Activity of Plasmodium vivax promoter elements in Plasmodium knowlesi, and a centromere-containing plasmid that expresses NanoLuc throughout the parasite life cycle

Abstract

Background: Plasmodium knowlesi is now the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species. Recent advances in the cultivation of P. knowlesi erythrocytic-stage parasites in vitro, transformation with exogenous DNA, and infection of mosquitoes with gametocytes from culture have opened up studies of this pathogen without the need for resource-intensive and costly non-human primate (NHP) models. For further understanding and development of methods for parasite transformation in malaria research, this study examined the activity of various trans-species transcriptional control sequences and the influence of Plasmodium vivax centromeric (pvcen) repeats in plasmid-transfected P. knowlesi parasites.

Methods: In vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated Plasmodium vivax or Plasmodium falciparum 5' UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey.

Results: Luciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, anti-malarial half-inhibitory concentration (IC₅₀) values of blood stages measured by NanoLuc activity proved comparable to IC₅₀ values measured by the standard SYBR Green method.

Conclusion: All three P. vivax promoters tested in this study functioned in P. knowlesi, whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new anti-malarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.

Keywords: Antimalarial drug response assays; Genetic transformation; Heterologous transfection; In vitro growth assays; Luciferase expression; Transgenic parasites.

Fig. 1

Plasmid constructs used to evaluate Plasmodium promoter sequences in P. knowlesi. Expression of hdhfr from the plasmids confers resistance to WR99210 and a fLuc or NanoLuc reporter. The pD-pfcam-Luc plasmid has the fLuc cassette driven by the P. falciparum calmodulin 5′ UTR (pfcam 5′) and the hdhfr cassette driven by the P. chabaudi dts 5′ UTR (pcdts 5′). pD-pvcam-Luc, pD-pvcrt-Luc and pD-pvhsp70-Luc have the 5′ UTR sequences of P. vivax calmodulin (pvcam 5′), chloroquine resistance transporter (pvcrt 5′) and heat shock protein 70 (pvhsp70 5′) driving fLuc expression, respectively, replacing the pfcam promoter from the pD-pfcam-Luc. Plasmids pvhsp70D-pfcam-Luc and pkef1D-pfcam-Luc have the 5′ UTR sequences of P. vivax heat shock protein 70 (pvhsp70 5′) and P. knowlesi elongation factor 1 α (pkef1-α 5′), respectively, replacing the pcdts promoter from the pD-pfcam-Luc to drive the expression of hdhfr. The pvcen-pvhsp70-D-NanoLuc plasmid includes P. vivax centromeric sequence repeats from chromosome 11 (Pv centromeric repeats) and has hdhfr-NanoLuc fusion expression driven by the P. vivax heat shock protein 70 5′ UTR (pvhsp70 5′). The arrows indicate directions of transcription

Fig. 2

Plasmodium knowlesi recognizes P. vivax promoter regions not recognized by P. falciparum. Plasmodium falciparum and P. knowlesi parasites were transformed in vitro by spontaneous DNA uptake from RBCs pre-loaded with plasmids pD-pfcam-Luc, pD-pvcam-Luc, pD-pvcrt-Luc, or pD-pvhsp70-Luc. Luciferase activity measurements were obtained after 72 h of parasite cultivation in the plasmid-loaded RBCs and were normalized relative to the activity obtained from parasites transformed with pD-pfcam-Luc. Values represent the mean ± standard error from three independent experiments

Fig. 3

Selection of drug-resistant P. knowlesi harbouring markers under the control of P. vivax regulatory sequences. Drug selection with 1 nM WR99210 was initiated 72 h after addition of plasmid-loaded RBCs to parasite cultures. A Parasitaemia counts by microscopy of cultures transfected with plasmids pvhsp70D-pfcam-Luc and pkef1D-pfcam-Luc over 16 days of drug selection. B Luminescence measurements from samples of the transformant cultures presented in A. C Parasitaemia counts by microscopy of cultures transfected with plasmid pvcen-pvhsp70-D-NanoLuc in three independent experiments. D Luminescence measurements from samples of the transformant cultures presented in C. LUs, luminescence units; mock, control transformation experiments performed in parallel without plasmid; parasitaemia, percentage of RBCs infected with P. knowlesi parasites (counts of 1000 RBCs)

Fig. 4

Bioluminescent P. knowlesi transformants complete the parasite life cycle in vivo. A Development of blood stage parasitaemia in a splenectomized non-naïve rhesus macaque (rhesus #1) infected with pvcen-pvhsp70-D-NanoLuc-transformed P. knowlesi. The red arrows indicate when mosquito feedings were performed; blue arrows indicate administration of chloroquine (50 mg/kg, oral). B Counts of oocysts in the mosquito midgut 7 days after blood feeding. C NanoLuc activities from midguts isolated from Feed #2 infected mosquitoes. D NanoLuc activities from sporozoites isolated from mosquitoes 10 days after either Feed #1 or Feed #2 on rhesus macaque DCID. E Blood stage parasitaemia developed in a splenectomized non-naïve rhesus macaque (rhesus #2) after bites of mosquitoes carrying infectious sporozoites. The blue arrows indicate chloroquine treatments (50 mg/kg, oral). F Copy numbers of the NanoLuc coding sequence relative to those of the single-copy P. knowlesi aldolase gene in blood-stage parasites from in vitro cultures and from blood samples of rhesus monkeys #1 and #2. Copy number results from each sample are presented relative to the copy number of pvcen-pvhsp70-D-NanoLuc-transformed P. knowlesi parasites cultivated under WR99210 selection pressure (black): cultivated parasites maintained 14 days in vitro without drug pressure (gray); parasites from rhesus #1 (blue); parasites from rhesus #2 (red). Error bars represent standard error of the mean; LUs, Luminescence units