{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,20]],"date-time":"2026-09-20T12:52:53Z","timestamp":1789908773876,"version":"4.0.1"},"posted":{"date-parts":[[2026,5,29]]},"group-title":"Microbiology","reference-count":43,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T00:00:00Z","timestamp":1780012800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["INV-039628"],"award-info":[{"award-number":["INV-039628"]}],"id":[{"id":"https:\/\/ror.org\/0456r8d26","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01 AI169892"],"award-info":[{"award-number":["R01 AI169892"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01 AI172066"],"award-info":[{"award-number":["R01 AI172066"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01 AI52533"],"award-info":[{"award-number":["R01 AI52533"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["S10 OD026929"],"award-info":[{"award-number":["S10 OD026929"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,29]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Gene expression analysis in malaria parasites has been used to define transcriptional regulatory networks but has been used less frequently to characterize parasite response to drug treatment or to show how parasites may evade killing. Here, we applied single-cell RNA sequencing (scRNA-seq) to hundreds of thousands of individually infected asynchronous red blood cells to evaluate the parasite\u2019s response to treatment with three chemotypes that can be used for treatment (artemisinin) or prophylaxis and treatment (atovaquone, ganaplacide). We found that each treatment gave rise to different cell populations with different transcriptional profiles. Comparing single cell transcription patterns in compound-treated cells, to transcript patterns observed previously with synchronized cells showed an enrichment of cells expressing gametocyte-associated genes after artemisinin treatment but fewer lifecycle perturbations after treatment with the two other compounds. In contrast, bulk analysis showed an enrichment of pyrimidine biosynthesis transcripts for atovaquone treatment. Our results show that scRNA-seq may be used to profile diverse drug responses across many lifecycle stages and to potentially classify drug classes.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>Determining the mechanism of action (MOA) of compounds with antimalarial activity remains a key activity in both drug development and drug resistance studies but remains challenging for some chemotypes. Here we highlight the potential of single cell transcriptional sequencing to augment the process of MOA deconvolution. We develop a new analytical pipeline that involves comparing single cell transcription patterns to existing profiles from synchronized parasites to comprehensively characterize life cycle stage enrichments that may be observed after chemical perturbations. We also show that transcriptional feedback regulation may be present for some drug classes.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.05.27.728291","type":"posted-content","created":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T08:50:16Z","timestamp":1780044616000},"source":"Crossref","is-referenced-by-count":2,"title":["Single-cell analysis of\n                  <i>Plasmodium falciparum<\/i>\n                  transcripts after drug perturbation identifies feedback regulation as well as increased transmission potential"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6323-0204","authenticated-orcid":false,"given":"Karla P.","family":"Godinez-Macias","sequence":"first","affiliation":[{"name":"Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4812-3168","authenticated-orcid":false,"given":"Jaeson","family":"Calla","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristen","family":"Jepsen","sequence":"additional","affiliation":[{"name":"Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4049-2113","authenticated-orcid":false,"given":"Elizabeth A.","family":"Winzeler","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA"},{"name":"Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026060109150884000_2026.05.27.728291v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41573-023-00772-9"},{"key":"2026060109150884000_2026.05.27.728291v1.2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.0000005"},{"key":"2026060109150884000_2026.05.27.728291v1.3","doi-asserted-by":"publisher","DOI":"10.1126\/science.1087025"},{"key":"2026060109150884000_2026.05.27.728291v1.4","doi-asserted-by":"crossref","unstructured":"Howick VM , Russell AJC , Andrews T , Heaton H , Reid AJ , Natarajan K , Butungi H , Metcalf T , Verzier LH , Rayner JC , Berriman M , Herren JK , Billker O , Hemberg M , Talman AM , Lawniczak MKN . 2019. The malaria cell atlas: single parasite transcriptomes across the complete Plasmodium life cycle. Science 365.","DOI":"10.1126\/science.aaw2619"},{"key":"2026060109150884000_2026.05.27.728291v1.5","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1038\/s41564-024-01905-5","article-title":"Genome-wide gene expression profiles throughout human malaria parasite liver stage development in humanized mice","volume":"10","year":"2025","journal-title":"Nat Microbiol"},{"key":"2026060109150884000_2026.05.27.728291v1.6","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)81845-0"},{"key":"2026060109150884000_2026.05.27.728291v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/s41588-021-00840-z"},{"key":"2026060109150884000_2026.05.27.728291v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-07466-1"},{"key":"2026060109150884000_2026.05.27.728291v1.9","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1000138107"},{"key":"2026060109150884000_2026.05.27.728291v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)00015-5"},{"key":"2026060109150884000_2026.05.27.728291v1.11","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-20805-w"},{"key":"2026060109150884000_2026.05.27.728291v1.12","doi-asserted-by":"crossref","unstructured":"Lee HJ , Georgiadou A , Otto TD , Levin M , Coin LJ , Conway DJ , Cunnington AJ . 