Mark F. Wiser

A chimeric protein produced from the fusion of a type-P4 ATPase, also called flippase, and a guanylate cyclase (P4GC) was originally described in ciliates and malaria parasites. An extensive search for P4GC homologs was carried out, and the protein’s structural features were determined by sequence alignments and homology modeling. P4GC is found in all alveolate lineages and in some stramenopile lineages. This suggests that the gene fusion event occurred in a common ancestor of stramenopiles and alveolates after the divergence of rhizarians, although alternative evolutionary scenarios cannot be excluded. This scenario necessitates the subsequent loss of P4GC in some stramenopile lineages. Alternatively, the gene fusion may have arisen in an early alveolate lineage and subsequently been transferred horizontally to an ancestor of oomycetes and bicosoecids, or vice versa. The sequence homology of the flippase module is not well preserved in some lineages, whereas the structure and sequence homology of the cyclase module is highly conserved in all lineages. These findings suggest that the cyclase module has been subject to stronger evolutionary constraints than the flippase module, while the latter may have undergone lineage-specific functional diversification. However, despite sequence divergence, homology modeling indicates that the predicted 3-dimensional structures of the flippase modules are highly conserved across all lineages, suggesting that structural constraints have preserved its functional role(s) within P4GC. Together, these findings indicate that P4GC has undergone lineage-specific functional diversification while maintaining a highly conserved guanylate cyclase module throughout alveolate and stramenopile evolution.

A chimeric protein produced from the fusion of a type-P4 ATPase, also called flippase, and a guanylate cyclase (P4GC) was originally described in ciliates and malaria parasites. An extensive search for P4GC homologues was carried out and structural features of the pro-tein were determined by sequence alignments and homology modeling. P4GC is found in all alveolate lineages and in some stramenopile lineages. This suggests that the gene fu-sion event occurred before the divergence of stramenopiles and alveolates, but after the di-vergence of rhizarians and stramenopiles. This scenario necessitates the subsequent loss of P4GC in some stramenopile lineages. Alternatively, and perhaps the more favorable ex-planation, the gene fusion occurred in an alveolate progenitor before diversification of cil-iates, dinoflagellates, and apicomplexans and then a horizontal gene transfer event oc-curred in a progenitor of oomycetes and bicosoecids, or vice versa. The sequence homology of the flippase module is not well preserved in some lineages, whereas the structure and sequence homology of the cyclase module is highly conserved in all lineages. Thus, P4GC may function primarily as a guanylate cyclase and secondarily as a flippase in some line-ages. However, the predicted 3-dimensional structure of the flippase module is highly conserved in all lineages and P4GC likely plays a supportive role in the function of the cyclase module. Furthermore, P4GC may have distinct roles in the various stramenopile and alveolate lineages.

Type-P5 ATPases are the least characterized among the P-type ATPases and this is especially true in the case of the malaria parasite. In this study, Spf1, a subtype-P5A ATPase of yeast, and ATP13A2, a subtype-P5B ATPase of humans, were used as templates to extensively characterize the sequences and structural features of haemosporidian type-P5 ATPases. Malaria parasites have both subtype-P5A and subtype-P5B ATPase genes and the structural features of the proteins recapitulate the known structures of subtype-P5A and subtype-P5B ATPases. Detailed structural analysis detected an additional α-helix in the P-domain of subtype-P5A ATPases, which is not found in subtype-P5B ATPases. This feature may be an additional signature to distinguish subtype-P5A and subtype-P5B ATPases, in addition to the previously described differences in the membrane loops of the N-terminal domain, the arm in the P-domain of subtype-P5A, and substrate differences. A notable difference in the type-P5 ATPases from the malaria parasite, as compared to the templates, is the insertion of multiple variable and low-complexity regions that form intrinsically disorganized loops. These loops may form a shroud-like structure that protects the core ATPase structure and/or participates in low-affinity interprotein interactions. Homology modeling did not provide definitive answers about the substrate specificity of the haemosporidian type-P5 ATPases. However, the haemosporidian subtype-P5A ATPase is likely an ER transmembrane dislocase as are the other subtype-P5A ATPases. In contrast, the subtype-P5B ATPases of the malaria parasite are not likely to be polyamine transporters in lysosomes, as have been described in fungi and metazoans. This suggests that subtype-P5B ATPases have undergone lineage-specific divergence in regard to their function(s).

