Name: generating genetically modified plasmodium berghei sporozoites
Uploaded: 2023-05-05T12:50:00
Description: Watch this Scientific Journal Video about Generating Genetically Modified Plasmodium berghei Sporozoites at JoVE.com

This work was supported by the National Institutes of Health grant AI168307 to SPK.&#160; 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.

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
<ol>
	<li>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 &#181;...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+evaluation+of+rodent+malaria+parasite+genomes+and+gene+expression.">A comprehensive evaluation of rodent malaria parasite genomes and gene expression.</a> BMC Biology. 12, 86 (2014).</li><li>Janse, C. J., Ramesar, J., Waters, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transfection+and+drug+selection+of+genetically+transformed+blood+stages+of+the+rodent+malaria+parasite+Plasmodium+berghei.">High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei.</a> Nature Protocols. 1 (1), 346-356 (2006).</li><li>Tripathi, A. K., Mlambo, G., Kanatani, S., Sinnis, P., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+gametocyte+culture+and+mosquito+infection+through+artificial+membrane+feeding.">Plasmodium falciparum gametocyte culture and mosquito infection through artificial membrane feeding.</a> Journal of Visualized Experiments. (161), e61426 (2020).</li><li>Pacheco, N. D., Strome, C. P., Mitchell, F., Bawden, M. P., Beaudoin, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+large-scale+isolation+of+Plasmodium+berghei+sporozoites+from+infected+mosquitoes.">Rapid, large-scale isolation of Plasmodium berghei sporozoites from infected mosquitoes.</a> The Journal of Parasitology. 65 (3), 414-417 (1979).</li><li>Kamiyama, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+protein+tagging+in+cells+with+split+fluorescent+protein.">Versatile protein tagging in cells with split fluorescent protein.</a> Nature Communications. 7, 11046 (2016).</li><li>Bailey, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guideline:+the+laboratory+diagnosis+of+malaria.+General+Haematology+Task+Force+of+the+British+Committee+for+Standards+in+Haematology.">Guideline: the laboratory diagnosis of malaria. General Haematology Task Force of the British Committee for Standards in Haematology.</a> British Journal of Haematology. 163 (5), 573-580 (2013).</li><li>Das, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+literature+review+of+microscopy+methods+reported+in+malaria+clinical+trials.">A systematic literature review of microscopy methods reported in malaria clinical trials.</a> The American Journal of Tropical Medicine and Hygiene. 104 (3), 836-841 (2020).</li><li>de Oca, M. M., Engwerda, C., Haque, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+berghei+ANKA+(PbA)+infection+of+C57BL/6J+mice:+a+model+of+severe+malaria.">Plasmodium berghei ANKA (PbA) infection of C57BL/6J mice: a model of severe malaria.</a> Methods in Molecular Biology. 1031, 203-213 (2013).</li><li>Musiime, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+that+a+real+oocyst?+Insectary+establishment+and+identification+of+Plasmodium+falciparum+oocysts+in+midguts+of+Anopheles+mosquitoes+fed+on+infected+human+blood+in+Tororo,+Uganda.">Is that a real oocyst? Insectary establishment and identification of Plasmodium falciparum oocysts in midguts of Anopheles mosquitoes fed on infected human blood in Tororo, Uganda.</a> Malaria Journal. 18 (1), 287 (2019).</li><li>Marques-da-Silva, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+type+I+interferon+signaling+in+hepatocytes+controls+malaria.">Direct type I interferon signaling in hepatocytes controls malaria.</a> Cell Reports. 40 (3), 111098 (2022).</li><li>Bowers, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+Plasmodium+sporozoites.">Cryopreservation of Plasmodium sporozoites.</a> Pathogens. 11 (12), 1487 (2022).</li><li>Zander, R. 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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...

