We acknowledge the support and facilities available through the Central Instrumentation Facility, School of Life Sciences, JNU. Financial support from the Department of Biotechnology (BT/PR28256/MED/29/1313/2018) to AB is also gratefully acknowledged. We thank Jose-Juan Lopez-Rubio for providing pL6eGFP and pUF1 plasmids. The funding agency has no role in the preparation and decision to publish the work. MS is a recipient of JRF-SRF Fellowship from the Council for Scientific and Industrial Research.

The P. falciparum parasite strain (NF54) was obtained from Alvaro Molina Cruz21,32,33. O+ human red blood cells used for the parasite culture were obtained from Rotary Blood Centre, New Delhi, India. The plasmids, pL6eGFP and pUF1, were obtained from Jose-Juan Lopez-Rubio. DSM267 was obtained from Margaret A. Phillips and Pradipsinh K. Rathod. WR99210 was provided by Jacobus Pharmaceutical Company.</...

Identifying Kinases Rewiring in Malaria Parasite: A Chemical Genetics Approach

The generation of a mutant transgenic parasite containing a hypomorphic allele of cdpk1 is based on the in vitro kinase activity data with recombinant enzymes. It is better to generate as many mutant recombinant proteins with different gatekeeper residues as possible. Since the P. falciparum genome is highly AT-rich, therefore, expression of recombinant proteins in E. coli may require codon optimization. This will help in a comprehensive evaluation of gatekeeper substitution o...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x phosphate inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>04906 845001</td>
			<td></td>
		</tr>
		<tr>
			<td>AflII</td>
			<td>NEB</td>
			<td>R0520S</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumax II&#160;</td>
			<td>Gibco</td>
			<td>11021-037</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GST antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>G7781</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse HRP</td>
			<td>Sigma-Aldrich</td>
			<td>A4416</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rabbit HRP</td>
			<td>Sigma-Aldrich</td>
			<td>A6154</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>NEB</td>
			<td>R3136S</td>
			<td></td>
		</tr>
		<tr>
			<td>BtgZ1</td>
			<td>NEB</td>
			<td>R0703S</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>Sorvall legend micro 17R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge ( For pelleting Bacterial cell)</td>
			<td>ThermoFisher Scientific</td>
			<td>Sorvall ST 8R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge ( For pelleting/processing parasite)</td>
			<td>ThermoFisher Scientific</td>
			<td>Sorvall ST 8R (TX-400 rotor)</td>
			<td></td>
		</tr>
		<tr>
			<td>DpnI</td>
			<td>NEB</td>
			<td>RO1765</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sorbitol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S1876</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli BLR(DE3) pLysS competent cells</td>
			<td>Sigma-Aldrich</td>
			<td>69956</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli DH5a Competent Cells</td>
			<td>Takara Bio</td>
			<td>9057</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation Cuvettes, 0.2 cm gap</td>
			<td>BioRad</td>
			<td>1652086</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>BioRad</td>
			<td>GenePulser Xcell</td>
			<td></td>
		</tr>
		<tr>
			<td>femtoLUCENTTM PLUS HRP chemiluminescent reagent</td>
			<td>G-Bioscience</td>
			<td>7860-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>ThermoFisher Scientific</td>
			<td>15750078</td>
			<td></td>
		</tr>
		<tr>
			<td>Giemsa Stain</td>
			<td>Himedia</td>
			<td>S011-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Sepharose 4B</td>
			<td>GE Healthcare</td>
			<td>17075601</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, Free Acid</td>
			<td>Merck</td>
			<td>391338</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxanthine</td>
			<td>Merck</td>
			<td>4010CBC</td>
			<td></td>
		</tr>
		<tr>
			<td>In-fusion HD cloning Kit</td>
			<td>Takara Bio</td>
			<td>1711641A</td>
			<td></td>
		</tr>
		<tr>
			<td>iQ SYBR Green Supermix</td>
			<td>BioRad</td>
			<td>1708880EDU</td>
			<td></td>
		</tr>
		<tr>
			<td>MBP, dephosphorylated</td>
			<td>&#160;Merck</td>
			<td>13-110</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>NEB</td>
			<td>R3189S</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleobond Xtra Maxi EF&#160;&#160;</td>
			<td>Takara Bio</td>
			<td>740424</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoSpin Gel and PCR clean-up Mini kit</td>
			<td>Takara Bio</td>
			<td>740609</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>p-nitrobenzyl mesylate (PNBM)</td>
			<td>Abcam</td>
			<td>Ab138910</td>
			<td></td>
		</tr>
		<tr>
			<td>Primestar Max DNA Polymerase</td>
			<td>Takara Bio</td>
			<td>R045A</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiaprep Spin Miniprep Kit&#160;</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>QuikChange II XL site-directed mutagenesis kit</td>
			<td>Agilent</td>
			<td>200521</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>ThermoFisher Scientific</td>
			<td>31800-105</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeI-HF</td>
			<td>NEB</td>
			<td>R3133S</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III First-Strand Synthesis kit</td>
			<td>ThermoFisher Scientific</td>
			<td>18080-051</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>ThermoFisher Scientific</td>
			<td>15224017</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiophosphate ester antibody</td>
			<td>Abcam</td>
			<td>Ab92570</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Author Spotlight: Identifying Compensatory Pathways in Malaria Parasites Containing Hypomorphic Allele of Essential Protein Kinases

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

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.

Recombinant WT CDPK1 is expressed as a fusion protein with an N-terminal Glutathione S-transferase (GST) tag and purified using GST affinity chromatography. The purified CDPK1 protein was detected through Western blot using anti-CDPK1 and anti-GST antibodies (Figure 1). The threonine gatekeeper residue (T145) in WT CDPK1 was replaced with Met and Ser using site-directed mutagenesis to generate CDPK1T145M and CDPK1T145S mutant recombinant proteins, respectively (Figure 2<...

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 ...

understanding-development-compensatory-pathways-mutant-malaria