ABSTRACT
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are important human pathogens that rely on translocation of type III secretion system (T3SS) effectors for subversion of signal transduction pathways and colonization of the mammalian gut mucosa. While a core set of effectors is conserved between EPEC and EHEC strains, a growing number of accessory effectors that were found at various frequencies in clinical and environmental isolates have been recently identified. Recent genome projects identified espV as a pseudogene in EHEC but a putative functional gene in EPEC strains E110019 and E22 and the closely related mouse pathogen Citrobacter rodentium. The aim of this study was to determine the distribution of espV among clinical EPEC and EHEC strains and to investigate its function and role in pathogenesis. espV was found in 16% of the tested strains. While deletion of espV from C. rodentium did not affect colonization dynamics or fitness in mixed infections, expression of EspV in mammalian cells led to drastic morphological alterations, which were characterized by nuclear condensation, cell rounding, and formation of dendrite-like projections. Expression of EspV in yeast resulted in a dramatic increase in cell size and irreversible growth arrest. Although the role of EspV in infection and its target host cell protein(s) require further investigation, the data point to a novel mechanism by which the T3SS subverts cell signaling.
Subject(s)
Bacterial Secretion Systems/genetics , Citrobacter rodentium/metabolism , Enterohemorrhagic Escherichia coli/genetics , Enteropathogenic Escherichia coli/genetics , Escherichia coli Infections/genetics , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Enterohemorrhagic Escherichia coli/metabolism , Enterohemorrhagic Escherichia coli/pathogenicity , Enteropathogenic Escherichia coli/metabolism , Enteropathogenic Escherichia coli/pathogenicity , Escherichia coli Infections/metabolism , Eukaryotic Cells/microbiology , Fluorescent Antibody Technique , Genes, Bacterial/genetics , HeLa Cells , Humans , Mice , Microscopy, Electron, Scanning , Molecular Sequence Data , VirulenceABSTRACT
A key strategy in microbial pathogenesis is the subversion of the first line of cellular immune defences presented by professional phagocytes. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC respectively) remain extracellular while colonizing the gut mucosa by attaching and effacing mechanism. EPEC use the type three secretion system effector protein EspF to prevent their own uptake into macrophages. EPEC can also block in trans the internalization of IgG-opsonized particles. In this study, we show that EspJ is the type three secretion system effector protein responsible for trans-inhibition of macrophage opsono-phagocytosis by both EPEC and EHEC. While EspF plays no role in trans-inhibition of opsono-phagocytosis, espJ mutants of EPEC or EHEC are unable to block uptake of opsonized sheep red blood cells (RBC), a phenotype that is rescued upon complementation with the espJ gene. Importantly, ectopic expression of EspJ(EHEC) in phagocytes is sufficient to inhibit internalization of both IgG- and C3bi-opsonized RBC. These results suggest that EspJ targets a basic mechanism common to these two unrelated phagocytic receptors. Moreover, EspF and EspJ target independent aspects of the phagocytic function of mammalian macrophages in vitro.
Subject(s)
Enterohemorrhagic Escherichia coli/metabolism , Escherichia coli O157/metabolism , Escherichia coli Proteins/metabolism , Macrophages/microbiology , Opsonin Proteins/immunology , Phagocytosis , Animals , COS Cells , Carrier Proteins/metabolism , Chlorocebus aethiops , Intracellular Signaling Peptides and Proteins , Macrophage-1 Antigen/immunology , TransfectionABSTRACT
EspF of enteropathogenic Escherichia coli targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified sorting nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and non-mitochondrial protein colocalization in infected epithelial cells.