Background
Malaria is a devastating global disease, responsible for approximately 500 million clinical cases and more than a million deaths per year [
1]. The most deadly form is caused by
Plasmodium falciparum, which has a complex life cycle. The parasite is delivered to the human bloodstream as sporozoites that develop in the salivary gland of infected mosquitoes. These motile forms enter the liver, transversing through several cells before establishing themselves in a hepatocyte. Within the hepatocyte, the parasite undergoes a large number of asexual divisions, eventually releasing tens of thousands of merozoites that are capable of attaching to and invading erythrocytes. Within the erythrocyte, the parasite again divides several times in a 48-hour timeframe, releasing 16 or more merozoites that invade new erythrocytes. This intraerythrocytic stage is responsible for the cyclic symptoms of human disease (reviewed in [
2]).
As a consequence of its life cycle,
P. falciparum must be able to recognize, bind to and invade at least the two human cell types of hepatocytes and erythrocytes. These events are defined by particular molecular interactions. For example, hepatocyte invasion requires binding of the parasite's circumsporozoite surface protein to heparan sulfate proteoglycan surface receptors [
3], although other
P. falciparum proteins have been implicated in this process [
4]. Many
P. falciparum proteins have been proposed to be involved in erythrocyte invasion, and the human receptors for a few of them have been characterized (for example, EBA-175/glycophorin A and EBA-140/glycophorin C, MSP1/band3); however, the receptors for most of the parasite proteins implicated in erythrocyte invasion remain to be identified (reviewed in [
5,
6]).
In both hepatocytes and erythrocytes, the parasite is contained in a membranous structure known as the parasitophorous vacuole. Within this structure, however, the parasite is not isolated from the host cell, and must import nutrients from the plasma and the host cell cytoplasm to survive [
7]. Particularly in the case of erythrocyte invasion,
P. falciparum also establishes an active export of its proteins to the cytoplasm and surface of the host cell, where they modify the surface's structural and adhesive properties (reviewed in [
8]). The presence of parasite proteins, such as PfEMP1 and other adhesins on the surface of erythrocytes, causes these infected cells to sequester to endothelia and placenta via interactions with surface receptor molecules such as CD36, chondroitin sulfate A, ICAM-1 and selectins, resulting in the evasion of these cells from the host immune system. In addition, several parasite proteins interact with human cytoskeletal proteins; for example, PfEMP3 interacts with spectrin and actin [
9], and both RESA and KAHRP interact with spectrin [
10,
11].
Previously, a large network of interactions among the proteins of
P. falciparum was generated [
12]. The results of a similar high-throughput yeast two-hybrid analysis aimed at detecting interactions between proteins of
P. falciparum and those of its human host are presented here. An initial dataset of over 2,200 putative interactions was curated to exclude interactions that are not likely to be relevant to the pathogenesis of the parasite, resulting in a final dataset of 456 interactions. Among these, a cluster of interactions involving
P. falciparum PFE1590w/ETRAMP5 and human apolipoproteins ApoA1, ApoB and ApoE was studied further.
Discussion
Protein-protein interactions underlie the critical processes of infectious diseases, determining the specificity, affinity and efficiency by which pathogenic organisms are able to invade the human host. In the case of the malaria parasite P. falciparum, several processes that are essential to pathogenesis depend on host-parasite interactions, such as entry into host cells, growth and division within these cells, and binding to the lining of vasculature. An effort to identify such interactions was undertaken through the use of a large-scale yeast two-hybrid screening approach. The initial dataset of putative interactions was pared down to 456 interactions deemed most likely to be bona fide ones. However, two-hybrid false positives remain in this pared down set and no complementary verification of these interactions by a biochemical approach, such as co-immunoprecipitation, has been carried out; thus, these interaction data should be interpreted cautiously.
The interactions between P. falciparum hypothetical protein PFE1590w and human apolipoproteins ApoA1, ApoB and ApoE were further studied and determined to be specific by yeast two-hybrid. Furthermore, the preferential interaction of PFE1590w with the ApoE ε3 and ApoE ε4 isoforms but not the ApoE ε2 isoform was observed.
