Evidence for mitochondrial-derived alternative oxidase in the apicomplexan parasite Cryptosporidium parvum: a potential anti-microbial agent target.

The observation that Plasmodium falciparum possesses cyanide insensitive respiration that can be inhibited by salicylhydroxamic acid (SHAM) and propyl gallate is consistent with the presence of an alternative oxidase (AOX). However, the completion and annotation of the P. falciparum genome project did not identify any protein with convincing similarity to the previously described AOXs from plants, fungi or protozoa. We undertook a survey of the available apicomplexan genome projects in an attempt to address this anomaly. Putative AOX sequences were identified and sequenced from both type 1 and 2 strains of Cryptosporidium parvum. The gene encodes a polypeptide of 336 amino acids and has a predicted N-terminal transit sequence similar to that found in proteins targeted to the mitochondria of other species. The potential of AOX as a target for new anti-microbial agents for C. parvum is evident by the ability of SHAM and 8-hydroxyquinoline to inhibit in vitro growth of C. parvum. In spite of the lack of a good candidate for AOX in either the P. falciparum or Toxoplasma gondii genome projects, SHAM and 8-hydroxyquinoline were found to inhibit the growth of these parasites. Phylogenetic analysis suggests that AOX and the related protein immutans are derived from gene transfers from the mitochondrial endosymbiont and the chloroplast endosymbiont, respectively. These data are consistent with the functional localisation studies conducted thus far, which demonstrate mitochondrial localisation for some AOX and chloroplastidic localization for immutans. The presence of a mitochondrial compartment is further supported by the prediction of a mitochondrial targeting sequence at the N-terminus of the protein and MitoTracker staining of a subcellular compartment in trophozoite and meront stages. These results give insight into the evolution of AOX and demonstrate the potential of targeting the alternative pathway of respiration in apicomplexans.

Erythrocyte detergent-resistant membrane proteins: their characterization and selective uptake during malarial infection.

Infection of human erythrocytes by the apicomplexan malaria parasite Plasmodium falciparum results in endovacuolar uptake of 4 host proteins that reside in erythrocyte detergent-resistant membranes (DRMs). Whether this vacuolar transport reflects selective uptake of host DRM proteins remains unknown. A further complication is that DRMs of vastly different protein and cholesterol contents have been isolated from erythrocytes. Here we show that isolated DRMs containing the highest cholesterol-to-protein ratio have low protein mass. Liquid chromatography, mass spectrometry, and antibody-based studies reveal that the major DRM proteins are band 3, flotillin-1 and -2, peroxiredoxin-2, and stomatin. Band 3 and stomatin, which reflect the bulk mass of erythrocyte DRM proteins, and all tested non-DRM proteins are excluded from the vacuolar parasite. In contrast, flotillin-1 and -2 and 8 minor DRM proteins are recruited to the vacuole. These data suggest that DRM association is necessary but not sufficient for vacuolar recruitment and there is active, vacuolar uptake of a subset of host DRM proteins. Finally, the 10 internalized DRM proteins show varied lipid and peptidic anchors indicating that, contrary to the prevailing model of apicomplexan vacuole formation, DRM association, rather than lipid anchors, provides the preferred criteria for protein recruitment to the malarial vacuole.

Erythrocyte G protein-coupled receptor signaling in malarial infection.

Erythrocytic mechanisms involved in malarial infection are poorly understood. We have found that signaling via the erythrocyte beta2-adrenergic receptor and heterotrimeric guanine nucleotide-binding protein (Galphas) regulated the entry of the human malaria parasite Plasmodium falciparum. Agonists that stimulate cyclic adenosine 3',5'-monophosphate production led to an increase in malarial infection that could be blocked by specific receptor antagonists. Moreover, peptides designed to inhibit Galphas protein function reduced parasitemia in P. falciparum cultures in vitro, and beta-antagonists reduced parasitemia of P. berghei infections in an in vivo mouse model. Thus, signaling via the erythrocyte beta2-adrenergic receptor and Galphas may regulate malarial infection across parasite species.