Growth rates of Static and Suspended parasites in neuraminidase-treated erythrocytes

High multiplication rates, and thus high parasite densities, are virulence features of P. falciparum infections³⁶. Cultivation of P. falciparum parasites in suspended cultures is known to favour a higher rate of multiplication^37–39. Therefore, it was of interest to determine if the change in invasion phenotypes observed when Dd2 and W2mef were maintained in a shaking incubator was also associated with an increase in growth rate. Furthermore, we sought to establish that Suspended Dd2 and W2mef can thrive in Nm-treated erythrocytes, in a similar manner as Dd2NM²⁸. Thus, ring stage parasites from 12-week Static and Suspended cultures were incubated with Nm-treated erythrocytes for two complete erythrocytic parasite cycles, under Static and Suspended conditions, respectively. Parallel cultures with normal untreated erythrocytes were established as references. Generally, suspended parasites grew better than Static parasites in untreated erythrocytes (Fig. 2a). However, statistical significance was only achieved at the end of cycle 1 for Dd2 and W2mef (cycle 1: P=0.007 and P=0.045 for Dd2 and W2mef, respectively; cycle 2: P=0.1302 and P=0.0965 for Dd2 and W2mef, respectively). In 3D7 however, the growth rate in Suspended parasites was higher than in Static cultures at the end of both cycles (P=0.012 and 0.007, respectively). In the Nm-treated erythrocytes (Fig. 2b), Static Dd2 and W2mef seemed incapable of establishing successful infections, with multiplication rates of about 1.0. (PMR=1.01 and PMR=0.96 at the end of cycle 2 for Dd2 and W2mef, respectively). In contrast, Suspended Dd2 and W2mef successfully thrived and established efficient infections (PMR=8.31 and PMR=12.93 at the end of cycle 2 for Dd2 and W2mef, respectively), with growth rates well above those in Static cultures (P<0.006 and 0.002 for Dd2 and W2mef, respectively). Both Static and Suspended 3D7 grew normally in Nm-treated erythrocyte (PMR=12.10 and 12.89 for ST and SP, respectively, at the end of cycle 2), with no statistically significant differences between ST and SP (P=0.403). Our results clearly demonstrate that Suspended parasites are capable of establishing successful infections in SA-deficient erythrocytes, confirming that these parasites are SA-independent.

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Figure 2

Suspended, but not Static Dd2 and W2mef, establish successful infection in Nm-treated erythrocytes. Static (ST) and Suspended (SP) parasites were cultivated in normal (a) and Nm-treated (b) erythrocytes for 2 asexual cycles. Parasitemias were determined by flow cytometry as percentage of ring-infected erythrocytes at the end of each cycle. Parasite multiplication rates (PMR) were estimated by dividing the parasitemia at the end of each cycle by the starting parasitemia. Data are presented as mean±standard errors of 2 independent experiments. *P-value<0.05 at 95% CI; Student t-test.

Sensitivity of Static and Suspended parasites to neuraminidase, chymotrypsin and trypsin treatment of erythrocytes

To determine the relative contributions of the major receptors to the invasion of Suspended cultures, parasites were incubated with erythrocytes treated with Nm, chymotrypsin or trypsin, which are enzymes that selectively remove receptors from the cell surfaces. Trypsin usually cleaves glycophorins A and C, but not B, while chymotrypsin cleaves glycophorin B and band 3^40–42. CR1 is sensitive to both trypsin and chymotrypsin⁴⁰, while basigin is resistant to both enzymes^13,40. Static Dd2 and W2mef parasites were highly sensitive to Nm treatment, with invasion of enzyme-treated erythrocytes reduced to less than 10% relative to untreated erythrocytes (Fig. 3a). Consistent with the switch in phenotype, suspended parasite cultures of Dd2 and W2mef were resistant to Nm treatment (Fig. 3a), with invasion reduced to only 75% relative to untreated erythrocytes (Fig. 3a). In the 3D7 cultures, there were no differences in the invasion of Nm-treated erythrocytes by both Static and Suspended parasites (Fig. 3a). Of interest, invasion by Suspended W2mef and Dd2 parasites were significantly more sensitive to chymotrypsin treatment compared to their corresponding Static cultures, (P=0.004 for W2mef and P<0.001 for Dd2; Fig. 3b). Conversely, invasion by Suspended W2mef and Dd2 cultures were slightly more resistant to trypsin treatment compared to the respective Static parasites (P=0.046 for W2mef and P=0.034 for Dd2; Fig. 3c). Sensitivities of Suspended 3D7 cultures to chymotrypsin and trypsin were not significantly different from the corresponding Static cultures (P=0.288 for chymotrypsin and P=0.526 for trypsin; Fig. 3b and c).

