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Immunology and Infection

Generating Genetically Modified Plasmodium berghei Sporozoites

Published: May 5th, 2023

DOI:

10.3791/64992

1Center for Tropical and Emerging Global Diseases, University of Georgia, 2Department of Cellular Biology, University of Georgia

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Despite advances in drug development and research into malaria prevention and treatment, the global disease burden of malaria remains high. Over half a million people die of malaria each year, with the highest levels of mortality seen among children living in malaria-endemic regions, such as sub-Saharan Africa1. Malaria is caused by the parasite Plasmodium, which is transmitted to humans through the bite of female Anopheles mosquitoes bearing the parasite in their salivary glands. The infectious stage of Plasmodium-the sporozoites-are deposited in the skin of the vertebrate hosts during a blood m....

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All research involving vertebrate animals in our laboratory was performed in compliance with the University of Georgia animal use guidelines and protocols.

1. Generation of P. berghei -infected mice

  1. Initiate blood-stage infection in male or female, 6-8-week-old C57BL/6 (B6) mice using wild-type P. berghei parasites. To do this, transfer cryopreserved P. berghei-infected blood (2 x 105 infected RBCs), reconstituted in 400 µ.......

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Determining the frequency and development of schizonts is critical for assuring that enough viable parasites are in the optimal stage for transfection. Immature schizonts can be differentiated from fully mature schizonts by the presence of fewer merozoites that do not fill the entire intracellular space of the RBC (Figure 1B). It is important to note that when making blood smears from cultured blood, infected RBCs may break open, resulting in the observation of free, extracellular merozoites.......

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We have used the above protocol in our laboratory to create several lines of transgenic P. berghei parasites. Though optimized for P. berghei, we have also successfully used this protocol to generate transgenic P. yoelii sporozoites. After injecting the transfected schizonts into mice, parasites are detectable typically no later than 3 d.p.i. in all groups, including the no plasmid control. Selection is started only once parasitemia has been detected to ensure the viability of parasites followi.......

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This work was supported by the National Institutes of Health grant AI168307 to SPK.  We thank the UGA CTEGD Flow Cytometry Core and the UGA CTEGD Microscopy Core. We also acknowledge the contributions of Ash Pathak, Anne Elliot, and the staff of UGA Sporocore in optimizing the protocol. We want to thank Dr. Daichi Kamiyama for valuable insights, discussion, and the parent plasmids containing GFP11 and GFP1-10. We would also like to thank members of the Kurup lab for their constant support, patience, and encouragement.

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NameCompanyCatalog NumberComments
30 G x 1/2" Syringe needleExel international26437
Alsever's solutionSigma-AldritchA3551-500ML
Amaxa Basic Parasite Nucleofector Kit 2LonzaVMI-1021
Avertin (2,2,2-Tribromoethanol)TCI AmericaT1420
Blood collection tubesBD bioscience365967for serum collection
C-Chip disposable hematocytometerINCYTODHC-N01-5
CellVeiw Cell Culture DishGreiner Bio-One627860
Centrifuge 5425Eppendorf5405000107
Centrifuge 5910REppendorf5910RFor gradient centrifugation
Delta Vision II - Inverted microscope systemOlympusIX-71
Dimethyl SulfoxideSigmaD5879-500ml
Fetal bovine serumGenClone25-525
GFP11 plasmidKurup LabpSKspGFP11Generated from PL0017 plasmid
Giemsa StainSigma-Aldritch48900-1L-F
Hepa GFP1-10 cellsKurup LabHepa GFP1-10Generated from Hepa 1-6 cells (ATCC Cat# CRL-1830)
Mouse SerumUsed for mosquito dissection media
NaClMillipore-SigmaSX0420-51.5 M and 0.15 M for percoll solution
Nucleofector IIAmaxa Biosystems (Lonza)Program U-033 used for RBC electroporation
Pasteur pipetteVWR14673-043
Penicillin/StreptomycinSigma-AldritchP0781-100ML
Percoll (Density gradient stock medium)Cytivia17-0891-02Details in protocol
PL0017 PlasmidBEI ResourcesMRA-786
Pyrimethamine (for oral administration)Sigma46706Preparation details: Add 17.5 mg Pyrimethamine to 2.5 mL of DMSO. Vortex, if needed to dissolve completely; Adjust pH of 225 mL of dH2O to 4 using HCL. Add Pyrimethamine in DMSO to water and bring to 250 mL. Add 10 g of sugar to encourage regular consumption of drugged water. Pyrimethamine is light sensitive. Use dark bottle or aluminum foil covered bottle when treating mice.
RPMI 1640Corning15-040-CV
SoftWoRx microscopy softwareApplied Precisionv6.1.3

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