ApiAP2 Factors as Candidate Regulators of Stochastic Commitment to Merozoite Production in Theileria annulata

Background

Differentiation of one life-cycle stage to the next is critical for survival and transmission of apicomplexan parasites. A number of studies have shown that stage differentiation is a stochastic process and is associated with a point that commits the cell to a change over in the pattern of gene expression. Studies on differentiation to merozoite production (merogony) in T. annulata postulated that commitment involves a concentration threshold of DNA binding proteins and an auto-regulatory loop.

Principal Findings

In this study ApiAP2 DNA binding proteins that show changes in expression level during merogony of T. annulata have been identified. DNA motifs bound by orthologous domains in Plasmodium were found to be enriched in upstream regions of stage-regulated T. annulata genes and validated as targets for the T. annulata AP2 domains by electrophoretic mobility shift assay (EMSA). Two findings were of particular note: the gene in T. annulata encoding the orthologue of the ApiAP2 domain in the AP2-G factor that commits Plasmodium to gametocyte production, has an expression profile indicating involvement in transmission of T. annulata to the tick vector; genes encoding related domains that bind, or are predicted to bind, sequence motifs of the type 5''-(A)CACAC(A) are implicated in differential regulation of gene expression, with one gene (TA11145) likely to be preferentially up-regulated via auto-regulation as the cell progresses to merogony.

Conclusions

We postulate that the Theileria factor possessing the AP2 domain orthologous to that of Plasmodium AP2-G may regulate gametocytogenesis in a similar manner to AP2-G. In addition, paralogous ApiAP2 factors that recognise 5''-(A)CACAC(A) type motifs could operate in a competitive manner to promote reversible progression towards the point that commits the cell to undergo merogony. Factors possessing AP2 domains that bind (or are predicted to bind) this motif are present in the vector-borne genera Theileria, Babesia and Plasmodium, and other Apicomplexa; leading to the proposal that the mechanisms that control stage differentiation will show a degree of conservation.     

Cryptosporidium parvum possesses a short-type replication protein A large subunit that differs from its host

Replication protein A (RPA) consisting of three subunits is a eukaryotic single-stranded DNA (ssDNA)-binding protein involved in DNA replication, repair and recombination. We report here the identification and characterization of a RPA large subunit (CpRPA1) gene from the apicomplexan Cryptosporidium parvum. The CpRPA1 gene encodes a 53.9-kDa peptide that is remarkably smaller than that from other eukaryotes (i.e. approximately 70 kDa) and is actively expressed in both free sporozoites and parasite intracellular stages. This short-type RPA large subunit has also been characterized from one other protist, Crithidia fasciculata. Three distinct domains have been identified in the RPA large subunit of humans and yeasts: an N-terminal protein interaction domain, a central ssDNA-binding area, and a C-terminal subunit-interacting region. Sequence analysis reveals that the short-type RPA large subunit differs from that of other eukaryotes in that only the domains required for ssDNA binding and heterotrimer formation are present. It lacks the N-terminal domain necessary for the binding of proteins mainly involved in DNA repair and recombination. This major structural difference suggests that the mechanism for DNA repair and recombination in some protists differs from that of other eukaryotes. Since replication proteins play an essential role in the cell cycle, the fact that RPA proteins of C. parvum differ from those of its host suggests that RPA be explored as a potential chemotherapeutic target for controlling cryptosporidiosis and/or diseases caused by other apicomplexans.     

The protozoan parasite Cryptosporidium parvum possesses two functionally and evolutionarily divergent replication protein A large subunits

