Effect of parasitic infection on dopamine biosynthesis in dopaminergic cells.

Infection by the neurotropic agent Toxoplasma gondii alters rodent behavior and can result in neuropsychiatric symptoms in humans. Little is understood regarding the effects of infection on host neural processes but alterations to dopaminergic neurotransmission are implicated. We have previously reported elevated levels of dopamine (DA) in infected dopaminergic cells however the involvement of the host enzymes and fate of the produced DA were not defined. In order to clarify the effects of infection on host DA biosynthetic enzymes and DA packaging we examined enzyme levels and activity and DA accumulation and release in T. gondii-infected neurosecretory cells. Although the levels of the host tyrosine hydroxylase (TH) and DOPA decarboxylase and AADC (DDC) did not change significantly in infected cultures, DDC was found within the parasitophorous vacuole (PV), the vacuolar compartment where the parasites reside, as well as in the host cytosol in infected dopaminergic cells. Strikingly, DDC was found within the intracellular parasite cysts in infected brain tissue. This finding could provide some explanation for observations of DA within tissue cysts in infected brain as a parasite-encoded enzyme with TH activity was also localized within tissue cysts. In contrast, cellular DA packaging appeared unchanged in single-cell microamperometry experiments and only a fraction of the increased DA was accessible to high potassium-induced release. This study provides some understanding of how this parasite produces elevated DA within dopaminergic cells without the toxic ramifications of free cytosolic DA. The mechanism for synthesis and packaging of DA by T. gondii-infected dopaminergic cells may have important implications for the effects of chronic T. gondii infection on humans and animals.

Petri Net representations in systems biology.

The mathematical structures known as Petri Nets have recently become the focus of much research effort in both the structural and quantitative analysis of all kinds of biological networks. This review provides a very brief summary of these interesting new research directions.

Identification of a nucleoside/nucleobase transporter from Plasmodium falciparum, a novel target for anti-malarial chemotherapy.

Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue protein, which we designate PfENT1, is predicted to contain 11 membrane-spanning segments and is a distantly related member of the widely distributed eukaryotic protein family the equilibrative nucleoside transporters (ENTs). However, it differs profoundly at the sequence and functional levels from its homologous counterparts in the human host. The parasite protein exhibits a broad substrate specificity for natural nucleosides, but transports the purine nucleoside adenosine with a considerably higher apparent affinity (K(m) 0.32+/-0.05 mM) than the pyrimidine nucleoside uridine (K(m) 3.5+/-1.1 mM). It also efficiently transports nucleobases such as adenine (K(m) 0.32+/-0.10 mM) and hypoxanthine (K(m) 0.41+/-0.1 mM), and anti-viral 3'-deoxynucleoside analogues. Moreover, it is not sensitive to classical inhibitors of mammalian ENTs, including NBMPR [6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, or nitrobenzylthioinosine] and the coronary vasoactive drugs, dipyridamole, dilazep and draflazine. These unique properties suggest that PfENT1 might be a viable target for the development of novel anti-malarial drugs.

Plasmodium falciparum: isolation and characterisation of a gene encoding protozoan GMP synthase.

The final step in guanylate nucleotide biosynthesis is catalysed by GMP synthase. This paper presents the first isolation of a gene encoding a protozoan GMP synthase. The deduced amino acid sequence from Plasmodium falciparum shares 40% identity with yeast GMP synthase and contains motifs conserved in catalysis. Expression of the gene is regulated through the parasite's development in human red blood cells with maximal expression during the point of DNA replication. Psicofuranine, which inhibits GMP synthase, interrupts parasite growth, supporting the role of this enzyme. These findings will aid development of inhibitors of purine salvage in malaria parasites.

Identification of the transcription initiation site of the asexually expressed rRNA genes of the malaria parasite Plasmodium berghei.

