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

<meta charset="utf8"></head><body>말라리아 기생충에서 키나아제 재배선 식별: 화학 유전학 접근법

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>저자 스포트라이트: Identifying comcomcomatory pathways in Malaria Parasites Containing Hypomorphic Allele of Essential Protein Kinases

식물 유사 키나아제의 저형성 대립유전자를 품고 있는 돌연변이 말라리아 기생충의 보상 경로 개발 이해

Plasmodium falciparum Kinase 저항성 메커니즘을 규명하기 위한 cdpk1 유전자좌의 원하는 변형을 통한 형질전환 기생충의 PCR 검증

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

142 조회수. 시작하려면 변형 된 Plasmodium falciparum 기생충을 원하는 약물로 치료하십시오. 기생충 성장의 2-3 일 후, 5 분 동안 2, 400 G의 마이크로 원심 분리기 튜브에 500 마이크로 리터의 배양물을 원심 분리합니다. 적혈구를 용해시키기 위해, 부유된 기생충 1밀리리터를 10마이크로리터의 10% 사포닌과 함께 섭씨 4도에서 5분 동안 배양합니다.세포를 원심 분리 ...

비디오: Plasmodium falciparum Kinase 저항성 메커니즘을 규명하기 위한 cdpk1 유전자좌의 원하는 변형을 통한 형질전환 기생충의 PCR 검증

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.