Very little is known about protozoan replication protein A (RPA), a heterotrimeric complex critical for DNA replication and repair. We have discovered that in medically and economically important apicomplexan parasites, two unique RPA complexes may exist based on two different types of large subunit RPA1. In this study, we characterized the single-stranded DNA binding features of two distinct types (i.e. short and long) of RPA1 subunits from Cryptosporidium parvum (CpRPA1A and CpRPA1B). These two proteins differ from human RPA1 in their intrinsic single-stranded DNA binding affinity (K) and have significantly lower cooperativity (omega). We also identified the RPA2 and RPA3 subunits from C. parvum, the latter of which had yet to be reported to exist in any protozoan. Using fluorescence resonance energy transfer technology and pull-down assays, we confirmed that these two subunits interact with each other and with CpRPA1A and CpRPA1B. This suggests that the heterotrimeric structure of RPA complexes may be universally conserved from lower to higher eukaryotes. Bioinformatic analyses indicate that multiple types of RPA1 are present in the other apicomplexans Plasmodium and Toxoplasma. Apicomplexan RPA1 proteins are phylogenetically more related to plant homologues and probably arose from a single gene duplication event prior to the expansion of the apicomplexan lineage. Differential expression during the life cycle stages in three apicomplexan parasites suggests that the two RPA1 types exercise specialized biological functions.     

Repeated secondary loss of adaptin complex genes in the Apicomplexa

The Apicomplexa include parasites of devastating medical and economic consequence. While obviously essential for their parasitic mechanism, the molecular machinery underpinning membrane-trafficking in many apicomplexans is poorly understood. One potentially key set of players, the adaptins, selects cargo for incorporation into trafficking vesicles. Four distinct adaptin (AP) complexes exist in eukaryotes; AP1 and AP3 are involved in transport between the trans-Golgi Network (TGN) and endosomes, AP4 in TGN to cell surface transport, and AP2 in endocytosis from the cell surface. Of particular interest is the involvement of AP1 in Toxoplasma rhoptry biogenesis. The recent completion of several apicomplexan genomes should jump-start molecular parasitological studies and provide systems-level insight into the apicomplexan adaptin machinery. However, many of the encoded adaptin proteins are annotated conservatively and not to the necessary complex or subunit level. Prompted by previous evidence suggesting the lack of AP3 in Plasmodium falciparum, we undertook homology-searching and phylogenetic analysis to produce a rigorously annotated set of adaptin subunits encoded in diverse apicomplexan genomes. We found multiple losses of adaptins across the phylum; in particular Theileria, Babesia, and Cryptosporidium, but surprisingly not Plasmodium, appear to have lost the entirety of the AP3 complex. The losses correlate with a degenerate Golgi body structure and are reminiscent of recently reported secondary losses of additional endocytic components (i.e. the ESCRTs) in several Apicomplexa. These data may indicate a relaxation of the selective pressure on the apicomplexan endocytic system and, regardless, should greatly facilitate future molecular cell biological investigation of the role of adaptins in these important parasites.     

A malaria parasite formin regulates actin polymerization and localizes to the parasite-erythrocyte moving junction during invasion

Malaria parasites invade host cells using actin-based motility, a process requiring parasite actin filament nucleation and polymerization. Malaria and other apicomplexan parasites lack Arp2/3 complex, an actin nucleator widely conserved across eukaryotes, but do express formins, another type of actin nucleator. Here, we demonstrate that one of two malaria parasite formins, Plasmodium falciparum formin 1 (PfFormin 1), and its ortholog in the related parasite Toxoplasma gondii, follows the moving tight junction between the invading parasite and the host cell, which is the predicted site of the actomyosin motor that powers motility. Furthermore, in vitro, the PfFormin1 actin-binding formin homology 2 domain is a potent nucleator, stimulating actin polymerization and, like other formins, localizing to the barbed end during filament elongation. These findings support a conserved molecular mechanism underlying apicomplexan parasite motility and, given the essential role that actin plays in cell invasion, highlight formins as important determinants of malaria parasite pathogenicity.     

Unusual properties of Plasmodium falciparum actin: new insights into microfilament dynamics of apicomplexan parasites

Plasmodium falciparum, the etiologic agent of malaria, is a facultative intracellular parasite of the phylum Apicomplexa. A limited turnover of microfilaments takes place beneath the parasite plasma membrane, but the cytoplasm of apicomplexans is virtually devoid of F-actin. We produced Plasmodium actin in yeast. Purified recombinant Plasmodium actin polymerized inefficiently unless both gelsolin and phalloidin were added. The resulting actin polymers appeared fragmented in the fluorescence microscope. Plasmodium actin bound DNaseI about 200 times weaker than bovine non-muscle actin. Our findings suggest that the unique properties of Plasmodium actin can explain some of the unusual features of apicomplexan parasite microfilaments.     

