{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:17:27Z","timestamp":1785403047728,"version":"3.56.0"},"reference-count":49,"publisher":"American Society for Microbiology","issue":"6","license":[{"start":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T00:00:00Z","timestamp":1782777600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T00:00:00Z","timestamp":1782777600000},"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":["R01AI163140"],"award-info":[{"award-number":["R01AI163140"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["R01GM144545"],"award-info":[{"award-number":["R01GM144545"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mSphere"],"published-print":{"date-parts":[[2026,6,30]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Parasitic trypanosomatids, such as\n                      <jats:italic toggle=\"yes\">Trypanosoma cruzi<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">Trypanosoma brucei<\/jats:italic>\n                      , and\n                      <jats:italic toggle=\"yes\">Leishmania spp<\/jats:italic>\n                      ., cause devastating tropical diseases, with\n                      <jats:italic toggle=\"yes\">T. cruzi<\/jats:italic>\n                      being increasingly recognized as a public health concern in the United States due to established sylvatic cycles and rising autochthonous transmission. Although\n                      <jats:italic toggle=\"yes\">T. brucei<\/jats:italic>\n                      has long served as the model trypanosomatid due to the array of molecular tools available for its study,\n                      <jats:italic toggle=\"yes\">T. cruzi<\/jats:italic>\n                      shares greater biological similarity with the majority of trypanosomatids regarding nutrient uptake, transmission strategy, and metabolism. Recent advancements in genetic tools have now enabled in-depth studies in\n                      <jats:italic toggle=\"yes\">T. cruzi<\/jats:italic>\n                      previously restricted to\n                      <jats:italic toggle=\"yes\">T. brucei<\/jats:italic>\n                      . Here, using the Small Hammerhead Aptazyme-Regulated Knockdown (SHARK) system, we report the characterization of cytoskeleton-associated protein 5.5 (CAP5.5) in\n                      <jats:italic toggle=\"yes\">T. cruzi<\/jats:italic>\n                      . To validate this phenotype, we introduce a novel method for simultaneous knockdown and rescue of a target protein using two tetracycline-responsive aptazymes. Knockdown of CAP5.5 revealed stage-specific roles in the maintenance of cell shape, organelle segregation, and proliferation. To quantify these phenotypes, we leveraged open-source, state-of-the-art segmentation software to develop a semi-automated, high-throughput image analysis pipeline. Our data suggest a potential role for CAP5.5 in repairing motility-related damage to the microtubule cytoskeletal array. These findings demonstrate the emerging utility of\n                      <jats:italic toggle=\"yes\">T. cruzi<\/jats:italic>\n                      as a robust model trypanosomatid.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        <jats:italic toggle=\"yes\">Trypanosoma cruzi<\/jats:italic>\n                        is the causative agent of Chagas disease, a neglected tropical disease that presents a growing public health concern. Historically, research into\n                        <jats:italic toggle=\"yes\">T. cruzi<\/jats:italic>\n                        biology has been hindered by a scarcity of genetic tools, forcing scientists to rely on models from related but biologically distinct parasites. In this study, we utilize genetic tools (the SHARK conditional knockdown system) to characterize a cytoskeletal protein, CAP5.5, revealing that it is required for the parasite to maintain its shape and divide during its motile stage. We further demonstrate the use of a dual-control system that allows simultaneous gene knockdown and rescue, alongside a new high-throughput 3D imaging pipeline. This work establishes a rigorous technical framework that empowers the community to conduct in-depth investigations of essential\n                        <jats:italic toggle=\"yes\">T. cruzi<\/jats:italic>\n                        genes directly, thus accelerating the discovery of potential therapeutic targets.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/msphere.00090-26","type":"journal-article","created":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T12:00:21Z","timestamp":1780056021000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["A dual-aptazyme system for genetic complementation reveals stage-specific roles for the\n                    <i>Trypanosoma cruzi<\/i>\n                    cytoskeleton-associated protein 5.5"],"prefix":"10.1128","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9889-8653","authenticated-orcid":false,"given":"Justin","family":"Wiedeman","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00te3t702","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for Tropical and Emerging Global Diseases, University of Georgia","place":["Athens, Georgia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ganesh Babu","family":"Malli Mohan","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00te3t702","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for Tropical and Emerging Global Diseases, University of Georgia","place":["Athens, Georgia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gonzalo","family":"Seminario-Mondejar","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00te3t702","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for Tropical and Emerging Global Diseases, University of Georgia","place":["Athens, Georgia"]},{"id":[{"id":"https:\/\/ror.org\/00te3t702","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Cellular