Host cell invasion by Plasmodium falciparum requires multiple molecular interactions between host receptors and parasite ligands. A family of parasite proteins, which contain the conserved thrombospondin structural repeat motif (TSR), has been implicated in receptor binding during invasion. In this study we have characterized the functional role of a TSR containing blood stage protein referred to as P. falciparum thrombospondin related apical merozoite protein (PfTRAMP). Both native and recombinant PfTRAMP bind untreated as well as neuraminidase, trypsin or chymotrypsin-treated human erythrocytes. PfTRAMP is localized in the rhoptry bulb and is secreted during invasion. Adhesion of microneme protein EBA175 with its erythrocyte receptor glycophorin A provides the signal that triggers release of PfTRAMP from the rhoptries. Rabbit antibodies raised against PfTRAMP block erythrocyte invasion by P. falciparum suggesting that PfTRAMP plays an important functional role in invasion. Combination of antibodies against PfTRAMP with antibodies against microneme protein EBA175 provides an additive inhibitory effect against invasion. These observations suggest that targeting multiple conserved parasite ligands involved in different steps of invasion may provide an effective strategy forthe development of vaccines against blood stage malaria parasites.
Plasmodium falciparum causes the most severe form of malaria in humans. All the clinical symptoms of malaria are attributed to the blood stage of the P. falciparum life cycle during which merozoites invade and multiply within host erythrocytes (Miller et al., 2002). The invasion of host erythrocytes is a complex process that is mediated by multiple molecular interactions between merozoite proteins and erythrocyte receptors (Cowman and Crabb, 2006; Gaur and Chitnis, 2011). Merozoite proteins that mediate interactions with host receptors during invasion are often localized in membrane bound apical organelles, referred to as micronemes and rhoptries, and are secreted to the merozoite surface during invasion (Cowman and Crabb, 2006; Gaur and Chitnis, 2011). Such parasite proteins often contain conserved amino acid motifs that are functionally important for receptor binding. Prominent P. falciparum proteins that are implicated in erythrocyte invasion include the family of erythrocyte binding antigens (EBA family) comprising of EBA175, EBA180, EBA140 and EBL1, which are localized in micronemes and contain conserved, cysteine-rich Duffy binding domains, and the family of reticulocyte binding-like homologous proteins (PfRH) comprising of PfRH1, PfRH2, PfRH4 and PfRH5, which are also localized in apical organelles and share homology with P. vivax reticulocyte binding proteins (PvRBP family) (Rayner et al., 2001; Triglia et al., 2009). A third group of invasion related proteins that has been identified from different stages of malaria parasites as well as other apicomplexan parasites such as Toxoplasma gondii contains the conserved thrombospondin structural repeat motif (TSR) (Naitza et al., 1998).
TSR containing proteins are found widely across different species including mammals. They commonly mediate adhesive functions involved in cell–cell and cell–matrix interactions and play functional roles in diverse processes such as cell motility and innate immunity (Adams and Tucker, 2000; Chen et al., 2000; Silverstein, 2002). TSR containing proteins that are expressed in different stages of malaria parasites include the circumsporozoite protein (CSP) and thrombospondin-related adhesive protein (TRAP) from sporozoites, thrombospondin related proteins MTRAP and TRAMP from merozoites, and CSP-TRAP related protein (CTRP) from ookinetes (Naitza et al., 1998). CSP, the major structural protein from the surface of plasmodium sporozoites, mediates interactions with hepatocytes and salivary glands during invasion (Cerami et al., 1992; Pancake et al., 1992; Frevert et al., 1993). TRAP, the other TSR containing sporozoite surface protein, also binds hepatocyte receptors and mediates gliding motility by linking with the parasite motor complex through its cytoplasmic domain (Muller et al., 1993; Sultan et al., 1997; Wengelnik et al., 1999; Bergman et al., 2003). In case of merozoites, the cytoplasmic domain of MTRAP binds the actin-binding protein aldolase to provide a link between the parasite motor complex and the merozoite surface (Baum et al., 2006).
Plasmodium falciparum TRAMP (PfTRAMP) shares homology with TSR domains from the mammalian proteins thrombospondin, properdin and brain angiogenesis inhibitor (Tan et al., 2002; Thompson et al., 2004). Attempts to delete P. berghei TRAMP (PbTRAMP) were reported to be unsuccessful suggesting that TRAMP is essential for parasite survival (Thompson et al., 2004). There are no reports on the functional characterization of PfTRAMP and its role in erythrocyte invasion remains to be elucidated. Here, we demonstrate that PfTRAMP is localized in the rhoptries and is secreted following attachment of merozoites with the host erythrocyte during invasion. We also demonstrate that PfTRAMP binds host erythrocytes and antibodies directed against PfTRAMP inhibit erythrocyte invasion and limit parasite growth in vitro. These observations suggest that PfTRAMP plays a critical role in erythrocyte invasion and support its inclusion in a recombinant subunit blood stage vaccine against P. falciparum.
