Functional Elements of EspF<sub>u</sub>, an Enterohemorrhagic <em>E. coli</em> Effector that Stimulates Actin Assembly: A Dissertation
Authors
Skehan, Brian M.Faculty Advisor
John M. LeongAcademic Program
MD/PhDUMass Chan Affiliations
Molecular Genetics and MicrobiologyDocument Type
Doctoral DissertationPublication Date
2009-06-17Keywords
Enterohemorrhagic Escherichia coliEscherichia coli Proteins
Microfilament Proteins
Actins
Carrier Proteins
Receptors
Cell Surface
Amino Acids, Peptides, and Proteins
Bacteria
Macromolecular Substances
Metadata
Show full item recordAbstract
Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an attaching and effacing pathogen that upon attachment to host cells, induce characteristic attaching and effacing lesions and formation of F-actin rich pedestals beneath sites of bacterial attachment. EHEC harbors a Type III secretion system through which it delivers dozens of effectors into the host cell. The two secreted effectors critical for EHEC-mediated actin pedestal formation are the translocated intimin receptor (Tir) and EspFU. EspFU consists of an N-terminal secretion signal and a C-terminus containing six tandem 47-residue proline-rich repeats, each of which can bind and activate the actin nucleation promoting factor N-WASP. Structural and functional analyses described here have identified the mechanism of N-WASP activation by EspFU and the minimal domains and specific residues required for this activity. While EspFU and Tir are the only bacterial effectors required for F-actin pedestal formation, recruitment of EspFU to Tir is mediated by an unidentified putative host factor. To identify the host factor responsible for linking these two effectors, a combination of in vitro and functional assays were used to identify the host factor, IRTKS and the residues required for these interactions were defined. Further, the presence of at least two 47-residue repeats in all characterized clinical isolates of canonical EHEC strains led us to address the minimal requirements for EspFU functional domains to promote recruitment to Tir and N-WASP activation. Here we show that two proline-rich elements of EspFU are required for recruitment of EspFU by IRTKS to sites of bacterial attachment. Furthermore, once artificially clustered at the membrane, a single N-WASP binding element of EspFU can induce actin pedestal formation.DOI
10.13028/y7mt-8035Permanent Link to this Item
http://hdl.handle.net/20.500.14038/31772Rights
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10.13028/y7mt-8035