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In vitro cultivation of the vascular phase of Sarcocystis capracanis and Sarcocystis tenella.

Sporozoites of Sarcocystis capracanis and S. tenella (Apicomplexa) penetrated all four cell types tested (bovine monocytes, BM; bovine pulmonary artery endothelial cells, CPA; Madin-Darby bovine kidney; and ovine monocytes). Sporozoites of S. tenella developed to meronts in BM and CPA; those of S. capracanis developed to meronts in BM only. Both species of Sarcocystis developed to large first-generation meronts followed by small meronts. At 40 to 50 days after inoculation (DAI) of sporozoites, considerably more merozoites of S. tenella were harvested from CPA (24.9 X 10(6) merozoites/75-cm2 flask; n = 4) than from BM (1.9 X 10(6) merozoites/75-cm2 flask; n = 4). Merozoites of S. capracanis were most numerous in BM at 88 to 100 DAI during which time 2.1 X 10(6) merozoites/75-cm2 flask (n = 4) were harvested.

Animals↗

Antigenic relationship between Plasmodium falciparum and Babesia bovis: reactivity with antibodies to culture-derived soluble exoantigens.

Antigenic similarities between Plasmodium and Babesia parasites of the phylum Apicomplexa have been previously demonstrated primarily by the serological cross reactivity observed in the indirect fluorescent antibody (IFA) test. We have now studied the antigenic relationship between the human malaria parasite, Plasmodium falciparum, and the hemoparasitic agent of cattle, Babesia bovis, using rabbit monospecific antibodies produced against individual culture-derived P. falciparum polypeptides and bovine polyspecific antibodies to B. bovis exoantigens. These respective antibodies were found to be distinctly cross reactive in the IFA test using infected erythrocytes (squirrel monkey--P. falciparum; bovine--B. bovis) as antigen substrates. Immunofluorescence was shown to be highly specific for parasite surfaces. Additionally, the degree of reactivity with soluble exoantigens contained in Plasmodium and Babesia culture supernatants was monitored by a two-site enzyme immunoassay employing the cross-reactive antibodies. Further evidence for antigenic cross reactivity between P. falciparum and B. bovis parasites was shown with the in vitro inhibition assay. Antibodies to P. falciparum and B. bovis were found to be highly inhibitory for the in vitro growth of P. falciparum in human erythrocytes.

Animals↗

The number of species of rodent coccidia and of other protozoa.

About 447 species of coccidia have been named from the 1687 living, known species of rodents; 207 host species, 92 host genera, and 15 host families are represented; this is about 12% of the known species of rodents. About 4600 species of apicomplexan protozoa have been named. Assuming that the same proportion of the total number of apicomplexan species has been named as of the coccidian species, there must actually be about 38,333 species of apicomplexan protozoa. There are 5.4 times as many protozoan genera as of apicomplexan genera. Assuming that the number of species in each genus is the same for all the protozoa as it is for the Apicomplexa, there may actually be 206,998 species of protozoa. This may be too conservative an estimate. Based on other criteria, an estimate of over 20 million species could be made.

Animals↗

Molecular factors responsible for host cell recognition and invasion in Plasmodium falciparum.

In Plasmodium falciparum, the rhoptries involved in the invasion process are a pair of flask-shaped organelles located at the apical tip of invading stages. They, along with the more numerous micronemes and dense granules, constitute the apical complex in Plasmodium and other members of the phylum Apicomplexa. Several proteins of varying molecular weight have been identified in P. falciparum rhoptries. These include the 225-, 140/130/110-, 80/60/40-, RAP-1 80-, AMA-1 80-, QF3 80-, and 55-kDa proteins. Some of these proteins are lost during schizont rupture and release of merozoites. Others such as the 140/130/110-kDa complex are transferred to the erythrocyte membrane during invasion. The ring-infected surface antigen (RESA), a 155-kDa polypeptide located in dense granules also associates with the erythrocyte membrane during invasion. Erythrocyte-binding studies have demonstrated that both the 140/130/110-kDa rhoptry complex and RESA bind to inside-out-vesicles (IOVs) prepared from human erythrocytes. The 140/130/110-kDa complex also binds to erythrocyte membranes prepared by hypotonic lysis. These proteins, however, do not bind to intact human erythrocytes. In a heterologous erythrocyte model, both the 140/130/110-kDa complex and RESA are shown to bind directly to mouse erythrocytes. Other studies have shown that RESA associates with spectrin in the erythrocyte cytoskeleton. We have recently developed a liposome-binding assay to demonstrate the lipophilic binding properties of the P. falciparum rhoptry complex of 140/130/110 kDa. The rhoptry complex binds to liposomes containing neutrally, positively, and negatively charged phospholipids. However, liposomes containing phosphatidylethanolamine compete effectively for rhoptry protein binding to mouse erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Plastids and protein targeting.

