Plasmodium RON11 triggers biogenesis of the merozoite rhoptry pair and is essential for erythrocyte invasion

David Anaguano, Opeoluwa Adewale-Fasoro, Grace S. Vick, Sean Yanik, James Blauwkamp, Manuel A. Fierro, Sabrina Absalon, Prakash Srinivasan, Vasant Muralidharan

Posted on: 3 April 2024

Preprint posted on 13 February 2024

An unusual single-rhoptry phenotype caused by the depletion of RON11 in Plasmodium parasites

Selected by Sara Chelaghma

Categories: cell biology


Protists comprise a large number of diverse unicellular eukaryotes that have evolved complex machinery to adapt to their environments and lifestyle. The protist phylum of Apicomplexa includes Plasmodium, the causative agent of malaria, Toxoplasma and Cryptosporidium, the causative agents of toxoplasmosis and cryptosporidiosis respectively, as well as other organisms of importance to human and veterinary health.

Plasmodium parasites invade, divide within, and egress from red blood cells (RBCs) which causes many of the clinical symptoms associated with malaria. For the parasites, key to successful invasion and subsequent host modifications are one of its secretory organelles termed rhoptries, also found in other apicomplexans. The RBC- invading cells of Plasmodium, termed merozoites, possess two rhoptries containing proteins concentrated at the neck called RONs and proteins concentrated at the bulb called ROPs. This study by Anaguano and colleagues sought to characterise a rhoptry-associated protein, RON11, which has a dual role in invasion and rhoptry biogenesis in Plasmodium merozoites. Depletion of RON11 leads to loss of one of the rhoptries, a phenotype reported for the first time in this study.

Key findings

  • RON11 is essential for parasite growth and invasion

The authors used the tetR-DOZI system for conditional knockdown of RON11. This strategy relies on the insertion of aptamer repeats in the 3’UTR of the gene of interest to control translation through the addition of anhydrous tetracycline (Atc). Using a combination of microscopy and flow cytometry, parasites were monitored through the different stages of the Plasmodium asexual cycle. This revealed that RON11 is especially important for merozoites, the invasive stage, as RON11-depleted merozoites were not able to progress onto the next stage where they form rings. Failure to form rings could either be due to an invasion defect or a rhoptry biogenesis defect. By using anti-RON11 antibodies, the authors could distinguish between a role in invasion or a role in biogenesis. Indeed, incubation of parasites with inhibitory anti–RON11 antibodies produced data consistent with the gene knockdown results suggesting that RON11 plays a direct role in red blood cell invasion.

  • RON11-deficient parasites make one rhoptry rather than two

To further dissect the role of RON11 in parasitic invasion, the authors sought to identify at what stage invasion fails. The depletion of RON11 did not affect merozoite attachment to RBCs. This led to the hypothesis that RON11 may be important for entry into RBCs rather than a role in initial contact. RON11 is associated with the membrane of rhoptries and could potentially be important for rhoptry biogenesis.” To test this, ultrastructural expansion microscopy was applied on RON11-depleted cells. At the schizont stage in which Plasmodiumparasites form individual merozoites, the authors observed no changes in the morphology or number of merozoites. However, the merozoites themselves appeared to have only one rhoptry, where wildtype cells always have two. The single rhoptry formed by the merozoites appeared morphologically unaffected and properly attached to the apical end of the cell. The single rhoptry also exhibited compartmentalisation of RONs and ROPs. Quantification of rhoptry content showed that cells without RON11 expressed half the amount of rhoptry content compared to wildtype parasites. The processing of rhoptry-associated proteins such as RAP1 was normal even after RON11 loss. This indicates that the observed defect in rhoptry biogenesis was not due to a general biogenesis defect of secretory organelles in the cells or failure to process and deliver rhoptry proteins. How then does RON11 affect rhoptry biogenesis?

  • RON11 triggers the biogenesis of the last rhoptry pair

The authors narrowed down the window in which RON11 is important to the final hours of schizont development. This led to the hypothesis that RON11 may play a role in the formation of the last rhoptry pair. During schizogony, rhoptries from de novo and associate with centriolar plaques (CP) to ensure correct segregation at a 1:1 ratio of rhoptry to CP. Once merozoite segmentation starts during the later stages of schizogony, however, the CP associates with two rhoptries rather than one and each merozoite acquires two rhoptries. Ultrastructural expansion microscopy revealed that RON11 loss impairs the formation of the second rhoptry in late schizogony, and consequently each merozoite obtains only one rhoptry.

What I liked about this study

Rhoptries are essential organelles for the success of apicomplexan parasites. However, little is known about their biogenesis and the proteins that orchestrate it. Previous attempts to study RON11 in Plasmodium bergheiwere hindered by the difficulty of disrupting this gene. Here the authors used the improved tetR-DOZI strategy to elucidate the role of RON11 in RBC invasion. The single rhoptry phenotype is intriguing, unique and is beautifully deciphered using expansion microscopy. This study brings us closer to understanding the adaptations that make Plasmodium successful in invading red blood cells. I particularly liked the use of expansion microscopy which, in addition to generating aesthetically beautiful images, is an essential tool when trying to resolve important biological questions.

Future directions/questions for authors

  • Rhoptry number varies in apicomplexans, do you have any theories why that’s the case?
  • Do you think that the two rhoptries in Plasmodium have the same protein content?
  • Do you plan on doing any proteomics to identify the differences between the single-rhoptry cells and wildtype ones?
  • How do you think RON11 is affecting rhoptry biogenesis? Is it the regions of the protein exposed to the cytosol or an interaction within the rhoptry lumen? and do you have plans to further dissect this in future work?


Tags: expansion microscopy, invasion, malaria


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Author's response

Vasant Muralidharan shared

– Rhoptry number varies in apicomplexans, do you have any theories why that’s the case? One theory is that these differences could be due to the host cells these apicomplexans invade or to put it another way, how many different host cells they invade. For example, Cryptosporidium only invades intestinal cells and hence only needs one rhoptry. On the other hand, Toxoplasma invades almost any nucleated cell and hence Toxoplasma may require several rhoptries to deal with variability in the host cells it may encounter.
– Do you think that the two rhoptries in Plasmodium have the same protein content? It is very likely that the two rhoptries are not identical. I think the asymmetry between rhoptries may have important implications for invasion.
– Do you plan on doing any proteomics to identify the differences between the single-rhoptry cells and wildtype ones? Yes, we are currently analysing proteomic experiments to determine the differences between the wildtype and single-rhoptry parasites.
– How do you think RON11 is affecting rhoptry biogenesis? Is it the regions of the protein exposed to the cytosol or an interaction within the rhoptry lumen? and do you have plans to further dissect this in future work? We are very curious about the loss of rhoptry protein production upon RON11 knockdown. This is very unusual because other organelles in Plasmodium do not control the transcription or translation of their proteins. For example, when the apicoplast is lost (via gene knockout or drug treatment), nuclear encoded apicoplast proteins are still made and trafficked into vesicles that are found in the cytoplasm. Why don’t we find rhoptry proteins in vesicles? Or packaged into the single rhoptry upon RON11 depletion? These data suggest the existence of a signalling axis, requiring RON11, from the rhoptry to the nucleus (or cytoplasm) that turns on rhoptry protein production when the second rhoptry is being made. We are pursuing this question by determining if there’s a loss of rhoptry protein transcription upon RON11 depletion OR if there’s a block in protein translation. We are also dissecting the role of the RON11 EF-hand domain, which can bind calcium, in the functions of RON11 as well as determining the interacting partners of RON11. Ongoing work will dissect the role of these proteins in rhoptry biogenesis and merozoite invasion.

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