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 bacterial-encoded 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

ExoUExoT

Translocators

ExoY ExoS

ExoU SycU(Chaperone)

ExoU

SpcU (chaperone)

Patatin

107-357

1 687

Activation domain

• 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

• 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 Two-

Hybrid System

Deletions of ExoU to be tested in

Two-Hybrid System

ExoU1 125

106

1258

91

50

32

69

56

97

76

37

SpcUExoU

Negative

Results

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)

SupernatantCell Pellet

70 Kd

+EGTA +EGTA

Wt

Levels of ExoU-HA detected Inside Cells (Cell Pellet) and Secreted (Supernatant) after Induction of Secretion (+EGTA)

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