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
] 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 ), 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
], and PFE1590w peptides have been detected by mass spectrometry in trophozoites and in the membrane of the infected erythrocytes [31
]. 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.
] 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
]. 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
]. 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
]. 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
]. 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.