Background

    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.

 

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.

References

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

 

doi: https://doi.org/10.1242/prelights.26268

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