Apicomplexan Species Research Articles

Building the Perfect Parasite: Cell Division in Apicomplexa

Apicomplexans are pathogens responsible for malaria, toxoplasmosis, and crytposporidiosis in humans, and a wide range of livestock diseases. These unicellular eukaryotes are stealthy invaders, sheltering from the immune response in the cells of their hosts, while at the same time tapping into these cells as source of nutrients. The complexity and beauty of the structures formed during their intracellular development have made apicomplexans the darling of electron microscopists. Dramatic technological progress over the last decade has transformed apicomplexans into respectable genetic model organisms. Extensive genomic resources are now available for many apicomplexan species. At the same time, parasite transfection has enabled researchers to test the function of specific genes through reverse and forward genetic approaches with increasing sophistication. Transfection also introduced the use of fluorescent reporters, opening the field to dynamic real time microscopic observation. Parasite cell biologists have used these tools to take a fresh look at a classic problem: how do apicomplexans build the perfect invasion machine, the zoite, and how is this process fine-tuned to fit the specific niche of each pathogen in this ancient and very diverse group? This work has unearthed a treasure trove of novel structures and mechanisms that are the focus of this review.

Widespread Intron Loss Suggests Retrotransposon Activity in Ancient Apicomplexans

Several facets of spliceosomal intron in apicomplexans remain mysterious. First, intron numbers vary across species by 2 orders of magnitude, indicating massive intron loss and/or gain. Second, previous studies have shown very different evolutionary patterns over different timescales, with 100-fold higher rates of intron loss/gain between genera than within genera. Third, the timing and dynamics of nearly complete intron loss in Cryptosporidium species, as well as reasons for retention of the few remaining introns, remain unknown. We compared intron positions in 785 orthologous genes between 3 moderate to intron-rich apicomplexan species. We estimate that the Theileria-Plasmodium ancestor had 4.5 times as many introns as modern Plasmodium species and 38% more than modern Theileria species, and that subsequent intron losses have outnumbered intron gains by 5.8 to 1 in Theileria and by some 56 to 1 in Plasmodium. Several patterns suggest that these intron losses occurred by recombination with reverse-transcribed mRNAs. Intriguingly, this finding suggests significant retrotransposon activity in the lineages leading to both Theileria and Plasmodium, in contrast to the dearth of known retrotransposons and intron loss within modern species from both genera. We also compared genomes from Cryptosporidium parvum and C. hominis and found no evidence of ongoing intron loss, nor of intron gain. By contrast, Cryptosporidium introns are less evolutionary conserved with Toxoplasma than are introns from other apicomplexans; thus the few remaining introns are not simply indispensable ancestral introns.

The methylerythritol phosphate pathway for isoprenoid biosynthesis in coccidia: Presence and sensitivity to fosmidomycin

The apicoplast is a recently discovered, plastid-like organelle present in most apicomplexa. The methylerythritol phosphate (MEP) pathway involved in isoprenoid biosynthesis is one of the metabolic pathways associated with the apicoplast, and is a new promising therapeutic target in Plasmodium falciparum. Here, we check the presence of isoprenoid genes in four coccidian parasites according to genome database searches. Cryptosporidium parvum and C. hominis, which have no plastid genome, lack the MEP pathway. In contrast, gene expression studies suggest that this metabolic pathway is present in several development stages of Eimeria tenella and in tachyzoites of Toxoplasma gondii. We studied the potential of fosmidomycin, an antimalarial drug blocking the MEP pathway, to inhibit E. tenella and T. gondii growth in vitro. The drug was poorly effective even at high concentrations. Thus, both fosmidomycin sensitivity and isoprenoid metabolism differs substantially between apicomplexan species.

