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

Roberto R Moraes Barros,Theresa Engels,Lynn Lambert,Tyler J Gibson,Jacqueline K Brockhurst,John H Adams,Jennifer S Armistead,Sachy Orr-Gonzalez,Miho Kaneko,Thomas E Wellems,Juliana M Sá,J Patrick Mershon,Whitney A Kite,Kittisak Thawnashom,Ramoncito L Caleon,Osamu Kaneko

doi:10.1186/s12936-021-03773-4

Abstract

BackgroundPlasmodium 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.MethodsIn 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.ResultsLuciferase 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.ConclusionAll 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.

Highlights

Plasmodium knowlesi is the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species
Plasmodium knowlesi parasites were cultivated in rhesus red blood cells as described [10]; blood samples were obtained according to the National Institutes of Health (NIH) Guidelines for Animal Care and Use, under an Animal Study Proposal (ASP) approved by the National Institute of Allergy and Infectious Diseases (NIAID) Animal Care and Use Committee (ACUC); rhesus blood was processed and plasma was removed as described [10]
Plasmodium vivax 5′ untranslated region (UTR) are broadly active in P. knowlesi but limited in P. falciparum To evaluate the cross-species activity of different Plasmodium gene regulatory sequences, a variety of plasmids were constructed for expression of firefly luciferase under control of P. vivax or P. falciparum 5′ UTR sequences

Summary

Introduction

Plasmodium knowlesi is 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. As with P. falciparum, transfection methods and the ability to infect mosquitoes with cultivated P. knowlesi parasites followed [8,9,10,11], and these are supporting new experimental studies without the need for resource-intensive and costly non-human primate (NHP) models. Fundamental biological differences between the Plasmodium species and factors affecting malaria control that are more accessible to investigation include: the period of the intraerythrocytic life cycle (24 h for P. knowlesi vs. 48 h for P. falciparum) [12, 13]; features of host cell preference and cell invasion mechanisms [14,15,16]; gametocyte development and transmission to mosquitoes [17]; and anti-malarial drug responses and mechanisms of resistance [18, 19]

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Discussion

Conclusion