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The NPPs are critical to parasite survival and, therefore, in the pursuit of novel therapeutics are an attractive drug target. The NPPs of the human parasite, <jats:italic>P. falciparum,<\/jats:italic> have been linked to the RhopH complex, with the monoallelic paralogues <jats:italic>clag3.1<\/jats:italic> and <jats:italic>clag3.2<\/jats:italic> encoding the protein RhopH1\/CLAG3 that likely forms the NPP channel-forming component. Yet curiously, the combined knockout of both <jats:italic>clag3<\/jats:italic> genes does not completely eliminate NPP function. The essentiality of the <jats:italic>clag3<\/jats:italic> genes is, however, complicated by three additional <jats:italic>clag<\/jats:italic> paralogs (<jats:italic>clag2, clag8<\/jats:italic> and <jats:italic>clag9<\/jats:italic>) in <jats:italic>P. falciparum<\/jats:italic> that could also be contributing to NPP formation.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Methods<\/jats:title>\n            <jats:p>Here, the rodent malaria species, <jats:italic>P. berghei<\/jats:italic>, was utilised to investigate <jats:italic>clag<\/jats:italic> essentiality since it contains only two <jats:italic>clag<\/jats:italic> genes, <jats:italic>clagX<\/jats:italic> and <jats:italic>clag9.<\/jats:italic> Allelic replacement of the regions encompassing the functional components of <jats:italic>P. berghei clagX<\/jats:italic> with either <jats:italic>P. berghei clag9<\/jats:italic> or <jats:italic>P. falciparum clag3.1<\/jats:italic> examined the relationship between the two <jats:italic>P. berghei clag<\/jats:italic> genes as well as functional orthology across the two species. An inducible <jats:italic>P. berghei clagX<\/jats:italic> knockout was created to examine the essentiality of the <jats:italic>clag3<\/jats:italic> ortholog to both survival and NPP functionality.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>It was revealed <jats:italic>P. berghei<\/jats:italic> CLAGX and CLAG9<jats:italic>,<\/jats:italic> which belong to two distinct phylogenetic clades, have separate non-complementary functions, and that <jats:italic>clagX<\/jats:italic> is the functional orthologue of <jats:italic>P. falciparum clag3.<\/jats:italic> The inducible <jats:italic>clagX<\/jats:italic> knockout in conjunction with a guanidinium chloride induced-haemolysis assay to assess NPP function provided the first evidence of CLAG essentiality to <jats:italic>Plasmodium<\/jats:italic> survival and NPP function in an in vivo model of infection.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusions<\/jats:title>\n            <jats:p>This work provides valuable insight regarding the essentiality of the RhopH1 <jats:italic>clag<\/jats:italic> genes to the NPPs functionality and validates the continued investigation of the RhopH complex as a therapeutic target to treat malaria infections.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/s12929-024-01105-7","type":"journal-article","created":{"date-parts":[[2025,2,3]],"date-time":"2025-02-03T00:02:31Z","timestamp":1738540951000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Utilisation of an in vivo malaria model to provide functional proof for RhopH1\/CLAG essentiality and conserved orthology with P. falciparum"],"prefix":"10.1186","volume":"32","author":[{"given":"Mitchell L.","family":"Trickey","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mrittika","family":"Chowdury","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Georgina","family":"Bramwell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8973-3344","authenticated-orcid":false,"given":"Natalie A.","family":"Counihan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5810-8063","authenticated-orcid":false,"given":"Tania F.","family":"de Koning-Ward","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,2,3]]},"reference":[{"key":"1105_CR1","unstructured":"WHO, World malaria report (2023). 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