Abundance of intrinsically unstructured proteins in P. falciparum and other apicomplexan parasite proteomes

Preliminary sequence analysis of Plasmodium falciparum has shown that the proteome of this organism is enriched in intrinsically unstructured proteins (IUPs), which are either completely disordered or contain large disordered regions. IUPs have been characterized as a unique class of proteins that plays an important role in biology and disease. In this study, the IUP contents in the proteomes of apicomplexan parasites, especially the proteome of P. falciparum and its various life cycle stages, have been evaluated with DisEMBL-1.4. Compared with other proteomes, apicomplexan species are extremely abundant in proteins containing long disordered regions, and the IUP contents in mammalian Plasmodium species are higher than in most other apicomplexan parasites. The proteome of the P. falciparum sporozoite appears to be distinct from the other life cycle stages in having an even higher content of disordered proteins. The abundance of IUPs in the P. falciparum proteome correlates with its enrichment in repetitive sequences. The structural plasticity of IUPs, which allows promiscuous binding interactions, may favour parasite survival both by inhibiting the generation of effective high affinity antibody responses and by facilitating the interactions with host molecules necessary for attachment and invasion of host cells.

Population genetic analysis and sub-structuring of Theileria parva in Uganda

In recent years the population structures of many apicomplexan parasites including Plasmodium spp., Toxoplasma gondii and Cryptospordium parvum have been elucidated. These species show a considerable diversity of population structure suggesting different strategies for transmission and survival in mammalian hosts. We have undertaken a population genetic analysis of another apicomplexan species ( Theileria parva) to investigate the levels of diversity of this parasite and the role of genetic exchange in three geographically separate populations. The principal hindrance to carrying out such a study on field isolates was the high proportion of blood samples that contain multiple genotypes, making it impossible to determine the genotypes of the parasites directly. This problem was overcome by sampling only young indigenous calves between 3 and 9 months of age in which approximately 60% of the T. parva infected calves contained a single/predominant allele at each locus, making it possible to undertake population genetic analyses. Blood samples were collected from calves in three geographically distinct regions of Uganda and were analysed using 12 polymorphic mini and microsatellite markers that were evenly dispersed across the four chromosomes. We have identified 84 multilocus genotypes (MLG) from these samples, indicating high levels of diversity in the parasite. Analysis of linkage disequilibrium between pairs of loci provides evidence that the population in Lira district had an epidemic structure. The population in Mbarara was substructured containing two genetically distinct sub-groups and the larger sub-group also had an epidemic population structure. The population from Kayunga was in linkage disequilibrium. Genetic distances and Wrights fixation index ( F ST) indicate that there is evidence for geographical sub-structuring between the Lira and the Kayunga populations.

Molecular and functional aspects of parasite invasion

Apicomplexan parasites have evolved an efficient mechanism to gain entry into non-phagocytic cells, hence challenging their hosts by the establishment of infection in immuno-privileged tissues. Gliding motility is a prerequisite for the invasive stage of most apicomplexans, allowing them to migrate across tissues, and actively invade and egress host cells. In the late 1960s, detailed morphological studies revealed that motile apicomplexans share an elaborate architecture comprising a subpellicular cytoskeleton and apical organelles. Since 1993, the development of technologies for transient and stable transfection have provided powerful tools with which to identify gene products associated with these structures and organelles, as well as to understand their functions. In combination with access to several parasite genomes, it is now possible to compare and contrast the strategies and molecular machines that have been selectively designed by distinct life stages within a species, or by different apicomplexan species, to optimize infection.

Pathology associated with endogenous development of haematozoa in birds from southeast Queensland

A study was undertaken on the pathology and associated schizont morphology of apicomplexan species of avian haematozoa. Some 32 birds from the families Artamidae, Meliphagidae, Oriolidae, Podargidae, Columbidae, Alcedinidae and Psittacidae were identified as having schizonts in various tissues. Based on blood stages observed, the probable relationship to tissue stages was considered. The majority of schizonts were referable to the genera Leucocytozoon and Haemoproteus. The comparative morphology of tissue stages previously described in the literature is discussed and the involvement of protozoa other than haematozoa considered. The naturally occurring infections in wild birds described in this study represent previously unreported data on the life-cycle stages involved. Some schizonts measured up to 640 μm. While pathological changes in some hosts were noticeable, in others no significant findings were observed. The role of endogenous stages in avian morbidity is discussed briefly.