PfTRAMP forms a dimer and undergoes proteolytic cleavage
PfTRAMP is characterized by the presence of a putative signal sequence at the amino terminus, a conserved thrombospondin structural repeat (TSR) in the ectodomain followed by a transmembrane domain and cytoplasmic region at the carboxyl end (Fig. 1A). Recombinant PfTRAMP (rPfTRAMP) expressed as a soluble protein in Escherichia coli migrates with the expected mobility of ∼ 33 kDa under reducing conditions and as a ∼ 66 kDa homodimer under non-reducing conditions on SDS-PAGE gels (Fig. 1B). Detection of PfTRAMP in merozoite lysates by Western blotting under reducing and non-reducing conditions using anti-PfTRAMP mouse sera shows that native PfTRAMP forms a disulfide-linked dimer in merozoites (Fig. 1C). PfTRAMP is known to undergo proteolytic cleavage by P. falciparum subtilisin-2(PfSUB2) at a site between the TSR and the transmembrane domain (Green et al., 2006) (Fig. 1A). Anti-PfTRAMP mouse and sera detect full-length monomeric PfTRAMP (∼ 37 kDa) and processed monomeric PfTRAMP (∼ 28 kDa) in reduced P. falciparum merozoite lysates by Western blotting (Fig. 1C). No bands were detected in lysates made from uninfected red blood cells (RBC) when probed with anti-PfTRAMP mouse sera (Fig. 1C).
Expression of PfTRAMP in asexual blood stage parasites
Immunofluorescence assays (IFA) using anti-PfTRAMP mouse sera demonstrated that PfTRAMP is expressed in all blood stages (Fig. 2A). The stage specific expression of PfTRAMP was further confirmed by reverse transcriptase-PCR analysis (RT-PCR analysis) using RNA isolated from tightly synchronized rings, trophozoites, schizonts and merozoites. PfTRAMP transcripts are detected in all stages with maximum expression in schizonts and merozoites (Fig. 2B). EBA175, which is transcribed only in late schizonts, was used as a control in the RT-PCR experiments. Transcripts for EBA175 were only detected in schizonts as expected (Fig. 2B). 18S RNA, which is expressed in all blood stages was used as a loading control to confirm that similar amounts of RNA isolated from different stages were used for RT-PCR analysis (Fig. 2B). Anti-PfTRAMP mouse and rabbit sera were used to detect PfTRAMP expression in parasite lysates made at different stages by Western blotting (Fig. 2C and Fig. S1). In ring and trophozoite stage parasites, only full-length PfTRAMP of 37 kDa was observed, while at schizont stage both the 37 kDa protein and the proteolytically processed 28 kDa fragment of PfTRAMP were observed (Fig. 2C). Anti-PfTRAMP mouse as well as rabbit sera did not react with any other protein in parasite lysates confirming the specific recognition of PfTRAMP by these sera.
PfTRAMP was found to localize at the apical end of merozoites in late stage schizonts as well as free merozoites, suggesting that PfTRAMP may be localized in one of the apical organelles (Fig. 2A). Expression of PfTRAMP was also detected in rings and trophozoites by IFA (Fig. 2A). Given that the apical organelles form only in late schizonts, it was not clear where PfTRAMP is localized in rings and trophozoites. Colocalization experiments were performed with P. falciparum trophozoites using rabbit sera raised against PfTRAMP and mouse sera raised against either P. falciparum BiP, which is known to be localized in the endoplasmic reticulum (ER)(Kumar et al., 1991), or mouse sera raised against P. falciparum GRASP, which is known to be localized in the Golgi (Struck et al., 2005). In trophozoite stage parasites, PfTRAMP was found to localize partially with BiP and partially with GRASP, suggesting that PfTRAMP is localized in the classical secretory organelles, namely, the ER and Golgi in trophozoites (Fig. S2).
PfTRAMP localizes to the rhoptry bulb in P. falciparum merozoites
PfTRAMP is localized in a punctate pattern at the apical ends of individual merozoites in mature schizonts and free merozoites (Fig. 2). PfTRAMP did not colocalize with microneme protein EBA175 (Sim et al., 1992) in late stage schizonts and merozoites, but overlapped with the rhoptry bulb marker PfRH2b (Duraisingh et al., 2003) (Fig. 3A). To further confirm its localization, PfTRAMP was expressed with a C-terminal fusion with green fluorescent protein (GFP) in P. falciparum3D7 under the control of the constitutive crt promoter. The expression pattern for PfTRAMP-GFP in transgenic parasites was similar to that observed for PfTRAMP in wild type parasites at different stages (Fig. 2A and Fig. S3A). Anti-GFP mouse sera specifically recognized a protein of 75 kDa corresponding to the molecular weight of PfTRAMP-GFP fusion protein in the transgenic parasites by Western blotting (Fig. S4). The distribution of PfTRAMP-GFP in trophozoites and schizonts as determined by anti-GFP mouse sera overlaps completely with the distribution of PfTRAMP as detected by anti-PfTRAMP rabbit sera indicating that native PfTRAMP and PfTRAMP-GFP have similar localization (Fig. S3B). PfTRAMP-GFP colocalized with the rhoptry bulb protein, Clag 3.1 (Kaneko et al., 2005), in late schizonts and merozoites but did not colocalize with the microneme marker PfAMA1 or merozoite surface protein PfMSP1 (Fig. 3B). Immuno-electron microscopy (IEM) using anti-PfTRAMP mouse antibodies confirmed that PfTRAMP is localized in the rhoptry bulb of P. falciparum merozoites (Fig. 4).
