Malian field isolates provide insight into Plasmodium malariae intra-erythrocytic development and invasion

Abstract

Plasmodium malariae is the third most prevalent human malaria parasite species and contributes significantly to morbidity. Nevertheless, our comprehension of this parasite's biology remains limited, primarily due to its frequent co-infections with other species and the lack of a continuous in vitro culture system. To effectively combat and eliminate this overlooked parasite, it is imperative to acquire a better understanding of this species. In this study, we embarked on an investigation of P. malariae, including exploring its clinical disease characteristics, molecular aspects of red blood cell (RBC) invasion, and host-cell preferences. We conducted our research using parasites collected from infected individuals in Mali. Our findings revealed anaemia in most of P. malariae infected participants presented, in both symptomatic and asymptomatic cases. Regarding RBC invasion, quantified by an adapted flow cytometry based method, our study indicated that none of the seven antibodies tested, against receptors known for their role in P. falciparum invasion, had any impact on the ability of P. malariae to penetrate the host cells. However, when RBCs were pre-treated with various enzymes (neuraminidase, trypsin, and chymotrypsin), we observed a significant reduction in P. malariae invasion, albeit not a complete blockade. Furthermore, in a subset of P. malariae samples, we observed the parasite's capability to invade reticulocytes. These results suggest that P. malariae employs alternative pathways to enter RBCs of different maturities, which may differ from those used by P. falciparum.

Fig 1. Characteristics of circulating parasites in P. malariae natural infections.

A) Percentage of intraerythrocytic parasite stages in circulation and representative pictures of each (Rings, Trophozoites, Schizonts) (n = 23). Error bars show SD; B) Merozoites per schizont (n = 23, each point represents the average of merozoites seen in all schizonts in a given sample); C) Number of gametocytes per 1000 WBCs per sample (n = 23); D) Transmission electron microscopy image of a schizont. Black arrows indicating knobs-like structures. Black, white and red scale bar = 4, 1 and 0.2 µm, respectively.

Fig 2. P. malariae replicates and matures in vitro.

A) Percentage of intraerythrocytic parasite stages in vitro (from 23 samples, n = 121 exploitable smears in total for all time points. n = 23 at 0 h, n = 19 at 12 h, n = 21 at 24 h, n = 17 at 48 h, n = 14 at 60 h, n = 12 at 72 h and n = 15 at 96 h). Error bars show SD; B) Representative flow cytometry layout of parasite development within RBCs stained with SybrGreen I (SG) and Mitotracker (MT). 8 samples were timely taken every 12 hours (Ctrl: stained non-infected RBCs, NV: Non-viable; R: Rings; T: Trophozoites; S: Schizonts). The sample displayed is a P. malariae monoinfection confirmed by qPCR; C) Number of merozoites per schizont (n = 55 schizonts counted from 11 different samples in which schizonts were found).

Fig 3. P. malariae field isolates invade RBCs in vitro.

A) Experimental work plan: magnetic separation of pRBCs with early stages (ES) or late stages (LS), incubation with stained RBCs (sRBCs) and diagram of flow cytometry plots to measure new RBC invasions; B) Representative flow cytometry layout of parasite development within RBCs stained with SybrGreen I (SG) and Cell Trace Far Red (CTFR); C) Quantification of new invasions after 24 h and 48 h of in vitro culture (n = 9). Lines join the same sample measured at the different time points. Each dot represents the mean of the results in duplicates.

Fig 4. Blocking agents show a partial effect on P. malariae RBC invasion.

A). Effect of antibodies in P. malariae invasion. Heparin (Hep, n = 10) was used as an inhibitory positive control. All data was normalized with a control (Ctrl) in which no antibody was used. CD35 (CR1, n = 4), CD55 (n = 4), CD71 (TR1, n = 4), CD108 (Sema7A, n = 5), CD147 (Basigin, n = 4), CD233 (Band3, n = 4), CD235a (GYPA, n = 5). B). Effect of enzymatically treated RBCs with Chymotrypsin (ChT), Trypsin (T); and Neuraminidase (NA) in P. malariae invasion (n = 6). Each dot represents the mean of the results in duplicates.

Fig 5. P. malariae is capable to invade reticulocytes.

A) Experimental workflow diagram; B) Representative flow cytometry plots with the two combinations tested (anti-CD71APC+SG; anti-CD71PE+MT). Ctrl: non-infected RBCs. EEF482: P. malariae monoinfection confirmed by qPCR; C) Percentage of parasitized reticulocytes (CD71+ cells). In red, same sample (EEF482) displaying successful reticulocyte invasions with both approaches (n = 8).

Fig 6. Model of hypothetical P. malariae pathways to entry into RBCs.

Potential binding partners based on database search and current knowledge in other Plasmodium spp. The enzymatic phenotypic profile of each receptor is indicated (R: resistant to pre-treatment; S: sensitive to RBC pre-treatment;? : unknown). The potential effect of blocking agents (i.e. heparin, antibodies) is also displayed. Proteins for which specific antibodies has been tested in this study are highlighted in a box. Other proteins that might have been affected by enzymatic treatment, but for which no antibody was tested, and that might play a role in P. malariae-RBC interactions are shown unboxed.