Pf155/RESA protein influences the dynamic microcirculatory behavior of ring-stage Plasmodium falciparum infected red blood cells

Abstract

Proteins exported by Plasmodium falciparum to the red blood cell (RBC) membrane modify the structural properties of the parasitized RBC (Pf-RBC). Although quasi-static single cell assays show reduced ring-stage Pf-RBCs deformability, the parameters influencing their microcirculatory behavior remain unexplored. Here, we study the dynamic properties of ring-stage Pf-RBCs and the role of the parasite protein Pf155/Ring-Infected Erythrocyte Surface Antigen (RESA). Diffraction phase microscopy revealed RESA-driven decreased Pf-RBCs membrane fluctuations. Microfluidic experiments showed a RESA-dependent reduction in the Pf-RBCs transit velocity, which was potentiated at febrile temperature. In a microspheres filtration system, incubation at febrile temperature impaired traversal of RESA-expressing Pf-RBCs. These results show that RESA influences ring-stage Pf-RBCs microcirculation, an effect that is fever-enhanced. This is the first identification of a parasite factor influencing the dynamic circulation of young asexual Pf-RBCs in physiologically relevant conditions, offering novel possibilities for interventions to reduce parasite survival and pathogenesis in its human host.

Figure 1. Membrane dynamics of ring-stage Pf-RBCs at physiological body temperature.

(A) RMS displacements, and (B) in-plane shear modulus values of parasite-free RBC, wild-type resa1+ and resa1-KO ring-stage Pf-RBCs. Open circles are experimental values and represent individual cell measurements. Significant differences are shown as * (p < 10⁻⁴) and * (p < 10⁻⁵) values.

Figure 2. Membrane fluctuations and in-plane shear modulus of ring-stage Pf-RBCs at body and febrile temperature.

RMS displacement histogram and in-plane shear modulus values (upper and lower panel, respectively) of (A), (D). parasite-free RBCs, (B), (E). resa1+, and (C), (F). resa1-KO ring-stage Pf-RBCs, measured at 37°C and 41°C. Open circles represent individual RBC measurements. Closed circles are shear modulus results obtained with optical tweezers and the same set of parasites shown for comparaison purposes. Significant differences are shown as * (p < 0.02) values.

Figure 3. Differences in the dynamic response of ring-stage Pf-RBCs expressing or not RESA.

Real-time snap-shots transit cell velocity of a representative example of all the experiments performed of (A) resa1+, and (B) resa1-KO ring-stage Pf-RBCs, forced to traverse through 3 μm successive constrictions in micro-sized channels at a constant pressure gradient of 0.24 Pa μm⁻¹. Pf-RBCs are labelled fluorescently using Thiazole orange. Yellow arrows indicate individual parasite-free RBC. (C). Illustrative example of all the transit cell velocity measurements performed. Each open circle represents the spatial position (μm) as a function of time (s) of individual resa1+ (red) and resa1-KO (green) Pf-RBCs compared to parasite-free RBCs (grey) calculated from Figure 3A and B.

Figure 4. Transit cell velocity of ring-stage Pf-RBCs at body temperature.

Transit cell velocities of (A) resa1+, (B) resa1-KO and (C) resa1-rev ring-stage Pf-RBCs compared to corresponding co-cultured parasite-free RBCs, passing through 3 μm constrictions in micro-sized channels. Measurements were performed at 37°C at a constant pressure gradient of 0.24 Pa μm⁻¹. Each open circle represents an individual cell measurement. Significant differences are shown as * (p < 10⁻⁹) values.

Figure 5. Transit cell velocity of ring-stage Pf-RBCs at body and febrile temperature.

Transit cell velocities of parasite-free RBCs (black), resa1+ (red), resa1-KO (blue) and resa1-rev (green) ring-stage Pf-RBCs, measured at 37°C and 41°C. Measurements were performed at a constant pressure gradient of 0.24 Pa μm⁻¹. Each open circle represents an individual cell measurement. Significant differences are shown as * (p < 0.01) values.

Figure 6. Incubation at febrile temperature increases retention rate by microspheres of RESA expressing Pf-RBCs (resa1-rev) and not of resa1-KO ring-stage Pf-RBCs.

(A) Synchronized cultures (15–18 h rings) of resa1-rev (red) and resa1-KO (blue) Pf-RBCs adjusted to 2% hematocrit and 10% parasitemia, were incubated at either at 37°C or 40°C for 3 h. Error bars represent the standard deviation. (B) Highly synchronized parasite cultures (12–15 h, 15–18 h, 18–21 h and 21–24 h after invasion) of resa1-rev (red) and resa1-KO (blue) Pf-RBCs adjusted to 2% hematocrit and 5% parasitemia, were incubated at 40°C for 2 h. Error bars represent the standard deviation. The p values for resa1-rev/resa1-KO are 0.07 for 12–15 h; 0.00066 for 15–18 h; 0.397 for 18–21 h, and 0.641 for 21–24 h.