Plastids with two bounding membranes--as exemplified by red algae, green algae, plants, and glaucophytes--derive from primary endosymbiosis; a process involving engulfment and retention of a cyanobacterium by a phagotrophic eukaryote. Plastids with more than two bounding membranes (such as those of euglenoids, dinoflagellates, heterokonts, haptopytes, apicomplexa, cryptomonads, and chlorarachniophytes) probably arose by secondary endosymbiosis, in which a eukaryotic alga (itself the product of primary endosymbiosis) was engulfed and retained by a phagotroph. Secondary endosymbiosis transfers photosynthetic capacity into heterotrophic lineages, has apparently occurred numerous times, and has created several major eukaryotic lineages comprising upwards of 42,600 species. Plastids acquired by secondary endosymbiosis are sometimes referred to as "second-hand." Establishment of secondary endosymbioses has involved transfer of genes from the endosymbiont nucleus to the secondary host nucleus. Limited gene transfer could initially have served to stabilise the endosymbioses, but it is clear that the transfer process has been extensive, leading in many cases to the complete disappearance of the endosymbiont nucleus. One consequence of these gene transfers is that gene products required in the plastid must be targeted into the organelle across multiple membranes: at least three for stromal proteins in euglenoids and dinoflagellates, and across five membranes in the case of thylakoid lumen proteins in plastids with four bounding membranes. Evolution of such targeting mechanisms was obviously a key step in the successful establishment of each different secondary endosymbiosis. Analysis of targeted proteins in the various organisms now suggests that a similar system is used by each group. However, rather than interpreting this similarity as evidence of an homologous origin, I believe that targeting has evolved convergently by combining and recycling existing protein trafficking mechanisms already existing in the endosymbiont and host. Indeed, by analyzing the multiple motifs in targeting sequences of some genes it is possible to infer that they originated in the plastid genome, transferred from there into the primary host nucleus, and subsequently moved into the secondary host nucleus. Thus, each step of the targeting process in "second-hand" plastids recapitulates the gene's previous intracellular transfers.

Animals↗

Babesia canis canis, Babesia canis vogeli, Babesia canis rossi: differentiation of the three subspecies by a restriction fragment length polymorphism analysis on amplified small subunit ribosomal RNA genes.

The parasites Babesia canis and Babesia gibsoni (phylum Apicomplexa) are responsible for canine babesiosis throughout the world. Babesia canis was previously described as a group of three biologically different subspecies, namely B. canis canis, B. canis vogeli, and B. canis rossi. We report partial sequences of small subunit ribosomal RNA gene (ssu-rDNA) of each subspecies amplified in vitro with primers derived from a semi-conserved region of the ssu-rDNA genes in other Babesia species. The polymerase chain reaction combined with a restriction fragment length polymorphism analysis, using HinfI and TaqI restriction enzymes, confirmed the separation of B. canis into three subspecies. These sequences were compared with previously published sequences of other Babesia species. A phylogenetic approach showed that the three subspecies of B. canis belong to the clade of Babesia species sensu stricto where B. canis canis clusters with B. canis rossi whereas B. canis vogeli might form a monophyletic group with the cluster B. divergens and B. odocoilei. Our results show that the three subspecies of B. canis can readily be differentiated at the molecular level and suggest that they might be considered as true species.

Animals↗

Characterization of an alpha-tubulin gene of Cryptosporidium parvum.

