Phosphoethanolamine-N-methyltransferase is a potential biomarker for the diagnosis of P. knowlesi and P. falciparum malaria

BACKGROUND: Plasmodium knowlesi is recognised as the main cause of human malaria in Southeast Asia. The disease is often misdiagnosed as P. falciparum or P. malariae infections by microscopy, and the disease is difficult to eliminate due to its presence in both humans and monkeys. P. knowlesi infections can rapidly cause severe disease and require prompt diagnosis and treatment. No protein biomarker exists for the rapid diagnostic test (RDT) detection of P. knowlesi infections. Plasmodium knowlesi infections can be diagnosed by PCR. METHODS AND PRINCIPAL FINDINGS: Phosphoethanolamine-N-methyltransferase (PMT) is involved in malaria lipid biosynthesis and is not found in the human host. The P. falciparum, P. vivax and P. knowlesi PMT proteins were recombinantly expressed in BL21(DE3) Escherichia coli host cells, affinity purified and used to raise antibodies in chickens. Antibodies against each recombinant PMT protein all detected all three recombinant proteins and the native 29 kDa P. falciparum PMT protein on western blots and in ELISA. Antibodies against a PMT epitope (PLENNQYTDEGVKC) common to all three PMT orthologues detected all three proteins. Antibodies against unique peptides from each orthologue of PMT, PfCEVEHKYLHENKE, PvVYSIKEYNSLKDC, PkLYPTDEYNSLKDC detected only the parent protein in western blots and P. falciparum infected red blood cell lysates or blood lysates spiked with the respective proteins. Similar concentrations of PfPMT and the control, PfLDH, were detected in the same parasite lysate. The recombinant PfPMT protein was detected by a human anti-malaria antibody pool. CONCLUSION: PMT, like the pan-specific LDH biomarker used in RDT tests, is both soluble, present at comparable concentrations in the parasite and constitutes a promising antimalarial drug target. PMT is absent from the human proteome. PMT has the potential as a biomarker for human malaria and in particular as the first P. knowlesi specific protein with diagnostic potential for the identification of a P. knowlesi infection.