{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T17:09:21Z","timestamp":1785431361930,"version":"3.56.0"},"reference-count":35,"publisher":"American Society for Microbiology","issue":"4","license":[{"start":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T00:00:00Z","timestamp":1777334400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T00:00:00Z","timestamp":1777334400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R21AI183196-01"],"award-info":[{"award-number":["R21AI183196-01"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mSphere"],"published-print":{"date-parts":[[2026,4,28]]},"abstract":"ABSTRACT<\/jats:title>\n \n \n \n Trypanosoma brucei<\/jats:italic>\n is a model unicellular parasite for cellular and molecular genetic studies, but tools for more multiplexed experiments are limited. Tetracycline (Tet)-inducible gene regulation has been a long-standing and effective approach for overexpression, RNAi, and genome-wide screens and has been foundational for studying essential genes that are required for biological processes and identifying potential drug targets. To achieve greater flexibility in experimental design, we capitalized upon previously described dual inducer systems that combined vanillic acid (Van) or cumate (Cym) with Tet as inducers. Here, we report the development of a triple inducible system combining Cym, Van, and Tet repressors to selectively regulate the expression of three genes within a single cell line called PHITER. To demonstrate independent control, we adapted the previously characterized Van\/Tet dual inducible RNAi complementation cell line, IBComp\n VaT<\/jats:sup>\n , for additional Cym inducible expression of an eGFP reporter. We provide evidence that each inducer operates independently with no evidence of leaky expression. In this system, Cym induction is specific and tunable. As proof of principle, we used triple induction for\n POLIB<\/jats:italic>\n RNAi, complementation with a recoded ectopic POLIB-PTP variant, and overexpression of the maxicircle helicase. We confirmed that Cym inducible PIF2 overexpression resulted in massive overreplication of maxicircles that is suppressed when\n POLIB<\/jats:italic>\n is silenced, thereby demonstrating a requirement for POLIB during replication stress. The triple inducible system enables the study of complex and pleiotropic phenotypes through a more sophisticated experimental design that could not previously be achieved and can be applied for the development of multiplexed genomic screens.\n <\/jats:p>\n \n IMPORTANCE<\/jats:title>\n \n Trypanosoma brucei<\/jats:italic>\n is a protist parasite that causes significant health and economic burden for sub-Saharan Africa and serves as a key model organism for a group of eukaryotes called the kinetoplastids.\n T. brucei<\/jats:italic>\n and related parasites contain an interlocked mtDNA network called kinetoplast DNA (kDNA), composed of maxicircles and minicircles. Although\n T. brucei<\/jats:italic>\n is highly genetically tractable, limitations exist for more complex experimental designs and multiplexed forward genetic screens. Here, we describe the first triple inducible system in\n T. brucei<\/jats:italic>\n or any eukaryote that combines three independent repressors (cumate [Cym], tetracycline [Tet], and vanillic acid [Van]) to selectively regulate three genes within a single cell line. We demonstrate the utility of the system using an eGFP reporter and robust RNAi complementation of kDNA polymerase POLIB. Furthermore, we investigate the role of POLIB in maxicircle replication by overexpressing the maxicircle helicase during\n POLIB<\/jats:italic>\n RNAi, revealing an early requirement for POLIB during replication stress.\n <\/jats:p>\n <\/jats:sec>\n <\/jats:sec>","DOI":"10.1128\/msphere.00596-25","type":"journal-article","created":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T13:00:13Z","timestamp":1774530013000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["A trypanosome trifecta: an independently tunable triple inducible system for genetic studies in\n Trypanosoma brucei<\/i>"],"prefix":"10.1128","volume":"11","author":[{"given":"Matt J.","family":"Romprey","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0072zz521","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, University of Massachusetts","place":["Amherst, 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Synthesis and in vitro anti-trypanosomal activity of vanillic acid and para-hydroxybenzoic acid on T. congolense. 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