Biology, University of Georgia","place":["Athens, Georgia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9144-287X","authenticated-orcid":true,"given":"Ronald Drew","family":"Etheridge","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00te3t702","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for Tropical and Emerging Global Diseases, University of Georgia","place":["Athens, Georgia"]},{"id":[{"id":"https:\/\/ror.org\/00te3t702","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Cellular Biology, University of Georgia","place":["Athens, Georgia"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1111\/jeu.12896"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ympev.2007.03.023"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0169-4758(99)01532-X"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1007\/s10863-016-9665-9"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1009254"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02097-14"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.01012-15"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2416009122"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2023.07.001"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1111\/boc.202100038"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.pt.2019.07.008"},{"key":"e_1_3_3_13_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2021.642271"},{"key":"e_1_3_3_14_2","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(89)90959-8"},{"key":"e_1_3_3_15_2","doi-asserted-by":"publisher","DOI":"10.1111\/mmi.12436"},{"key":"e_1_3_3_16_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00239-004-0272-8"},{"key":"e_1_3_3_17_2","doi-asserted-by":"publisher","DOI":"10.1152\/physrev.00029.2002"},{"key":"e_1_3_3_18_2","doi-asserted-by":"publisher","DOI":"10.1016\/s0166-6851(01)00296-1"},{"key":"e_1_3_3_19_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0106777"},{"key":"e_1_3_3_20_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0079581"},{"key":"e_1_3_3_21_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.protis.2009.05.003"},{"key":"e_1_3_3_22_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41597-022-01452-w"},{"key":"e_1_3_3_23_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0009141"},{"key":"e_1_3_3_24_2","doi-asserted-by":"publisher","DOI":"10.3389\/fnmol.2023.1270849"},{"key":"e_1_3_3_25_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0197541"},{"key":"e_1_3_3_26_2","doi-asserted-by":"publisher","DOI":"10.1111\/mmi.13986"},{"key":"e_1_3_3_27_2","doi-asserted-by":"publisher","unstructured":"Pachitariu M Rariden M Stringer C. 2025. Cellpose-SAM: superhuman generalization for cellular segmentation. Bioinformatics. doi:10.1101\/2025.04.28.651001","DOI":"10.1101\/2025.04.28.651001"},{"key":"e_1_3_3_28_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12859-021-04344-9"},{"key":"e_1_3_3_29_2","doi-asserted-by":"publisher","DOI":"10.3390\/microorganisms9112234"},{"key":"e_1_3_3_30_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2019.00445"},{"key":"e_1_3_3_31_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1112631"},{"key":"e_1_3_3_32_2","doi-asserted-by":"publisher","DOI":"10.15698\/mic2021.09.758"},{"key":"e_1_3_3_33_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1013105"},{"key":"e_1_3_3_34_2","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.e04-06-0457"},{"key":"e_1_3_3_35_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2012.08008.x"},{"key":"e_1_3_3_36_2","doi-asserted-by":"publisher","DOI":"10.1155\/2012\/520380"},{"key":"e_1_3_3_37_2","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.03556-22"},{"key":"e_1_3_3_38_2","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.00531-23"},{"key":"e_1_3_3_39_2","doi-asserted-by":"publisher","DOI":"10.1038\/nmat4396"},{"key":"e_1_3_3_40_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1011270"},{"key":"e_1_3_3_41_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.104.3.439"},{"key":"e_1_3_3_42_2","doi-asserted-by":"publisher","DOI":"10.1038\/ncb3481"},{"key":"e_1_3_3_43_2","doi-asserted-by":"publisher","DOI":"10.1017\/S0031182018001828"},{"key":"e_1_3_3_44_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1009588"},{"key":"e_1_3_3_45_2","doi-asserted-by":"publisher","DOI":"10.1155\/2014\/893272"},{"key":"e_1_3_3_46_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.protis.2007.09.001"},{"key":"e_1_3_3_47_2","doi-asserted-by":"publisher","DOI":"10.1016\/s0378-1119(97)00348-x"},{"key":"e_1_3_3_48_2","doi-asserted-by":"publisher","DOI":"10.1128\/mSphere.00313-20"},{"key":"e_1_3_3_49_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12859-017-1934-z"},{"key":"e_1_3_3_50_2","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2075"}],"container-title":["mSphere"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/msphere.00090-26","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/msphere.00090-26","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,10]],"date-time":"2026-07-10T13:01:56Z","timestamp":1783688516000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/msphere.00090-26"}},"subtitle":[],"editor":[{"given":"Ira J.","family":"Blader","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,6,30]]},"references-count":49,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2026,6,30]]}},"alternative-id":["10.1128\/msphere.00090-26"],"URL":"https:\/\/doi.org\/10.1128\/msphere.00090-26","relation":{},"ISSN":["2379-5042"],"issn-type":[{"value":"2379-5042","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,30]]},"assertion":[{"value":"2026-02-11","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-04-15","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-05-29","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e00090-26"}}