The start site of the A-type ribosomal RNA transcription units of the rodent malaria parasite, Plasmodium berghei, has been identified. The two A-type units cannot be distinguished within the transcription unit, yet exist as single copies on different chromosomes. Gene transcription initiates 820 bp upstream of the A-type small subunit (SSU) ribosomal gene and two major processing sites were mapped 610 and 611 nucleotides upstream of the SSU in the external transcribed spacer region. Surprisingly the nucleotide sequence of the DNA region containing the putative ribosomal promoter lacked repetitive DNA sequences typical of ribosomal promoters. This region was further analysed by computer using programs designed to reveal sequence-dependent structural features. Comparison of DNA curvature, duplex stability and pattern of twist angle variation revealed a striking degree of conservation between the ribosomal promoters from Plasmodium and other eukaryotes.

Targeting the shikimate pathway in the malaria parasite Plasmodium falciparum.

The shikimate pathway presents an attractive target for malaria chemotherapy. Three shikimic acid analogs exhibited different effects on Plasmodium falciparum growth. (6R)-6-Fluoro-shikimate and (6S)-6-fluoro-shikimate inhibited growth (50% inhibitory concentrations, 1.5 x 10(-5) and 2.7 x 10(-4) M, respectively), whereas 2-fluoro-shikimate had no effect. para-Aminobenzoic acid abrogated the inhibition, demonstrating that the shikimate pathway was specifically targeted.

Inhibition of Plasmodium falciparum protein synthesis. Targeting the plastid-like organelle with thiostrepton.

The human malaria parasite Plasmodium falciparum has two extrachromosomal DNAs associated with organelles whose function is unclear. Both genomes encode ribosomal RNAs (rRNAs) that are distinct from the nuclear-encoded rRNAs. Secondary structure analysis of all the P. falciparum rRNAs indicates that only the large subunit (LSU) rRNA encoded by the plastid-like genome is the target for thiostrepton. Indeed we find that thiostrepton inhibits growth of the parasite in the micromolar range which is 10-fold below concentrations with observable effects on total protein synthesis. We have further examined selective effects of thiostrepton on the plastid function by comparing differential effects of the drug on cytoplasmic and organellar encoded transcripts. Treatment with either thiostrepton or rifampin, an inhibitor of organellar and eubacterial RNA polymerase, both showed disappearance of organellar-encoded RNA transcripts within 6 h of treatment while transcripts of a nuclear-encoded mRNA remained constant for at least 8 h of treatment. Hence, we show a selective effect on organelle function that is suggestive of interference in the protein synthesis apparatus of the plastid. Sensitivity of P. falciparum to thiostrepton confirms that the plastid-like genome is essential for the erythrocytic cycle and presents a novel therapeutic site for this class of antibiotics.

Parasite diversity in an endemic region for avian malaria and identification of a parasite causing penguin mortality.

Understanding the population structure of Plasmodium parasites is essential for malaria intervention. A survey of parasites in vectors and host infections was conducted in an area of intense mortality due to malaria in a captive penguin (Spheniscus demersus) colony, using a novel method for identification of Plasmodium species by amplification of ribosomal sequences in DNA or RNA. Three phylogenetically distinct groups of avian Plasmodium were detected in mosquitoes (Culex) collected at the study site (Baltimore Zoo, Baltimore, MD) during a period of high transmission. One of the three clades of Plasmodium was found to be prevalent in penguins monitored through the malaria transmission season and consistent with morphological identification as Plasmodium relictum. This parasite sequence was directly associated with the death of a penguin. Thus, a complete transmission cycle is defined at this site. Phylogenetic comparison of ribosomal sequences to an authenticated reference strain of Plasmodium relictum indicates that this is not the parasite causing death in the penguins, suggesting that different parasites may be morphologically indistinguishable.

Structural features of the large subunit rRNA expressed in Plasmodium falciparum sporozoites that distinguish it from the asexually expressed subunit rRNA.

