Transcript ExoU Secretion - University of Nebraska Omaha

Defining the role of the
Pseudomonas
aeruginosa chaperone
SpcU in Secretion of
ExoU
Kampalli, S.B., J. Bigelow, A.
Dale, M. Seil, E. Suellentrop
and D. W. Rowen.
Department of Biology,
University of Nebraska at
Omaha, Omaha, NE
Background
Type III secretion systems (TTSS) are a
virulence factor of many pathogenic
bacteria that mediates the secretion and
translocation of specific bacterialencoded toxins or effectors into the
cytoplasm of eukaryotic host cells.
Some effectors require a chaperone for
efficient secretion and translocation.
The role of chaperones of Type III
effectors appears to be varied and is not
fully understood. The effector ExoU of
P. aerginosa has be shown to be bound
by a likely chaperone called SpcU, but
the role of SpcU has not be defined. In
this study, we hope to characterize the
role of SpcU in the secretion of ExoU.
Type III Secretion
Machinery
ExoT
ExoU
ExoS
ExoY
Translocators
ExoU
SycU
(Chaperone)
ExoU
• An effector/exotoxin secreted by Type
III system
• Has an patatin-like phospholipase
domain and a proposed activation
domain
• Amino terminus is thought to contain
secretion signal and chaperone-binding
domain
SpcU (chaperone)
107-357
687
1
Patatin
Activation domain
SpcU
• Small acidic protein (137
residues) similar to other
chaperones
• Observed to bind to ExoU
(requires N-terminal amino acids
3-123 of ExoU)
• Proposed roles of chaperones
– Protect effector from degradation
– Keep effector in secretion
competent state (unfolded)
– Delivery of effector to secretion
apparatus
– Delivery of effector to translocation
factors
Goals of This Study
• Determine if SpcU protects ExoU
from degradation inside bacterial
cell
• Determine if SpcU helps bring
ExoU to secretion apparatus
• Determine if SpcU plays a role in
translocation of ExoU
Mapping SpcU binding domain
within ExoU with Yeast TwoHybrid System
ExoU
1
125
106
91
69
Deletions of
ExoU to be
tested in TwoHybrid
System
50
Results
32
125
8
37
56
76
97
ExoU
SpcU
Negative
Monitoring Levels of ExoU in
Bacterial Cytoplasm and
Secreted into Media
• Tagging full length or mutant forms of ExoU
with HA epitope on C-terminus
• Induce expression of ExoU and Type III
secretion system in P. aeruginosa cells with
or without SpcU
• Detect levels with anti-HA antibody in
Western blots in cell pellet (cell) associated
fraction or in supernatant (secreted)
Levels of ExoU-HA detected Inside Cells
(Cell Pellet) and Secreted (Supernatant)
after Induction of SecretionWt(+EGTA)
+EGTA
+EGTA
70 Kd
Cell Pellet
Supernatant
Detecting Translocation of
ExoU into MDCK cells
• Developing procedure to monitor
translocation of ExoU into MDCK animal
cells
• In preliminary tests, we observed
cytopathic effects 6 hours after addition of
wild type PA103 strain to MDCK cells at a
MOI of 20
• In future, we will monitor translocation by
using ExoU-cya fusions and measuring
cAMP produced by the Ca2+ dependent
adenylate cyclase activity of Cya
MDCK cells –
No Infection
MDCK cells –
Infected with PA103
Summary
• Seeking to define role of SpcU in
secretion and translocation of ExoU
• Constructing plasmids to identify
region of ExoU to which SpcU binds
by using the yeast two-hybrid
method
• Observed secretion of HA-tagged full
length ExoU. Will use HA-tagging to
monitor levels of ExoU mutants
inside cells and secreted into
supernantant
• Observed cytopathic effects of
translocation of full length ExoU into
MDCK cells. Plan to use cya fusions
to monitor translocation of ExoU
mutants into MDCK cells
• Constructing SpcU knockout mutant
strain