Close

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

Mirko Singer, Friedrich Frischknecht

Posted on: 9 December 2020

Preprint posted on 22 October 2020

Article now published in Cellular Microbiology at http://dx.doi.org/10.1111/cmi.13321

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

Selected by Mariana De Niz

Categories: cell biology

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.

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.

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

Read preprint (No Ratings Yet)

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.

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the cell biology category:

Leukocytes use endothelial membrane tunnels to extravasate the vasculature

Werner J. van der Meer, Abraham C.I. van Steen, Eike Mahlandt, et al.

Selected by 08 December 2024

Felipe Del Valle Batalla

Cell Biology

Platelet-derived LPA16:0 inhibits adult neurogenesis and stress resilience in anxiety disorder

Thomas Larrieu, Charline Carron, Fabio Grieco, et al.

Selected by 04 December 2024

Harvey Roweth

Neuroscience

Investigating Mechanically Activated Currents from Trigeminal Neurons of Non-Human Primates

Karen A Lindquist, Jennifer Mecklenburg, Anahit H. Hovhannisyan, et al.

Selected by 04 December 2024

Vanessa Ehlers

Neuroscience

preLists in the cell biology category:

November in preprints – the CellBio edition

This is the first community-driven preList! A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. Categories include: 1) cancer cell biology 2) cell cycle and division 3) cell migration and cytoskeleton 4) cell organelles and organisation 5) cell signalling and mechanosensing 6) genetics/gene expression

 



List by Felipe Del Valle Batalla et al.

BSCB-Biochemical Society 2024 Cell Migration meeting

This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.

 



List by Reinier Prosee

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

preLights peer support – preprints of interest

This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.

 



List by preLights peer support

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.

 



List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage
Close