Comparative analysis of stage specific gene regulation of apicomplexan parasites: Plasmodium falciparum and Toxoplasma gondii.

Apicomplexans comprise some of the most life threatening parasites infecting human and livestock and includes Plasmodium and Toxoplasma, the causative agents of malaria and toxoplasmosis respectively, in humans as well as Neospora caninum (abortion in livestock, neosporosis in dogs), Cryptosporidium (Diarrheal cryptosporidiosis and opportunistic infections in AIDS patients) and Eimeria (poultry coccidiosis). These parasites are characterized by a complex life cycle usually alternating between sexual and asexual cycles in different hosts. The need to adapt to different host environments demands a tight regulation of gene expression during parasite development. Therefore, the understanding of parasite biology will facilitate the control of the infection and the disease. In this review we emphasize the progress made so far in gene regulation in two medically important parasites, namely Plasmodium falciparum and Toxoplasma gondii, as well as other less known apicomplexan. The genome of both Plasmodium and Toxoplasma has been sequenced and since then there has been a significant progress in understanding the molecular mechanisms that control stage specific gene expression in the two parasites. In addition, the information gained in each of the parasite can be used in studying mechanisms that are still elusive in the other apicomplexans that are not readily available. Additionally, they can serve as model system for other disease causing Apicomplexan parasites.

Role of cis-regulatory elements on the ring-specific hrp3 promoter in the human parasite Plasmodium falciparum.

Identification of promoter elements responsible for regulation of gene expression has been hampered by the AT richness of P. falciparum intergenic regions. Nested deletions of histidine-rich protein 3 (hrp3) promoter suggested the presence of a multipartite ring-specific element. Linker scanning (LS) of this ring-specific promoter showed that the alteration of several promoter regions decreased the luciferase activity compared to the wild-type configuration, indicating that these regions played a role in gene expression. No homology was observed by comparison of putative regulatory elements of other genes identified by bioinformatic analysis with the hrp3 enhancer, implying a different mechanism of gene regulation by the hrp3 promoter. LS and deletion analysis of the 5' untranslated region (UTR) of the hrp3 suggested that this region contains elements which interact with promoter elements to regulate gene expression. Analysis of the intron in the UTR region suggested that this region does not play a role in stage specificity in the hrp3 promoter. Together, our results indicate the presence of multiple mechanisms of gene regulation in the parasite.

Plasmodium falciparum: Preinitiation complex occupancy of active and inactive promoters during erythrocytic stage.

Over 80% of Plasmodium falciparum genes are developmentally regulated during the parasite's life cycle with most genes expressed in a "just in time" fashion. However, the molecular mechanisms of gene regulation are still poorly understood. Analysis of P. falciparum genome shows that the parasite appears to encode relatively few transcription factors homologous to those in other eukaryotes. We used Chromatin immunoprecipitation (ChIP) to study interaction of PfTBP and PfTFIIE with stage specific Plasmodium promoters. Our results indicate that PfTBP and PfTFIIE are bound to their cognate sequence in active and inactive erythrocytic-expressed promoters. In addition, TF occupancy appears to extend beyond the promoter regions, since PfTBP interaction with the coding and 3' end regions was also detected. No PfTBP or PfTFIIE interaction was detected on csp and pfs25 genes which are not active during the erythrocytic asexual stage. Furthermore, PfTBP and PfTFIIE binding did not appear to correlate with histone 3 and/or 4 acetylation, suggesting that histone acetylation may not be a prerequisite for PfTBP or PfTFIIE promoter interaction. Based on our observations we concluded that the PfTBP/PfTFIIE-containing preinitiation complex (PIC) would be preassembled on promoters of all erythrocytic-expressed genes in a fashion independent of histone acetylation, providing support for the "poised" model. Contrary to the classical model of eukaryotic gene regulation, PIC interaction with Plasmodium promoters occurred independent of transcriptional activity and to the notion that chromatin acetylation leads to PIC assembly.

The metabolic activation of 2-aminofluorine, 4-aminobiphenyl, and benzidine by cytochrome P-450-107S1 of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic pathogen of the human urinary bladder. Similar to rat liver S9, the cell-free extract from P. aeruginosa caused significant increase of histidine reversion numbers with the Salmonella typhimurium tester strain TA98 in the Ames Salmonella mutagenicity assay in the presence of either 2-aminofluorene, 4-aminobiphenyl, or benzidine procarcinogens. The presence of cytochrome P-450 protein in the cell-free extract was demonstrated by the carbon monoxide difference spectrum. We employed gene knockout technology to inactivate one of the three known putative cytochrome P-450 genes of P. aeruginosa, namely CYP107S1, which we postulated to be the most likely to induce activation. The ampicillin resistant gene from PUC19 DNA confers carbenicillin resistance to P. aeruginosa. We inserted a synthetic ampicillin gene flanked by 40 base-pairs of the 5' and 3' untranslated region of the CYP gene by electroporating the synthetic gene into electrocompetent P. aeruginosa cells. CYP107S1 knockout strains were selected on 1000 microg/ml carbenicillin plates. A single cloned carbenicillin resistant colony was isolated and used to determine its mutagenic capacity using Ames Salmonella mutagenicity assay. The results showed that Salmonella TA98 tester strain returned the number of revertants to its baselines level indicating the lack of metabolic activation of procarcinogens in the P. aeruginosa CYP107S1 knockout cell-free extract. In addition, the characteristic cytochrome P-450 peak determined by the carbon monoxide difference spectrum was completely absent in the cell-free extract from this CYP107S1 knockout strain bacterium. Homologous recombination of the synthetic ampicillin gene on the CYP 107S1 P-450 locus was confirmed by PCR on purified genomic DNA extracted from the knockout bacterium. The metabolic activation of tested procarcinogens is, therefore, carried out by CYP107S1 in P. aeruginosa.