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Figure 3

Differential sensitivities of Static and Suspended parasites to enzyme treatment of erythrocytes. Invasion efficiencies of Static (ST) and Suspended (SP) into erythrocytes treated with neuraminidase (250mU), chymotrypsin (1mg/ml) and trypsin (1mg/ml) were compared for P. falciparum Dd2, W2mef and 3D7 strains. Invasion rates were determined by flow cytometry as percentage of ring-infected erythrocytes after approximately 14hours’ incubation of schizonts with target erythrocytes, and expressed as invasion efficiency relative to invasion of untreated erythrocytes. Data are presented as mean±standard errors of three independent experiments performed in triplicates. *P-value<0.05 at 95% CI; Student t-test.

Sialic acid-independent invasion in switched Suspended parasites is CR1-dependent

The switch in Dd2 and W2mef when cultured on neuraminidase-treated erythrocytes is known to be associated with an increased expression of RH4^30,32, whose receptor is CR1^10–12. Therefore, we examined the effect of anti-CR1 antibodies on invasion efficiency of Static and Suspended cultures into untreated or Nm-treated erythrocytes. Anti-CR1 did not significantly inhibit invasion of any of the parasite strains into untreated erythrocytes compared to isotype control IgY antibodies (P>0.100 for all comparisons; Fig. 4a and b). Furthermore, since Static Dd2 and W2mef cultures invade Nm-treated erythrocytes poorly, anti-CR1 antibodies had no significant impact on invasion relative to control IgY (P>0.100 for all comparisons; Fig. 4a). However, for Suspended cultures, invasion of both Dd2 and W2mef parasites into Nm-treated erythrocytes was substantially abrogated by anti-CR1 antibodies compared to control IgY (P=0.004 and P=0.003 for Dd2 and W2mef, respectively; Fig. 4b). For 3D7 parasites, invasion into Nm-treated erythrocytes was significantly inhibited by anti-CR1 antibodies compared to IgY for both Static and Suspended cultures (P=0.001 and P<0.001 respectively; Fig. 4a and b).

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Figure 4

Phenotypic switching of Suspended cultures is highly dependent on complement receptor 1 (CR1). Anti-CR1 antibodies (12µg/mL) inhibited invasion of Suspended (SP) parasites into Nm-treated erythrocytes but not untreated erythrocytes. Invasion rates were determined by flow cytometry as percentage of ring-infected erythrocytes after approximately 14hours’ incubation and expressed as invasion efficiency relative to invasion into untreated erythrocytes. Data are presented as mean±standard errors of three independent experiments performed in triplicates. *P-value<0.05 at 95% CI compared to IgY control; student t-test.

To confirm that these observations were specific to CR1, similar experiments were performed with anti-basigin antibodies, and the results showed that anti-basigin antibodies abrogated invasion of both Static and Suspended Dd2 and W2mef parasites, and the inhibition was similar in both untreated and Nm-treated erythrocytes (Supplementary Figure ^S3). Taken together, these data suggest that SA-independent invasion in Suspended Dd2 and W2mef cultures is significantly dependent on CR1 receptor usage.

