pcr verification of transgenic parasite with desired modification of the cdpk1 locus for elucidating plasmodium falciparum kinase resistance mechanisms

<meta charset="utf8"></head><body>זיהוי חיווט מחדש של קינאזות בטפיל מלריה: גישה גנטית כימית

Malaria is one of the leading infectious diseases that is responsible for millions of deaths every year, especially in children below 5 years of age1. There is no clinically available vaccine against malaria. Moreover, Plasmodium falciparum, the deadliest human malaria parasite, is known to have acquired resistance against the frontline drug called artemisinin2,3,4,5. There is an urgent necessity to identify new drug targets and novel strategies that can be quickly deployed to avoid the spread of artemisinin resistance worldwide. A better understanding of the mechanism of drug action and the molecular basis of compensatory pathways can help in devising better strategies to avoid the development of drug resistance.
Protein kinases belonging to the family of Calcium Dependent Protein Kinases (CDPKs) are crucial at many stages of parasite development during erythrocytic, sexual, and pre-erythrocytic stages6. During asexual replication of P. falciparum, CDPK5 is known to play a critical role in the exit of merozoites from mature schizonts7. Conditional deletion of CDPK5 does not allow schizonts to release merozoites into the bloodstream, leading to the death of the parasite. The molecular mechanism of CDPK5-mediated parasite egress is not completely understood and is thought to involve protein kinase G (PKG) as an upstream regulator7,8. CDPK7 is essential for the maturation of ring-stage parasites to trophozoite9. CDPK4 is another member of the CDPK family that is critical for sexual stage development within mosquitoes. It is involved in multiple steps during the exflagellation process and is therefore critical for the formation of free, motile, male gametes10,11,12,13. CDPK1 phosphorylate components of inner membrane complex and rhoptry14,15. Moreover, conditional deletion of CDPK1 results in hypo-phosphorylation of proteins involved in invasion of RBC16. CDPK1 has been shown to regulate the discharge of apical organelles during the invasion process17. CDPK1 also helps in the egress of merozoites from infected RBC by phosphorylating SERA5 protease18. Phosphorylated CDPK1 is preferentially localized at the apical end of merozoite, where it may interact with other parasite proteins required for the invasion process19,20. CDPK1 is also involved in the formation of male and female gametes, which is a critical step for malaria transmission and the sexual development of the parasite within mosquitoes21.
The chemical genetics approach has been historically used for the identification of functional roles of mammalian kinases22,23. The approach utilizes the substitution of a residue at the gatekeeper position with an amino acid of a different side chain. The change of gatekeeper residue modulates the size of an adjacent ATP pocket that, in turn, changes the accessibility of chemical compounds called bumped kinase inhibitors (BKIs) towards the mutant enzyme compared to the wild type. Substitution of a bulky residue at the gatekeeper position with a smaller residue allows access to BKIs, while reverse substitution makes the kinase resistant to BKIs. In some cases, the substitution of gatekeeper residue is associated with a decrease in the kinase activity of the target enzyme, making the modification intolerant for functional studies24,25. The negative effect of gatekeeper substitution on the enzyme activity can be reversed by generating suppressor mutation at a second site in the intolerant kinase25. A chemical genetics approach has been utilized to study the function of Apicomplexan protein kinases. The wild-type CDPK4 containing a smaller gatekeeper residue was selectively inhibited by bumped kinase inhibitors BKI-1 and 1294, leading to a block in male gametogenesis and oocyst development11,12. Substitution of a small gatekeeper residue in CDPK4 with a bulky methionine residue led to a decrease in BKI-mediated inhibition in exflagellation11. CDPK1 of Toxoplasma gondii, an Apicomplexan parasite closely related to the malaria parasite, was shown to be involved in motility and invasion of host cells by tachyzoites26,27. A smaller gatekeeper pocket of the wild-type TgCDPK1 was exploited to design specific inhibitors that decreased the disease pathogenesis in animal models28,29. Involvement of P. falciparum Protein Kinase G (PKG) in late schizogonic development and gamete formation was demonstrated by inhibiting the activity of the enzyme using specific pharmacological inhibitors30,31. Substitution of threonine at the gatekeeper position with glutamine (T618Q) greatly decreased the sensitivity of the mutant enzyme to the inhibitors, resulting in normal gametogenesis and schizont-to-ring progression30,31.
Most of the previous studies have utilized a chemical genetics approach for the functional characterization of target kinases11,12,22,23,26,30,31 however, we have adopted this approach to understand the development of compensatory mechanisms in parasites that harbor hypomorphic allele of a protein kinase. We earlier showed that substitution of wild-type gatekeeper residue in CDPK1 with methionine (T145M) results in ~ 47% decrease in the transphosphorylation potential of the mutant enzyme32. A mutant parasite containing the hypomorphic allele of cdpk1 (cdpk1t145m) is pre-adapted for the reduced activity of CDPK1 by transcriptional rewiring of other CDPK family members. We believe that this strategy may be used in general to elucidate the compensatory mechanisms in mutant parasites containing hypomorphic alleles of essential protein kinases. Other kinases that partially compensate for the function of the target kinase may be simultaneously inhibited along with the target kinase to prevent the development of drug resistance against individual kinases. This may serve as a better strategy for malaria control.

