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

Summary

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.

Abstract

One of the mechanisms for subverting the effect of drugs by 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.

Introduction

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 artemisinin^2,3,4,5. There is an urgent necessity to identify new drug targets and novel strategies that can be quickly deployed to avoid the spread of artemisinin ....

Protocol

The P. falciparum parasite strain (NF54) was obtained from Alvaro Molina Cruz^21,32,33. 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.

Discussion

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.......

Materials

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