Enzyme treatment of erythrocyte and invasion assays

Enzymatic treatment of erythrocytes and invasion assays were done as previously described⁵, with few modifications. Briefly, O⁺ erythrocytes were treated with 250mU/mL of neuraminidase (Nm) from vibrio cholera (Sigma Aldrich, St. Louis, Missouri), 1mg/ml α-chymotrypsin (pretreated with 1-chloro-3-tosylamido-7-amino-2-heptanone, to remove trypsin activity) from bovine pancreas (Sigma Aldrich) or 1mg/ml trypsin from bovine pancreas (Sigma Aldrich). After incubation for 1hour at 37°C with gentle shaking, erythrocytes were washed with RPMI, and subsequently stained with (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (5(6)CFDA-SE; 20µM, Invitrogen), a cytoplasmic fluorescent stain, prior to assay plating to differentiate them (acceptor cells) from uninfected erythrocytes in parasite inoculum. Late stage parasites were mixed with acceptor cells in a 1:1 ratio at 2% hematocrit in 96-well titre plates and incubated overnight at 37°C in a mixed gas environment.

To determine the role of CR1 and basigin (CD147) as receptors for invasion phenotype switching, experiments were set up in the presence of chicken anti-CR1 antibodies or immunoglobulin Y (IgY) control (12µg/mL, Gallus Immunotech, Fergus, Canada), and mouse monoclonal anti-CD147 antibodies (clone MEM-M6/6; Abcam, USA) or control mouse monoclonal IgG1 (10μg/mL; Abcam, USA), respectively. All experiments were setup in triplicate in 96-well plates under static conditions and repeated at least once. Plates were incubated overnight (~14hours) at 37°C with mixed gas. The cultures were then stained with Hoechst 33342 (5µM, Sigma Aldrich) after incubation to differentiate parasitized erythrocytes from uninfected ones, and invasion levels determined by flow cytometry (LSR Fortessa X-20, BD) as previously described^5,47. The percentage of erythrocytes that were dual positive for CFDA and Hoechst 33342 was recorded as the invasion rate.

Erythrocyte surface expression of SA and CR1 was determined in Static and Suspended cultures by staining with SA-specific mouse anti-human glycophorin A (GPA; clone E4, 0.2mg/ml, Santa Cruz biotechnology, Inc.) and mouse anti-human CR1 (clone J3D3; 0.1mg/ml, Santa Cruz biotechnology, Inc.) monoclonal antibodies. Phycoerythrin (PE)-conjugated goat polyclonal anti-mouse antibodies (0.2mg/ml; ThermoFisher Scienctific) were used as secondary antibodies. Data were analyse with Flowjo software V10.

Determination of parasite growth rate in untreated and neuraminidase-treated erythrocytes

Parasite cultures were synchronized by treatment with 5% sorbitol to select for ring stages⁴⁸. For growth in untreated erythrocytes parasitemia levels were adjusted to approximately 1% with untreated erythrocytes. For growth in Nm-treated erythrocytes, parasitized erythrocytes were treated with neuraminidase (250mU) for 1hour at 37°C. This was done to prevent SA-dependent reinvasion of uninfected erythrocytes present in parasite inoculum. In addition, uninfected erythrocytes required to sub-culture parasites were also treated with neuraminidase. The parasitemia levels were subsequently adjusted to approximately 1% with the uninfected Nm-treated erythrocytes. All parasitemia levels were confirmed by flow cytometry. The parasites were then cultured for two complete asexual cycles (96hours). At the end of each cycle, parasitemia were determined by flow cytometry and growth rates calculated by dividing the parasitemia at the end of the cycle by the starting parasitemia.

We are grateful to Kathy Moch and David J. Haynes of the Walter Reed Army Institute of Research, Silver Spring MD, and Louis Miller and Lubin Jiang of the National Institutes of Health, Bethesda, MD for various contributions to this work. This work was supported by funds from a World Bank African Centres of Excellence grant (ACE02-WACCBIP: Awandare) and a DELTAS Africa grant (DEL-15-007: Awandare). Prince Nyarko and Reuben Ayivor were supported by WACCBIP-World Bank ACE Masters and PhD fellowships, respectively, while Yaw Aniweh was supported by a WACCBIP-DELTAS postdoctoral fellowship. The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)’s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa’s Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust [107755/Z/15/Z: Awandare) and the UK government. The views expressed in this publication are those of the author(s) and not necessarily those of AAS, NEPAD Agency, Wellcome Trust or the UK government.