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.&#160;PbGFP11 traffics the 11th &#946;-strand of super-folder green fluorescent protein (GFP), GFP11,&#160;into the parasitophorous vacuole (PV) generated in the host hepatocytes. PbGFP11&#160;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&#160;to enhance the resulting fluorescence signal. Upon staining PbGFP11&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 G x 1/2&#34; Syringe needle</td>
			<td>Exel international</td>
			<td>26437</td>
			<td></td>
		</tr>
		<tr>
			<td>Alsever&#39;s solution</td>
			<td>Sigma-Aldritch</td>
			<td>A3551-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Amaxa Basic Parasite Nucleofector Kit 2</td>
			<td>Lonza</td>
			<td>VMI-1021</td>
			<td></td>
		</tr>
		<tr>
			<td>Avertin (2,2,2-Tribromoethanol)</td>
			<td>TCI America</td>
			<td>T1420</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood collection tubes</td>
			<td>BD bioscience</td>
			<td>365967</td>
			<td>for serum collection</td>
		</tr>
		<tr>
			<td>C-Chip disposable hematocytometer</td>
			<td>INCYTO</td>
			<td>DHC-N01-5</td>
			<td></td>
		</tr>
		<tr>
			<td>CellVeiw Cell Culture Dish</td>
			<td>Greiner Bio-One</td>
			<td>627860</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5425</td>
			<td>Eppendorf</td>
			<td>5405000107</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5910R</td>
			<td>Eppendorf</td>
			<td>5910R</td>
			<td>For gradient centrifugation</td>
		</tr>
		<tr>
			<td>Delta Vision II - Inverted microscope system</td>
			<td>Olympus</td>
			<td>IX-71</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma</td>
			<td>D5879-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>GenClone</td>
			<td>25-525</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP11 plasmid</td>
			<td>Kurup Lab</td>
			<td>pSKspGFP11</td>
			<td>Generated from PL0017 plasmid</td>
		</tr>
		<tr>
			<td>Giemsa Stain</td>
			<td>Sigma-Aldritch</td>
			<td>48900-1L-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepa GFP1-10 cells</td>
			<td>Kurup Lab</td>
			<td>Hepa GFP1-10</td>
			<td>Generated from Hepa 1-6 cells (ATCC Cat# CRL-1830)</td>
		</tr>
		<tr>
			<td>Mouse Serum</td>
			<td></td>
			<td></td>
			<td>Used for mosquito dissection media</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Millipore-Sigma</td>
			<td>SX0420-5</td>
			<td>1.5 M and 0.15 M for percoll solution</td>
		</tr>
		<tr>
			<td>Nucleofector II</td>
			<td>Amaxa Biosystems (Lonza)</td>
			<td></td>
			<td>Program U-033 used for RBC electroporation</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>VWR</td>
			<td>14673-043</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldritch</td>
			<td>P0781-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (Density gradient stock medium)</td>
			<td>Cytivia</td>
			<td>17-0891-02</td>
			<td>Details in protocol</td>
		</tr>
		<tr>
			<td>PL0017 Plasmid</td>
			<td>BEI Resources</td>
			<td>MRA-786</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrimethamine (for oral administration)</td>
			<td>Sigma</td>
			<td>46706</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Corning</td>
			<td>15-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>SoftWoRx microscopy software</td>
			<td>Applied Precision</td>
			<td>v6.1.3</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating genetically modified 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.

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...

Watch this Scientific Journal Video about Generating Genetically Modified Plasmodium berghei Sporozoites at JoVE.com

Generating Genetically Modified Plasmodium berghei Sporozoites

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.

Generating Genetically Modified 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 ...