Purine Salvage Pathways in the Apicomplexan Parasite Toxoplasma gondii

We have exploited a variety of molecular genetic, biochemical, and genomic techniques to investigate the roles of purine salvage enzymes in the protozoan parasite Toxoplasma gondii. The ability to generate defined genetic knockouts and target transgenes to specific loci demonstrates that T. gondii uses two (and only two) pathways for purine salvage, defined by the enzymes hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK). Both HXGPRT and AK are single-copy genes, and either one can be deleted, indicating that either one of these pathways is sufficient to meet parasite purine requirements. Fitness defects suggest both pathways are important for the parasite, however, and that the salvage of adenosine is more important than salvage of hypoxanthine and other purine nucleobases. HXGPRT and AK cannot be deleted simultaneously unless one of these enzymes is provided in trans, indicating that alternative routes of functionally significant purine salvage are lacking. Despite previous reports to the contrary, we found no evidence of adenine phosphoribosyltransferase (APRT) activity when parasites were propagated in APRT-deficient host cells, and no APRT ortholog is evident in the T. gondii genome. Expression of Leishmania donovani APRT in transgenic T. gondii parasites yielded low levels of activity but did not permit genetic deletion of both HXGPRT and AK. A detailed comparative genomic study of the purine salvage pathway in various apicomplexan species highlights important differences among these parasites.

The Cryptosporidium oocyst wall protein is a member of a multigene family and has a homolog in Toxoplasma.

Coccidian parasites are transmitted via a fecal oocyst stage that is exceptionally resistant to environmental stress and harsh chemical treatments, which allows parasites to stably persist outside a host. Because of its oocyst durability Cryptosporidium parvum is a significant water- and food-borne pathogen of humans, as well as animals of agricultural importance. To date, only one apicomplexan oocyst membrane protein has been identified, Cryptosporidium oocyst wall protein 1 (COWP1). COWP1 has a highly cysteine-rich periodicity due to arrays of two apicomplexan-specific motifs, designated the type I and type II domains. In this study, exhaustive BLAST screening of a complete C. parvum genome sequence database resulted in identification of eight additional genes encoding similar arrays of cysteine-rich type I and/or type II domains. Transcript expression analysis revealed that all COWP genes are abundantly expressed at a time when developing oocysts are observed, roughly 48 to 72 h after inoculation of in vitro cultures. A monoclonal antibody recognizing COWP8 specifically localized to the C. parvum oocyst wall, supporting the hypothesis that multiple COWPs play a role in the oocyst wall structure. BLAST screening of the Toxoplasma gondii genome sequence database resulted in identification of a gene encoding at least one COWP homolog (TgOWP1), and this multiexon sequence information was used to isolate a full-length cDNA. Exhaustive screening of Plasmodium sp. genome sequence databases by using COWP genes as BLAST queries failed to detect similar proteins in PLASMODIUM: We therefore propose that the COWP family of proteins have a structural role in apicomplexan species that produce durable shed cysts capable of surviving environmental stress.

A new modular protein of Cryptosporidium parvum, with ricin B and LCCL domains, expressed in the sporozoite invasive stage.

The recombinant SA35 peptide has been described as an antigenic portion of a larger Cryptosporidium parvum protein. We identified and characterized the encoding Cpa135 gene and the entire protein, Cpa135. The Cpa135 gene was found to consist of a single exon of 4671 bp, and the mRNA transcribed in the sporozoites was identified. The predicted 1556 amino-acid protein showed the presence of domains which are widely conserved also in other unrelated phylogenetic groups (i.e. a ricin B and a LCCL motif). Comparison of Cpa135 sequence with genomic and protein databases revealed many related genes in other apicomplexan species and high homology with CCP2 protein from Plasmodium yoelii and Plasmodium berghei. The Cpa135 protein was identified and localized by using a monoclonal antibody (Mab) directed against the SA35 antigen (anti-SA35). In oocyst-sporozoite lysate, the anti-SA35 MAb recognized a 135 kDa protein that forms a protein complex larger than 200 kDa, which is mediated by disulfide bridges. Cpa135 synthesis was up-regulated during the excystation process. After host-cell invasion, Cpa135 gene expression was undetectable up to 48 h, whereas mRNA synthesis was newly observed at 72 h post-infection. The Cpa135 protein was localized in the apical complex, and it was found to be secreted by sporozoites during their gliding. Cpa135 persisted during the intracellular stages of the parasite, and it defined the boundaries of the parasitophorous vacuole in the infected cells. The unique array of domains and the homology with other apicomplexan proteins indicate that the Cpa135 protein is representative of a new family of proteins.

