Malaria parasites are unusual, early-diverging protozoans with non-canonical cell cycles.  They do not undergo binary fission, but divide primarily by schizogony.  This is a mode of replication involving asynchronous production of multiple nuclei within the same cytoplasm, culminating in a single mass cytokinesis event.  The rate and efficiency of parasite replication is fundamentally important to malarial disease, which tends to be severe in hosts with high parasite loads.  Here, we have studied for the first time the dynamics of schizogony in two human malaria parasite species, Plasmodium falciparum and Plasmodium knowlesi.  These differ in their cell-cycle length, the number of progeny produced and the genome composition, among other factors.  Comparing them could therefore yield new information about the parameters and limitations of schizogony.  We report that the dynamics of schizogony differ significantly between these two species, most strikingly in the gap phases between successive nuclear replications, which are longer in P. falciparum and shorter, but more heterogenous, in P. knowlesi.  In both species, gaps become longer as schizogony progresses, whereas each period of active replication grows shorter.  In both species there is also extreme variability between individual cells, with some schizonts producing many more nuclei than others, and some individual nuclei arresting their replication for many hours while adjacent nuclei continue to replicate.  The efficiency of schizogony is probably influenced by a complex set of factors in both the parasite and its host cell.

Parasite culture and transfection for ectopic expression of thymidine kinase

P. falciparum parasites were maintained in vitro in human O+ erythrocytes at 4% haematocrit in RPMI 1640 medium supplemented with 25mM HEPES (Sigma-Aldrich), 0.25% sodium bicarbonate, 50 mg/L hypoxanthine standard procedures [31].  P. knowlesi parasites were maintained similarly, maintained at 2% haematocrit instead of 4% and supplemented with 22.2 mM glucose and 10 % horse serum instead of human serum.

The P. falciparum 3D7 strain that expresses thymidine kinase has been previously described [10].   A similar thymidine-kinase-expressing P. knowlesi strain was created by transfecting the same plasmid into the A1-H.1 strain, essentially as previously described [32].  Late-stage P. knowlesi parasites were enriched using Histodenz and 10 µl of schizonts mixed in a transfection cuvette (Lonza) with 100 µl of P3 solution (Lonza) containing 30 µg of plasmid. Transfection was carried out using program FP158 (Amaxa Nucleofector, Lonza), followed by immediate transfer into 500 µl of complete culture media mixed with 190 µl uninfected erythrocytes. The transfection mix was incubated at 37°C while shaking at 800 rpm in a thermomixer for 30mins, before being transferred into a 6-well plate, gassed and incubated for one parasite life cycle. Selection was then applied with 100 nM pyrimethamine (Santa Cruz Biotechnology Inc) and daily media changes for 3 days, then routine maintenance until transgenic parasites appeared

Synchronisation for timecourse experiments

Mature schizont cultures at >6% parasitaemia were synchronised using 55% Nycodenz (Alere technologies AS) [33].  Cultures were centrifuged and media removed to leave 2ml of media and blood, which was layered gently on top of 5 mL of prewarmed 55% Nycodenz, then centrifuged at 1300g for 5 mins. The floating schizont layer was collected and added to a wash buffer containing incomplete RPMI, 4% haematocrit for the final culture volume, and 1.5 mM ‘Compound 2’ (4-[7-[(dimethylamino)methyl]-2-(4-fluorophenyl)imidazo[1,2-a]pyridine-3-yl]pyrimidin-2-amine) [34]), centrifuged (800g for 5 mins), resuspended in complete RPMI and 1.5 mM Compound 2 and incubated at 37°C for 2 h. Cultures were centrifuged (800xg for 5 mins) and supernatant removed. Cultures were washed in prewarmed incomplete media and resuspended in complete RPMI to allow reinvasion. Cultures were split into 5ml aliquots in 50ml falcon tubes and placed in an orbital shaker at 37°C for 1 h to increase reinvasion rate. After reinvasion, the cultures were pooled together, centrifuged and Nycodenz treated again, now retaining the bottom layer containing newly reinvaded ring stages. This layer was washed in incomplete media and resuspended in compete RPMI, marking timepoint 0 hours post invasion (hpi).

Pulse labelling with modified nucleotides

Cultures for single-labelled timecourses were pulse-labelled with 10 mM ethyl-deoxyuridine (EdU) for 30 mins at 1 h intervals. Cultures for double-labelling experiments were first pulse-labelled with 10 mM EdU for 30 mins at specific timepoints, then washed twice in prewarmed incomplete RPMI and resuspended in complete media that was incubated alongside the cultures throughout the experiments, to avoid any disturbance of cell cycle dynamics caused by switching into fresh media.  At specific timepoints, cultures were labelled with a second nucleotide, 5-bromo-2’-deoxyuridine (BrdU), at 200 mM for 30 mins at 37°C. Immediately after this label, blood smears were made, air dried, fixed in 2% paraformaldehyde for 5 mins, washed in PBS, washed in dH20, air dried and stored at 4°C.