An enhancer-like region regulates hrp3 promoter stage-specific gene expression in the human malaria parasite Plasmodium falciparum.

The asexual blood stage of Plasmodium falciparum is comprised of morphologically distinct ring, trophozoite and schizont stages. Each of these developmental stages possesses a distinct pattern of gene expression. Regulation of P. falciparum gene expression is thought to occur, at least in part, at the promoter level. Previously, we have found that although the hrp3 mRNA is only seen in ring-stage parasites, deletion of a specific sequence in the 5' end of the promoter region decreased ring-stage expression of hrp3 and enabled detection of its transcripts in trophozoite-stage parasites. In order to investigate this stage specific regulation of gene expression, we employed a series of nested deletions of the 1.7-kb hrp3 promoter. Firefly luciferase gene was used as a reporter to evaluate the role of promoter sequences in gene regulation. Using this approach, we identified a ring-stage specific regulatory region on the hrp3 promoter located between -1.7 kb and -1.1 kb from the ATG initiation codon. Small 100-150 bp truncations on this enhancer-like region failed to uncover discrete regulatory sequences, suggesting the multipartite nature of this element. The data presented in this study demonstrate that stage specific promoter activity of the hrp3 gene in P. falciparum blood stage parasites is supported, at least in-part, by a small promoter region that can function in the absence of a larger chromosomal context.

Plasmodium falciparum: hrp3 promoter region is associated with stage-specificity and episomal recombination.

The asexual blood stage of Plasmodium falciparum in the human host is comprised of morphologically distinct ring, trophozoite and schizont stages, each of which possesses a distinct pattern of gene expression. Episomal promoter recombination has been recently reported in malaria parasites. We aim to investigate the nature of this process, and its relationship with promoter activity by employing a series of nested deletions of the ring-specific hrp3 promoter. Our results showed a discrete promoter region that is preferentially used for recombination. The P. falciparum hrp3 mRNA is only seen in ring-stage parasites but deletion of the recombination region was associated with decreased ring-stage expression and concurrent detection of transcripts in trophozoite-stage parasites. Our results describe a ring-stage specific regulatory region possibly involved in episomal promoter recombination, suggesting that common sequences might mediate both processes.

A host-targeting signal in virulence proteins reveals a secretome in malarial infection.

Malaria parasites secrete proteins across the vacuolar membrane into the erythrocyte, inducing modifications linked to disease and parasite survival. We identified an 11-amino acid signal required for the secretion of proteins from the Plasmodium falciparum vacuole to the human erythrocyte. Bioinformatics predicted a secretome of >320 proteins and conservation of the signal across parasite species. Functional studies indicated the predictive value of the signal and its role in targeting virulence proteins to the erythrocyte and implicated its recognition by a receptor/transporter. Erythrocyte modification by the parasite may involve plasmodial heat shock proteins and be vastly more complex than hitherto realized.

Transcription termination factor reb1p causes two replication fork barriers at its cognate sites in fission yeast ribosomal DNA in vivo.

Polar replication fork barriers (RFBs) near the 3' end of the rRNA transcriptional unit are a conserved feature of ribosomal DNA (rDNA) replication in eukaryotes. In the mouse, in vivo studies indicate that the cis-acting Sal boxes required for rRNA transcription termination are also involved in replication fork blockage. On the contrary, in the budding yeast Saccharomyces cerevisiae, the rRNA transcription termination factors are not required for RFBs. Here we characterized the rDNA RFBs in the fission yeast Schizosaccharomyces pombe. S. pombe rDNA contains three closely spaced polar replication barriers named RFB1, RFB2, and RFB3 in the 3' to 5' order. The transcription termination protein reb1 and its two binding sites, present at the 3' end of the coding region, were required for fork arrest at RFB2 and RFB3 in vivo. On the other hand, fork arrest at the strongest RFB1 barrier was independent of the above transcription termination factors. Therefore, RFB2 and RFB3 resemble the barriers present in the mouse rDNA, whereas RFB1 is similar to the budding yeast RFBs. These results suggest that during evolution, cis- and trans-acting factors required for rRNA transcription termination became involved in replication fork blockage also. S. pombe is suggested to be a transitional species in which both mechanisms coexist.

Cooperative domains define a unique host cell-targeting signal in Plasmodium falciparum-infected erythrocytes.

When the malaria parasite Plasmodium falciparum infects an erythrocyte, it resides in a parasitophorous vacuole and remarkably exports proteins into the periphery of its host cell. Two of these proteins, the histidine-rich proteins I and II (PfHRPI and PfHRPII), are exported to the erythrocyte cytoplasm. PfHRPI has been linked to cell-surface "knobby" protrusions that mediate cerebral malaria and are a frequent cause of death. PfHRPII has been implicated in (i) the production of hemozoin, the black pigment associated with disease, as well as (ii) interactions with the erythrocyte cytoskeleton. Here we show that a tripartite signal that is comprised of an endoplasmic reticulum-type signal sequence followed by a bipartite vacuolar translocation signal derived from HRPII and HRPI exports GFP from the parasitophorous vacuole to the host cytoplasm. The bipartite vacuolar translocation signal is comprised of unique, peptidic (approximately equal to 40-aa) sequences. A domain within it contains the signal for export to "cleft" transport intermediates in the host erythrocyte and may thereby regulate the pathway of export to the host cytoplasm. A signal for posttranslational, vacuolar exit of proteins has hitherto not been described in eukaryotic secretion.