 687 1 S142A 679 155369 121 1-687 1-679 121-679 Δ155-369 HA

18. Summary and Conclusions 1.Mapping of Residues Required for Interaction via Two-hybrid –Have constructed and tested several N- and C- terminal deletions in yeast two-hybrid system –Preliminary results suggest residues on both N- and C- terminus are required for interaction –Working to confirm 2.Stability and Localization of ExoU. –Preliminary experiment showed levels of ExoU with MDL are lower in cells lacking SpcU –Constructing HA-tagged ExoU mutants –Will examine levels and localization of ExoU +/- SpcU

19. Future Directions Confirm two-hybrid results by testing association between ExoU deletion mutants and SpcU in bacterial cells with coimmunoprecipitation experiments Demonstrate interaction of SpcU with both N- and C-terminus of ExoU

20. Acknowldegements Funding –INBRE Grant Several Undergraduate Student researchers helped with project Summer, 2006 Summer, 2007 Spring, 2006