Additional remarks phenotype | Mutant/mutation
In the 'promoter-swap' mutant the RON4 promoter replaced by the promoter of PBANKA_1443300 (msp9). This promoter is active in asexual blood stages (in schizonts) but is silent in mosquito stages, such as oocysts and sporozoites. In addition the mutant expresses GFP under the constitutive eef1a promoter.
Protein (function)
RON4 is a rhoptry neck protein expressed in rhoptries of merozoites and sporozoites. Unsuccessful attempts to knock-out the ron4 gene (RMgm-686) indicates an essential function in blood stages. A number of rhoptry neck proteins (RONs), including RON2, RON4, and RON5 are secreted and inserted into the target cellular membrane as a complex.
Phenotype
Sporozoite numbers from midguts and hemolymph were not significantly different from those of control parasites indicating that RON4 is not critical for sporozoite formation, maturation inside oocysts, and release to the hemocoel. In contrast, the mean numbers of sporozoites residing in salivary glands were reduced 27-fold. Sporozoites showed reduced adhesion and gliding motility.
38% of control wild type hemolymph sporozoites start gliding, while 55% remain floating without attachment to the glass slide. In contrast, approximately 85% of RON4-cKD hemolymph sporozoites drifted. These results indicate that RON4 is required for hemolymph sporozoite attachment to the glass slide. Accordingly, only 9% of RON4-cKD hemolymph sporozoites show gliding, which is 4-fold less than wild type gliding sporozoites,
When embedded in Matrigel, 78% of control wild-type hemolymph sporozoites move continuously through the matrix, whereas only 15% of RON4-cKD hemolymph sporozoites display a circular mode of motility.
These results indicate that RON4 is mainly required for sporozoite attachment and are also involved in the onset of sporozoite movement.
Additional information
From the Abstract of the paper:
'Components of the merozoite rhoptry neck protein complex are also expressed in sporozoites, namely, RON2, RON4, and RON5, suggesting that invasion mechanism elements might be conserved between these infective stages. Recently, we demonstrated that RON2 is required for sporozoite invasion of mosquito salivary gland cells and mammalian hepatocytes, using a sporozoite stage-specific gene knockdown strategy in the rodent malaria parasite model, Plasmodium berghei. Here, we use a coimmunoprecipitation assay and oocyst-derived sporozoite extracts to demonstrate that RON2, RON4, and RON5 also form a complex in sporozoites. The sporozoite stage-specific gene knockdown strategy revealed that both RON4 and RON5 have crucial roles during sporozoite invasion of salivary glands, including a significantly reduced attachment ability required for the onset of gliding. Further analyses indicated that RON2 and RON4 reciprocally affect trafficking to rhoptries in developing sporozoites, while RON5 is independently transported.'
In a second paper (PMID: 37272704) the role of RON4 in liver stages is examined. From thispaper: 'During merozoite invasion of erythrocytes, rhoptry neck protein (RON) 2, RON4, and RON5 are secreted to the host cell membrane to form the RON complex, which interacts with AMA1 on the parasite membrane to create a moving junction. The inability to target gene disruption of ron2, ron4, ron5, or ama1 indicates that these genes are crucial for merozoite invasion. To elucidate the roles of RON2, RON4, and RON5 in sporozoites, we developed a sporozoite stage-specific knockdown system using the promoter swapping method. Repression of RON2, RON4, or RON5 expression in sporozoites demonstrated that the proteins are crucial for sporozoite invasion of salivary glands. The importance of RON2 and RON4 in sporozoite invasion of salivary glands was confirmed by a conditional knockout system based on DiCre recombinase. In this study, the detailed functions of RON4 in sporozoite transmission to the liver were analyzed in vivo and in vitro using RON4 conditional knockdown (RON4-cKD) sporozoites, in which RON4 protein expression was suppressed to undetectable levels. The results revealed that RON4 has multiple roles during the sporozoite crossing of sinusoidal cells to arrive at hepatocytes, the adhesion of hepatocytes, and the invasion of hepatocytes, all of which are essential steps for sporozoite transmission from the mosquito to the mammalian liver.'
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