{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,29]],"date-time":"2026-06-29T19:49:22Z","timestamp":1782762562840,"version":"3.54.5"},"posted":{"date-parts":[[2026,6,26]]},"group-title":"Genetics","reference-count":91,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T00:00:00Z","timestamp":1782432000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,26]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  <jats:italic>Orientia tsutsugamushi<\/jats:italic>\n                  is a mite-transmitted obligate intracellular bacterium that causes the potentially deadly zoonosis, scrub typhus. The absence of genetic tools for\n                  <jats:italic>Orientia<\/jats:italic>\n                  have limited studies of the microbe-host interactions that underlie scrub typhus. To address this gap, we developed a protocol for transforming and achieving allelic exchange in\n                  <jats:italic>O. tsutsugamushi<\/jats:italic>\n                  str. Ikeda. From evaluating multiple cell lines and antibiotics, we found that contact-inhibited EA.hy926 human endothelial-like cells best supported\n                  <jats:italic>Orientia<\/jats:italic>\n                  replication and that chloramphenicol was an effective selection marker. We engineered a homologous recombination cassette encoding a codon-modified version of the\n                  <jats:italic>O. tsutsugamushi ank13<\/jats:italic>\n                  gene (OTT_RS04140) (CM\n                  <jats:italic>ank13<\/jats:italic>\n                  ) and its promoter alongside genes for mScarlet-I and chloramphenicol acetyltransferase under control of the\n                  <jats:italic>O. tsutsugamushi tsa22<\/jats:italic>\n                  -up and\n                  <jats:italic>tsa56<\/jats:italic>\n                  -down promoters, respectively. A PCR product encompassing the cassette and chromosomal flanking regions was transformed into\n                  <jats:italic>O. tsutsugamushi<\/jats:italic>\n                  via electroporation or CaCl\n                  <jats:sub>2<\/jats:sub>\n                  , the latter of which better preserved bacterial and host cell viability. EA.hy926 cells inoculated with transformed\n                  <jats:italic>O. tsutsugamushi<\/jats:italic>\n                  were grown in glass-bottom plates in the presence of chloramphenicol and imaged by live-cell microscopy to identify cultures containing mScarlet-I positive bacteria, which could be maintained in perpetuity. Chromosomal integration of the CM\n                  <jats:italic>ank13<\/jats:italic>\n                  cassette and loss of wild-type\n                  <jats:italic>ank13<\/jats:italic>\n                  were verified by PCR and nanopore sequencing. This report establishes platforms for genetically manipulating\n                  <jats:italic>O. tsutsugamushi<\/jats:italic>\n                  and building additional genetic tools to investigate this globally significant pathogen.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>IMPORTANCE<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Orientia tsutsugamushi<\/jats:italic>\n                    causes scrub typhus, a globally emerging rickettsiosis that can have a high mortality rate and has been a known human disease since the fourth century. Of the genera of obligate intracellular bacterial pathogens that cause human disease,\n                    <jats:italic>Orientia<\/jats:italic>\n                    is the only one for which genetic tools have not been developed. This has limited understanding of\n                    <jats:italic>O. tsutsugamushi<\/jats:italic>\n                    -host dynamics that drive the bacterium\u2019s pathobiology and hindered development of novel treatment or protection strategies against scrub typhus. Here, we successfully transformed and achieved allelic exchange in\n                    <jats:italic>O. tsutsugamushi<\/jats:italic>\n                    . Transgenic bacteria were selected via antibiotic resistance, validated by PCR and nanopore sequencing, and visualized by immunofluorescence and live-cell fluorescence imaging. Our report includes detailed descriptions of empirically determined host cell cultivation, multiplicity of infection, transformation, and selection conditions to provide a foundation on which other researchers can build. Overall, this work begins to establish a genetic toolbox for\n                    <jats:italic>O. tsutsugamushi<\/jats:italic>\n                    .\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.06.22.733791","type":"posted-content","created":{"date-parts":[[2026,6,27]],"date-time":"2026-06-27T12:30:29Z","timestamp":1782563429000},"source":"Crossref","is-referenced-by-count":0,"title":["Transformation and allelic exchange in\n                  <i>Orientia tsutsugamushi<\/i>"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0299-7380","authenticated-orcid":false,"given":"Paige E.","family":"Allen","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jason R.","family":"Hunt","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Travis J.","family":"Chiarelli","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2778-5066","authenticated-orcid":false,"given":"Jason A.","family":"Carlyon","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026062912300806000_2026.06.22.733791v1.1","doi-asserted-by":"crossref","first-page":"107151","DOI":"10.1016\/j.ijid.2024.107151","article-title":"Global and regional seroprevalence, incidence, mortality of, and risk factors for scrub typhus: A systematic review and meta-analysis","volume":"146","year":"2024","journal-title":"Int J Infect Dis"},{"key":"2026062912300806000_2026.06.22.733791v1.2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0005838"},{"key":"2026062912300806000_2026.06.22.733791v1.3","doi-asserted-by":"publisher","DOI":"10.1186\/s13071-015-0784-7"},{"key":"2026062912300806000_2026.06.22.733791v1.4","doi-asserted-by":"crossref","unstructured":"Maina AN , Farris CM , Odhiambo A , Jiang J , Laktabai J , Armstrong J , Holland T , Richards AL , O\u2019Meara WP . 2016. 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