Rhoptry neck protein 2 expressed in Plasmodium sporozoites plays a crucial role during invasion of mosquito salivary glands

Abstract

Malaria parasite transmission to humans is initiated by the inoculation of Plasmodium sporozoites into the skin by mosquitoes. Sporozoites develop within mosquito midgut oocysts, first invade the salivary glands of mosquitoes, and finally infect hepatocytes in mammals. The apical structure of sporozoites is conserved with the infective forms of other apicomplexan parasites that have secretory organelles, such as rhoptries and micronemes. Because some rhoptry proteins are crucial for Plasmodium merozoite infection of erythrocytes, we examined the roles of rhoptry proteins in sporozoites. Here, we demonstrate that rhoptry neck protein 2 (RON2) is also localized to rhoptries in sporozoites. To elucidate RON2 function in sporozoites, we applied a promoter swapping strategy to restrict ron2 transcription to the intraerythrocytic stage in the rodent malaria parasite, Plasmodium berghei. Ron2 knockdown sporozoites were severely impaired in their ability to invade salivary glands, via decreasing the attachment capacity to the substrate. This is the first rhoptry protein demonstrated to be involved in salivary gland invasion. In addition, ron2 knockdown sporozoites showed less infectivity to hepatocytes, possibly due to decreased attachment/gliding ability, indicating that parts of the parasite invasion machinery are conserved, but their contribution might differ among infective forms. Our sporozoite stage‐specific knockdown system will help to facilitate understanding the comprehensive molecular mechanisms of parasite invasion of target cells.

Detection of the Rhoptry Neck Protein Complex in Plasmodium Sporozoites and Its Contribution to Sporozoite Invasion of Salivary Glands

“…3C). These results demonstrate that RON4 and RON5 have crucial roles required for sporozoite invasion of salivary glands, similarly to RON2 (18).…”

“…4A). In contrast, approximately 85% of RON4-cKD and RON5-cKD hemolymph sporozoites drifted, similar to the phenotypes of RON2-cKD hemolymph sporozoites (18). These results indicate that RON4 and RON5 are required for hemolymph sporozoite attachment to the glass slide similarly to RON2.…”

“…Two independent clones were isolated for each transgenic parasite and designated RON4 conditional knockdown (RON4-cKD) and RON5 conditional knockdown (RON5-cKD). For the generation of control parasites, the native promoter regions were replaced with the ron4 promoter (RON4-cont) and the pb rhoptry associated protein 1 (rap1) promoter (RON5-cont), similar to the creation of RON2-control parasites (18). The DNA insertion at the correct genomic locus of transgenic parasites was confirmed by genomic Southern blotting using a DNA probe corresponding to the drug-resistant cassette region ( Fig.…”

The Sporozoite's Journey Through the Mosquito: A Critical Examination of Host and Parasite Factors Required for Salivary Gland Invasion

“…The rhoptry neck protein 2 (RON2) was found to be highly expressed in oocyst sporozoites with transcripts present in hemolymph and salivary gland sporozoites (Ishino et al, 2019). Using a promoter swapping approach to restrict RON2 transcription to the intraerythrocytic stage, RON2-deficient parasites failed to invade the salivary glands as sporozoite numbers recovered from this organ were 20-fold lower in comparison to controls, while the numbers of parasites collected from midgut and hemolymph were the same as the controls (Ishino et al, 2019). When tested for gliding motility, the velocity of circular movement of RON2-deficient sporozoites was comparable to controls, suggesting the machinery for gliding motility is not affected by the lack of RON2 (Ishino et al, 2019).…”

The dimerisable Cre recombinase allows conditional genome editing in the mosquito stages of Plasmodium berghei

“…Various strategies have previously been developed for conditional mutagenesis in Plasmodium, including recombinase-based gene deletion [2], regulatable promoters [3], promoter swap strategies with known [4][5][6][7] or synthetic promoters [8], and mRNA or protein destabilization systems [9][10][11][12]. While most of these strategies are stage-specific, others require a promoter that is expressed by distinct stages of the parasite, thus limiting the investigation of essential genes in multiple developmental stages.…”