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The EPEC type III secretion system injects ~21 effectors into host enterocytes. EspZ, an effector secreted early in infection, delays host cell death, regulates effector translocation (rheostat function), and is essential for virulence; an isogenic non-polar\n                      <jats:italic toggle=\"yes\">espZ<\/jats:italic>\n                      mutant (\u0394\n                      <jats:italic toggle=\"yes\">espZ<\/jats:italic>\n                      ) causes increased death of epithelial cells relative to wild-type EPEC. EspZ has two predicted transmembrane (TM) domains separated by an 8\u201310 aa loop region. To systematically characterize EspZ functional domains, we generated plasmid-encoded variants (pM1\u2013pM16) to sequentially replace 3\u20135 amino acid (aa) blocks of the protein and assessed their ability to complement \u0394\n                      <jats:italic toggle=\"yes\">espZ<\/jats:italic>\n                      . Changes in three regions (M4:\n                      <jats:sup>37<\/jats:sup>\n                      GTSRSV\n                      <jats:sup>42<\/jats:sup>\n                      \u2192\n                      <jats:sup>37<\/jats:sup>\n                      AAAAAA\n                      <jats:sup>42<\/jats:sup>\n                      ; M12:\n                      <jats:sup>74<\/jats:sup>\n                      LGLGI\n                      <jats:sup>78<\/jats:sup>\n                      \u2192\n                      <jats:sup>74<\/jats:sup>\n                      AVAAA\n                      <jats:sup>78<\/jats:sup>\n                      ; M13:\n                      <jats:sup>81<\/jats:sup>\n                      GVLGG\n                      <jats:sup>85<\/jats:sup>\n                      \u2192\n                      <jats:sup>81<\/jats:sup>\n                      VAAAV\n                      <jats:sup>85<\/jats:sup>\n                      ) impaired \u0394\n                      <jats:italic toggle=\"yes\">espZ<\/jats:italic>\n                      complementation for cytoprotection. M4 and M13 changes, but not M12 alterations, abrogated EspZ secretion. Transfected epithelial cells ectopically expressing the M4 variant, but not the M12 and M13 variants, were protected from \u0394\n                      <jats:italic toggle=\"yes\">espZ<\/jats:italic>\n                      infection-induced heightened death. However, the M12 variant was comparable to WT EspZ for the rheostat function, suggesting that EspZ has a rheostat-independent cytoprotective role. The M12 and M13 alterations overlap with a tandem GXXXG (glycine zipper) motif, which is known to promote homotypic and heterotypic protein-protein interactions. ToxLuc assays confirmed that EspZ TM2 localizes to the membrane and promotes homotypic interactions, and targeted alteration of the glycine zipper abolished self-association. Collectively, these data implicate two internal sequences (M4, M13) in EspZ secretion, confirm membrane targeting of TM1 and TM2, and implicate the TM2 glycine zipper in EspZ self-association and host cytoprotection.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Enteropathogenic\n                        <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                        (EPEC) causes severe diarrhea and mortality in young children. There are limited therapeutic options for EPEC diarrhea, and there are no approved vaccines. EPEC injects several proteins into intestinal cells, and the changes mediated by these proteins lead to disease. One such protein, EspZ, enters human cells, fine-tunes the injection apparatus, and delays the death of the host cells. The EspZ regions critical for its functions and for its ability to promote disease are not well-defined. Our studies show that two regions (M4, M13) are necessary for EspZ delivery into human cells, and a third region (M12) is required for keeping host cells alive. We also show that a feature called a \u201cglycine zipper\u201d in the region overlapping with M12 and M13 facilitates protein-protein associations that may be critical for function. Our studies shed critical knowledge on the role of EspZ in EPEC-induced diarrhea.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.00943-26","type":"journal-article","created":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T13:01:06Z","timestamp":1789131666000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["A transmembrane region in EPEC EspZ promotes oligomerization and contributes to survival of infected intestinal epithelial cells"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-2190-3759","authenticated-orcid":false,"given":"Katie","family":"Cocchi","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03m2x1q45","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Animal and Comparative Biomedical Sciences, University of Arizona","place":["Tucson, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shylaja","family":"Ramamurthy","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03m2x1q45","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Animal and Comparative Biomedical Sciences, University of Arizona","place":["Tucson, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6276-9208","authenticated-orcid":false,"given":"Jennifer Lising","family":"Roxas","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03m2x1q45","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Animal and Comparative Biomedical Sciences, University of Arizona","place":["Tucson, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bryan Angelo P.","family":"Roxas","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03m2x1q45","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Animal and Comparative Biomedical Sciences, University of Arizona","place":["Tucson, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michele","family":"Chu-Pilli","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03m2x1q45","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Animal and Comparative Biomedical Sciences, University of Arizona","place":["Tucson, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cole","family":"Puetz","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03m2x1q45","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Animal and Comparative Biomedical Sciences, University of Arizona","place":["Tucson, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1540-9821","authenticated-orcid":true,"given":"Samuel K.","family":"Campos","sequence":"additional","affiliation":[{"name":"Department of Biology, Morgan State University","place":["Baltimore, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3144-0610","authenticated-orcid":true,"given":"Gayatri","family":"Vedantam","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03m2x1q45","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Animal and Comparative Biomedical Sciences, University of Arizona","place":["Tucson, USA"]},{"id":[{"id":"https:\/\/ror.org\/03m2x1q45","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Immunobiology, University of Arizona","place":["Tucson, USA"]},{"id":[{"id":"https:\/\/ror.org\/03m2x1q45","id-type":"ROR","asserted-by":"publisher"}],"name":"BIO5 Institute for Collaborative Research, University of Arizona","place":["Tucson, USA"]},{"id":[{"id":"https:\/\/ror.org\/00xb4cb83","id-type":"ROR","asserted-by":"publisher"}],"name":"Southern Arizona VA Healthcare System","place":["Tucson, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9258-9865","authenticated-orcid":true,"given":"V. 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