Gene knock-in as a tool to phenotype clinically relevant varient alleles for studies on malaria pathobiology: proof of concept using the plasmodium knowlesi normocyte binding protein Xa gene

Abstract

The zoonotic parasite, Plasmodium knowlesi, is one of five human malaria species. P. knowlesi is geographically restricted to locations of the long-tailed and pig-tailed macaque, indigenous across South-East Asia (SEA). Initial research showed that P. knowlesi was present in the human population in Malaysian Borneo, with subsequent studies confirming P. knowlesi throughout SEA. P. knowlesi cases were shown to be both severe (10%) and lethal (2%), with hyperparasitaemia correlating with severe malaria. Recent work has identified a polymorphism in the essential P. knowlesi normocyte binding protein Xa (Pknbpxa) gene that associates with high parasitaemia. The aims of this study were firstly to enhance and standardise the isolation of parasite DNA from a Biobank of frozen clinical isolates. Alongside this we aimed to take P. knowlesi whole genome sequence data and identify further mutations in the Pknbpxa gene linked to severe disease and express these in an in vitro P. knowlesi experimental line. Detailed here is the optimisation of the Whatman-Plasmodipur method to purify P. knowlesi DNA from a Biobank of frozen clinical samples. This resulted in 13/22 samples returning greater than 70% P. knowlesi DNA and within this, 8 samples were suitable for genome sequencing. Genome sequence data representing cluster type 2 was used to inform the synthesis and construction of a Pknbpxa synthetic gene representing clinical alleles. Single-crossover homologous recombination was used to replace the native Pknbpxa with this synthetic copy, containing polymorphisms associated with high parasitaemia. This was subsequently transfected into the P. knowlesi A.1-H.1 clone via nucleofection, resulting in an experimental line expressing clinically relevant mutations within the essential PkNBPXa invasion gene. The construction of this translational approach enables functional examination for mutation involvement in parasite erythrocyte invasion and contribution to severe disease.