A gene encoding an alpha-tubulin of Cryptosporidium parvum was isolated and characterized. It had no introns, and encoded a 441-amino acid protein whose predicted ORF represented a typical alpha-tubulin protein with a MW of 50.5 kDa. This tubulin had an amino acid sequence similarity with Apicomplexa Plasmodium falciparum and Toxoplasma gondii higher than 88% and shared a number of conserved motifs.

Animals↗

Alpha-proteobacterial relationship of apicomplexan lactate and malate dehydrogenases.

We have cloned and sequenced a lactate dehydrogenase (LDH) gene from Cryptosporidium parvum (CpLDH1). With this addition, and that of four recently deposited alpha-proteobacterial malate dehydrogenase (MDH) genes, the phylogenetic relationships among apicomplexan LDH and bacterial MDH were re-examined. Consistent with previous studies, our maximum likelihood (ML) analysis using the quartet-puzzling method divided 105 LDH/MDH enzymes into five clades, and confirmed that mitochondrial MDH is a sister clade to those of y-proteobacteria, rather than to alpha-proteobacteria. In addition, a Cryptosporidium parvum MDH (CpMDH1) was identified from the ongoing Cryptosporidium genome project that appears to belong to a distinct clade (III) comprised of 22 sequences from one archaebacterium, numerous eubacteria, and several apicomplexans. Using the ML puzzling test and bootstrapping analysis with protein distance and parsimony methods, the resulting trees not only robustly confirmed the alpha-proteobacterial relationship of apicomplexan LDH/MDH, but also supported a monophyletic relationship of CpLDH1 with CpMDHI. These data suggest that, unlike most other eukaryotes, the Apicomplexa may be one of the few lineages retaining an alpha-proteobacterial-type MDH that could have been acquired from an ancestral alpha-proteobacterium through primary endosymbiosis giving rise to the mitochondria, or through an unknown lateral gene transfer (LGT) event.

Alphaproteobacteria↗

Identification of a second rRNA gene unit in the Perkinsus andrewsi genome.

Perkinsus species are parasitic protozoa of mollusks, currently classified within the Perkinsozoa, a recently established phylum that is basal to the Apicomplexa and Dinozoa. Ribosomal RNA (rRNA) genes and their intergenic spacers have been used to support the taxonomy of Perkinsus species, the description of new species, and to develop molecular probes for their detection and identification. We previously described ultrastructure, behavior in culture, and partial sequence of the rRNA locus of a Perkinsus species isolated from the baltic clam Macoma balthica. The rRNA genes and intergenic spacers of this Perkinsus isolate differed from those described in the currently accepted species to a degree that led to its designation as a new species, Perkinsus andrewsi. In this study, we identify an additional rRNA gene unit (rRNA-B) in the P. andrewsi holotype, and report the complete sequences of both rRNA gene units. Except for the 5.8S, all regions of the rRNA-B gene unit exhibited sequence differences from that initially described (rRNA-A). Each rRNA gene unit is arranged in a "head-to-tail" tandem repeat. This is the first report demonstrating two distinct rRNA units in a Perkinsus species.

Animals↗

Ellobiopsids of the genus Thalassomyces are alveolates.

Ellobiopsids are multinucleate protist parasites of aquatic crustaceans that possess a nutrient absorbing 'root' inside the host and reproductive structures that protrude through the carapace. Ellobiopsids have variously been affiliated with fungi, 'colorless algae', and dinoflagellates, although no morphological character has been identified that definitively allies them with any particular eukaryotic lineage. The arrangement of the trailing and circumferential flagella of the rarely observed bi-flagellated 'zoospore' is reminiscent of dinoflagellate flagellation, but a well-organized 'dinokaryotic nucleus' has never been observed. Using small subunit ribosomal RNA gene sequences from two species of Thalassomyces, phylogenetic analyses robustly place these ellobiopsid species among the alveolates (ciliates, apicomplexans, dinoflagellates and relatives) though without a clear affiliation to any established alveolate lineage. Our trees demonstrate that Thalassomyces fall within a dinoflagellate + apicomplexa + Perkinsidae + "marine alveolate group 1" clade, clustering most closely with dinoflagellates. However, the poor statistical support for branches within this region indicates that additional data will be needed to resolve relationships among these taxa.

