Under conditions of high wall shear stress, several PfEBA and PfRH ligands are important for malaria Plasmodium falciparum blood-stage growth

Abstract

Malaria kills over 600,000 people annually, with all clinical symptoms arising from blood-stage infection. Plasmodium falciparum blood-stage replication happens primarily in the blood circulation, bone marrow, and spleen, where there are flow-generated forces, yet most in vitro growth assays are carried out in static conditions. We systematically tested the effect of orbital shaking on growth and linked it to the wall shear stress forces generated by the resultant fluid motion. Strikingly, there is a critical shaking speed, below which growth rates are reduced and above which growth increases. Forces at this critical speed correspond to previously measured forces in the microvasculature. Red blood cell invasion depends on two families of parasite attachment proteins, the Plasmodium falciparum erythrocyte-binding antigen and the Plasmodium falciparum reticulocyte-binding protein. Using a panel of knockouts, we show for the first time that several of these ligands have greater importance in high wall shear stress conditions, highlighting the importance of understanding the effect of fluid motion on parasite biology.IMPORTANCEMalaria parasite growth occurs in dynamic environments like blood circulation, where fluid forces impact red blood cells and parasites. Yet, most laboratory growth assays are conducted in static environments, failing to replicate these forces. We explored the effects of growing the parasites on orbital shakers, which generate biologically relevant forces, and found that shaking speed critically impacts parasite growth, with reduced growth at speeds that mimic forces in the microvasculature. Importantly, using these conditions revealed invasion phenotypes not observed under static conditions. Understanding how fluid dynamics influence parasite growth offers a new approach to investigating malaria pathogenesis, with the potential to improve the development of therapeutic interventions.

Keywords: PfEBA; PfRH; Plasmodium falciparum; bloodstream infections; growth assay; host cell invasion; malaria; orbital shaker.

Fig 1

Shaking speed affects the growth rate of wild-type P. falciparum lines NF54, 3D7, and Dd2. All data were collected for samples cultured in a six-well plate, 5 mL culture volume, and a 4% HCT. (a) A schematic to represent visually what the motion of the blood looks like at the conditions for which growth was compared. In static conditions, the blood is sedimented at the base of the well; at 45 rpm, the fluid just starts to move over the sedimented blood; 90 rpm is just above the critical clustering speed, and the RBCs are largely confined to the vortex. At 180 rpm the conditions are highly turbulent, and the RBCs are completely mixed through the medium. The simulated mean WSS is shown for the conditions where the blood is still sedimented. (b) The growth rate is shown as the parasite erythrocyte multiplication rate (PEMR). The data displayed are from all experiments run under the indicated conditions across multiple assays. The lines between conditions indicate the significantly different conditions (t-test) at a greater than 5% significance level. (c) Graphs represent the mean frequency of different numbers of parasites in a single RBC for a given speed for each wild-type line. The frequency of single-infected, double-infected, triple-infected, quadruple-infected, and higher-infected rings is shown as a fraction of the total infected ring-stage parasites. The number of rings per erythrocyte was measured using the peak intensity of the ring-stage infected samples and the number of counts in each peak. Data sets without clearly defined peaks were excluded, which is why there are no data for Dd2 at 180 rpm.

Fig 2

The type of culture container and hematocrit affects the growth rate of wild-type lines of P. falciparum. (a) Schematics illustrate the different cultural conditions that were compared. (b) The wild-type lines NF54, 3D7, and Dd2 were tested. All cultures were kept at a 4% HCT. The mean growth rate is shown as the parasite erythrocyte multiplication rate (PEMR). The experiment was performed in triplicate technical repeats; each point shows a PEMR measured for a single invasion cycle for an individual well. The lines between conditions indicate the significantly different conditions (t-test or rank sum) at a greater than 5% level of significance. (c) Growth rates from panel b for the round wells plotted against the normalized shaking speed. The shaking speed was normalized to the rotation speed at which the blood was first seen to cluster at the center of the well (Fig. S2a). (d) Schematics illustrate the different hematocrit conditions that were compared. (e) All cultures were kept in a 5 mL culture volume in a six-well plate. All data presented in the figure were collected in parallel. The mean growth rate is shown as the PEMR; each point shows a PEMR measured for a single invasion cycle for an individual well. The lines between conditions indicate the conditions that were significantly different (t-test) at a greater than 5% level of significance.

Fig 3

Knocking out specific invasion ligands shows flow-dependent effects. Experiments were carried out to compare PfEBA and PfRH knockout lines constructed in the NF54 background growth rates under static and detrimental shaking conditions at 90 rpm; two clones of each line were tested wherever possible. All cultures were grown at 4% HCT in 5 mL in a six-well plate. The top box plot shows the mean parasite erythrocyte multiplication rate (PEMR) of the knockout lines. The central red line shows the median, with the top and bottom of the box at the 25th and 75th percentiles and the whiskers showing the total range of the data. The bottom bar plot shows the relative difference in growth in shaking compared to static for each line. The error bars show the standard deviation. The lines between conditions indicate the significantly different conditions (t-test or rank sum) at a greater than 5% level of significance.

Fig 4

Evaluating if changes in growth rate are due to changes in invasion rate. All cultures were kept at a 4% HCT in a 5 mL culture in a six-well plate. All data presented in the figure were collected in parallel. Wild-type lines 3D7 and NF54 were tested along with ∆PfEBA175c2 and ∆PfRH4c1, which are in the NF54 background. Measurements were taken before and after a 3.5 h incubation window. The percentage of rings and schizonts was measured for each well before and after the invasion window. The change in newly invaded RBC rings was calculated by subtracting the percentage of rings present before incubation from the percentage of rings measured after invasion. The change in schizonts was calculated by subtracting the percentage of schizonts present after incubation from the percentage of rings measured before invasion. The invasion rate was calculated by dividing the change in the rings by the change in schizonts. Anything with an invasion rate of about 35 was excluded as an outlier, as there is a maximum of 32 merozoites per schizont. (a) Box plot comparing the invasion rates measured for the different lines across the shaking speed; each point is a single invasion cycle for an individual well. The lines between conditions indicate the conditions that were significantly different (t-test) at a greater than 5% level of significance. (b) The change in schizonts, change in rings, and invasion rate were compared to the previous growth rate measurements. All measurements were normalized to the static measurement to allow comparison. No data were collected for the growth rate of the knockout lines at 45 or 190 rpm. (c) Scatter plot comparing the normalized growth rates to the normalized change in schizonts, change in rings, and invasion rate.

Fig 5

Summary of effects of shaking on P. falciparum growth. (a) Cartoon of the relation we have shown between increased orbital rotation speed and growth in round wells. Alongside are the observed changes in fluid motion. (b) Summary of the factors determining fluid motion when a round well is on an orbital shaker.