{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T19:40:07Z","timestamp":1768506007723,"version":"3.49.0"},"posted":{"date-parts":[[2023,7,6]]},"group-title":"Microbiology","reference-count":30,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,7,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Rickettsiae are Gram-negative obligate intracellular parasites of numerous eukaryotes. Human pathogens of the Transitional Group (TRG), Typhus Group (TG), and Spotted Fever Group (SFG) rickettsiae infect blood-feeding arthropods, have dissimilar clinical manifestations, and possess unique genomic and morphological attributes. Lacking glycolysis, rickettsiae pilfer numerous metabolites from host cytosol to synthesize peptidoglycan and lipopolysaccharide (LPS). For LPS, O-antigen immunogenicity varies between SFG and TG pathogens; however, lipid A proinflammatory potential is unknown. We previously demonstrated that\n                  <jats:italic>R. akari<\/jats:italic>\n                  (TRG),\n                  <jats:italic>R. typhi<\/jats:italic>\n                  (TG), and\n                  <jats:italic>R. montanensis<\/jats:italic>\n                  (SFG) produce lipid A with long 2\u2019 secondary acyl chains (C16 or C18) compared to short 2\u2019 secondary acyl chains (C12) in\n                  <jats:italic>R. rickettsii<\/jats:italic>\n                  (SFG) lipid A. To further probe this structural heterogeneity and estimate a time point when shorter 2\u2019 secondary acyl chains originated, we generated lipid A structures for two additional SFG rickettsiae (\n                  <jats:italic>R. rhipicephali<\/jats:italic>\n                  and\n                  <jats:italic>R. parkeri<\/jats:italic>\n                  ) utilizing Fast Lipid Analysis Technique adopted for use with tandem mass spectrometry (FLAT\n                  <jats:sup>n<\/jats:sup>\n                  ). FLAT\n                  <jats:sup>n<\/jats:sup>\n                  allowed analysis of lipid A structure directly from host cell-purified bacteria, providing substantial improvement over lipid A chemical extraction. FLAT\n                  <jats:sup>n<\/jats:sup>\n                  -derived structures indicate SFG rickettsiae diverging after\n                  <jats:italic>R. rhipicephali<\/jats:italic>\n                  evolved shorter 2\u2019 secondary acyl chains. Bioinformatics analysis of\n                  <jats:italic>Rickettsia<\/jats:italic>\n                  LpxL late acyltransferases revealed discrete active sites and hydrocarbon rulers for long versus short 2\u2019 secondary acyl chain addition. While the significance of different lipid A structures for diverse\n                  <jats:italic>Rickettsia<\/jats:italic>\n                  pathogens is unknown, our success using FLAT\n                  <jats:sup>n<\/jats:sup>\n                  will facilitate determining how structural heterogeneity impacts interactions with host lipid A receptors and overall inflammatory potential.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>IMPORTANCE<\/jats:title>\n                  <jats:p>\n                    Deforestation, urbanization, and homelessness lead to spikes in Rickettsioses. Vector-borne human pathogens of Transitional Group (TRG), Typhus Group (TG), and Spotted Fever Group (SFG) rickettsiae differ by clinical manifestations, immunopathology, genome composition, and morphology. We previously showed that lipid A (or endotoxin), the membrane anchor of Gram-negative bacterial lipopolysaccharide (LPS), structurally differs in\n                    <jats:italic>R. rickettsii<\/jats:italic>\n                    (later-evolving SFG) relative to\n                    <jats:italic>R. montanensis<\/jats:italic>\n                    (basal SFG),\n                    <jats:italic>R. typhi<\/jats:italic>\n                    (TG), and\n                    <jats:italic>R. akari<\/jats:italic>\n                    (TRG). As lipid A structure influences recognition potential in vertebrate LPS sensors, further assessment of\n                    <jats:italic>Rickettsia<\/jats:italic>\n                    lipid A structural heterogeneity is needed. Here, we sidestepped the difficulty of\n                    <jats:italic>ex vivo<\/jats:italic>\n                    lipid A chemical extraction by utilizing FLAT\n                    <jats:sup>n<\/jats:sup>\n                    , a new procedure for generating lipid A structures directly from host cell-purified bacteria. These data confirm later-evolving SFG pathogens synthesize structurally distinct lipid A. Our findings impact interpreting immune responses to different\n                    <jats:italic>Rickettsia<\/jats:italic>\n                    pathogens and utilizing lipid A adjuvant or anti-inflammatory properties in vaccinology.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2023.07.06.547954","type":"posted-content","created":{"date-parts":[[2023,7,7]],"date-time":"2023-07-07T00:30:15Z","timestamp":1688689815000},"source":"Crossref","is-referenced-by-count":1,"title":["Structural determination of\n                  <i>Rickettsia<\/i>\n                  lipid A without chemical extraction confirms shorter acyl chains in later-evolving Spotted Fever Group pathogens"],"prefix":"10.64898","author":[{"given":"Hyojik","family":"Yang","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Victoria I.","family":"Verhoeve","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Courtney E.","family":"Chandler","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shreeram","family":"Nallar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4409-0453","authenticated-orcid":false,"given":"Greg A.","family":"Snyder","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robert K.","family":"Ernst","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5447-7264","authenticated-orcid":false,"given":"Joseph J.","family":"Gillespie","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2023071205273717000_2023.07.06.547954v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-27838-9"},{"key":"2023071205273717000_2023.07.06.547954v1.2","doi-asserted-by":"crossref","unstructured":"Pilgrim J , Thongprem P , Davison HR , Siozios S , Baylis M , Zakharov E V , Ratnasingham S , deWaard JR , Macadam CR , Smith MA , Hurst GDD . 2021. 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