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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.

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defining the role of the pseudomonas aeruginosa chaperone spcu in secretion of exou
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
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

ExoU

ExoT

ExoS

ExoY

Translocators

SycU

(Chaperone)

ExoU

Type III Secretion Machinery
slide4
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

slide5
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
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
Mapping SpcU binding domain within ExoU with Yeast Two-Hybrid System

ExoU

Deletions of ExoU to be tested in Two-Hybrid System

1

125

106

91

69

50

Results

32

Negative

125

8

37

56

76

97

ExoU

SpcU

monitoring levels of exou in bacterial cytoplasm and secreted into media
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 Secretion (+EGTA)

Wt

+EGTA

+EGTA

70 Kd

Cell Pellet

Supernatant

detecting translocation of exou into mdck cells
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
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