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 meal and travel through the bloodstream to infect liver cells, where they undergo mandatory development (constituting pre-erythrocytic malaria) prior to infecting the erythrocytes. The infection of the erythrocytes initiates the blood-stage of malaria and is responsible for the entirety of the morbidity and mortality associated with the disease2,3.
The obligate nature of the pre-erythrocytic development of Plasmodium has made it an attractive target for prophylactic vaccine and drug development efforts4. A prerequisite for studying the biology of pre-erythrocytic malaria, as well as the development of vaccines or drugs targeting the liver stage, is access to Plasmodium sporozoites. Furthermore, our ability to generate genetically modified Plasmodium sporozoites has been instrumental in the success of such research endeavors5,6,7,8,9. Transgenic Plasmodium lines expressing fluorescent or luminescent reporter proteins have allowed us to track their development in vivo and in vitro10,11. Genetically attenuated parasites (GAPs), generated through the deletion of multiple genes in Plasmodium, are also some of the most promising vaccine candidates12,13.
Rodent and non-human primate malaria models have helped us understand the mechanisms of host-parasite interactions in human malaria due to the similarities in biology and life cycle among Plasmodium species14. The use of Plasmodium species that infect rodents, but not humans (e.g., P. berghei) allows the maintenance of the complete parasite life cycle and the generation of infectious sporozoites for studying liver-stage malaria in a controlled, biosafety level 1 setting. A variety of separate protocols already exist for the generation of transgenic blood-stage Plasmodium parasites15, infection of mosquitoes16, and isolation of sporozoites17. Here, we outline a comprehensive protocol combining these methodologies in order to generate and isolate transgenic P. berghei sporozoites, utilizing the novel transgenic strain PbGFP11 as an example. PbGFP11 traffics the 11th β-strand of super-folder green fluorescent protein (GFP), GFP11, into the parasitophorous vacuole (PV) generated in the host hepatocytes. PbGFP11 is used in conjunction with transgenic hepatocytes (Hepa1-6 background) expressing residues that constitute the GFP 1-10 fragment (GFP1–10) in the cytoplasm (Hepa GFP1–10 cells). PbGFP11 reports PV lysis in the host hepatocytes through self-complementation and the reformation of functional GFP and the green fluorescence signal18. Of note, GFP11 is encoded as a series of seven tandem sequences in PbGFP11 to enhance the resulting fluorescence signal. Upon staining PbGFP11 sporozoites with the cytoplasmic dye CellTrace Violet (CTV), we can track the parasites. The lysis of such CTV-stained intracellular parasites itself results in leakage of CTV into the host cell cytoplasm and staining of the host cell. In addition to visualizing and distinguishing the lysis of Plasmodium PV and/or the parasite in host hepatocytes, this system can be reliably used to study the immune pathways responsible for either of these processes, through the genetic or therapeutic perturbation of the molecular components of such pathways.
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
2. Generation of schizonts in culture
3. Transfection of Plasmodium schizonts
4. Selection of the transfected parasites
5. Infection of mosquitoes with the transgenic lines
6. Collection of sporozoites
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 in the blood smear (Figure 1). Such merozoites do not count toward the assessment of the frequency of schizonts in step 2.5.
The removal of salivary glands from mosquitoes can be challenging if the user is unfamiliar with mosquito physiology or small-scale dissections. During the isolation of salivary glands from mosquitoes, note that the translucence of the glands can be used to differentiate them from the opaque mosquito debris (Figure 2). The counting of sporozoites following disruption of the glands is most efficient at 400x magnification, and the use of phase-contrast allows for easier identification of the sporozoites within the counting chamber.
We have shown that over 90% of the Plasmodium in hepatocytes are possibly eliminated through cell-intrinsic immune mechanisms23. Therefore, we expect parasites in a considerable number of hepatocytes to undergo lysis. As a tool to determine the lysis of Plasmodium or its PV in the host hepatocytes, we generated transgenic hepatocytes expressing the GFP1–10 subunit (Hepa-GFP1–10) and transgenic P. berghei expressing and trafficking the GFP11 subunit to its PV (Pb-GFP11). The GFP1–10 fragment, which contains the residues that constitute the GFP chromophore, is nonfluorescent by itself and fluoresces only upon associating with GFP11, potentially through self-complementation. In addition to functionally validating transgenesis in Plasmodium, the generation of green fluorescence signal in the Pb-GFP11 infected Hepa-GFP1–10 host cells indicated the lysis of the PV (Figure 3). Wild-type P. berghei-infected Hepa-GFP1–10 cells or Pb-GFP11-infected wild-type Hepa1–6 cells failed to generate any green fluorescence signal (data not shown). We consider the lysis of the PV to be a key preceding step in the destruction of liver stage Plasmodium. The Pb-GFP11 sporozoites were also stained with CTV to visualize the parasites in the hepatocytes. CTV is expected to leak into the host cell only upon lysis of the parasite itself (Figure 3). The dispersed CTV signal observed in the host hepatocyte likely indicates the lysis of the parasite within the hepatocyte. The close overlap between the CTV and GFP signals is expected with the concomitant lysis of both the PV and the parasite. This system allows us to query the distinct contributions of the individual host molecules in innate and cell-intrinsic immune pathways in modulating the lysis of Plasmodium or its PV.
Figure 1: Plasmodium berghei schizonts. Representative light microscopy images depicting P. berghei schizonts in parasitized mouse blood culture (16 h) stained with Giemsa stain. Arrows indicate fully matured (A) or immature (B) schizonts. Scale bars: 5 µm . Please click here to view a larger version of this figure.
