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Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis
Published on: May 23, 2021
Identification and characterization of Ibe, a novel type III effector protein of A/E pathogens targeting human IQGAP1
Christoph Buss1, Daniel Müller, Christian Rüter
1Institut für Infektiologie, Zentrum für Molekularbiologie der Entzündung (ZMBE), Westfälische Wilhelms-Universität Münster, Münster, Germany.
Abstract:
Enteropathogenic Escherichia coli (EPEC), atypical enteropathogenic Escherichia coli (ATEC) and enterohemorrhagic Escherichia coli (EHEC) belong to the family of attaching and effacing (A/E) pathogens. Pathogenicity is mediated by subversion of host cell functions involving type III secretion system (TTSS)-dependent effector proteins. In this study, we have identified and characterized a novel TTSS-dependent effector protein encoded at the 5'-end of the locus of enterocyte effacement (LEE) pathogenicity island (PAI) of ATEC strain 3431-4/86 (O8:H(-)). Using affinity purification we identified IQGAP1, a scaffolding protein involved in the regulation of the actin cytoskeleton, as a putative host cell target. Accordingly, we termed the novel effector protein 'Ibe' for IQGAP1-binding effector. The interaction of Ibe and IQGAP1 was confirmed by co-immunoprecipitation from ATEC-infected cells and immunofluorescence analysis, which revealed colocalization of Ibe and IQGAP1 in ATEC-induced pedestals and actin-rich membrane ruffles. This suggests that the putative effector function of Ibe is mediated via IQGAP1. The Ibe-independent recruitment of IQGAP1 to ATEC-induced pedestals implies a general role for IQGAP1 in the subversion of host cell functions during infection. Homologues of the novel effector Ibe are widely distributed among EPEC, ATEC and EHEC strains but are not necessarily genetically linked to the LEE as they have occasionally also been found to be encoded within lambdoid prophages.
Insights
Researchers discovered a new bacterial protein, Ibe, used by atypical enteropathogenic Escherichia coli (ATEC) to manipulate host cells. This protein targets IQGAP1, a key regulator of the cell
Area of Science:
- Microbiology and Molecular Pathogenesis
- Bacterial Genetics and Virulence Factors
- Cell Biology and Host-Pathogen Interactions
Background:
- Enteropathogenic Escherichia coli (EPEC), atypical EPEC (ATEC), and enterohemorrhagic EHEC are attaching and effacing (A/E) pathogens.
- Pathogenicity relies on type III secretion system (TTSS)-dependent effector proteins that subvert host cell functions.
- The locus of enterocyte effacement (LEE) pathogenicity island (PAI) encodes key virulence factors in APEC.
Purpose of the Study:
- To identify and characterize novel TTSS-dependent effector proteins from ATEC.
- To elucidate the host cell targets and functions of these novel effectors.
- To investigate the distribution and genetic linkage of these effectors in A/E pathogens.
Main Methods:
- Affinity purification to identify host cell targets of TTSS-dependent effectors.
- Co-immunoprecipitation assays to confirm protein-protein interactions.
- Immunofluorescence microscopy to visualize protein localization in infected host cells.
Main Results:
- A novel TTSS-dependent effector, termed 'Ibe' (IQGAP1-binding effector), was identified in ATEC strain 3431-4/86.
- Ibe directly interacts with IQGAP1, a host scaffolding protein regulating the actin cytoskeleton.
- Ibe and IQGAP1 colocalize in ATEC-induced pedestals and membrane ruffles, indicating Ibe's effector function via IQGAP1.
- IQGAP1 is recruited independently of Ibe, suggesting a broader role in ATEC infection.
- Ibe homologues are widespread among EPEC, ATEC, and EHEC, found both within and outside the LEE, including lambdoid prophages.
Conclusions:
- The novel effector Ibe targets the host protein IQGAP1, mediating subversion of host cell functions during ATEC infection.
- IQGAP1 plays a significant role in host cell manipulation by ATEC, independent of Ibe.
- The widespread distribution and variable genomic location of Ibe homologues highlight the diverse evolutionary strategies of A/E pathogens.
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