The genome of
P. falciparum encodes 13 members of the ETRAMP protein family, which are predicted to have an amino-terminal signal peptide and a single transmembrane domain [
21]. ETRAMPs are conserved among
Plasmodium species, although PFE1590w does not have a clear orthologue in the rodent
Plasmodium species. Unlike other ETRAMPs, PFE1590w is abundant in negatively charged residues, and has a 50-residue insertion rich in serines and prolines between the signal peptide and the transmembrane domain, resulting in its annotation as an atypical ETRAMP [
21]. The gene encoding PFE1590w is expressed during the intraerythrocytic cycle [
21,
28,
29] as well as in liver stages (Cate Speake and Patrick Duffy, personal communication). A transfected myc-tagged version of PFE1590w localizes to the periphery of the parasite in late ring and early trophozoite-infected erythrocytes, suggesting a parasitophorous vacuole membrane localization for this protein inside the infected erythrocytes [
22]. Phobius [
30], a programme that predicts the orientation of membrane proteins, predicts that residues 136–181 are cytoplasmic (Figure
1A), suggesting that the carboxy-terminal domain involved in the interactions described in this study could be exposed to the cytoplasm of the host cell. Since apolipoprotein-containing particles are internalized by the hepatocyte [
23], if PFE1590w also localizes to the parasitophorous vacuole during the liver stages, the interactions between PFE1590w and apolipoproteins could take place in the hepatocyte.
The mRNA for PFE1590w has also been detected in gametocytes and in sporozoites [
21,
29], and PFE1590w peptides have been detected by mass spectrometry in trophozoites and in the membrane of the infected erythrocytes [
31,
32]. Parasite inhibitory antibodies against PFE1590w are present in the sera of individuals that have suffered malaria infections, supporting the idea that PFE1590w might be a surface protein, although it is also possible that this immunity is a result of exposure of the immune system of the host to the protein upon lysis of the parasite [
33]. Therefore, besides being localized to the parasitophorous vacuole membrane, PFE1590w could be present on the plasma membrane of the sporozoite or the membrane of the infected red blood cell, where it could interact with plasma apolipoproteins.
Other ETRAMPs have been shown to be involved in interactions with host proteins.
Plasmodium yoelii UIS3, a rodent ETRAMP essential for parasite development during liver stages that is orthologous to
P. falciparum PF13_0012, interacts with liver fatty-acid binding protein (L-FABP) [
34]. UIS3 localizes to the parasitophorous vacuole membrane within the hepatocyte [
35], and binds L-FABP via its carboxy-terminal domain, which is predicted to be exposed to the cytoplasm of the host cell cytoplasm. Down-regulation of L-FABP by RNAi greatly inhibits the growth of parasites in cultured hepatoma cells, whereas overexpression of L-FABP promotes growth [
34]. An interaction between human L-FABP and
P. falciparum PFD0090c was observed in this study. PFD0090c is a hypothetical protein of the pHIST family[
36], and is predicted to be exported to the cytoplasm of the host cell. In addition, PFD0090c has also been found by mass spectrometry on the surface of the infected erythrocyte [
31].
