Fluorescent tagging of Plasmodium circumsporozoite protein allows imaging of sporozoite formation but blocks egress from oocysts

Mirko Singer, Friedrich Frischknecht

Preprint posted on 22 October 2020

Article now published in Cellular Microbiology at

Looking with a magnifying glass: In-depth investigation of Plasmodium CSP

Selected by Mariana De Niz

Categories: cell biology


    Transmission of malaria occurs when Plasmodium sporozoites are inoculated into the skin of the mammalian host during the probing phase of the mosquito bite. The circumsporozoite protein (CSP) is the major surface protein of Plasmodium sporozoites. CSP is pivotal for sporozoite formation within oocysts, for their egress from oocysts, for entry into the mosquito salivary glands, migration in the mammalian host skin and entry into the mammalian liver. Antibodies against CSP have been shown to lead to a block in migration in the skin, and reduction of liver cell invasion. Moreover, part of CSP has been used to develop the RTS,S AS01 liver stage malaria vaccine. Despite the known relevance of CSP for malaria infections, the mechanisms by which it facilitates sporozoite formation, oocyst egress and hepatocyte invasion are not yet fully understood. Several past studies have given insight into how deletions of different domains impact on morphology and infective capacities of sporozoites. To better understand CSP function, in their current work, Singer and Frischknecht generated a series of parasites expressing full-length versions of CSP internally fused to GFP to investigate CSP localization during Plasmodium berghei sporozoite formation.

Figure 1. Tagging of Plasmodium circumsporozoite protein allows imaging of sporozoite formation but blocks egress from oocysts. Left panel shows a model of oocyst development. Right panels show oocysts with mature sporozoites.


Key findings and developments

Reporter line generation and confirmation of CSP-GFP fusions.

The authors began by generating 5 parasite lines expressing CSP-GFP fusion proteins, considering the multiple functions of the CSP domain. The lines produced were:

  1. GFP-GPI parasites, expressing a protein consisting of the signal peptide of CSP, GFP and the C-terminal 22 amino acids of CSP corresponding to the GPI-anchor sequence.
  2. SP-GFP-CSP-add parasites, expressing a CSP-GFP fusion protein with the GFP placed between the signal peptide (SP) and the N-terminus of CSP. This line expressed SP-GFP-CSP in addition to the endogenous CSP.
  3. SP-GFP-CSP-rep parasites, whereby the endogenous csp was replaced by the sp-gfp-csp
  4. R-GFP-CSP parasites, where the GFP was placed between the repeat region and TSR of CSP. This was in addition to endogenous CSP.
  5. TSR-GFP-CSP parasites, where the GFP was placed between the TSR and the GPI-anchor. This was in addition to endogenous CSP.

The lines generated comparable number of oocysts at the infected mosquito midgut and ‘immature’ sporozoites as WT parasites, although some lines showed no ‘mature’ sporozoite accumulation in the salivary glands. Moreover, all but the two SP-GFP-CSP lines showed the expected surface localization of the fusion proteins, while the GPI-GFP line also showed small vesicular localization in the proximity of the plasma membrane. The authors then confirmed that the fused proteins were indeed GFP-CSP fusions by western blotting. Together, the authors emphasize this as a key point: altogether, all membrane-localizing CSP versions as extra copies (and SP-GFP-CSP-rep) can form sporozoites, but fail to egress the oocyst, pointing towards an important role for CSP in sporozoite egress.

Phenotypic characterization

The authors then investigated sporozoite formation by the different parasite lines, by electron microscopy. Their main finding was that all parasite lines developed normally in a manner reminiscent of WT parasites, and fully formed sporozoites could be detected in late stage oocysts within the mosquito. In terms of gliding motility, most of the reporter lines were able to attach more robustly than WTs at day 25 post-infection – suggesting that they continue to mature within oocysts. Although the authors hypothesized that R-GFP-CSP and SP-GFP-CSP-rep lines would be infective to livers, intravenous injection of at least half-a-million sporozoites into mice did not result in blood stage infection.