Molecular cloning of the 82-kDa heat shock protein (HSP90) of Toxoplasma gondii associated with the entry into and growth in host cells

Among the monoclonal antibodies (mAb) against Toxoplasma gondii, mAb Tg485 specifically reacted with an 82-kDa cytoplasmic protein of tachyzoites. The protein was secreted from extracellular tachyzoites, but was not released into the parasitophorous vacuole after invasion. The cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg485. The full-length cDNA was amplified by the 5′-RACE method and sequenced. The deduced amino acid sequence of the 82kDa protein reacting with Tg485 revealed a polypeptide of 708 amino acids showing significant homology to the heat shock protein 90 (HSP90) family of other organisms, especially to those of apicomplexan species. Treatment with geldanamycin, a drug known to interfere with HSP90 function, did not affect the secretion of TgHSP90 from extracellular tachyzoites, but the entry of the tachyzoites into host cells and the intracellular growth of the parasite were significantly disturbed.

Multiple Splice Variants Encode a Novel Adenylyl Cyclase of Possible Plastid Origin Expressed in the Sexual Stage of the Malaria Parasite Plasmodium falciparum

We report the characterization of an unusual adenylyl cyclase gene from Plasmodium falciparum, here designated PfACalpha. The level of mRNA expression is maximum during development of gametocytes (the sexual blood stage of the parasite life cycle). The gene is highly interrupted by 22 introns, and reverse transcriptase-PCR analysis revealed that there are multiple mRNA splice variants. One intron has three alternative 3'-splice sites that confer the potential to encode distinct forms of the enzyme using alternative start codons. Deduced amino acid sequences predict membrane-spanning regions, the number of which can vary between two and six depending on the splice variant. Expression of a synthetic form of two of these variants in Xenopus oocytes and in Dictyostelium adenylyl cyclase-deficient mutants, confirms that PfACalpha is a functional adenylyl cyclase. These results identify a novel mechanism in P. falciparum for the generation of multiple isoforms of a key, membrane-bound signaling molecule from a single genomic copy. Comparisons of the catalytic domains of PfACalpha and a second putative P. falciparum adenylyl cyclase (PfACbeta) with those from other species reveal an unexpected similarity with adenylyl cyclases from certain prokaryotes including the cyanobacteria (blue green algae). In addition, the presence of an unusual active site substitution in a position that determines substrate specificity, also characteristic of these prokaryotic forms of the enzyme, further suggests a plastid origin for the Plasmodium cyclases.

Population structures and the role of genetic exchange in the zoonotic pathogen Cryptosporidium parvum.

Apicomplexan protozoan parasites include some of the most globally important human and animal pathogens, all of which have obligatory sexual cycles in their definitive hosts. Despite their importance and the relevance of understanding the population genetic structure and role of genetic exchange in generating diversity, population genetic analysis has largely been restricted to Plasmodium spp. and Toxoplasma gondii. These species show a considerable diversity of population structure suggesting different strategies for transmission and survival in mammalian hosts. We have undertaken a population genetic analysis of a further apicomplexan species (Cryptosporidium parvum) to extend our understanding of the diversity of genetic structures and test whether it has a clonal population structure. Nothing is known about the population structure of this parasite. We have analyzed 180 parasite isolates from both humans and cattle derived from a single discrete geographical area, using three minisatellite and four microsatellite markers that define 38 multilocus genotypes. Analysis of linkage disequilibria between pairs of loci combined with measures of genetic distance and similarity provides evidence that the sample comprises four genetically isolated populations. One group of human isolates consists primarily of two closely related multilocus genotypes (clonal), while the major subtypes of a second group, common to both humans and animals, show a panmictic population structure. The data provide an important step in understanding the role of genetic exchange in these parasites, which is an essential prerequisite for determining the value of multilocus genotyping for the analysis of sources of human infection as well as future molecular epidemiological studies.

