Abstract
BackgroundPlasmodium 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 findingsPhosphoethanolamine-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.ConclusionPMT, 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.
Highlights
The Plasmodium genus includes over a hundred species that infect vertebrate hosts including birds, rodents, reptiles, amphibians and simians, by dipteran vectors [1]
PMT, like the pan-specific lactate dehydrogenase (LDH) biomarker used in rapid diagnostic test (RDT) tests, is both soluble, present at comparable concentrations in the parasite and constitutes a promising antimalarial drug target
The orthologues of PMT, from P. falciparum, P. vivax and P. knowlesi were recombinantly expressed in BL21(DE3) E. coli host cells as histidine tagged proteins and the proteins were affinity purified, using a TALON1 resin (Fig 1)
Summary
The Plasmodium genus includes over a hundred species that infect vertebrate hosts including birds, rodents, reptiles, amphibians and simians, by dipteran vectors [1]. Four Plasmodium species, P. falciparum, P. vivax, P. ovale and P. malariae are infective and transmissible to humans by natural mosquito bites. In 2004, Singh et al [5] detected P. knowlesi infections in a human population in Malaysia and subsequently P. knowlesi has been included as the fifth human infecting species [6]. It remains unclear if natural transmission between humans is common and the species is still considered a zoonosis [7,8,9].
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