Does the Chaperone SpcU Mask the Membrane Localization Domain of the

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
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
Pathogenicity of P. aeruginosa.
• Virulence factors
– Capsule – biofilm formation
– Antibiotic resistance
– Secretion of the exotoxins/effectors by Type III
secretion system.
• Four known exotoxins: ExoS, ExoT, ExoU, ExoY
Type III secretion
Mammalian Cells
Pseudomonas
Secretion and
Translocation
Type III secretion machinery
ExoT
ExoU
ExoS
ExoY
Chaperone
Exotoxin
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
Secretion
1
107-357
I Patatin
Chaperone
binding?
S142
687
MLD
C-Terminus
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
Secretion
1
107-357
I Patatin
Chaperone
binding?
S142
687
MLD
C-Terminus
First Objective of This Study
1. Determine residues of ExoU required for
SpcU interaction
– Testing whether residues on both the Nterminus 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
ExoU
GAL AD
GAL BD
SpcU
Transcription of
B-gal gene
GAL UAS
Promoter
ExoU-BD + AD vector
Reporter gene
Truncations of ExoU made to Map
residues required for SpcU interaction
S142A
1
687
39
N-terminus
deletions
57
98
121
1
679
654
605
C-terminus
deletions
552
687
369
C-terminus
only
552
1
155
687
369
552
Internal
deletions
Preliminary Results with N-terminal
deletions
S142A
1
687
39
57
98
121
369
552
Blue color
Detected
B-gal
Act
+++
33.4
++
13.7
-
0.8
-
0.5
-
0.4
+
5.7
+*
ND
Preliminary Results with C-terminal and
Internal deletions
1
S142A
687
679
654
605
551
155
369
552
Blue color
Detected
B-gal
Act
+++
33.4
-
0.1
-
ND
-
ND
-
0.1
+
8.2
+*
ND
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 Cterminus
• 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
PA0103
wt
70 Kd
PAO103
exsA
Low
levels
of
SpcU
Ongoing Work
• 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 DMLD
-
+++
-
+++
ExoU Mutants to be Tested
1
S142A
687 HA
679
1-687
1-679
121
121-679
155
369
Δ155-369
Summary and Conclusions
1. Mapping of Residues Required for
Interaction via Two-hybrid
–
–
–
Have constructed and tested several N- and Cterminal 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 Nand C-terminus of ExoU
Acknowldegements
• Funding
– INBRE Grant
• Several Undergraduate
Student researchers
helped with project
Spring, 2006
Summer, 2007
Summer, 2006