Type-P5 ATPases are the least characterized among the P-type ATPases and this is especially true in the case of the malaria parasite. In this study, Spf1, a subtype-P5A ATPase of yeast, and ATP13A2, a subtype-P5B ATPase of humans, were used as templates to extensively characterize the sequences and structural features of haemosporidian type-P5 ATPases. Malaria parasites have both subtype-P5A and subtype-P5B ATPase genes and the structural features of the proteins recapitulate the known structures of subtype-P5A and subtype-P5B ATPases respectively. Detailed structural analysis detected an addition-al alpha-helix in the P-domain of subtype-P5A ATPases which is not found in subtype-P5B ATPases. This feature may be an additional signature to distinguish subtype-P5A and subtype-P5B ATPases in addition to the differences in the membrane loops of the N-terminal domain, the arm in the P-domain of subtype-P5A, and substrate differences. A notable difference in the type-P5 ATPases from the malaria parasite, as compared to the templates, is the insertion of multiple regions of variable and low-complexity regions that form intrinsically disorganized loops. These loops may form a shroud-like structure that protects the core ATPase structure and/or participates in low-affinity interprotein interactions. Homology modeling did not provide definitive answers about the substrate specificity of the haemosporidian type-P5 ATPases. However, the haemosporidian sub-type-P5A ATPase is likely an ER transmembrane dislocase as are the other subtype-P5A ATPases. In contrast, the subtype-P5B ATPases of the malaria parasite are not likely to be polyamine transporters in lysosomes, as have been described in fungi and metazoans. This suggests that subtype-P5B ATPases have undergone lineage specific divergence in regard to their function(s).

Two related P-type ATPases, designated as ATPase1 and ATPase3, were identified in Plasmodium falciparum. These two ATPases exhibit very similar gene and protein structures and are most similar to P5B-ATPases. There are some differences in the predicted substrate-binding sites of ATPase1 and ATPase3 that suggest different functions for these two ATPases. Orthologues of ATPase3 were identified in all Plasmodium species, including the related Hepatocystis and Haemoproteus. ATPase3 orthologues could also be identified in all apicomplexan species, but no clear orthologues were identified outside of the Apicomplexa. In contrast, ATPase1 orthologues were only found in the Laverania, avian Plasmodium species, and Haemoproteus. ATPase1 likely arose from a duplication of the ATPase3 gene early in the evolution of malaria parasites. These results support a model in which early malaria parasites split into two clades. One clade consists of mammalian malaria parasites and Hepatocystis but excludes P. falciparum and related Laverania. The other clade includes Haemoproteus, avian Plasmodium species, and Laverania. This contrasts to recent models that suggest all mammalian malaria parasites form a monophyletic group, and all avian malaria parasites form a separate monophyletic group. ATPase1 may be a useful taxonomic/phylogenetic character for the phylogeny of Haemosporidia.

Two related P-type ATPases designated as ATPase1 and ATPase3 have been identified in Plasmodium falciparum. These two ATPases exhibit very similar gene and protein structures and are most similar to P5B-ATPases. There are some differences in the predicted substrate-binding sites of ATPase1 and ATPase3 which suggest different functions for these two ATPases. Orthologues of ATPase3 were identified in all Plasmodium species including the related Hepatocystis and Haemoproteus. ATPase3 orthologues could also be identified in all apicomplexan species, but no clear orthologues were identified outside of the Apicomplexa. In contrast, ATPase1 orthologues were only found in the Laverania, avian Plasmodium species, and Haemoproteus. ATPase1 likely arose from a duplication of the ATPase3 gene early in the evolution of malaria parasites. These results support a model in which early malaria parasites split into two clades. One clade consists of mammalian malaria parasites and Hepatocystis but excludes P. falciparum and related Laverania. The other clade includes Haemoproteus, avian Plasmodium species, and Laverania. This contrasts to recent models which suggest all mammalian malaria parasites form a phylogenetic group and all avian malaria parasites form a separate phylogenetic group. ATPase1 may be a useful taxonomic/phylogenetic character for the phylogeny of Haemosporidia.