Molecular cloning of ribosomal P protein in Toxoplasma gondii and the availability to detect antibody against recombinant protein in toxoplasmosis patients.

Among the panel of monoclonal antibodies (mAb) against Toxoplasma gondii, mAb of Tg621 (Tg621) clone blotted 38 kDa protein which localized in the cytoplasm of tachyzoites by immunofluorescence microscopy. The protein was not released into the parasitophorous vacuole during or after invasion. The cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg621. The full length cDNA sequence was completed with 5'-RACE as 1,592 bp, which contained open reading frame of 942 bp. The deduced amino acid sequence of Tg621 consisted of a polypeptide of 313 amino acids, with significant homology to ribosomal P proteins (RPP) of other organisms especially high to those of apicomplexan species. The expressed and purified TgRPP was assayed in western blot with the sera of toxoplasmosis patients and normal sera, which resulted in the 74.0% of positive reactions in toxoplasmosis patients whereas 8.3% in normal group. Therefore, the antibody formation against TgRPP in toxoplasmosis patients was regarded as specific for T. gondii infection and suggested a potential autoantibody.

Mining the Plasmodium genome database to define organellar function: what does the apicoplast do?

Apicomplexan species constitute a diverse group of parasitic protozoa, which are responsible for a wide range of diseases in many organisms. Despite differences in the diseases they cause, these parasites share an underlying biology, from the genetic controls used to differentiate through the complex parasite life cycle, to the basic biochemical pathways employed for intracellular survival, to the distinctive cell biology necessary for host cell attachment and invasion. Different parasites lend themselves to the study of different aspects of parasite biology: Eimeria for biochemical studies, Toxoplasma for molecular genetic and cell biological investigation, etc. The Plasmodium falciparum Genome Project contributes the first large-scale genomic sequence for an apicomplexan parasite. The Plasmodium Genome Database (http://PlasmoDB.org) has been designed to permit individual investigators to ask their own questions, even prior to formal release of the reference P. falciparum genome sequence. As a case in point, PlasmoDB has been exploited to identify metabolic pathways associated with the apicomplexan plastid, or 'apicoplast' - an essential organelle derived by secondary endosymbiosis of an alga, and retention of the algal plastid.

Profiling the malaria genome: a gene survey of three species of malaria parasite with comparison to other apicomplexan species

We have undertaken the first comparative pilot gene discovery analysis of approximately 25 000 random genomic and expressed sequence tags (ESTs) from three species of Plasmodium, the infectious agent that causes malaria. A total of 5482 genome survey sequences (GSSs) and 5582 ESTs were generated from mung bean nuclease (MBN) and cDNA libraries, respectively, of the ANKA line of the rodent malaria parasite Plasmodium berghei, and 10 874 GSSs generated from MBN libraries of the Salvador I and Belem lines of Plasmodium vivax, the most geographically wide-spread human malaria pathogen. These tags, together with 2438 Plasmodium falciparum sequences present in GenBank, were used to perform first-pass assembly and transcript reconstruction, and non-redundant consensus sequence datasets created. The datasets were compared against public protein databases and more than 1000 putative new Plasmodium proteins identified based on sequence similarity. Homologs of previously characterized Plasmodium genes were also identified, increasing the number of P. vivax and P. berghei sequences in public databases at least 10-fold. Comparative studies with other species of Apicomplexa identified interesting homologs of possible therapeutic or diagnostic value. A gene prediction program, Phat, was used to predict probable open reading frames for proteins in all three datasets. Predicted and non-redundant BLAST-matched proteins were submitted to InterPro, an integrated database of protein domains, signatures and families, for functional classification. Thus a partial predicted proteome was created for each species. This first comparative analysis of Plasmodium protein coding sequences represents a valuable resource for further studies on the biology of this important pathogen.