Animals↗

Location, location, location: trafficking and function of secreted proteases of Toxoplasma and Plasmodium.

The Apicomplexan parasites Toxoplasma gondii and Plasmodium species are obligate intracellular parasites that rely upon unique secretory organelles for invasion and other specialized functions. Data is emerging that proteases are critical for the biogenesis of micronemes and rhoptries, regulated secretory organelles reminiscent of dense core granules and secretory lysosomes of higher eukaryotes. Proteases targeted to the Plasmodium food vacuole, a unique organelle dedicated to hemoglobin degradation, are also critical to parasite survival. Thus study of the targeting and function of the proteases of the Apicomplexa provides a fascinating model system to understand regulated secretion and secretory organelle biogenesis.

Animals↗

Phosphoproteomic Analysis of GAP50-Deficient Parasites Reveals the Inner Membrane Complex Is Involved in Ion Regulation in Toxoplasma gondii.

The Alveolata group of organisms is characterized by a vesicular structure located beneath the plasma membrane. In apicomplexa, this structure is known as the inner membrane complex (IMC). The IMC acts as a scaffold during the budding of the daughter parasite and is utilized as a rigid base for the glideosome. In this study, we discovered that the phosphatase activity of GAP50 is crucial for its function in the biogenesis of the IMC. Through further phosphoproteomic analysis of parasites lacking GAP50, we identified that an NIPA family transporter is localized in the IMC. Knocking out TgNIPA1 significantly impairs the robust growth of the parasites. Additionally, expressing TgNIPA1 in a Salmonella strain lacking magnesium transporters could rescue the growth phenotype of bacteria under low magnesium conditions, indicating that it functions as an active magnesium transporter. Our results strongly suggest that the flattened vesicles of the IMC may play important roles in ion regulation in T. gondii.

Toxoplasma↗

Polymorphic secreted kinases are key virulence factors in toxoplasmosis.

The majority of known Toxoplasma gondii isolates from Europe and North America belong to three clonal lines that differ dramatically in their virulence, depending on the host. To identify the responsible genes, we mapped virulence in F(1) progeny derived from crosses between type II and type III strains, which we introduced into mice. Five virulence (VIR) loci were thus identified, and for two of these, genetic complementation showed that a predicted protein kinase (ROP18 and ROP16, respectively) is the key molecule. Both are hypervariable rhoptry proteins that are secreted into the host cell upon invasion. These results suggest that secreted kinases unique to the Apicomplexa are crucial in the host-pathogen interaction.

Alleles↗

Ligands of the peripheral benzodiazepine receptor are potent inhibitors of Plasmodium falciparum and Toxoplasma gondii in vitro.

The increase in resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands the development of new antimalarial agents. In this quest, we have found that ligands to the peripheral benzodiazepine receptor such as flurazepam, an agonist of the benzodiazepine family, and PK11195, an antagonist derived from isoquinoline, were active against Plasmodium falciparum. These two compounds effectively and rapidly inhibited parasite growth in vitro, irrespective of parasite resistance to chloroquine and mefloquine. Treatment with both drugs induced a sharp and consistent decline in parasitemia, a complete inhibition of parasite replication, and the destruction of parasites within the host red blood cells. Using electron microscopy, we showed that dramatic morphological changes, involving swollen endoplasmic reticulum and the reduction of hemozoin, were consistent with parasite death. The potent activities of flurazepam and PK11195 were also evaluated for antagonist or synergistic effects with currently used antimalarial drugs such as chloroquine and mefloquine. Moreover, flurazepam was found to be active against Toxoplasma gondii, another member of the phylum Apicomplexa. Taken together, our results indicated that benzodiazepines could be considered promising candidates in the treatment of both malaria and toxoplasmosis.

Animals↗

Antileishmanial activity of the terpene nerolidol.