PfTRAMP is secreted from the rhoptries during invasion
Exposure of P. falciparum merozoites to a low K+ environment as found in blood plasma has been shown to trigger a rise in cytosolic Ca2+ levels leading to secretion of microneme proteins such as EBA175 and PfAMA1 to the merozoite surface (Singh et al., 2010). Subsequently, adhesion of EBA175 and its homologues with their erythrocyte receptors provides the signal that triggers the release of rhoptry proteins (Singh et al., 2010). We tested if PfTRAMP was also released by this two-step mechanism. P. falciparum merozoites were isolated in buffer mimicking the intracellular ionic environment of erythrocytes (IC buffer: 5 mM NaCl, 140 mM KCl, 1 mM EGTA, 5.6 mM glucose, 1 mM MgCl2, 25 mM Hepes, pH 7.2) and transferred to either buffer mimicking the extracellular environment (EC buffer: 140 mM NaCl, 5 mM KCl,1 mM CaCl2, 5.6 mM glucose, 1 mM MgCl2, 25 mM Hepes, pH 7.2) or EC buffer containing red blood cell membranes (EC buffer + M). Anti-PfTRAMP mouse sera were used to detect PfTRAMP released in merozoite supernatants under each condition by Western blotting (Fig. 5A). IC, EC and EC + M buffers were spiked with equal amounts (5 μg) of bovine serum albumin (BSA). Supernatants were probed with anti-BSA mouse sera to confirm that equal amounts of merozoite supernatants were assayed for presence of PfTRAMP under different conditions. Cytoplasmic proteins NapL and actin were detected in merozoite pellets using specific sera to control for number of merozoites used in each condition. NapL was also detected in merozoite supernatants to control for merozoite lysis. Transfer of merozoites from IC buffer to EC + M buffer triggered release of PfTRAMP in merozoite supernatants. In contrast, the microneme protein AMA1 is secreted following transfer of merozoites from IC to EC buffer. No further increase of AMA1 in supernatants is detected following transfer to EC + M buffer. These observations suggest that the release of PfTRAMP requires attachment of P. falciparum merozoites with receptors on RBC membranes as shown previously for other rhoptry proteins (Singh et al., 2010). A replicate of the secretion experiment with similar observations has been included in Supplementary Information (Fig. S5).
We also tested the translocation and discharge of PfTRAMP from cytochalasin D-treated merozoites following attachment to erythrocytes by IFA. PfTRAMP (green) is detected on the apical surface of merozoites at the point of contact with the erythrocyte (Fig. 5B). PfTRAMP localizes close to the microneme protein PfAMA1, which is also secreted to the merozoite surface and has been shown to localize to the junction between the merozoite and erythrocyte (Richard et al., 2010). PfTRAMP is also seen in tubular whorl-like structures secreted by the attached merozoites (Fig. 5B) as observed previously for other rhoptry proteins from P. falciparum (Riglar et al., 2011) and T. gondii (Hakansson et al., 2001). In contrast, the microneme protein PfAMA1 is only seen at the point of contact between the merozoite and target erythrocyte.
PfTRAMP has erythrocyte binding activity
The dimeric and monomeric forms of recombinant PfTRAMP were separated by size exclusion chromatography (Fig. 6A, i). Dimeric and monomeric fractions of PfTRAMP were separated on SDS-PAGE and stained with Commasie to confirm their purity (Fig. 6A, ii). Purified dimeric and monomeric PfTRAMP were used for erythrocyte binding assays (EBAs). Following incubation of recombinant dimeric and monomeric PfTRAMP with erythrocytes to allow binding, erythrocytes with bound proteins were separated from unbound proteins by centrifugation through an oil layer. Bound proteins were then eluted with 1.5 M NaCl and the eluates containing bound proteins were separated on SDS-PAGE gels under reducing conditions and probed with anti-PfTRAMP mouse sera by Western blotting. Both dimeric and monomeric PfTRAMP bind human erythrocytes (Fig. 6A, iii). Recombinant PfTRAMP as well as native PfTRAMP from P. falciparum culture supernatants were also tested for binding to untreated as well as neuraminidase, chymotrypsin and trypsin-treated erythrocytes to define their binding specificity. Both recombinant dimeric and monomeric PfTRAMP as well as native PfTRAMP from parasite culture supernatants bound normal as well as neuraminidase, chymotrypsin and trypsin-treated erythrocytes (Fig. 6B and Fig. S6). Recombinant PfF2 (receptor-binding F2 region of EBA-175) and native EBA-175, which binds sialic acid residues on trypsin-sensitive glycophorin A (glyA) (Camus and Hadley, 1985), and PvRII (receptor-binding region II of P. vivax Duffy binding protein), which binds the chymotrypsin-sensitive Duffy antigen (Chitnis and Miller, 1994), were used as controls for the enzyme treatments (Fig. 6B). Recombinant PfF2 does not bind neuraminidase or trypsin-treated erythrocytes, whereas recombinant PvRII does not bind chymotrypsin-treated erythrocytes. Both dimeric and monomeric recombinant PfTRAMP as well as native PfTRAMP did not bind rat erythrocytes, confirming that the binding observed with human erythrocytes was specific. EBAs were also performed with native and recombinant AMA1 (Fig. S7), which is known not to bind to untreated human erythrocytes, as a negative control. Neither recombinant AMA1 nor native AMA1 bound untreated human erythrocytes. Erythrocyte binding observed in case of PfTRAMP is thus specific. Binding of native PfTRAMP and rPfTRAMP to erythrocytes was also detected by IFA and flow cytometry. Enzyme-treated erythrocytes showed higher levels of binding with PfTRAMP both by IFA and flow cytometry (Fig. 6C and D). Furthermore, to elucidate whether binding of PfTRAMP involves carbohydrates moieties, we tested binding of recombinant PfTRAMP to O-glycosidase (O-Gly) treated red blood cells. O-glycosidase treatment significantly reduces binding of PfTRAMP to erythrocytes, indicating that carbohydrate moieties present on the ectodomain of erythrocyte membrane glycoproteins may serve as receptors for PfTRAMP (Fig. 6B). Recombinant PfF2, which binds sialic acid residues on glycophorin A (Camus and Hadley, 1985), also did not bind O-glycosidase-treated erythrocytes. In contrast recombinant PvRII, which binds the peptide backbone of Duffy antigen (Chitnis et al., 1996), bound O-glycosidase treated erythrocytes.