Several lines of evidence suggest that apolipoproteins might have a role in the pathogenesis of the malaria parasite. First, apolipoproteins have been reported to inhibit invasion of hepatocytes by
Plasmodium sporozoites by competing with the most abundant protein on the surface of sporozoites, circumsporozoite protein, for binding to HepG2 cultured liver cells, and delaying liver-mediated clearance of circumsporozoite protein from circulation [
37]. Second, apolipoprotein E-enriched β-very low density lipoprotein inhibits invasion of HepG2 cells by sporozoites of a rodent
Plasmodium species,
P. berghei, and mice with high levels of circulating apolipoproteins have lower hepatic parasite loads. Based on this evidence, Sinnis
et al. [
37] postulated that invasion of hepatocytes by the parasite and lipoprotein uptake by the liver share a common mechanism that is likely mediated by binding to heparan sulfate proteoglycans, the physiological receptors of apolipoproteins on the surface of the hepatocytes. Third, studies of human populations have revealed correlations between the apolipoprotein E genotype of the human host and the susceptibility to malaria infection; although the ApoE ε3 allele is the most frequent worldwide, the ApoE ε4 allele, which is possibly the ancestral one, has an extremely high frequency in malaria endemic areas, including sub-Saharan Africa and Papua New Guinea [
38,
39]. In addition, Gambian children homozygous for the ApoE ε2 allele are more likely to suffer early malaria infection [
25], while heterozygotes carrying the ApoE ε3 and ε4 alleles are more likely to suffer severe malaria, including cerebral malaria and severe anaemia [
26]. These apparently contradictory data are compatible with the notion that children who suffer infections earlier in life develop protection against severe malaria [
27]. While these results are based on a small number of individuals, they suggest that the ApoE genotype of the human host influences the outcome of malaria infection. The data presented here that indicates a selective interaction between PFE1590w and ApoE alleles ε3 and ε4 is consistent with the notion that this interaction might be related to malaria pathogenesis. It is possible that the PFE1590w – ApoE interaction is important for cerebral malaria and severe anaemia, and thus individuals carrying ApoE ε3 and ε4 alleles are more likely to develop these symptoms.
The relevance of the PFE1590w – apolipoprotein interaction for the parasite's ability to infect or develop within human host cells remains to be determined. Apolipoproteins are synthesized in the liver, and are cleared from circulation by the liver. The interaction may be involved in the invasion of liver cells by sporozoites, or alternatively, the interaction could be important for the parasite's survival inside the red blood cell or the hepatocyte. As in the case of the
P. yoelii UIS3 – L-FABP interaction, PFE1590w might be involved in the uptake of lipids, in particular cholesterol, from the host.
Toxoplasma gondii, an apicomplexan parasite related to
P. falciparum, critically depends on host cholesterol from low-density plasma lipoproteins for survival, which it acquires by co-opting the host endocytosis pathway and sequestering lysosomes into its parasitophorous vacuole [
40‐
42]. Although the proteins responsible for cholesterol acquisition in
T. gondii have not been identified, it is likely that they are also localized to the parasitophorous membrane [
40‐
42]. Thus, both
P. falciparum and
Toxoplasma gondii might have similar mechanisms of nutrient acquisition from the host cell.
Other interactions between
P. falciparum and human proteins that may shed light on parasite invasion and other processes were identified in this study. For example, in a screen against the liver human library, an interaction was observed between a fragment of
P. falciparum EXP-1 (PF11_0224) and overlapping fragments of human Programmed Cell Death 6-Interacting protein (PDCD6IP, or ALIX) (Additional file
1). This interaction was shown to be reproducible and specific by yeast two-hybrid (data not shown). EXP-1 is a well-characterized integral membrane protein localized to the parasitophorous vacuole [
43,
44]. PDCD6IP/ALIX is a class E vacuolar sorting protein involved in the transport of cargo proteins by the multivesicular body for incorporation into intralumenal vesicles; fusion between endosomes and the vacuole results in the localization of cargo proteins to the vacuolar lumen [
45]. In addition, PDCD6IP/ALIX is used by several viruses, including HIV-1, for exiting infected cells [
46]. Although the interaction was identified in the high-throughput screens against the liver library, EXP-1 is also expressed in blood stages, and PDCD6IP/ALIX has been found by mass spectrometry in the cytoplasm of the erythrocyte [
47], suggesting that the interaction could take place in either the hepatocyte or the erythrocyte. It is possible that this interaction is involved in acquisition of vesicle contents by the parasite.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MV participated in the generation of the P. falciparum Y2H libraries and data analysis and curation, and drafted the manuscript. AM retested the interactions and carried out the Western blots. DJL participated in the generation of the P. falciparum Y2H libraries and data analysis. RC and RB managed the database of interactions. REH, SS and SF conceived the study and participated in its coordination. All authors read and approved the final manuscript.