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. 2013 Jan 8;110(2):531-6.
doi: 10.1073/pnas.1216457110. Epub 2012 Dec 24.

Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

Affiliations

Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

Robert W Moon et al. Proc Natl Acad Sci U S A. .

Abstract

Research into the aetiological agent of the most widespread form of severe malaria, Plasmodium falciparum, has benefitted enormously from the ability to culture and genetically manipulate blood-stage forms of the parasite in vitro. However, most malaria outside Africa is caused by a distinct Plasmodium species, Plasmodium vivax, and it has become increasingly apparent that zoonotic infection by the closely related simian parasite Plasmodium knowlesi is a frequent cause of life-threatening malaria in regions of southeast Asia. Neither of these important malarial species can be cultured in human cells in vitro, requiring access to primates with the associated ethical and practical constraints. We report the successful adaptation of P. knowlesi to continuous culture in human erythrocytes. Human-adapted P. knowlesi clones maintain their capacity to replicate in monkey erythrocytes and can be genetically modified with unprecedented efficiency, providing an important and unique model for studying conserved aspects of malarial biology as well as species-specific features of an emerging pathogen.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Adaptation of the P. knowlesi A1 strain to continuous culture in human erythrocytes. (A) Strategy used to adapt the parasites to growth exclusively in human RBC. (B) Analysis of the A1-H(E) line to detect the first signs of stable growth in human RBC. Thin films were probed with an anti-RBC ghost antibody (green), which labels both human cells and cynomolgus cells, plus a monoclonal anti-human Band 3 antibody (red), which labels only human cells. Parasitized human and cynomolgus RBC, identified by staining parasite nuclei with DAPI, are indicated with a single and double white arrow, respectively. (Scale bar, 10 μm.) (C) Adaptation primarily involves improvements in invasion of human RBC. Purified schizonts of the nonadapted A1-C.1 clone (Top) or adapted A1-H.1 clone (Middle) were added to cynomolgus or human RBC to obtain an initial parasitaemia of 1%. Parasitaemia values were determined immediately after schizont rupture was complete and new ring stages had formed (4.5 h), and again at 24 h when the new generation of parasites had reached schizont stage. Note that no residual schizonts were present in any of the cultures by 10 h after initiation of the experiments, demonstrating efficient egress in all cases. Error bars, ± 1 SEM. Giemsa stained images (Bottom) show representative parasite stages from 0-, 4.5-, and 24-h time points. (Scale bar, 2 μm.)
Fig. 2.
Fig. 2.
In vitro growth of human-adapted P. knowlesi is Duffy-dependent. Purified P. knowlesi A1-H.1 clone schizonts were added in triplicate to washed RBC from 33 volunteer blood donors to attain a parasitaemia of ∼1%. Parasitaemia values at were then monitored by FACS over the ensuing two intraerythrocytic growth cycles (48 h), and mean average of fold growth rates plotted against (A) ABO blood group, or (B) Duffy phenotype [Fya+, Fyb+, Fy(a+b+), Fy(a−b−)]. Black bars indicate mean growth rate in each blood type, and an asterisk denotes Duffy-positive blood groups supporting growth rates significantly different from those in Duffy-negative blood groups (two-tailed t test, P value ≤ 0.001). Growth was highly dependent on Duffy positivity, but showed no dependence on ABO blood type. Note that the four blood samples in A that show low growth rates are the Duffy-negative [Fy(a−b−)] samples.
Fig. 3.
Fig. 3.
High efficiency transfection of human-adapted P. knowlesi. Purified mature P. knowlesi clone A1-H.1 schizonts (∼1 ×ばつ 108 per cuvette) were electroporated in triplicate with either PkconGFPep or no DNA (mock transfection). The transfected parasites were supplemented with 150 μL fresh human RBC, returned to culture, and monitored daily. (A) Time-dependent change in total parasitaemia and total proportion of GPF+ parasites determined by fluorescence microscopy. Arrow, point of addition of the selection drug WR99210 (2.5 nM). Over 50% of the PkconGFPep-transfected parasites were visibly GFP+ by day 2, increasing to ∼100% by day 4. Error bars denote ± 1 SEM. (B) Light microscopic images of live PkconGFPep-transfected parasites taken on the indicated days post transfection, stained with Hoechst 33342 (merge of GFP, Hoechst, and brightfield views). GFP+ and GFP− parasites are indicated (with green and black arrowheads, respectively). (C) Giemsa-stained thin films of the same cultures taken on the same days posttransfection as in B. (Scale bar, 10 μm.)
Fig. 4.
Fig. 4.
Rapid genomic integration of linear DNA constructs in human-adapted P. knowlesi. P. knowlesi clone A1-H.1 schizonts were transfected in triplicate with either linearized PkconGFPp230p or no DNA (mock transfection) and monitored daily. (A) Schematic of introduction of linearized PkconGFPp230p into the P. knowlesi genome. The construct, containing both eGFP and hdhfr expression cassettes, is predicted to integrate into the Pkp230p locus via single cross-over homologous recombination with the targeting sequence (hatched). Locations of primers used for PCR analysis are marked with thick black arrows. (B) Time-dependent change in total parasitaemia and total proportion of GFP+ parasites following transfection. The red arrow indicates the point of addition of the selection drug WR99210, and the black arrows and numbers above indicate points at which cultures were diluted, and the fold-dilution. Over 50% of the PkconGFPp230p-transfected parasites were GFP+ by day 2. Parasite replication rate was low for the first 10 d of culture (presumably because of selection of integrants) before increasing to normal rates. Error bars denote ± 1 SEM. (C) Diagnostic PCR analysis of three independent PkconGFPp230p transfected lines (T1–T3) on day 16 after transfection, as well a parasite clone derived by limiting dilution. (Top) Amplification of a band specific for the wild-type p230p locus (primers ol145 and ol146). (Middle) Amplification of a band expected only following correct integration of PkconGFPp230p into the Pkp230p locus (primers ol145 and ol144). (Bottom) Control reaction (with primers ol75 and ol76, specific for an irrelevant gene) expected to produce a product in all parasites. (D) Light microscopic image of the PkconGFPp230p clone showing GFP expression in a segmented schizont, stained with Hoechst 33342. The fluorescent images were produced from a deconvoluted z-stack and displayed as extended focus images. (Scale bar, 2 μm.)

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