PfTRAMP is conserved in P. falciparum field isolates
The gene encoding PfTRAMP was PCR-amplified using gene-specific primers and genomic DNA isolated from diverse P. falciparum laboratory strains and field isolates as template. DNA sequencing indicated that PfTRAMP from all the P. falciparum isolates tested had the identical amino acid sequence. The P. falciparum laboratory strain FCR3 showed a single nucleotide change at position 549 where a T→C transition was observed. However, this change in nucleotide does not cause any change in the PfTRAMP amino acid sequence. The amino acid sequence of PfTRAMP from four diverse P. falciparum laboratory strains and six field isolates is thus highly conserved. PCR typing of the P. falciparum laboratory strains and field isolates by nested PCR based on polymorphic markers PfMSP1 and PfMSP2 confirmed the presence of diverse single P. falciparum genotypes in the samples tested (Table S1).
Antibodies against PfTRAMP block erythrocyte invasion by P. falciparum
IgGs purified from rabbit sera raised against dimeric recombinant PfTRAMP were tested for inhibition of invasion into normal, neuraminidase and chymotrypsin-treated erythrocytes by P. falciparum in growth inhibition assays (GIAs) (Fig. 7). Anti-PfTRAMP IgGs inhibited invasion of normal erythrocytes by P. falciparum 3D7 by ∼ 22% at the highest concentration tested (10 mg ml−1). Anti-PfTRAMP IgGs inhibited invasion of chymotrypsin-treated and neuraminidase-treated erythrocytes by P. falciparum 3D7 by ∼ 35% and ∼ 40% respectively (Fig. 7). Higher inhibitory activity for invasion into enzyme-treated erythrocytes indicates that PfTRAMP plays a major role in invasion of these enzyme-treated erythrocytes by binding to residual neuraminidase and chymotrypsin resistant receptors. Anti-PfTRAMP IgGs were also tested in combination with anti-PfF2 (EBA-175) rabbit IgGs for inhibition of invasion into untreated (Un) and chymotrypsin (Chy) treated erythrocytes. Individually, anti-PfTRAMP IgGs (5 mg ml−1) and anti-PfF2 IgGs (5 mg ml−1) inhibit invasion of untreated erythrocytes by P. falciparum 3D7 by ∼ 12% and ∼ 21%, respectively, whereas combination of anti-PfTRAMP and anti-PfF2 IgGs at 5 mg ml−1 each inhibit erythrocyte invasion by P. falciparum 3D7 by ∼ 38% (Fig. 8A). Anti-PfTRAMP and anti-PfF2 IgGs have significantly higher invasion inhibitory activity against P. falciparum 3D7 invading chymotrypsin-treated erythrocytes (∼ 28% and ∼ 40% respectively at 5 mg ml−1) compared with invasion of untreated erythrocytes. Combination of anti-PfTRAMP and anti-PfF2 IgGs yields inhibits invasion of chymotrypsin-treated erythrocytes by P. falciparum 3D7 by ∼ 70% (Fig. 8A). Antibodies raised against PfTRAMP and PfF2 also inhibit invasion by P. falciparum 7G8 with similar efficiency (Fig. 8B). The inhibition of invasion achieved by combining antibodies against PfTRAMP and PfF2 is significantly higher than inhibition by either antibodies to PfTRAMP or PfF2 alone (P < 0.02) in case of both 3D7 and 7G8. Combination of antibodies against PfTRAMP and PfF2 thus seems to provide an additive inhibitory effect against these parasite strains. The invasion inhibitory activity of anti-PfTRAMP and anti-PfF2 IgG was also tested against P. falciparum Dd2, which primarily uses sialic acid dependent erythrocyte invasion pathways. P. falciparum Dd2 uses sialic acid residues on both glycophorin A as well as trypsin-resistant glycophorin B as receptors for invasion (Jude et al., 1999). Anti-PfTRAMP IgG inhibits invasion of untreated erythrocytes by P. falciparum Dd2 poorly compared with invasion inhibitory activity of anti-PfF2 IgG (Fig. 8C). Addition of anti-PfTRAMP IgG to anti-PfF2 IgG does not significantly increase the invasion inhibitory activity against P. falciparum Dd2 compared with that of anti-PfF2 IgG alone (Fig. 8C).