Localization during sporozoite formation

Investigation of the fluorescence signal during sporozoite formation within dissected mosquito midguts using spinning disc confocal microscopy  showed that the signal of all parasite lines except the two SP-GFP-CSP lines, could be found at the plasma membrane of the oocyst, delineating the invagination of the plasma membrane, which precedes sporozoite formation. In the SP-GFP-CSP line, the signal in early oocysts was not at the plasma membrane, nor was it cytoplasmic, but rather within a structure reminiscent of the ER. Later in sporozoite formation the induction of plasma membrane curvature of budding sporozoites was readily observable in the R-GFP-CSP, TSR-GFP-CSP and GFP-GPI expressing parasites. In the TSR-GFP-CSP line, the plasma membrane was strongly stained but weak GFP fluorescence was also detected in the ER. Overall, in all parasite lines, fully developed sporozoites could be detected. Sporozoites in fully matured oocysts labelled similar to those found in isolated sporozoites. In late oocysts of the SP-GFP-CSP-rep line, further unusual structures could be found, while only few normal sporozoites could be detected, which might explain their lack of infectivity in mice. Finally, the authors succeeded in labelling microtubules with SiR-tubulin within oocysts, and they show that microtubules appear after initial bud formation during early sporogony.

What I like about this preprint

I like this preprint because it provides new tools to understand a vital component of the Plasmodium parasite, CSP, which has proven to be key for infection and transmission. The tools generated by the authors help further understand the function of CSP, and revealed interesting functions of some GFP-CSP fusions.


  1. Singer and Frischknecht, Fluorescent tagging of Plasmodium circumsporozoite protein allows imaging of sporozoite formation but blocks egress from oocysts, bioRxiv,2020


Posted on: 9 December 2020


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Author's response

Mirko Singer and Freddy Frischknecht shared

Open questions 

1.You focused your work mostly on the role of CSP during parasite development within the mosquito. You described the infection outcome of intravenous infections with R-GFP-CSP and SP-GFP-CSP-rep parasite lines. Were you able to observe the behaviour of all 5 lines your produced in the host skin and liver (either in vivo or in vitro), to determine the differences that might affect infection outcomes?

A: Investigating hemolymph derived sporozoites in the skin or liver only makes partially sense as these sporozoites never see any of these during natural transmission and salivary gland sporozoites differ considerably in their infective capacity from those isolated from the oocysts or hemolymph. We therefore did not investigate skin and liver. However, we have recently also made new lines that are capable to enter the salivary glands. We will investigate these parasites in more detail in skin and liver and will report this in a follow-up manuscript.

2.The motility phenotypes you describe are very interesting. Is the altered adhesion you observe in some of the mutants the sole result of the modification of CSP? Is there a chance that further alterations could have occurred that have an impact on motility?

A: It is indeed a fair assumption that also other factors might play a role here, as sporozoites seem to partially mature as they stay in the oocysts. They never are as mature as those that reside in salivary glands though. Yet, it is possible that some factors important for adhesion of sporozoites and hence for gliding motility are expressed during this prolonged stay in the oocysts.

3.Deriving from both questions above, and given that you mentioned the role of CSP in liver entry, what happens to the various parasite lines when you inject them either intravenously or intradermally into mice?

A: We only injected two parasite lines i.v. as we believe this experiment is only partially interesting. It does show that entry to the liver is impaired as both lines did not infect the animals.

4.Are the phenotypes you observe conserved if instead of GFP you used a smaller/different tag?

A: This is a good question, likely a small tag would have less impact, but we did not generate one, as we wanted to see the protein in living sporozoites. Our motivation is twofold: (i) we want to better understand CSP function as this clearly is one of the most important proteins of Plasmodium and (ii) we want to investigate sporozoite formation. For the former, we could do with a smaller tag, but for the latter we really need a fluorescent tag to see the dynamic changes.

5.You describe in your work, labyrinthine structures. Have these been described in other biological systems? Can you expand more on what their potential function might be in Plasmodium?

A: Not in other organisms as far as we know. They could server multiple purposes e.g. (i) exchange or uptake of small molecules, e.g. lipids or nutrients, (ii) expansion of surface area during rapid membrane extension prior to sporozoite formation.

6.Are the findings you have presented here, conserved across Plasmodium species, including the human-infective ones? Namely, are the different mechanisms affected by CSP, equally relevant to all Plasmodium species?

A: We can’t say for sure, but most likely, yes. Previous work indicates that CSP is involved in host cell preference for the exoerythrocytic stage, which is different in bird infecting Plasmodium species despite the strong conservation of CSP sequence.

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