The developmentally regulated transcription of at least two distinct sets of nuclear-encoded ribosomal RNAs is detected in Plasmodium species. The identification of functional differences between the two sets of rRNAs is of interest. To facilitate the search for such differences, we have identified the 5.8S and 28S rRNAs from Plasmodium falciparum that are expressed in the sporozoite stage (S gene) of the parasites' life cycle in the mosquito host and compare them to transcripts expressed in the red blood cells (A gene) of the vertebrate host. This completes the first set of A- and S-type nuclear-encoded rRNA genes for a Plasmodium species. Analysis of the predicted secondary structures of the two units reveals the majority of differences between the A- and S-type genes occur in regions previously known to be variable. However, the predicted secondary structure of both 28S rRNAs indicates 11 positions within conserved areas that are not typical of eucaryotic rRNAs. Although the A-type gene resembles almost all eucaryotes, being atypical in only 4 of the 11 positions, the S gene is variant in 8 of the 11 positions. In three of these positions, the S-type gene resembles the consensus nucleotides for the 23S rRNA from Eubacteria and/or Archaea. A few differences occur in regions associated with ribosome function, in particular the GTPase site where the S-type differs in a base pair and loop from all known sequences. Further, the identification of compensatory changes at conserved points of interactions between the 5.8S-28S rRNAs indicates that transcripts from A- and S-units should not be interchangeable.

The ribosomal DNA loci in Plasmodium falciparum accumulate mutations independently.

Homogeneity of rDNA sequence within a cell is maintained by mechanisms working at the DNA level. The imperative to maintain homogeneity is thought to result from pressure to maintain the sequence of the rRNA transcript. We have investigated the extent of sequence variation within and between members of a species that is unable to utilize some standard mechanisms of rDNA sequence correction. We have compared the sequence of the internal transcribed spacer (ITS1) located between the 18 S rRNA and 5.8 S rRNA genes of five different loci of a single Plasmodium falciparum genotype. The ITS1 sequences are identical at 80 to 91% of the positions among the three asexually expressed genes (A-types) and 75% between the two genes expressed during sporogony (S-types), with only 42 to 57% identity between the types. This is rather startling in that the differences described here for a single genome are greater than those normally seen when comparing rDNA units from distantly related organisms. We observe an apparent conservation of secondary structure within ITS1 sequences from the different transcription units, which would reflect a level of selection at the rRNA but the organism seems to be quite tolerant of primary sequence variation. Investigation of the mature coding region within the 18 S rRNA genes did not reveal sequence variation within A- and S-types from a single genotype. However, comparison of the 18 S rRNA coding region from 17 geographically distinct strains reveals up to 10% sequence variation within a 400 nucleotide region. Hence homogeneity of rRNA units within a species does not seem to be an imperative driven totally by selection at the RNA level. The extraordinary maintenance of homogeneity within rDNA units normally seen within a species appears to have significance beyond those that can be ascribed to the events involved in processing, assembly and function of the ribosome.

Plasmodium: genus-conserved primers for species identification and quantitation.

Stable RNAs have regions of primary sequence that are nearly identical in every member of the Plasmodium genus and not found in the host or in other common pathogens. Several "genus-conserved" sequences, which flank hypervariable regions, were identified within the small subunit ribosomal RNA of Plasmodium species. Primers based on these conserved sequences permit amplification of species- or possibly even strain-specific sequences from samples of unknown composition. As an example of this approach, sequences from the four human malaria species were successfully recovered from Giemsa-stained blood smears, including two different sequences for Plasmodium ovale (of 91.5% similarity). This type of information is useful for epidemiological and phylogenetic analysis of any malaria species. We show that amplification of rRNA-derived sequences behaves in a competitive fashion during the cycles of polymerase amplification and therefore target sequences from Plasmodium species are amplified in proportion to their abundance in the sample. There are several implications of this finding. (1) The proportion of different products resulting from amplification from samples with mixed infections is closely related to the proportion of infecting species. (2) Direct quantitation of parasite nucleic acids within a sample can be derived when known amounts of competitor RNA are added to the RT/PCR reaction. (3) Amplification of rRNA sequences, using genus-specific primers, allows one to monitor the development of the parasite in the mosquito.

The cytoplasmic ribosomal RNAs of Plasmodium spp.