Plasmodium falciparum proteins such as PfCSP, PfTRAP, PfMTRAP, PfTRAMP and PfCTRP, which contain conserved thrombospondin structural repeat (TSR) motifs, mediate interactions with diverse host tissues at different stages of the parasite life cycle. Here, we have characterized PfTRAMP, a TSR containing parasite protein that is expressed in P. falciparum merozoites and is implicated in the process of erythrocyte invasion (Thompson et al., 2004). PfTRAMP, which is localized at the apical end of merozoites, is proteolytically cleaved and released from the merozoite surface during invasion (Green et al., 2006). PfTRAMP was previously reported to localize to micronemes (Thompson et al., 2004; Green et al., 2006). Here, we have demonstrated unequivocally that PfTRAMP colocalizes with the rhoptry bulb markers, PfRH2b and Clag 3.1, and does not colocalize with microneme markers EBA175 and PfAMA1. The localization of PfTRAMP to the rhoptry bulb in P. falciparum merozoites was confirmed by IEM using anti-PfTRAMP mouse sera (Fig. 4). We also demonstrate that PfTRAMP is expressed in all blood stages including rings, trophozoites, schizonts and merozoites. PfTRAMP transcripts are observed in rings, trophozoites and schizonts although the level of transcripts appears to be maximum in late schizonts and merozoite stages (Fig. 2B). These observations are in line with previous microarray studies that reported low level expression of PfTRAMP in rings and trophozoites and higher expression in schizonts (Le Roch et al., 2003). In stages such as rings and trophozoites in which rhoptries have not yet formed, PfTRAMP is found in the ER and Golgi. PfTRAMP localizes to the rhoptries in merozoites once they form in late schizonts. This is unusual as invasion related proteins that localize to apical organelles are usually expressed in late schizont stages. However, there are reports of some rhoptry proteins such as RAMA and 110 kDa rhoptry protein, which, like PfTRAMP, are expressed in early stages including rings and trophozoites (Sam-Yellowe et al., 1988; Topolska et al., 2004). Like PfTRAMP, RAMA and 110 kDa rhoptry protein are also localized to ER and Golgi in rings and trophozoites and translocate to the rhoptries in schizonts once the rhoptries are formed (Sam-Yellowe et al., 1988; Topolska et al., 2004).
Parasite proteins localized to the micronemes and rhoptries are secreted to the merozoite surface in response to external signals in a two-step process during erythrocyte invasion (Singh et al., 2010). In the first step, exposure of merozoites to a low K+ environment as found in blood plasma triggers release of Ca2+ from intracellular stores leading to a rise in cytosolic Ca2+ levels (Singh et al., 2010). Increase in cytosolic Ca2+ levels triggers the release of microneme proteins such as EBA175 and PfAMA1 to the merozoite surface (Singh et al., 2010). In the next step, binding of microneme proteins such as EBA175 and its homologues with their erythrocyte receptors provides the signal that triggers the release of rhoptry proteins to the merozoite surface (Singh et al., 2010). Similar to other rhoptry proteins such as PfRH2b and Clag 3.1, PfTRAMP is secreted when P. falciparum merozoites are transferred from buffer mimicking intracellular ionic conditions (IC) to buffer mimicking extracellular ionic conditions containing RBC membranes (EC + M), which provides receptors such as glycophorin A for interaction with secreted microneme proteins such as EBA175. The external signal that triggers secretion of PfTRAMP from merozoites is thus consistent with PfTRAMP being localized to the rhoptries. The secretion of PfTRAMP from merozoites following attachment with human erythrocytes was also observed by IFA. Cytochalasin D treated merozoites attach to erythrocytes, reorient apically and a junction develops between their apical end and the erythrocyte at the point of contact (Miller et al., 1979). However, invasion is arrested at this step and does not proceed (Miller et al., 1979). The microneme protein PfAMA1 has been previously localized to the junction between the invading merozoite and target erythrocyte (Richard et al., 2010). Both PfAMA1 and PfTRAMP were found at the apical surface of the merozoite at the point of contact with the target erythrocyte. In addition, PfTRAMP was found to be secreted into the erythrocyte in whorl-like structures as reported previously for other rhoptry proteins from P. falciparum (Riglar et al., 2011) and similar to the evacuoles observed in case of T. gondii (Hakansson et al., 2001). The secretion of PfTRAMP from merozoites following attachment with erythrocyte receptors as observed here is consistent with its localization in rhoptries.
Given the presence of the cell adhesive TSR motif in PfTRAMP, we explored the potential of PfTRAMP to bind host erythrocytes. Both native PfTRAMP and recombinant PfTRAMP form disulfide-linked homodimers. Both the monomeric and dimeric recombinant PfTRAMP bound erythrocytes (Fig. 6A). In addition to normal erythrocytes, recombinant PfTRAMP also bound neuraminidase, trypsin and chymotrypsin-treated erythrocytes. Treatment of erythrocytes with O-glycosidase greatly reduced binding by PfTRAMP (Fig. 6B), suggesting that PfTRAMP binds O-linked carbohydrate moieties on erythrocyte surface glycoprotein. The identity of the glycoprotein remains to be determined. In a recent study (Uchime et al., 2012), the authors were unable to show the binding of recombinant PfTRAMP to erythrocytes. PfTRAMP was expressed in inclusion bodies and refolded in vitro in this study. The inability to bind erythrocytes may be due to incorrect folding of recombinant PfTRAMP used in that study (Uchime et al., 2012). Recombinant PfTRAMP used here was produced as a soluble cytoplasmic protein in E. coli, suggesting that it may be naturally folded in its native structure. PfTRAMP was purified from supernatants of E. coli lysates by metal affinity chromatography and ion-exchange chromatography and did not require in vitro refolding.