2018. Transcriptomic studies of malaria: a paradigm for investigation of systemic host-pathogen interactions. Microbiol Mol Biol Rev 82.","DOI":"10.1128\/MMBR.00071-17"},{"key":"2026060109150884000_2026.05.27.728291v1.13","unstructured":"10x Genomics. Chromium single cell V(D)J reagent kits with feature barcoding technology for cell surface protein, document number CG000186 rev A, 2019."},{"key":"2026060109150884000_2026.05.27.728291v1.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.pt.2021.01.009"},{"key":"2026060109150884000_2026.05.27.728291v1.15","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-15440-4"},{"key":"2026060109150884000_2026.05.27.728291v1.16","doi-asserted-by":"crossref","unstructured":"LaMonte G , Lim MY , Wree M , Reimer C , Nachon M , Corey V , Gedeck P , Plouffe D , Du A , Figueroa N , Yeung B , Bifani P , Winzeler EA. 2016. Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) confer multidrug resistance. mBio 7.","DOI":"10.1128\/mBio.00696-16"},{"key":"2026060109150884000_2026.05.27.728291v1.17","doi-asserted-by":"crossref","first-page":"2877","DOI":"10.1093\/jac\/dkae300","article-title":"In vitro evaluation of ganaplacide\/lumefantrine combination against Plasmodium falciparum in a context of artemisinin resistance","volume":"79","year":"2024","journal-title":"J Antimicrob Chemother"},{"key":"2026060109150884000_2026.05.27.728291v1.18","doi-asserted-by":"crossref","unstructured":"Zheng D , Liu T , Yu S , Liu Z , Wang J , Wang Y . 2024. Antimalarial mechanisms and resistance status of artemisinin and its derivatives. Trop Med Infect Dis 9.","DOI":"10.3390\/tropicalmed9090223"},{"key":"2026060109150884000_2026.05.27.728291v1.19","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2164-9-398"},{"key":"2026060109150884000_2026.05.27.728291v1.20","doi-asserted-by":"crossref","first-page":"58","DOI":"10.12688\/wellcomeopenres.15194.1","article-title":"Progression of the canonical reference malaria parasite genome from 2002-2019","volume":"4","year":"2019","journal-title":"Wellcome Open Res"},{"key":"2026060109150884000_2026.05.27.728291v1.21","doi-asserted-by":"publisher","DOI":"10.1038\/S41587-023-01767-Y"},{"key":"2026060109150884000_2026.05.27.728291v1.22","doi-asserted-by":"publisher","DOI":"10.1101\/gr.203711.115"},{"key":"2026060109150884000_2026.05.27.728291v1.23","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2019.05.031"},{"key":"2026060109150884000_2026.05.27.728291v1.24","doi-asserted-by":"publisher","DOI":"10.1186\/s12864-024-10364-5"},{"key":"2026060109150884000_2026.05.27.728291v1.25","doi-asserted-by":"crossref","first-page":"44","DOI":"10.3390\/ph4010044","article-title":"Blocking Plasmodium falciparum malaria transmission with drugs: the gametocytocidal and sporontocidal properties of current and prospective antimalarials","volume":"4","year":"2010","journal-title":"Pharmaceuticals"},{"key":"2026060109150884000_2026.05.27.728291v1.26","doi-asserted-by":"crossref","unstructured":"Dembele L , Gupta DK , Lim MY , Ang X , Selva JJ , Chotivanich K , Nguon C , Dondorp AM , Bonamy GMC , Diagana TT , Bifani P . 2018. Imidazolopiperazines kill both rings and dormant rings in wild-type and K13 artemisinin-resistant Plasmodium falciparum in vitro. Antimicrob Agents Chemother 62.","DOI":"10.1128\/AAC.02235-17"},{"key":"2026060109150884000_2026.05.27.728291v1.27","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dks504"},{"key":"2026060109150884000_2026.05.27.728291v1.28","doi-asserted-by":"crossref","first-page":"841833","DOI":"10.3389\/fcimb.2022.841833","article-title":"Novel highlight in malarial drug discovery: aspartate transcarbamoylase","volume":"12","year":"2022","journal-title":"Front Cell Infect Microbiol"},{"key":"2026060109150884000_2026.05.27.728291v1.29","doi-asserted-by":"crossref","first-page":"e0397622","DOI":"10.1128\/spectrum.03976-22","article-title":"A conserved metabolic signature associated with response to fast-acting anti-malarial agents","volume":"11","year":"2023","journal-title":"Microbiol Spectr"},{"key":"2026060109150884000_2026.05.27.728291v1.30","doi-asserted-by":"publisher","DOI":"10.1038\/nature01097"},{"key":"2026060109150884000_2026.05.27.728291v1.31","doi-asserted-by":"publisher","DOI":"10.1101\/gr.2218604"},{"key":"2026060109150884000_2026.05.27.728291v1.32","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0801993105"},{"key":"2026060109150884000_2026.05.27.728291v1.33","doi-asserted-by":"crossref","unstructured":"Luth MR , Godinez-Macias KP , Chen D , Okombo J , Thathy V , Cheng X , Daggupati S , Davies H , Dhingra SK , Economy JM , Edgar RCS , Gomez-Lorenzo MG , Istvan ES , Jado JC , LaMonte GM , Melillo B , Mok S , Narwal SK , Ndiaye T , Ottilie S , Palomo Diaz S , Park H , Pena S , Rocamora F , Sakata-Kato T , Small-Saunders JL , Summers RL , Tumwebaze PK , Vanaerschot M , Xia G , Yeo T , You A , Gamo FJ , Goldberg DE , Lee MCS , McNamara CW , Ndiaye D , Rosenthal PJ , Schreiber SL , Serra G , De Siqueira-Neto JL , Skinner-Adams TS , Uhlemann AC , Kato N , Lukens AK , Wirth DF , Fidock DA , Winzeler EA . 2024. Systematic in vitro evolution in Plasmodium falciparum reveals key determinants of drug resistance. 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