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The Cag T4SS outer membrane core complex (OMCC) consists of an outer membrane cap (OMC), a periplasmic ring (PR), and a stalk. CagX and CagY span both the OMC (14 copies) and PR (17 copies) subassemblies of the OMCC. CagX is structurally related to VirB9 proteins found in other bacterial T4SSs, except for a unique 130-amino-acid region, designated as the periplasmic insertion (PI) domain. In this study, we investigated the contributions of individual CagX domains to Cag T4SS activity and OMCC assembly by engineering\n                      <jats:italic toggle=\"yes\">H. pylori<\/jats:italic>\n                      strains that produce only the CagX OMC domain, only the CagX PR domain, the CagX OMC and PR domains produced as independent proteins (Split CagX), or a CagX protein lacking the PI domain (CagX \u0394131\u2013260). We found that the OMC, PR, and PI domains of CagX are each essential for Cag T4SS activity. Electron microscopy analyses revealed that the Split CagX mutant could assemble the Cag T4SS PR (17 copies of CagX and CagY), and the CagX \u0394131\u2013260 mutant could assemble partially intact OMCCs. Crosslinking mass spectrometry analysis showed that the CagX \u0394131\u2013260 protein retained spatial relationships with the OMCC components CagY and CagM. An intact CagX protein was required for the association of CagW, CagH, CagI, and CagL with the OMCC. These results provide novel insights into the contributions of individual CagX domains to the assembly and activity of the Cag T4SS.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/iai.00699-25","type":"journal-article","created":{"date-parts":[[2026,6,10]],"date-time":"2026-06-10T13:44:41Z","timestamp":1781099081000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Contributions of conserved and species-specific CagX (VirB9) domains to the assembly and function of the\n                    <i>Helicobacter pylori<\/i>\n                    Cag type IV secretion system"],"prefix":"10.1128","volume":"94","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6366-0570","authenticated-orcid":false,"given":"Chiamaka D.","family":"Okoye","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05dq2gs74","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center","place":["Nashville, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7202-4701","authenticated-orcid":true,"given":"Arwen E.","family":"Frick-Cheng","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00jmfr291","id-type":"ROR","asserted-by":"publisher"}],"name":"Life Sciences Institute, University of Michigan","place":["Ann Arbor, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wilhelm","family":"Salmen","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00jmfr291","id-type":"ROR","asserted-by":"publisher"}],"name":"Life Sciences Institute, University of Michigan","place":["Ann Arbor, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sirena C.","family":"Tran","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05dq2gs74","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center","place":["Nashville, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"W. 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