Reduction and compaction in the genome of the apicomplexan parasite Cryptosporidium parvum

The complete genome of the apicomplexan parasite Cryptosporidium parvum reveals many new insights into apicomplexan biology and evolution, as well as the general process of genome reduction in parasites. The genome is globally compacted, but gene loss seems to be focused, in particular in relation to organelles. Massive losses of mitochondrial genes have taken place and there is no evidence of any plastid-related genes, providing a useful tool for examining putative plastid proteins in Plasmodium and other apicomplexans.     

Evolution of Cryptosporidium parvum lactate dehydrogenase from malate dehydrogenase by a very recent event of gene duplication

We have expressed the L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (malDH) genes from the apicomplexan Cryptosporidium parvum (CpLDH1 and CpMalDH1) as maltose-binding protein (MBP) fusion proteins in Escherichia coli. The substrate specificities, enzymatic kinetics, and oligomeric states of these two parasite enzymes have been characterized. By taking advantage of recently completed and ongoing apicomplexan genome sequencing projects, we identified additional MalDH genes from Plasmodium spp., Toxoplasma gondii, and Eimeria tenella that were previously unavailable. All apicomplexan MalDHs appeared to be cytosolic and no organellar homologs were identified from the completely sequenced P. falciparum genome and other ongoing apicomplexan genome-sequencing projects. Using these expanded apicomplexan LDH and MalDH sequence databases, we reexamined their phylogenetic relationships and reconfirmed their relationship to alpha-proteobacterial MalDHs. All LDH and MalDH enzymes from apicomplexans were monophyletic within the LDH-like MalDH group (i.e., MalDH resembling LDH) as a sister to alpha-proteobacterial MalDHs. All apicomplexan LDHs, with the exception of CpLDH1, formed a separate clade from their MalDH counterparts, indicating that these LDHs were evolved from an ancestral apicomplexan MalDH by a gene duplication coupled with functional conversion before the expansion of apicomplexans. Finally, CpLDH1 was consistently placed together with CpMalDH1 within the apicomplexan MalDH cluster, confirming an early working hypothesis that CpLDH1 was probably evolved from the same ancestor of CpMalDH1 by a very recent gene duplication that occurred after C. parvum diverged from other apicomplexans.     

The non-photosynthetic plastid in malarial parasites and other apicomplexans is derived from outside the green plastid lineage.

The discovery of a non-photosynthetic plastid genome in Plasmodium falciparum and other apicomplexans has provided a new drug target, but the evolutionary origin of the plastid has been muddled by the lack of characters, that typically define major plastid lineages. To clarify the ancestry of the plastid, we undertook a comprehensive analysis of all genomic characters shared by completely sequenced plastid genomes. Cladistic analysis of the pattern of plastid gene loss and gene rearrangements suggests that the apicomplexan plastid is derived from an ancestor outside of the green plastid lineage. Phylogenetic analysis of primary sequence data (DNA and amino acid characters) produces results that are generally independent of the analytical method, but similar genes (i.e., rpoB and rpoC) give similar topologies. The conflicting phylogenies in primary sequence data sets make it difficult to determine the the exact origin of the apicomplexan plastid and the apparent artifactual association of apicomplexan and euglenoid sequences suggests that DNA sequence data may be an inappropriate set of characters to address this phylogenetic question. At present we cannot reject our null hypothesis that the apicomplexan plastid is derived from a shared common ancestor between apicomplexans and dinoflagellates. During the analysis, we noticed that the Plasmodium tRNA-Met is probably tRNA-fMet and the tRNA-fMet is probably tRNA-Ile. We suggest that P. falciparum has lost the elongator type tRNA-Met and that similar to metazoan mitochondria there is only one species of methionine tRNA. In P. falciparum, this has been accomplished by recruiting the fMet-type tRNA to dually function in initiation and elongation. The tRNA-Ile has an unusual stem-loop in the variable region. The insertion in this region appears to have occurred after the primary origin of the plastid and further supports the monophyletic ancestory of plastids.