<meta charset="utf8"></head><body>מחבר זרקור: זיהוי מסלולי פיצוי בטפילי מלריה המכילים אלל היפומורפי של קינאזות חלבון חיוניות

הבנת התפתחותם של מסלולי פיצוי בטפיל מלריה מוטנטי בעל אלל היפומורפי של קינאזות דמויות צמחים

Drug resistance is a major challenge in combating malaria. Our study aims to identify compensatory pathways in malaria parasites containing hypomorphic allele of essential protein kinases. Targeting two kinases simultaneously may be a better strategy that avoids developing drug resistance against individual kinases.Gene editing using CRISPR-Cas9 has profoundly benefited malaria research and has been instrumental performing allelic exchanges, endogenous tagging, conditional knockout, and knockdown of target gene. The molecular mechanism of drug resistance, understanding target gene function and study of host parasite interaction can now be studied with greater ease. The current experimental challenges include heterologous protein expression in E.coli due to the high richness of the plasmodium genome.Additionally, of target effect of CRISPR-Cas9 complicated data interpretation and led to erroneous results. Low transcription efficiency with the malaria parasite increases the time required for generating the desired genetic modification. Our findings suggests that targeting a single kinase in the malaria parasite may lead to compensatory over expression of other kinases.This raises new question about the potential for adaptive resistance and whether dual kinase inhibition could effectively prevent such adaptation, opening avenues for the combination therapeutic strategies. In the future, our laboratory will target other essential protein kinases of malaria parasites using chemical genetics. Transcriptional rewiring in the mutant parasites will help in identifying compensatory pathways that may be simultaneously targeted to prevent development of drug resistance against individual kinases.

אימות PCR של טפיל מהונדס עם שינוי רצוי של מוקד cdpk1 להבהרת מנגנוני עמידות פלסמודיום פלציפרום קינאז

pcr-verification-transgenic-parasite-with-desired-modification-cdpk1

Research

JoVE Journal

Biology

PCR Verification of Transgenic Parasite with Desired Modification of the cdpk1 Locus for Elucidating Plasmodium falciparum Kinase Resistance Mechanisms

122 Views. כדי להתחיל, לטפל טפילים פלסמודיום falciparum הפך עם התרופות הרצויות. לאחר יומיים-שלושה של גידול טפילים, צנטריפוגה 500 מיקרוליטר של תרבית בצינור מיקרו צנטריפוגה ב 2, 400 גרם במשך חמש דקות. כדי לשקר את RBCs, לדגור מיליליטר אחד של טפילים resuspended עם 10 מיקרוליטר של 10% saponin בא...

Video: אימות PCR של טפיל מהונדס עם שינוי רצוי של מוקד cdpk1 להבהרת מנגנוני עמידות פלסמודיום פלציפרום קינאז

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases

One of the mechanisms for subverting the effect of drugs by&#160;the malaria parasite is through rewiring of its transcriptome. The effect is more pronounced for target genes belonging to the multigene family. Plasmodium falciparum protein kinases belonging to the CDPK family are essential for blood stage development. As such, CDPKs are considered good targets for the development of anti-malarial compounds. The chemical genetics approach has been historically used to elucidate the function of protein kinases in higher eukaryotes. It requires the substitution of gatekeeper residue for another amino acid with a different side chain through genetic manipulation. Amino acid substitution at the gatekeeper position modulates the activity of a protein kinase and changes its susceptibility to a specific class of compounds known as bumped kinase inhibitors (BKIs) that help in the functional identification of the target gene. Here, we have exploited the chemical genetics approach to understand compensatory mechanisms evolved by a mutant parasite harboring a hypomorphic allele of cdpk1. Overall, our approach helps in identifying compensatory pathways that may be simultaneously targeted to prevent the development of drug resistance against individual kinases.

Chemical genetics involves the substitution of a gatekeeper residue with an amino acid containing a different side chain at the target locus. Here, we have generated a mutant parasite containing a hypomorphic allele of cdpk1 and identified compensatory pathways adopted by the parasite in the mutant background.

One of the mechanisms for subverting the effect of drugs by&#160;the malaria parasite is through rewiring of its transcriptome. The effect is more ...