The failure of efforts to knock out the gene that encodes TRAMP in P. berghei (Thompson et al., 2004) suggests that it is essential for parasite survival. The observation that PfTRAMP binds normal and enzyme-treated erythrocytes indicates that PfTRAMP may play an important role in mediating invasion by alternative sialic acid/glycophorin A independent invasion pathways. We tested the ability of IgGs purified from rabbit sera raised against dimeric recombinant PfTRAMP to inhibit erythrocyte invasion by P. falciparum in vitro in GIAs. Anti-PfTRAMP IgG inhibited erythrocyte invasion by P. falciparum 3D7 by ∼ 22% at IgG concentration of 10 mg ml−1. The efficiency of inhibition was higher for invasion of into neuraminidase and chymotrypsin-treated erythrocytes (40% and 35% respectively), which suggests that PfTRAMP plays an important role in invasion of enzyme-treated erythrocytes that have fewer invasion pathways available. Previous studies have shown that EBA175 plays an important role in mediating invasion even in parasite strains that can use sialic acid/glycophorin A independent invasion pathways (Jiang et al., 2011). We therefore tested the ability of a combination of rabbit IgGs against PfTRAMP and PfF2 to inhibit growth of P. falciparum 3D7 and 7G8, which use multiple invasion pathways. Combination of IgGs against PfTRAMP and PfF2 provides an additive effect in inhibition of growth of P. falciparum 3D7 and 7G8. The inhibitory effect of combinations of IgGs against EBA175 and PfTRAMP was significantly higher for invasion into chymotrypsin-treated erythrocytes compared with normal erythrocytes (Fig. 8). This implies that EBA175 and PfTRAMP play major roles in invasion of chymotrypsin-treated erythrocytes. P. falciparum Dd2 primarily uses sialic acid dependent invasion pathways. PfTRAMP, which binds a neuraminidase-resistant receptor, thus appears to play only a minor role in invasion of erythrocytes by P. falciparum Dd2.
Sequence analysis of PfTRAMP derived from diverse P. falciparum laboratory strains and field isolates indicates that it is highly conserved. The late release of PfTRAMP from merozoites in the invasion process following attachment with erythrocytes may limit its exposure to the host immune system. The lack of immune pressure may be responsible for the high level of conservation observed in PfTRAMP. The observations that PfTRAMP is highly conserved, plays a significant role in erythrocyte invasion by binding to erythrocyte receptors during invasion, and elicits invasion inhibitory antibodies that provide an additive effect when combined with antibodies against other invasion related parasite proteins such as EBA175, provides support for its inclusion in a multi-component subunit vaccine against blood stages of P. falciparum.
Cloning, expression and purification of the ecto-domain of PfTRAMP and generation of specific antisera
A codon optimized synthetic gene encoding the ecto-domain of PfTRAMP (PFL0870w, amino acid residues 26–307) fused to a C-terminal 6-His tag was cloned in the expression vector pET28a and transferred to E. coli Rosetta DE3 pLysS (Novagen, USA). The recombinant E. coli cells were grown to mid-log phase and expression of PfTRAMP was induced at OD600 of 0.8 with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 4 hours. E. coli cells were collected by centrifugation, lysed by sonication and recombinant PfTRAMP was purified from the soluble lysate by metal affinity chromatography using Ni-NTA matrix (Qiagen, the Netherlands) followed by anion exchange chromatography using Q Sepharose (GE Healthcare, USA). Dimeric and monomeric fractions of recombinant PfTRAMP were further purified by gel permeation chromatography on a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare).
Reverse transcript analysis of PfTRAMP
RNA was isolated from tightly synchronized ring, trophozoite and schizont stages of P. falciparum 3D7 using RNeasy mini-columns (Qiagen, Germany) following saponin lysis according to the manufacturer's instructions. RNA (∼ 500 ng) from each stage was used for cDNA preparation using SuperScript II kit (Invitrogen, USA). The stage specific cDNA was used as template to detect transcripts for PfTRAMP, EBA175 and 18S RNA with gene specific oligonucleotide primers and Taq DNA polymerase (Fermentas, USA) by polymerase chain reaction (PCR). Sequences for oligonucleotide primers used for PCR for PfTRAMP, EBA175 and 18S RNA were as follows: PfTRAMP Fwd 5′-AATGCATGCAAGCTTTTACAAAAGACAAAATTTC-3′ and PfTRAMP Rev 5′-GACAAGCTTCTAATAAAAAATATGATACAATATAGTTATT-3′, EBA-175 Fwd 5′-AAT TTCTGTAAAATATTGTGACCATATG-3′ and EBA-175 Rev 5′-GATACTGCACAACACAGATTTCTTG-3′, 18S Fwd 5'-CCGCCCGTCGCTCCTACCG-3′ and 18S Rev 5′-CCTTGTTACGACTTCTCCTTCC-3′.
Transient expression of PfTRAMP fused with green fluorescent protein (GFP) in P. falciparum
DNA fragment encoding amino acid residues 1 to 352 of PfTRAMP was amplified by polymerase chain reaction (PCR) using primers PfTRAMPGFPF (5′-ATCAGATCTATGATTGATGTGTTGTTAAATAAAAC-3′) and PfTRAMPGFPR (5′-CATCCTAGGGTCGTACATATAACGACCAGC-3′) with P. falciparum 3D7genomic DNA as template. The amplified PCR fragment was cloned in the vector pHH2 at the BglII and AvrII restriction sites to create a fusion of PfTRAMP with green fluorescence protein (GFP) (Reed et al., 2000; Wickham et al., 2001). The PfTRAMP-GFP cassette was excised with XhoI and cloned in vector pARL1a at the XhoI site downstream of the crt promoter (Marti et al., 2004). P. falciparum 3D7 ring stage parasites were electroporated with 100 μg of purified pARL1-PfTRAMP-GFP plasmid DNA as described previously (Crabb and Cowman, 1996). P. falciparum transfectants were selected using 10 nM WR99210 (Reed et al., 2000). Expression of PfTRAMP-GFP in transformed P. falciparum 3D7 blood stage parasites was confirmed by Western blotting using anti-GFP sera (Roche, USA) and observed by IFA.
