Mutant/mutation

Protein (function)
From the paper: '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.'

Phenotype
Expression of mCherry-tagged RON4 in oocyst-derived sporozoites  was demonstrated by western blot using anti-RON4 and anti-mCherry antibody staining. Two bands corresponding to full-length RON4 (about 100 kDa) and a processed form (about 60 kDa) were observed on western blots, both of which were shifted due to the successful addition of an mCherry-tag at the C-terminus. Inoculating sporozoites collected from mosquito salivary glands to C57BL/6 mice demonstrated that RON4-mCherry-expressing sporozoites could normally infect and proliferate within hepatocytes. By confocal fluorescence microscopy, RON4-mCherry signals were confirmed to localize at the apical end of sporozoites residing in salivary glands, consistent with a rhoptry localization To examine RON4-mCherry expression in LS parasites, RON4-mCherry sporozoites collected from salivary glands were inoculated onto the human hepatoma cell line, HepG2. At 24 and 48 hours post-inoculation, RON4-mCherry signals were undetectable in LS parasites. At 72-hour post-inoculation, RON4-mCherry signals were detected as dots corresponding to the tip of mature liver merozoites.

Additional information
In vitro infection experiments using a hepatoma cell line revealed that secreted RON4 is involved in sporozoite adhesion to hepatocytes and has an important role in the early steps of hepatocyte infection. In addition, in vitro motility assays indicated that RON4 is required for sporozoite attachment to the substrate and the onset of migration. These findings indicate that RON4 is crucial for sporozoite migration toward and invasion of hepatocytes via attachment ability and motility.

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