Figure 2: Mosquito salivary glands and sporozoites. Light microscopy images depicting salivary glands isolated from Pb-GFP11-infected female Anopheles mosquitoes. (A) Image of a mosquito head with the salivary glands (arrow) still intact during dissection, prior to its removal and further processing (scale bar: 1 mm). (B,C) Representative images of salivary glands at lower (B) and higher (C) magnifications (scale bars: 0.1 mm and 0.04 mm, respectively). Sporozoites can be seen both inside and outside of the gland (arrow). Please click here to view a larger version of this figure.
Figure 3: Detecting the lysis of Plasmodium and its parasitophorous vacuole in host hepatocytes. Differential interference contrast (DIC) and pseudo-colored immunofluorescence images with overlay depicting Hepa-GFP1–10 hepatocytes infected with Pb-GFP11 sporozoites. Sporozoites were stained with CTV prior to the infection (scale bar: 10 µm). A total of 1 x 106 Hepa-GFP1–10 hepatocytes were plated in a 35/10 mm glass-bottom culture dish and inoculated with 3 x 105 PbGFP11 sporozoites 4 h after plating. Images were taken with an inverted fluorescent microscope at 600x magnification and at 16 h post-infection. Abbreviations: DIC = differential interference contrast. Please click here to view a larger version of this figure.
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 following electroporation. Additionally, when preparing for drug selection, acidification of the water with hydrochloric acid, bringing the pH down to 4, may be necessary for pyrimethamine to fully dissolve. We expect complete clearance of the parasites from the no plasmid control group, while the mice inoculated with the transfectants remain infected. Clearance in the control mice typically occurs 5-8 days after the initiation of pyrimethamine treatment. Of note, B6 mice infected with P. berghei may show signs of experimental cerebral malaria and succumb to death (follow humane endpoints according to institutional animal use guidelines), typically in the interval of 6-12 d.p.i. This can be avoided by performing drug selection of the transfectants in TCRaKO mice25, or by transferring the parasites undergoing selection at 5 d.p.i. to new B6 recipients and continuing selection in the latter. Transgenesis in Plasmodium can be verified using a variety of approaches, such as genetic screens, phenotypic assessment, or gain or loss of specific functions.
It is important to note that the selection of a suitable plasmid is crucial for the successful transfection and retention of the introduced DNA in the parasite. Plasmids may also show limited efficacy in achieving transgenesis across different Plasmodium species. For example, the pSKspGFP11 plasmid (based on PL0017; BEI resources) we utilized to generate PbGFP11 can be used to efficiently generate transgenic P. yoelli as well, but not P. chabaudi. To generate pSKspGFP11, we replaced the GFP mutant3 sequence in the parent pL0017 plasmid (BEI resources) with seven tandem sequences of the 11th β-strand of super-folder GFP, GFP11 (GFP11-7X)18. Although linearization of the plasmids before transfection allows better genomic integration, both linear and circular plasmids show similar transfection efficiencies using our protocol. The Plasmodium lines generated by transfection with circular plasmids also appear to maintain their transgenic properties over the course of the generation of sporozoites in the mosquitoes.
Notably, only female mosquitoes can harbor and transmit Plasmodium. Following the isolation of mosquitoes, it is recommended to not provide the isolated mosquitoes with a source of sugar water at this stage. Starving them in this manner would increase the chance of any male mosquitoes that may have been inadvertently transferred dying, and the females feeding the blood more efficiently from the infected mice. We typically return the sugar water 1 day after transferring the infection from mice to mosquitoes. It is noteworthy that the timeframe for the development and maturation of the sporozoite in mosquitoes may vary among transgenic Plasmodium lines. Therefore, it is important to assess sporozoite numbers in salivary glands at multiple time points after infection before a protocol specific for each transgenic line is established.
The authors have nothing to disclose.
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.
30 G x 1/2" Syringe needle | Exel international | 26437 | |
Alsever's solution | Sigma-Aldritch | A3551-500ML | |
Amaxa Basic Parasite Nucleofector Kit 2 | Lonza | VMI-1021 | |
Avertin (2,2,2-Tribromoethanol) | TCI America | T1420 | |
Blood collection tubes | BD bioscience | 365967 | for serum collection |
C-Chip disposable hematocytometer | INCYTO | DHC-N01-5 | |
CellVeiw Cell Culture Dish | Greiner Bio-One | 627860 | |
Centrifuge 5425 | Eppendorf | 5405000107 | |
Centrifuge 5910R | Eppendorf | 5910R | For gradient centrifugation |
Delta Vision II – Inverted microscope system | Olympus | IX-71 | |
Dimethyl Sulfoxide | Sigma | D5879-500ml | |
Fetal bovine serum | GenClone | 25-525 | |
GFP11 plasmid | Kurup Lab | pSKspGFP11 | Generated from PL0017 plasmid |
Giemsa Stain | Sigma-Aldritch | 48900-1L-F | |
Hepa GFP1-10 cells | Kurup Lab | Hepa GFP1-10 | Generated from Hepa 1-6 cells (ATCC Cat# CRL-1830) |
Mouse Serum | Used for mosquito dissection media | ||
NaCl | Millipore-Sigma | SX0420-5 | 1.5 M and 0.15 M for percoll solution |
Nucleofector II | Amaxa Biosystems (Lonza) | Program U-033 used for RBC electroporation | |
Pasteur pipette | VWR | 14673-043 | |
Penicillin/Streptomycin | Sigma-Aldritch | P0781-100ML | |
Percoll (Density gradient stock medium) | Cytivia | 17-0891-02 | Details in protocol |
PL0017 Plasmid | BEI Resources | MRA-786 | |
Pyrimethamine (for oral administration) | Sigma | 46706 | Preparation 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 1640 | Corning | 15-040-CV | |
SoftWoRx microscopy software | Applied Precision | v6.1.3 |