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New and improved drugs are required for the treatment and ultimate eradication of malaria. The efficacy of front-line therapies is now threatened by emerging drug resistance; thus, new tools to support the development of drugs with a lower propensity for resistance are needed. Here, we describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium drug targets. Proof-of-concept studies focused on interrogating the antimalarial drug target, Plasmodium falciparum lysyl tRNA synthetase ( Pf KRS). Saturation mutagenesis was used to construct a plasmid library encoding all conceivable mutations within a 20-residue span at the base of the Pf KRS ATP-binding pocket. The superior transfection efficiency of Plasmodium knowlesi was exploited to generate a high coverage parasite library expressing Pf KRS bearing all possible amino acid changes within this region of the enzyme. The selection of the library with Pf KRS inhibitors, cladosporin and DDD01510706, successfully identified multiple resistance-conferring substitutions. Genetic validation of a subset of these mutations confirmed their direct role in resistance, with computational modeling used to dissect the structural basis of resistance. The application of ResMAP to inform the development of resistance-resilient antimalarials of the future is discussed., Importance: An increase in treatment failures for malaria highlights an urgent need for new tools to understand and minimize the spread of drug resistance. We describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium spp, the causative agent of malaria. Saturation mutagenesis was used to generate a mutation library containing all conceivable mutations for a region of the antimalarial-binding site of a promising drug target, Plasmodium falciparum lysyl tRNA synthetase ( Pf KRS). Screening of this high-coverage library with characterized Pf KRS inhibitors revealed multiple resistance-conferring substitutions including several clinically relevant mutations. Genetic validation of these mutations confirmed resistance of up to 100-fold and computational modeling dissected their role in drug resistance. We discuss potential applications of this data including the potential to design compounds that can bypass the most serious resistance mutations and future resistance surveillance., Competing Interests: The authors declare no conflict of interest.

Plasmodium knowlesi is the only Plasmodium that causes zoonotic disease among the Plasmodium that cause infection in humans. It is fatal due to its short asexual growth cycle within 24 h. Lactate dehydrogenase (LDH), an enzyme that catalyzes the final step of glycolysis, is a biomarker for diagnosing infection by Plasmodium spp. parasite. Therefore, this study aimed to efficiently produce the soluble form of P. knowlesi LDH (PkLDH) using a bacterial expression system for studying malaria caused by P. knowlesi . Recombinant pET-21a(+)- PkLDH plasmid was constructed by inserting the PkLDH gene into a pET-21a(+) expression vector. Subsequently, the recombinant plasmid was inserted into the protein-expressing Escherichia coli Rosetta(DE3) strain, and the optimal conditions for overexpression of the PkLDH protein were established using this strain. We obtained a yield of 52.0 mg/L PkLDH from the Rosetta(DE3) strain and confirmed an activity of 483.9 U/mg through experiments. This methodology for high-efficiency PkLDH production can be utilized for the development of diagnostic methods and drug candidates for distinguishing malaria caused by P. knowlesi .

Plasmodium lactate dehydrogenase (pLDH) is a common target in malaria rapid diagnostic tests (RDTs). These commercial antibody capture assays target either Plasmodium falciparum-specific pLDH (PfLDH), P. vivax-specific pLDH (PvLDH), or a conserved epitope in all human malaria pLDH (PanLDH). However, there are no assays specifically targeting P. ovale, P. malariae or zoonotic parasites such as P. knowlesi and P. cynomolgi. A malaria multiplex array, carrying the specific antibody spots for PfLDH, PvLDH, and PanLDH has been previously developed. This study aimed to assess potential cross-reactivity between pLDH from various Plasmodium species and this array. We tested recombinant pLDH proteins, clinical samples for P. vivax, P. falciparum, P. ovale curtisi, and P. malariae; and in vitro cultured P. knowlesi and P. cynomolgi. P. ovale-specific pLDH (PoLDH) and P. malariae-specific pLDH (PmLDH) cross-reacted with the PfLDH and PanLDH spots. Plasmodium Knowlesi-specific pLDH (PkLDH) and P. cynomolgi-specific pLDH (PcLDH) cross-reacted with the PvLDH spot, but only PkLDH was recognized by the PanLDH spot. Plasmodium ovale and P. malariae can be differentiated from P. falciparum by the concentration ratios of PanLDH/PfLDH, which had mean (range) values of 4.56 (4.07-5.16) and 4.56 (3.43-6.54), respectively, whereas P. falciparum had a lower ratio of 1.12 (0.56-2.61). Plasmodium knowlesi had a similar PanLDH/PvLDH ratio value, with P. vivax having a mean value of 2.24 (1.37-2.79). The cross-reactivity pattern of pLDH can be a useful predictor to differentiate certain Plasmodium species. Cross-reactivity of the pLDH bands in RDTs requires further investigation.

Plasmodium lactate dehydrogenase (pLH) is one of the enzymes in glycolysis with potential target for chemotherapy. This study aimed to clone, overexpress and characterize soluble recombinant lactate dehydrogenase from Plasmodium knowlesi in a bacterial system. Synthetic P. knowlesi lactate dehydrogenase ( Pk -LDH) gene was cloned into pET21a expression vector, transformed into Escherichia coli strain BL21 (DE3) expression system and then incubated for 18 h, 20 °C with the presence of 0.5 mM isopropyl β-d-thiogalactoside in Terrific broth supplemented with Magnesium sulfate, followed by protein purifications using Immobilized Metal Ion Affinity Chromatography and size exclusion chromatography (SEC). Enzymatic assay was conducted to determine the activity of the enzyme. SDS-PAGE analysis revealed that protein of 34 kDa size was present in the soluble fraction. In SEC, a single peak corresponding to the size of Pk -LDH protein was observed, indicating that the protein has been successfully purified. From MALDI-TOF analysis findings, a peptide score of 282 was established, which is significant for lactate dehydrogenase from P. knowlesi revealed via MASCOT analysis. Secondary structure analysis of CD spectra indicated 79.4% α helix and 1.37% β strand structure. Specific activity of recombinant Pk -LDH was found to be 475.6 U/mg, confirming the presence of active protein. Soluble Pk -LDH that is biologically active was produced, which can be used further in other malaria studies.