Affiliation:
1. Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College, London United Kingdom, London, United Kingdom
2. Functional Proteomics Group, Chester Beatty Laboratories, Institute of Cancer Research, London, United Kingdom
3. Department of Chemistry, Imperial College, London United Kingdom, London, United Kingdom
4. University of California at San Diego, La Jolla, California, USA
Abstract
ABSTRACT
Tyrosine phosphorylation is key for signal transduction from exogenous stimuli, including the defense against pathogens. Conversely, pathogens can subvert protein phosphorylation to control host immune responses and facilitate invasion and dissemination. The bacterial effectors EspJ and SeoC are injected into host cells through a type III secretion system by enteropathogenic and enterohemorrhagic
Escherichia coli
(EPEC and EHEC, respectively),
Citrobacter rodentium
, and
Salmonella enterica
, where they inhibit Src kinase by coupled amidation and ADP-ribosylation.
C. rodentium
, which is used to model EPEC and EHEC infections in humans, is a mouse pathogen triggering colonic crypt hyperplasia (CCH) and colitis. Enumeration of bacterial shedding and CCH confirmed that EspJ affects neither tolerance nor resistance to infection. However, comparison of the proteomes of intestinal epithelial cells isolated from mice infected with wild-type
C. rodentium
or
C. rodentium
encoding catalytically inactive EspJ revealed that EspJ-induced ADP-ribosylation regulates multiple nonreceptor tyrosine kinases
in vivo
. Investigation of the substrate repertoire of EspJ revealed that in HeLa and A549 cells, Src and Csk were significantly targeted; in polarized Caco2 cells, EspJ targeted Src and Csk and the Src family kinase (SFK) Yes1, while in differentiated Thp1 cells, EspJ modified Csk, the SFKs Hck and Lyn, the Tec family kinases Tec and Btk, and the adapter tyrosine kinase Syk. Furthermore, Abl (HeLa and Caco2) and Lyn (Caco2) were enriched specifically in the EspJ-containing samples. Biochemical assays revealed that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, controls immune responses and the Src/Csk signaling axis.
IMPORTANCE
Enteropathogenic and enterohemorrhagic
Escherichia coli
(EPEC and EHEC, respectively) strains cause significant mortality and morbidity worldwide.
Citrobacter rodentium
is a mouse pathogen used to model EPEC and EHEC pathogenesis
in vivo
. Diarrheal disease is triggered following injection of bacterial effectors, via a type III secretion system (T3SS), into intestinal epithelial cells (IECs). While insights into the role of the effectors were historically obtained from pathological, immunologic, or cell culture phenotypes, subtle roles of individual effectors
in vivo
are often masked. The aim of this study was to elucidate the role and specificity of the ADP-ribosyltransferase effector EspJ. For the first time, we show that the
in vivo
processes affected by a T3SS effector can be studied by comparing the proteomes of IECs extracted from mice infected with wild-type
C. rodentium
or an
espJ
catalytic mutant. We show that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, regulates the host immune response
in vivo
.
Funder
Wellcome Trust
RCUK | Medical Research Council
Publisher
American Society for Microbiology
Cited by
18 articles.
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