Immunofluorescence

All slides were incubated in 0.2% Triton X-100 for 15 mins and washed in PBS for 5 mins.

Single-nucleotide EdU-labelled slides were incubated in blocking solution (1% BSA in PBS) for 30 mins and then 1:100 anti-centrin antibody (clone 20H5, Millipore) in blocking solution for 1 h at room temperature. Slides were washed three times in blocking solution. EdU signal was detected with click chemistry. Slides were incubated in click reaction buffer (0.845mM Tris HCl pH 8.8, 1mM CuSO4, 2.5 mM Alexa Fluorescent Azide 594, freshly dissolved 75mM ascorbic acid) for 1 h at room temperature. Slides were washed in blocking solution three times and then incubated with the secondary antibody for centrin detection, goat anti-mouse Alexa 488 (Molecular Probes) for 1 h at room temperature. Slides were washed twice in PBS, incubated with 2 mg/ml 4′,6-diamidino-2-phenylindole (DAPI) for 10 mins, washed in PBS, mounted using 20 ml Prolong Diamond Antifade (Molecular Probes) and set overnight at room temperature.

Double-labelled EdU/BrdU slides were incubated with 0.2% Triton-X for 15 mins, washed in PBS for 5 mins, incubated in 1M HCl for 1 h and washed in PBS for 5 mins.  EdU labels were “clicked” as above.  Slides were washed with PBS three times. Slides were then incubated with 20 mM non-fluorescent dye Azidomethylphenosulphide (Sigma) for 30 mins at room temperature to block remaining EdU residues. Slides were incubated with blocking solution for 30 mins. Primary immuno-detection of BrdU was with rat anti BrdU BU1/75 (ICR1) antibody (1:100 dilution, Abcam).  Slides were washed in blocking solution three times and incubated with goat anti-rat Alexa 488 secondary antibody (1:500 dilution, Molecular probes) at room temperature for 1 h; washed, incubated with DAPI and mounted as above.

Data analysis and statistics

100 parasites were counted for each timepoint of the single-nucleotide-labelled timecourses, and 50 parasites were counted for double-labelled timecourses.  For the ‘stalling’ experiment, 20 parasites were counted for each category of schizont maturity.  Images were classified regarding presence and number of centrin foci and the presence and pattern of nucleotide labelling (BrdU/EdU) within the nucleus.  Data were plotted using Graphpad Prism and the statistical significance of differences between groups of data was calculated via Mann Whitney tests or analysis of variance.

DNA fibre spreading

DNA fibre spreading was performed as previously described [22].  Cultures were labelled with 10mM EdU for 10 mins, then 100 mM BrdU for 10 mins. 2ml of saponin-released parasites was pipetted near the top of a glass slide and allowed to dry for ~5 mins, until sticky but not dry. 7 ml of spreading buffer (20 mM TrisHCl pH 7.4, 50 mM EDTA pH 8, 0.5% SDS) was added and stirred gently with a pipette tip to release the DNA. The slide was incubated for 2 mins and then tilted (15°) to let drop run slowly down the slide, producing a constant stretching factor of 2.59kb/mM [23]. Slides were air dried, fixed in MeOH: acetic acid (3:1), air dried and stored at 4°C.

Detection of EdU and BrdU in DNA fibre spreads

Fibre spreads were denatured with 1M HCl for 75 mins, washed three times in PBS and blocked with blocking solution (1% BSA and 0.1% Tween 20 in PBS).  Immuno-detection of double labelled fibre spreads was done with EdU click chemistry and then antibodies diluted in blocking solution, each incubated with a coverslip on top in a humid chamber at room temperature for 1h. Slides were incubated with click reaction buffer as above, followed with three washes in blocking buffer. Slides were then incubated in blocking solution for 30 mins. Primary immuno-detection for BrdU was done with rat anti BrdU BU1/75 (ICR1) antibody (1:100 dilution, Abcam), together with mouse anti ssDNA (clone16-19) antibody (Millipore, 1:300 dilution). The secondary antibodies (Molecular Probes) were goat anti-rat coupled to Alexa 488 (1:500 dilution) and goat anti-mouse coupled to Alexa 405 (1:500 dilution). Slides were washed three times in PBS and mounted using 20ml Prolong Diamond Antifade (Molecular Probes), set overnight at room temperature. Single-labelled fibre spreads were detected above but without click chemistry.

Image Acquisition and Processing of DNA fibre data

Image acquisition was via a Nikon Microphot SA microscope equipped with a Qimaging Retiga R6 camera. Images were acquired with a 100X oil objective (Leica, 1.30 na) where 1 mm = 28.65 pixels, which  corresponds to 69.8 bp per pixel (DNA stretching factor 2kb/ mm for DNA combing) and 90.40bp per pixel (DNA stretching factor 2.59kb/mm for DNA spreading). Observation of long DNA fibres required the capture and assembly of adjacent fields. Replication tracts and fibre lengths were measured manually using ImageJ software. Statistical analysis and graphs of BrdU tract length and replication velocities were performed using GraphPad Prism.