Immuno-blot analysis of parasite proteins
Synchronized P. falciparum blood stage parasites were collected by centrifugation at ring, trophozoite and schizont stages, treated with saponin (0.05%), washed three times with PBS. Pellets were lysed in lysis buffer [50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% SDS, 1× protease inhibitor cocktail (Roche, USA)] and total protein concentration was measured using bicinchoninic acid (BCA) kit (Peirce, USA). 100 μg of total protein from each stage was then boiled in SDS-PAGE buffer with or without β-mercaptoethanol and separated on 12% SDS-PAGE gels. The proteins were transferred to 0.45 μm nitrocellulose membrane, blocked in 5% skimmed milk in PBS overnight at 4°C and probed with anti-PfTRAMP mouse sera at 1:200 dilution and horseradish peroxidase conjugated goat anti-mouse IgG antibodies (Sigma Aldrich, USA) at 1:2000 dilution. The blot was developed using ECL Plus Western Blotting Detection Reagents (GE Healthcare) using manufacturer's protocol.
Immunofluorescence assay to localize PfTRAMP
Transfected P. falciparum blood stage cultures expressing PfTRAMP-GFP were synchronized with sorbitol as described above, stained with diamidino-2-phenylindole (DAPI) and visualized at different stages using Nikon Ti 2000 microscope (Nikon, Japan). For colocalization experiments P. falciparum blood stage parasite smears were also probed with anti-Clag3.1 mouse sera (kindly provided by Dr Osamu Kaneko, Japan) or anti-PfRH2b rabbit sera as rhoptry markers, anti-PfAMA1 rabbit sera or anti-EBA175 rabbit sera as microneme markers and anti-PfMSP1 rabbit sera as a merozoite surface marker at dilution of 1:100. For colocalization experiments with ER and Golgi markers, anti-BiP mouse sera and anti-GRASP mouse sera respectively were used at a dilution of 1:100.
To detect secretion of PfTRAMP during erythrocyte invasion, P. falciparum merozoites were isolated as described earlier (Singh et al., 2010), treated with 1 μM cytochalasin D for 10 min, incubated with uninfected erythrocytes for 5 min and fixed as described earlier (Riglar et al., 2011). After fixation and washing, PfTRAMP was detected by IFA using anti-PfTRAMP mouse sera as primary antibody and Alexa Fluor 488 conjugated goat anti-mouse IgG antibodies as the secondary antibody. Similarly, PfAMA1 was detected using anti-PfAMA1 rabbit sera followed by Alexa-Fluor 594 conjugated goat anti-rabbit IgG antibodies. Images were acquired on a Nikon A1R Confocal microscope (Nikon, Japan) using NIS-elements software and processed using Adobe Photoshop CS2.
Plasmodium falciparum schizonts and merozoites were isolated as described earlier (Singh et al., 2010), washed with PBS, fixed with 4% para-formaldehyde, 0.04% glutaraldehyde in PBS at 4°C for 1 h, embedded in LR white, and infiltrated with a cryo-preservative and plasticizer (2.3 M sucrose, 20% polyvinyl pyrrolidone). After freezing in liquid nitrogen, samples were sectioned with a Leica Ultracut UCT cryo-ultramicrotome (Leica Microsystems Inc., Bannockburn, IL) at −60°C. Ultrathin sections were blocked with 5% fetal bovine serum and 5% normal goat serum in PBS for 30 min, incubated with anti-PfTRAMP mouse sera or pre-immune mouse sera diluted 1:100 in blocking buffer, washed thoroughly and incubated with 18 nm colloidal gold-conjugated goat anti-mouse IgG antibodies (Jackson Immuno Research Laboratories, USA) for 1 h. Sections were stained with 0.3% uranyl acetate, 1.7% methyl cellulose and visualized under JEOL 1200EX transmission electron microscope (JEOL, USA). Control sections were stained with pre-immune sera as the primary antibody.
Sequence analysis of PfTRAMP from P. falciparum laboratory strains and field isolates
Frozen aliquots of P. falciparum field isolates and laboratory strains were obtained from the Malaria Parasite Bank at the National Institute of Malaria Research (NIMR), Delhi or Malaria Research and Reference Reagent Resource Center (MR4), USA. Parasites were thawed and cultured using standard protocols. Parasite cultures were harvested at 2–5% parasitemia and genomic DNA was isolated as described previously (Schlichtherle et al., 2000). Genotyping by PCR amplification of gene fragments encoding polymorphic regions of blood stage proteins such as block 2 of PfMSP1 and blocks 2 and 3 of PfMSP2 was used to type the P. falciparum field isolates (Jude et al., 1999). Sequences of the primers used for PCR as well as PCR amplification conditions were identical to those described earlier (Smythe et al., 1991; Snewin et al., 1991; Kyes et al., 1997). Genomic DNA from the parasites was also used as template for PCR to amplify the gene encoding PfTRAMP using the following gene specific primers: PfTRAMPflF (5′-ATGATTGATGTGTTGTTAAATAAAAC-3′); PfTRAMPflR (5′-CTAGTCGTACATATAACGACCAGC-3′). The PCR products were cloned and sequenced. Sequence alignments and comparisons were performed using ClustalW.