Data mining parasite genomes: haystack searching with a computer.

A number of genomes of parasitic organisms are presently being sequenced in the public domain, including Plasmodium falciparum, Leishmania major and Trypanosoma brucei with the likelihood of at least expressed sequence tag (EST) projects for several filarial and apicomplexan species. The early and timely release of sequence data to the community via the World Wide Web (www), and the public databases, (EMBL and GENBANK), forms an invaluable resource. Data mining, or 'haystack searching' this resource is becoming more fruitful to all members of the scientific community as the volume of data, diversity of genomes sampled, and accessibility increase.

Protists on the Web

Apicomplexan parasites have evolved an efficient mechanism to gain entry into non-phagocytic cells, hence challenging their hosts by the establishment of infection in immuno-privileged tissues. Gliding motility is a prerequisite for the invasive stage of most apicomplexans, allowing them to migrate across tissues, and actively invade and egress host cells. In the late 1960s, detailed morphological studies revealed that motile apicomplexans share an elaborate architecture comprising a subpellicular cytoskeleton and apical organelles. Since 1993, the development of technologies for transient and stable transfection have provided powerful tools with which to identify gene products associated with these structures and organelles, as well as to understand their functions. In combination with access to several parasite genomes, it is now possible to compare and contrast the strategies and molecular machines that have been selectively designed by distinct life stages within a species, or by different apicomplexan species, to optimize infection.

Plastids are widespread and ancient in parasites of the phylum Apicomplexa

Current evidence supports the presence of a non-photosynthetic chloroplast-like organelle in several apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii. This apicomplexan organelle, referred to here as the plastid, may have been acquired through a primary or secondary endosymbiosis of a photosynthetic organism. Alternatively, apicomplexan plastids may have been acquired through several independent endosymbiotic events, as appears to be the case for the acquisition of chloroplasts by dinoflagellates. The likelihood of multiple origins of an apicomplexan plastid is enhanced by the close evolutionary relatedness of apicomplexan and dinoflagellate taxa. In this study, we have tested the hypothesis that apicomplexan plastids are derived from a single ancient ancestor. Two lines of evidence supporting this hypothesis are presented. First, this study supports the widespread presence of plastid DNA in apicomplexan species. Second, the topologies of the phylogenetic trees derived from plastid and nuclear-encoded rRNA gene sequences suggest the co-evolution of the DNAs localised in these two compartments. Taken together, these data support a single ancient lineage for the plastids of parasites in the phylum Apicomplexa.

Evaluation of grazing lands of Zimbabwe using the AVHRR normalised difference vegetation index

African Cape buffalo (Syncerus caffer) is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo) has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB) and sequencing of 18S rDNA genes, that in an area where buffalo and cattle co-graze and there is a heavy tick challenge, T. sp. (buffalo) can frequently be isolated in culture from cattle leukocytes. We also show that T. sp. (buffalo), which is genetically very closely related to T. parva, according to 18s rDNA sequence, has a conserved orthologue of the polymorphic immunodominant molecule (PIM) that forms the basis of the diagnostic ELISA used for T. parva serological detection. Closely related orthologues of several CD8 T cell target antigen genes are also shared with T. parva. By contrast, orthologues of the T. parva p104 and the p67 sporozoite surface antigens could not be amplified by PCR from T. sp. (buffalo), using conserved primers designed from the corresponding T. parva sequences. Collectively the data re-emphasise doubts regarding the value of rDNA sequence data alone for defining apicomplexan species in the absence of additional data. ‘Deep 454 pyrosequencing’ of DNA from two Theileria sporozoite stabilates prepared from Rhipicephalus appendiculatus ticks fed on buffalo failed to detect T. sp. (buffalo). This strongly suggests that R. appendiculatus may not be a vector for T. sp. (buffalo). Collectively, the data provides further evidence that T. sp. (buffalo). is a distinct species from T. parva.