Does the Chaperone SpcU Mask the Membrane Localization Domain of the Pseudomonas aeruginosa Type

III Toxin ExoU?

Presented by: Donald RowenWork mainly of Master Student

– Suresh Kampalli

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

• 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.

Type III secretionMammalian Cells

Pseudomonas

Secretion and Translocation

Type III secretion machineryExoU

ExoT

ExoY ExoS

Exotoxin Chaperone

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

107-3571

687

MLD

C-TerminusS142

ISecretion

Chaperone binding?

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

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

107-3571

687

MLD

C-TerminusS142

ISecretion

Chaperone binding?

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

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 ofB-gal gene

ExoU-BD + AD vector

Truncations of ExoU made to Map residues required for SpcU interaction

679

654

605

552

1

C-terminusdeletions

369 687

552C-terminus

only

39

57

98

121

6871

N-terminusdeletions

S142A

155 369 687

552Internal

deletions

1

Preliminary Results with N-terminal deletions

369

552

39

57

98

121

6871 S142A

Blue colorDetected

B-gal Act

+++

++

-

-

-

+

+*

33.4

13.7

0.8

0.5

0.4

5.7

ND

Preliminary Results with C-terminal and Internal deletions

6871 S142A

Blue colorDetected

B-gal Act

+++

-

-

-

-

+

+*

33.4

0.1

ND

ND

0.1

8.2

ND

679

654

605

551

155 369

552

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.

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

• 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

Pellet Supernatant Pellet Supernatant

ExoU - full length - +++ + +/-

ExoU MLD - +++ - +++

Ongoing Work

ExoU Mutants to be Tested

6871 S142A

679

155 369

121

1-687

1-679

121-679

Δ155-369

HA

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

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

Acknowldegements

• Funding– INBRE Grant

• Several Undergraduate Student researchers helped with project

Summer, 2006Summer, 2007

Spring, 2006