in vitro culturing of plasmodium falciparum and sorbitol synchronization for transfection to unravel compensatory mechanisms using cdpk1

<meta charset="utf8"></head><body>Identificación de la reconexión de quinasas en el parásito de la malaria: un enfoque de genética química

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>Autor destacado: Identificación de vías compensatorias en parásitos de la malaria que contienen alelos hipomórficos de proteínas quinasas esenciales

Comprender el desarrollo de vías compensatorias en un parásito mutante de la malaria que alberga un alelo hipomórfico de quinasas similares a las plantas

Cultivo in vitro de Plasmodium falciparum y sincronización de sorbitol para la transfección con el fin de desentrañar mecanismos compensatorios utilizando CDPK1

in-vitro-culturing-plasmodium-falciparum-sorbitol-synchronization-for

Research

JoVE Journal

Biology

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

178 vistas. Para comenzar, cultive la cepa NF54 de Plasmodium falciparum en hematocrito al 2% de glóbulos rojos O + humanos en medio RPMI completo suplementado. Para la sincronización del sorbitol, granule los parásitos asíncronos en un tubo cónico de 50 mililitros a 2.300 G durante tres minutos a temperatura ambiente. Después de desechar el sobrenadante, incubar los glóbulos rojos parasitados con nueve volúmenes de sorbitol al 5% durante 10 minutos a 37 grados c...

Video: Cultivo in vitro de Plasmodium falciparum y sincronización de sorbitol para la transfección con el fin de desentrañar mecanismos compensatorios utilizando CDPK1

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 ...

Investigación

Biología

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

Transfección de parásito palúdico en etapa de anillo con plásmidos funcionales para recablear el transcriptoma

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

Verificación por PCR de parásitos transgénicos con la modificación deseada del locus cdpk1 para dilucidar los mecanismos de resistencia a la quinasa Plasmodium falciparum

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

Limitación de la dilución para la obtención de parásitos clonales transgénicos Plasmodium para combatir la resistencia a los medicamentos en la malaria