Characterisation of the Type III Secretion System EspO effector family in enteric pathogens
Author(s)
Constantinou, Nicholas
Type
Thesis
Abstract
Many enteric pathogens employ a type III secretion system to translocate effector
proteins directly into the host cell cytoplasm, where they subvert signalling pathways of the
intestinal epithelium. Once inside the cell, effector proteins may interact with host cell
proteins to control various cellular processes. This enables enteric pathogens to establish
their infective niche within the human host.
The T3SS effectors EspO of enterohaemorrhagic Escherichia coli and Citrobacter
rodentium, OspE of Shigella flexnerie and SopO of Salmonella enterica Serovar
Typhimurium are known to interact with integrin-linked kinase to enhance cellular
attachment to the substratum. In this project we demonstrate that the EspO family of
effectors also interacts with the anti-apoptotic regulator Hax-1 and the co-chaperone
DnaJB6.
Employing fluorescence microscopy we found that ectopically expressed EspO
resides on large cytosolic vacuoles, which stained positive for the endoplasmic reticulum
marker CREB3. Moreover, ectopic expression of EspO changed the cellular distribution of
endogenous Hax-1, which co-localized with the EspO vacuoles. We found that the EspO
orthologs are anti apoptotic, protecting cells from apoptosis triggered by multiple inducers
including staurosporine (a global kinase inhibitor), tunicamycin (induces unfolded protein
response) and thapsigargin (interferes with Ca2+ homeostasis). Additionally, EspO protects
HeLa cells from the apoptosis induced in EPEC infections. Hax-1 depleted HeLa cells by
siRNA showed that the anti apoptotic activity of the EspO orthologs is Hax-1 dependent.
Using the C. rodentium in-vivo model of EHEC / EPEC infections, we found that
deletion of espO results in decreased levels of hyperplasia similar to the uninfected control.
The mutant strain also showed low levels of neutrophils and CD4+T cells, whereas the
EspO complemented strain showed a dramatic increase in both inflammatory cells. These
results suggest new mechanisms unique to the T3SS EspO family of effectors, which might
shed new insights regarding the infection strategies followed by enteric pathogens. These
results suggest new mechanisms unique to the T3SS EspO family of effectors, which might
shed new insights regarding the infection strategies followed by enteric pathogens.
proteins directly into the host cell cytoplasm, where they subvert signalling pathways of the
intestinal epithelium. Once inside the cell, effector proteins may interact with host cell
proteins to control various cellular processes. This enables enteric pathogens to establish
their infective niche within the human host.
The T3SS effectors EspO of enterohaemorrhagic Escherichia coli and Citrobacter
rodentium, OspE of Shigella flexnerie and SopO of Salmonella enterica Serovar
Typhimurium are known to interact with integrin-linked kinase to enhance cellular
attachment to the substratum. In this project we demonstrate that the EspO family of
effectors also interacts with the anti-apoptotic regulator Hax-1 and the co-chaperone
DnaJB6.
Employing fluorescence microscopy we found that ectopically expressed EspO
resides on large cytosolic vacuoles, which stained positive for the endoplasmic reticulum
marker CREB3. Moreover, ectopic expression of EspO changed the cellular distribution of
endogenous Hax-1, which co-localized with the EspO vacuoles. We found that the EspO
orthologs are anti apoptotic, protecting cells from apoptosis triggered by multiple inducers
including staurosporine (a global kinase inhibitor), tunicamycin (induces unfolded protein
response) and thapsigargin (interferes with Ca2+ homeostasis). Additionally, EspO protects
HeLa cells from the apoptosis induced in EPEC infections. Hax-1 depleted HeLa cells by
siRNA showed that the anti apoptotic activity of the EspO orthologs is Hax-1 dependent.
Using the C. rodentium in-vivo model of EHEC / EPEC infections, we found that
deletion of espO results in decreased levels of hyperplasia similar to the uninfected control.
The mutant strain also showed low levels of neutrophils and CD4+T cells, whereas the
EspO complemented strain showed a dramatic increase in both inflammatory cells. These
results suggest new mechanisms unique to the T3SS EspO family of effectors, which might
shed new insights regarding the infection strategies followed by enteric pathogens. These
results suggest new mechanisms unique to the T3SS EspO family of effectors, which might
shed new insights regarding the infection strategies followed by enteric pathogens.
Version
Open Access
Date Issued
2013-05
Date Awarded
2013-07
Advisor
Frankel, Gad
Sponsor
Biotechnology and Biological Sciences Research Council (Great Britain)
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)