In Vitro Culturing of Plasmodium falciparum and Sorbitol Synchronization for Transfection to Unravel Compensatory Mechanisms Using CDPK1

To begin, culture the NF54 strain of Plasmodium falciparum in 2%hematocrit of O+human RBCs in supplemented complete RPMI medium. For sorbitol synchronization, pellet the asynchronous parasites in a 50 milliliter conical tube at 2, 300 G for three minutes at room temperature. After discarding the supernatant, incubate the parasitized red blood cells with nine volumes of 5%sorbitol for 10 minutes at 37 degrees Celsius.Following incubation, pellet the red blood cells as demonstrated earlier, and discard the sorbitol. Wash the sorbitol treated parasites with incomplete RPMI and RPMI media without AlbuMAX and sodium bicarbonate. Subsequently incubate them in complete RPMI to obtain the ring stage parasites.

תרבית חוץ גופית של פלסמודיום פלציפרום וסנכרון סורביטול לטרנספקציה לפענוח מנגנוני פיצוי באמצעות CDPK1

Transfection of Ring Stage Malarial Parasite with Plasmids Functional for Rewiring the Transcriptome

To begin, obtain the sorbitol synchronized ring stage parasites of plasmodium falciparum. Prepare 50 milliliters of 2x cytomix buffer and dilute it 1 to 1 with sterile water. Then, wash 100 microliters of packed ring stage parasitized RBCs twice with 5 milliliters of 1X cytomix buffer and centrifuge them at 994G for three minutes at room temperature.After removing the buffer, add 50 micrograms of each plasmid into the cells, followed by an appropriate volume of 2x buffer. Adjust the volume to 400 microliters with 1x buffer before transferring the prepared solution into 0.2 centimeter cuvettes for each transfection. Then, electroporate the cells carefully.Following electroporation, mix and incubate the cells with complete RPMI medium in a T25 flask with a total volume of 15 milliliters. Next, centrifuge the transfected parasites in a 50 milliliter conical tube at 994G for three minutes. After removing the supernatant, wash the parasites with 10 milliliters of incomplete RPMI.Culture them with fresh complete RPMI for two days, replenishing the medium after 24 hours. After 48 hours, remove the complete RPMI from each flask. Resuspend the culture in complete RPMI added with drugs.Incubate the culture in a T75 flask along with 400 microliters of fresh red blood cells for a fortnight On day 14, aliquot 200 microliters of the transfected culture into a 1.5 milliliter microcentrifuge tube. Centrifuge the tube at 500G for five minutes at room temperature. After aspirating the supernatant, transfer the cells onto a slide and place another slide on it at a 45 degree angle to make a smear.Following methanol fixation, immerse the slide in Giemsa stain for 20 minutes. Wash the slide under running water and dry it thoroughly. Observe the slide under a light microscope at 100x magnification after applying immersion oil.Once the parasites are visualized, replenish the medium daily for two to three days.

טרנספקציה של טפיל מלריה בשלב הטבעתי עם פלסמידים פונקציונלי לחיווט מחדש של התמליל

To begin, treat the transformed Plasmodium falciparum parasites with the desired drugs. Following two to three days of parasite growth, centrifuge 500 microliters of culture in a micro centrifuge tube at 2, 400 G for five minutes. To lyse the RBCs, incubate one milliliter of the resuspended parasites with 10 microliters of 10%saponin at four degrees Celsius for five minutes.After centrifuging the cells, discard the supernatant to obtain the parasite pellet. Wash the parasite pellet with one milliliter of PBS and resuspend the pellet in 50 microliters of DNAse and RNAse-free water. Heat the resuspended pellet at 95 degrees Celsius for five minutes, and centrifuge at 16, 200 G for 10 minutes at four degrees Celsius.Transfer the supernatant containing parasite DNA to a fresh tube.

Limiting Dilution for Obtaining Clonal Transgenic Plasmodium Parasites for Combating Drug Resistance in Malaria

To begin, verify the sequence of the desired genes isolated from the drug-treated Plasmodium falciparum parasites. After diluting the parasitized RBCs, add an appropriate volume to achieve a total of 50 parasites in 10 milliliters of media, ensuring 2%hematocrit. Then, add 100 microliters of this media containing 50 parasites to each well of the 96-well plate.Place the plate in an airtight Secador flushed with mixed gas. And incubate at 37 degrees Celsius, replenishing the media every two days. Replace the media with complete RPMI containing 2%hematocrit every seventh day until the parasites appear.Tilt the 96-well plate to a 45-degree angle to let the red blood cells settle down. Carefully remove the media from each well using a multi-channel pipette. Observe a color change to yellow or dark red in wells containing parasites, which contrasts with the pink color of media in parasite-free wells.Next, take out a small amount of culture from the well exhibiting the color change and prepare a smear on a labeled slide. After performing Giemsa staining, observe the slide under a microscope. Transfer the contents of the well where parasites were observed into a T25 flask and incubate to let the parasites grow for four to five days.Once the parasitemia reaches two to 3%saponin lyse 500 microliters of parasite culture. And extract the parasite genetic material.