Trickey Mitchell L, Chowdury Mrittika, Bramwell Georgina, Counihan Natalie A, de Koning-Ward Tania F
School of Medicine, Deakin University, Geelong, Australia.
Institute for Mental and Physical Health and Clinical Translation (IMPACT), Deakin University, Geelong, Australia.
J Biomed Sci. 2025 Feb 3;32(1):13. doi: 10.1186/s12929-024-01105-7.
Malaria parasites establish new permeation pathways (NPPs) at the red blood cell membrane to facilitate the transport of essential nutrients from the blood plasma into the infected host cell. 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, P. falciparum, have been linked to the RhopH complex, with the monoallelic paralogues clag3.1 and clag3.2 encoding the protein RhopH1/CLAG3 that likely forms the NPP channel-forming component. Yet curiously, the combined knockout of both clag3 genes does not completely eliminate NPP function. The essentiality of the clag3 genes is, however, complicated by three additional clag paralogs (clag2, clag8 and clag9) in P. falciparum that could also be contributing to NPP formation.
Here, the rodent malaria species, P. berghei, was utilised to investigate clag essentiality since it contains only two clag genes, clagX and clag9. Allelic replacement of the regions encompassing the functional components of P. berghei clagX with either P. berghei clag9 or P. falciparum clag3.1 examined the relationship between the two P. berghei clag genes as well as functional orthology across the two species. An inducible P. berghei clagX knockout was created to examine the essentiality of the clag3 ortholog to both survival and NPP functionality.
It was revealed P. berghei CLAGX and CLAG9, which belong to two distinct phylogenetic clades, have separate non-complementary functions, and that clagX is the functional orthologue of P. falciparum clag3. The inducible clagX knockout in conjunction with a guanidinium chloride induced-haemolysis assay to assess NPP function provided the first evidence of CLAG essentiality to Plasmodium survival and NPP function in an in vivo model of infection.
This work provides valuable insight regarding the essentiality of the RhopH1 clag genes to the NPPs functionality and validates the continued investigation of the RhopH complex as a therapeutic target to treat malaria infections.
疟原虫在红细胞膜上建立新的渗透途径（NPPs），以促进必需营养物质从血浆转运到受感染的宿主细胞中。NPPs对寄生虫的存活至关重要，因此，在寻找新型治疗方法的过程中，它是一个有吸引力的药物靶点。人类疟原虫恶性疟原虫的NPPs与RhopH复合体有关，单等位基因旁系同源物clag3.1和clag3.2编码可能构成NPP通道形成成分的RhopH1/CLAG3蛋白。然而，奇怪的是，clag3两个基因的联合敲除并没有完全消除NPP功能。然而，恶性疟原虫中另外三个clag旁系同源物（clag2、clag8和clag9）可能也有助于NPP的形成，这使得clag3基因的必要性变得复杂。
在这里，啮齿动物疟原虫伯氏疟原虫被用来研究clag的必要性，因为它只包含两个clag基因，clagX和clag9。用伯氏疟原虫clag9或恶性疟原虫clag3.1对等位基因替换伯氏疟原虫clagX功能成分所在区域，研究了伯氏疟原虫两个clag基因之间的关系以及两个物种之间的功能直系同源性。构建了可诱导的伯氏疟原虫clagX敲除模型，以研究clag3直系同源物对生存和NPP功能的必要性。
研究发现，属于两个不同系统发育分支的伯氏疟原虫CLAGX和CLAG9具有独立的非互补功能，并且clagX是恶性疟原虫clag3的功能直系同源物。可诱导的clagX敲除结合氯化铵诱导的溶血试验来评估NPP功能，为CLAG对疟原虫在体内感染模型中的存活和NPP功能的必要性提供了首个证据。
这项工作为RhopH1 clag基因对NPPs功能的必要性提供了有价值的见解，并验证了将RhopH复合体作为治疗疟疾感染的靶点继续进行研究的合理性。