Endocytosis is a quintessential feature of eukaryotes, and the emergence of endocytosis played a major role in the origin and evolution of eukaryotes. During the early evolution of eukaryotes, phagocytosis and the digestion of prey (i.e., bacteria) combined with the endocytosis of macromolecules opened a new source of nutrients beyond osmotrophy. Pathogenic and commensal protozoa have retained endocytosis as a major mechanism of nutrient acquisition even though, in theory, nutrients could be obtained from the host through osmotrophy. Nearly all pathogenic protozoa exhibit endocytosis and have lysosomal-like compartments that function as digestive vacuoles, and endocytosis appears to play a major role in the acquisition of nutrients. Cryptosporidium is a possible exception that may not exhibit endocytosis. Phagotrophy, however, is only observed in parasites of the intestinal lumen and appears to have been lost in blood and tissue parasites. Overall, the basic features of endocytosis and lysosomes are similar to other eukaryotes. Nonetheless, adaptation to the host has generated some novel features that are specific to certain protozoan lineages.

The malaria parasite resides within erythrocytes during one stage of its life cycle. During this intraerythrocytic period, the parasite ingests the erythrocyte cytoplasm and digests approximately two-thirds of the host cell hemoglobin. This digestion occurs within a lysosome-like organelle called the digestive vacuole. Several proteases are localized to the digestive vacuole and these proteases sequentially breakdown hemoglobin into small peptides, dipeptides, and amino acids. The peptides are exported into the host cytoplasm via the chloroquine-resistance transporter and an amino acid transporter has also been identified on the digestive vacuole membrane. The environment of the digestive vacuole also provides appropriate conditions for the biocrystallization of toxic heme into non-toxic hemozoin by a poorly understood process. Hemozoin formation is an attribute of Plasmodium and Haemoproteus and is not exhibited by other intraerythrocytic protozoan parasites. The efficient degradation of hemoglobin and detoxification of heme likely plays a major role in the high level of replication exhibited by malaria parasites within erythrocytes. Unique features of the digestive vacuole and the critical importance of nutrient acquisition provide therapeutic targets for the treatment of malaria.

The malaria parasite resides within erythrocytes during one stage of its life cycle. During this intraerythrocytic period, the parasite ingests the erythrocyte cytoplasm and digests approximately two-thirds of the host cell hemoglobin. This digestion occurs within a lysosome-like organelle called the digestive vacuole. Several proteases are localized to the digestive vacuole and these proteases sequentially breakdown hemoglobin into small peptides, dipeptides, and amino acids. The peptides are exported into the host cytoplasm via the chloroquine-resistance transporter and an amino acid transporter has also been identified on the digestive vacuole membrane. The environment of the digestive vacuole also provides appropriate conditions for the biocrystallization of toxic heme into non-toxic hemozoin by a poorly understood process. Hemozoin formation is an attribute of Plasmodium and Haemoproteus and is not exhibited by other intraerythrocytic protozoan parasites. The efficient degradation of hemoglobin and detoxification of heme likely plays a major role in the high level of replication exhibited by malaria parasites within erythrocytes. Unique features of the digestive vacuole and the critical importance of nutrient acquisition provide therapeutic targets for the treatment of malaria.

Plasmodium falciparum can cause a severe disease with high mortality. A major factor contributing to the increased virulence of P. falciparum, as compared to other human malarial parasites, is the sequestration of infected erythrocytes in the capillary beds of organs and tissues. This sequestration is due to the cytoadherence of infected erythrocytes to endothelial cells. Cytoadherence is primarily mediated by a parasite protein expressed on the surface of the infected erythrocyte called P. falciparum erythrocyte membrane protein-1 (PfEMP1). PfEMP1 is embedded in electron-dense protuberances on the surface of the infected erythrocytes called knobs. These knobs are assembled on the erythrocyte membrane via exported parasite proteins, and the knobs function as focal points for the cytoadherence of infected erythrocytes to endothelial cells. PfEMP1 is a member of the var gene family, and there are approximately 60 antigenically distinct PfEMP1 alleles per parasite genome. Var gene expression exhibits allelic exclusion, with only a single allele being expressed by an individual parasite. This results in sequential waves of antigenically distinct infected erythrocytes and this antigenic variation allows the parasite to establish long-term chronic infections. A wide range of endothelial cell receptors can bind to the various PfEMP1 alleles, and thus, antigenic variation also results in a change in the cytoadherence phenotype. The cytoadherence phenotype may result in infected erythrocytes sequestering in different tissues and this difference in sequestration may explain the wide range of possible clinical manifestations associated with severe falciparum malaria.