{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T18:22:18Z","timestamp":1785435738794,"version":"3.56.0"},"posted":{"date-parts":[[2026,6,13]]},"group-title":"Microbiology","reference-count":57,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,13]],"date-time":"2026-06-13T00:00:00Z","timestamp":1781308800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"award":["R01AI185119"],"award-info":[{"award-number":["R01AI185119"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]},{"award":["T34GM136498"],"award-info":[{"award-number":["T34GM136498"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Lipopolysaccharide (LPS) is highly immunostimulatory, yet it is evolutionarily conserved among many obligate intracellular bacteria for unknown reasons. We report a forward genetic screen to identify factors required for survival of the tick-borne obligate cytosolic pathogen\n                  <jats:italic>Rickettsia parkeri<\/jats:italic>\n                  in primary macrophages. The most critical factors were WecA and RmlD, which synthesize O-antigen, the outermost layer of LPS.\n                  <jats:italic>wecA<\/jats:italic>\n                  and\n                  <jats:italic>rmlD<\/jats:italic>\n                  mutants grew at similar rates to wild type bacteria in epithelial cells, yet in macrophages they were targeted by guanylate binding proteins (GBPs) and they hyperactivated inflammasomes. Survival of O-antigen-deficient mutants was restored &gt;1,000-fold in macrophages lacking Caspases-1 and -11, interferon signaling, and nitric oxide production, suggesting a multifaceted role for O-antigen in protecting against innate immunity. O-antigen was essential for causing disease in mice and protected\n                  <jats:italic>R. parkeri<\/jats:italic>\n                  against complement\n                  <jats:italic>in vitro<\/jats:italic>\n                  . Despite O-antigen being known as a major target of antibodies, mice immunized with O-antigen-deficient mutants were protected from a lethal rechallenge, suggesting that protection can be elicited independently of O-antigen-targeting antibodies. Together, these findings help resolve a paradox as to why obligate cytosolic bacteria evolutionarily maintain LPS despite it being immunostimulatory, which is that it serves as a multifunctional shield against innate immunity.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance:<\/jats:title>\n                  <jats:p>\n                    Eukaryotic innate immune systems evolved to detect conserved microbial structures as danger signals of infection. Intracellular pathogens, in turn, evolved to hide from innate immunity, yet these mechanisms remain incompletely understood. Here, we performed an unbiased forward genetic screen in macrophages that identified lipopolysaccharide O-antigen as a critical virulence determinant in the tick-borne obligate cytosolic pathogen\n                    <jats:italic>Rickettsia parkeri<\/jats:italic>\n                    . We found that O-antigen shields the bacteria from multiple innate immune defenses, including guanylate-binding proteins, inflammasomes, nitric oxide, and complement. These findings reveal why a highly immunostimulatory molecule such as lipopolysaccharide is maintained by an obligate intracellular pathogen and establish O-antigen as a central determinant of\n                    <jats:italic>Rickettsia<\/jats:italic>\n                    cytosolic survival with implications for vaccine development.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.06.12.731980","type":"posted-content","created":{"date-parts":[[2026,6,14]],"date-time":"2026-06-14T04:55:16Z","timestamp":1781412916000},"source":"Crossref","is-referenced-by-count":0,"title":["A genetic screen identifies O-antigen as essential for\n                  <i>Rickettsia parkeri<\/i>\n                  survival in macrophages by protecting from inflammasomes and interferon-stimulated genes"],"prefix":"10.64898","author":[{"given":"Han","family":"Sun","sequence":"first","affiliation":[{"name":"Department of Microbiology and Molecular Medicine, School of Medicine, University of California, Irvine; Irvine, CA 92617 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alejandro A.","family":"Guzman","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Medicine, School of Medicine, University of California, Irvine; Irvine, CA 92617 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1385-8757","authenticated-orcid":false,"given":"Thomas P.","family":"Burke","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Medicine, School of Medicine, University of California, Irvine; Irvine, CA 92617 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026061607051865000_2026.06.12.731980v1.1","doi-asserted-by":"publisher","DOI":"10.1086\/381894"},{"key":"2026061607051865000_2026.06.12.731980v1.2","doi-asserted-by":"publisher","DOI":"10.1086\/592254"},{"key":"2026061607051865000_2026.06.12.731980v1.3","first-page":"1","article-title":"Centers for Disease Control & Prevention (CDC) Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever and Other Spotted Fever Group Rickettsioses, Ehrlichioses, and Anaplasmosis-United States","volume":"65","year":"2016","journal-title":"Mortality Weekly Report: Recommendations and Reports"},{"key":"2026061607051865000_2026.06.12.731980v1.4","doi-asserted-by":"publisher","DOI":"10.3201\/eid2205.151824"},{"key":"2026061607051865000_2026.06.12.731980v1.5","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0131080"},{"key":"2026061607051865000_2026.06.12.731980v1.6","first-page":"e03463","article-title":"Unique strain of Rickettsia parkeri associated with the hard tick Dermacentor parumapertus Neumann in the western United States","volume":"83","year":"2017","journal-title":"Applied and Environmental Microbiology"},{"key":"2026061607051865000_2026.06.12.731980v1.7","doi-asserted-by":"crossref","unstructured":"Burke, T. 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