Currently we have a limited understanding of the biology of plasmodium infection of the liver. This protocol has helped us generate a lot of transgenic lines that has helped answer some critical questions about the biology of the organism. A fundamental understanding of the parasite and its biology are required to develop new tools and approaches to combat the infection of malaria.The method of transgenesis that we are describing here has helped us decipher some key and complex biology of the organism as well as the host. Demonstrating this procedure will be Carson Bowers, my laboratory manager. After generating P.Bergehei infected mice and euthanizing them, collect two milliliters of blood from two infected mice into five milliliters of Elsevier solution in a sterile environment.Centrifuge the blood at 450 G for eight minutes at room temperature. Discard the supernatant and re-suspend the cell pellet in 10 milliliters of complete RPMI media. Centrifuge the cell suspension again and re-suspend the pellet in 25 milliliters of complete RPMI media.Transfer the cell suspension to a T75 culture flask with a filter cap and incubate with gentle shaking to keep the cells in suspension. At the end of the incubation, collect 500 microliters of the sample. Centrifuge and re-suspend the pellet in 10 microliters of complete RPMI media.Next, prepare a thin blood smear. Stain the blood smear with Giemsa stain and determine the frequency of schizonts. For optimal transfection efficiency, 60 to 70%of the parasites should be in the schizont stage.Prepare a gradient centrifugation buffer by reconstituting 13 milliliters of density gradient stock medium in a 50 milliliter centrifugation tube. Transfer 25 milliliters of blood culture to a fresh 50 milliliter centrifugation tube. Then carefully layer 20 milliliters of gradient centrifugation buffer to the bottom of the centrifugation tube, using a narrow glass pipette to create a gradient column and ensure a clear division between the buffer and the media.Centrifuge the prepared buffer and media for 20 minutes at 450 G with no break at room temperature. Using a pasture pipette, carefully remove the schizont layer at the interface of the culture media and the gradient centrifugation buffer. Place it in a new 50 milliliter centrifugation tube.Re-suspend the isolated schizonts in complete RPMI media and increase the total volume to 40 milliliters. After centrifuging and discarding the supernatant, re-suspend the schizont in 10 milliliters of complete RPMI media. Then transfer one milliliter of this solution into a 1.5 milliliter micro centrifuge tube for each transfection and pellet the infected cells by centrifugation at 16, 000 G for five seconds.Next, combine 100 microliters of the electroporation buffer at room temperature with five micrograms of target plasmid DNA in a 1.5 milliliter micro centrifuge tube. After centrifuging the infected cells, remove the supernatant and carefully re-suspend the cells in the prepared nucleoefector solution plus plasmid DNA. Then transfer the suspension into electroporation, cuvettes, and transfect using a suitable nucleofector program.Next, add 100 microliters of complete RPMI media to the cuvette. Transfer the entire solution into a 1.5 milliliter micro centrifuge tube and place it on ice. Verify the parasitemia levels in the mice inoculated with the transfected schizont daily from two days post inoculation.Once the infection is confirmed, start drug selection by administering the drug orally and drinking water offered ad libitum. Monitor parasitemia using blood smears starting from six days after the initiation of pyromethymine treatment. When parasitemia reaches at least 1%transfer the parasites to naive B6 mice to create blood stage parasite stocks.Once parasitemia reaches 2%to 5%generate stocks and cryopreserve them. One day before mosquito infection, separate the female mosquitoes by placing a glove filled with water heated to 37 degrees Celsius on top of the cage containing both male and female mosquitoes. Use an insect aspirator to remove the female mosquitoes attracted to the heat source and transfer them to a smaller cage for infection.On the day of mosquito feeding and infection, determine parasitemia in the infected B6 mice. Parasitemia between 2%to 5%is optimal for mosquito infection. After verifying the parasitemia, transfer the infection to the female mosquitoes by placing the anesthetized mice on the caged female mosquitoes under sternal recumbency.Manually spread the front and back legs to provide the greatest surface area for mosquito feeding. Leave the infected mice in each cage for 15 minutes, checking every five minutes to ensure the mice are not awake. Once the feeding is complete and the mice are euthanized, dispose of them safely following the institution's guidelines.To verify successful infection within the mosquitoes, isolate the mosquitoes midguts by removing the terminal abdominal segment with forceps 10 days post-infection. Then view the midguts under polarized light to detect the presence of oocysts. At 18 days post-infection, dissect five to 10 mosquitoes to evaluate the number of sporozoites present.To isolate and count the sporozoites, carefully remove the head of the mosquito while applying pressure to the thorax with forceps or a fine dissecting needle. The salivary glands will remain attached to the removed head. Next, remove any additional mosquito debris from the salivary glands and place them in a micro centrifuge tube containing 400 microliters of mosquito dissection media.Then disrupt the isolated salivary glands by passing them through a 30 gauge needle syringe 15 to 20 times, Afterward, place 10 microliters of the disrupted salivary glands in mosquito dissection media into a hemocytometer and count. Finally, re-suspend the salivary glands in the desired concentration of mosquito dissection media or PBS for injection into mice or long-term cryo-preservation. Like microscopy images depicting mature and immature P.berghei schizont in parasitized mouse blood cultures are presented.Like microscopy images of a mosquito head with the salivary glands still intact during dissection, and the dissected salivary gland under lower and higher magnifications are shown. The generation of green fluorescence signal in the PB, GFP 11 infected HEPA GFP one to 10 host cells indicated the lysis of the parasitophorous vacuole. It's important to carefully remove the chaisson layer without disrupting the gradient interface.Also, it takes a bit of practice to reliably remove the salivary glands along with the head during spores light isolation. Following isolation, transgenic sporozoites can be cryo-preserved or used fresh for in vivo or in vitro infection studies.

generating-genetically-modified-plasmodium-berghei-sporozoites

Research

JoVE Journal

Immunology and Infection

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases show Chagas disease up to three decades or later. Autoimmunity and parasite persistence are competing theories to explain the pathogenesis of Chagas disease 1, 2. To separate roles played by parasite persistence and autoimmunity in Chagas disease we inoculate the T. cruzi in the air chamber of fertilized eggs. The mature chicken immune system is a tight biological barrier against T. cruzi and the infection is eradicated upon development of its immune system by the end of the first week of growth 3. The chicks are parasite-free at hatching, but they retain integrated parasite mitochondrial kinetoplast DNA (kDNA) minicircle within their genome that are transferred to their progeny. Documentation of the kDNA minicircle integration in the chicken genome was obtained by a targeted prime TAIL-PCR, Southern hybridizations, cloning, and sequencing 3, 4. The kDNA minicircle integrations rupture open reading frames for transcription and immune system factors, phosphatase (GTPase), adenylate cyclase and phosphorylases (PKC, NF-Kappa B activator, PI-3K) associated with cell physiology, growth, and differentiation 3, 5-7, and other gene functions. Severe myocarditis due to rejection of target heart fibers by effectors cytotoxic lymphocytes is seen in the kDNA mutated chickens, showing an inflammatory cardiomyopathy similar to that seen in human Chagas disease. Notably, heart failure and skeletal muscle weakness are present in adult chickens with kDNA rupture of the dystrophin gene in chromosome 1 8. Similar genotipic alterations are associated with tissue destruction carried out by effectors CD45+, CD8&gamma;&delta;+, CD8&alpha; lymphocytes. Thus this protozoan infection can induce genetically driven autoimmune disease.

The inoculation of Trypanosoma cruzi in fertile eggs prior to incubation renders the parasite kDNA minicircle integration in embryo cells genome. Crossbreeding reveals the vertical transfer of the mutations to progeny. The kDNA integrates into coding regions at several chromosomes and the chickens die with an inflammatory autoimmune heart disease.

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...