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.
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
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