Erythrocyte binding assays
Erythrocyte binding assays were performed using recombinant proteins or culture supernatants of P. falciparum 3D7 schizont-infected erythrocytes as described previously (Gaur et al., 2007). Briefly, culture supernatants or recombinant proteins were incubated with human erythrocytes at 37°C following which the suspension was centrifuged through dibutyl phthalate. The supernatant and oil were removed by aspiration. Bound parasite proteins were eluted from the erythrocytes with 1.5 M NaCl. The eluted fractions were analysed for the presence of PfTRAMP by Western blotting using anti-PfTRAMP mouse sera. Alternatively, bound PfTRAMP on surface of erythrocytes was detected using anti-PfTRAMP rabbit sera and Alexa 488 conjugated goat anti-rabbit IgG antibodies by flow cytometry or fluorescence microscopy. EBAs were also performed with neuraminidase, chymotrypsin and trypsin-treated erythrocytes. Erythrocytes were treated with trypsin, chymotrypsin and neuraminidase as described earlier (Gaur et al., 2003). For O-glycosidase treatment erythrocytes were incubated with 200 U μl−1 of O-glycosidase for 90 minutes at 37°C followed by washing with incomplete RPMI three times.
Detection of proteins secreted from P. falciparum merozoites by Western blotting
Plasmodium falciparum merozoites were isolated in buffer mimicking the intracellular ionic environment in erythrocytes (IC buffer:5 mM NaCl, 140 mM KCl, 1 mM EGTA, 5.6 mM glucose, 1 mM MgCl2, 25 mM Hepes, pH 7.2) as described earlier (Singh et al., 2010). Merozoites isolated in IC buffer were collected by centrifugation and resuspended in IC buffer or buffer mimicking extracellular ionic environment (EC buffer: 140 mM NaCl, 5 mM KCl,1 mM CaCl2, 5.6 mM glucose, 1 mM MgCl2, 25 mM Hepes, pH 7.2) or EC buffer containing erythrocyte membranes (EC + M). BSA (5 μg) was added to each buffer to control for amount of supernatant used for detection of secreted proteins. Following incubation at 37°C in each buffer condition, merozoite supernatants were collected by centrifugation and aliquots were separated by SDS-PAGE, transferred to nitrocellulose and probed by Western blotting for presence of PfTRAMP using anti-PfTRAMP mouse sera at 1:200 dilution, followed by horseradish peroxidase conjugated goat anti-mouse IgG antibodies (Sigma, USA) diluted 1:2000. Rabbit sera raised against the P. falciparum cytoplasmic proteins NapL (Chandra et al., 2005) and actin were used at a dilution of 1:2000 to probe merozoite pellet lysates and supernatants by Western blotting to control for numbers of merozoites used and merozoite lysis respectively. Anti-BSA mouse sera were used to detect BSA, which was added as control to each buffer, to confirm that equal amounts of supernatants were used for each buffer condition. Anti-AMA-1 rabbit sera, anti-actin rabbit sera and anti-BSA mouse sera were used at dilutions of 1:2000, 1:500 and 1:1000, respectively, for Western blotting. Erythrocyte membranes were prepared as described previously (Singh et al., 2010).
Generation of sera in rabbits and IgG purification
Recombinant PfTRAMP produced as described above and recombinant PfF2 produced as described previously (Pandey et al., 2002) were formulated with complete or incomplete Freund's adjuvant and used for immunization of New Zealand White rabbits to generate specific antisera. Rabbits were primed with 100 μg of recombinant PfTRAMP and PfF2 formulated with complete Freund's adjuvant followed by two boosts with recombinant PfTRAMP and PfF2 formulated with incomplete Freund's adjuvant on days 28 and 56. Sera were collected on days 42, 70 and 98. IgGs were purified from rabbit sera raised against PfTRAMP and PfF2 on protein G Sepharose columns (GE Healthcare) as per manufacturer's instructions, dialysed against RPMI1640 medium and concentrated by diafiltration to a concentration of ∼ 20 mg ml−1 prior to use in GIAs. The purity of rabbit IgG was confirmed by separation on SDS-PAGE gels and detection by Coomassie staining (Fig. S8).
Growth inhibition assays
Purified anti-PfTRAMP rabbit IgG were tested at different concentrations for inhibition of erythrocyte invasion by P. falciparum 3D7, 7G8and Dd2. P. falciparum schizonts were purified using Percoll as previously described (Lambros and Vanderberg, 1979) diluted to 0.3% parasitemia with untreated and enzyme-treated erythrocytes at 2% haematocrit and incubated in RPMI 1640 medium with different concentrations of IgG purified from pre-immune rabbit sera or rabbit sera raised against PfTRAMP or PfF2. Parasitemia was estimated by detecting newly infected erythrocytes by flow cytometry following staining with ethidium bromide after incubation for 40 h (Tham et al., 2009; Sahar et al., 2011). The inhibition efficiency was calculated as follows:
The invasion inhibitory activities of anti-PfTRAMP IgGs and anti-PfF2 IgGs were tested individually and in combination at different concentrations. GIAs were performed in duplicate and were repeated three times.
This work was supported in part by a Program Support Grant from the Department of Biotechnology (DBT), Government of India, EVIMalaR and MalSig grants from the European Commission and a grant from Cellex Foundation. C.E.C. and D.G. are recipients of the TATA Innovation Fellowship and Ramalingaswami Fellowship, respectively, from DBT. We thank Dr Osamu Kaneko, Nagasaki University for providing anti-Clag 3.1 sera, Dr Pawan Malhotra, ICGEB, New Delhi, for providing anti-BiP and anti-GRASP sera, Dr C. R. Pillai, NIMR, Delhi for providing P. falciparum field isolates and the staff of the Animal Facilities at ICGEB, New Delhi for their assistance.