The activity of nerolidol, a sesquiterpene used as a food-flavoring agent and currently under testing as a skin penetration enhancer for the transdermal delivery of therapeutic drugs, was evaluated against Leishmania species. Nerolidol inhibited the growth of Leishmania amazonensis, L. braziliensis, and L. chagasi promastigotes and L. amazonensis amastigotes with in vitro 50% inhibitory concentrations of 85, 74, 75, and 67 microM, respectively. The treatment of L. amazonensis-infected macrophages with 100 microM nerolidol resulted in 95% reduction in infection rates. Inhibition of isoprenoid biosynthesis, as shown by reduced incorporation of [2-(14)C]mevalonic acid (MVA) or [1-(14)C]acetic acid precursors into dolichol, ergosterol, and ubiquinone, was observed in nerolidol-treated promastigotes. This drug effect can be attributed to the blockage of an early step in the mevalonate pathway, since incorporation of the precursor [1(n)-(3)H]farnesyl pyrophosphate in polyisoprenoids is not inhibited by nerolidol. L. amazonensis-infected BALB/c mice were treated with intraperitoneal doses of 100 mg/kg/day for 12 days or topically with 5 or 10% ointments for 4 weeks. Significant reduction of lesion sizes in nerolidol treated mice was observed for both treatment routes. However, long-term follow up indicated that the disease was not cured in this highly susceptible animal model. Nonetheless, the in vitro activity of nerolidol against these parasites may prove a useful tool for the development of new drugs for the treatment of leishmaniasis. In addition, biosynthesis of dolichols with 11 and 12 isoprene units was identified in Leishmania, as described for other trypanosomatids and Apicomplexa.

Animals↗

Molecular karyotype analysis of Cryptosporidium parvum: evidence for eight chromosomes and a low-molecular-size molecule.

We report improved separation of chromosome-sized DNA molecules of the coccidian parasite Cryptosporidium parvum with contour-clamped homogeneous electric fields (CHEF). We used scanning densitometry to determine that the most likely number of chromosomes is eight. Molecular probes consisting of cloned genes were used to distinguish each of five bands visible on CHEF gels. We have also identified a low-molecular-size DNA molecule possibly related to the 35-kb circular DNAs found in other Apicomplexa.

Animals↗

Plasmodium falciparum AMA1 binds a rhoptry neck protein homologous to TgRON4, a component of the moving junction in Toxoplasma gondii.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) coimmunoprecipitates with the Plasmodium homologue of TgRON4, a secreted rhoptry neck protein of Toxoplasma gondii that migrates at the moving junction in association with TgAMA1 during invasion. PfRON4 also originates in the rhoptry necks, suggesting that this unusual collaboration of micronemes and rhoptries is a conserved feature of Apicomplexa.

Animals↗

Multiple functionally redundant signals mediate targeting to the apicoplast in the apicomplexan parasite Toxoplasma gondii.

Most species of the protozoan phylum Apicomplexa harbor an endosymbiotic organelle--the apicoplast--acquired when an ancestral parasite engulfed a eukaryotic plastid-containing alga. Several hundred proteins are encoded in the parasite nucleus and are posttranslationally targeted to the apicoplast by a distinctive bipartite signal. The N-terminal 20 to 30 amino acids of nucleus-encoded apicoplast targeted proteins function as a classical signal sequence, mediating entry into the secretory pathway. Cleavage of the signal sequence exposes a transit peptide of variable length (50 to 200 amino acids) that is required for directing proteins to the apicoplast. Although these peptides are enriched in basic amino acids, their structural and functional characteristics are not well understood, which hampers the identification of apicoplast proteins that may constitute novel chemotherapeutic targets. To identify functional domains for a model apicoplast transit peptide, we generated more than 80 deletions and mutations throughout the transit peptide of Toxoplasma gondii ferredoxin NADP+ reductase (TgFNR) and examined the ability of these altered transit peptides to mediate proper targeting and processing of a fluorescent protein reporter. These studies revealed the presence of numerous functional domains. Processing can take place at multiple sites in the protein sequence and may occur outside of the apicoplast lumen. The TgFNR transit peptide contains at least two independent and functionally redundant targeting signals, each of which contains a subdomain that is required for release from or proper sorting within the endoplasmic reticulum. Certain deletion constructs traffic to multiple locations, including the apicoplast periphery, the rhoptries, and the parasitophorous vacuole, suggesting a common thread for targeting to these specialized compartments.

Amino Acid Sequence↗