Plasmodium spp maintain several structurally distinct sets of ribosomal RNA genes whose expression is developmentally regulated. This feature sets them apart from all other eukaryotes studied to date. In this review, Thomas McCutchan, Jun Li, Glenn McConkey, John Rogers and Andy Waters give an account of the progress in our understanding of this unusual phenomenon as it relates to the biology of the parasite. They also outline an interesting turnabout in scientific direction involving the use of the parasite as an important new model for the study of the eukaryotic ribosome.

Transition of Plasmodium vivax ribosome types corresponds to sporozoite differentiation in the mosquito.

Two distinct small subunit ribosomal RNA (SSUrRNA) genes were amplified from the genomic DNA of Plasmodium vivax. Comparison of the two coding sequences reveals an overall divergence of 14.5% and most differences are clustered into the regions known to diverge rapidly in all eukaryotic SSUrRNAs. Oligonucleotides complementary to unique sequences of each gene have been used to distinguish the transcripts expressed either at schizogony in human blood (A gene) or at sporogony in the mosquito (C gene). These oligonucleotides were also used to monitor turnover of ribosomes during parasite development in mosquitoes. Transcripts of the A gene were predominant in the infected human blood and engorged mosquitoes but disappeared within 24 h after feeding. Expression of the C gene in mosquitoes was not detected until day 6 after the blood meal. A period of rapid accumulation of the C type rRNA from day 6 to day 8 corresponds to differentiation of individual sporozoites within the oocyst. Possible functional implications relating to the timing of this transition are discussed.

Auxotrophs of Plasmodium falciparum dependent on p-aminobenzoic acid for growth.

The isolation of auxotrophic strains of a parasite offers new opportunities for studying parasitology. We have isolated cloned lines of Plasmodium falciparum that, unlike the parent line from which they were derived, rely on exogenous p-aminobenzoic acid (PABA) for growth. Isolation involved random mutagenesis of a cloned line of P. falciparum and subsequent selection of PABA-dependent parasites. Both parent and PABA-dependent clones were analyzed for PABA uptake and synthesis. Each clone takes up comparable amounts of PABA from the medium. The parent line, clone 3D7, can synthesize PABA de novo, whereas the PABA-dependent clones cannot. The requirement of exogenous PABA for growth by the auxotrophic strains coupled with their inability to synthesize PABA indicates that normal parasite growth can be completely supported by either synthesis or salvage. This work further clarifies the relationship between the availability of PABA and success of the parasite, an issue of debate from classic studies showing reduced parasite load in individuals on milk-fed diets.

The generation of genetic diversity in malaria parasites.

The genetic diversity among malaria parasites is considerable, and much scientific investigation has focused on the ramifications of this diversity. Here we have discussed types of genetic change that are related to phenotypic changes in the parasite. To date, these changes have not been observed to result from precise mechanisms like the antigenic diversity seen in trypanosomes. Rather, the parasite seems to depend upon less precise mechanisms for generating diversity in combination with the large number of organisms that accumulate during infection as a reservoir for selection.

TFIIIA binds with equal affinity to somatic and major oocyte 5S RNA genes.

Current models for the differential control of expression of Xenopus somatic and oocyte 5S RNA genes suggest that an impaired ability to bind TFIIIA contributes to the inactivation of oocyte 5S RNA genes in somatic cells. The somatic 5S RNA gene is transcribed more efficiently than the major oocyte 5S RNA gene in S-150 extracts of mature oocytes. However, this differential transcription efficiency is not determined simply by the relative affinity for binding of a positive transcription factor, TFIIIA. We have compared the abilities of somatic, major oocyte, and minor oocyte 5S RNA genes to bind TFIIIA using both a standard footprint competition assay and an indirect DNase protection assay. This indirect DNase protection assay permits the direct comparison of TFIIIA binding to two templates in one reaction. Both assay methods indicate that the major oocyte 5S RNA gene and the somatic 5S RNA gene bind TFIIIA with equal affinity. As a further control, we have confirmed earlier work indicating that the minor oocyte gene binds TFIIIA with a reduced affinity. Binding of TFIIIA to these three 5S RNA genes results in a different pattern of protection of each gene. We suggest that slight differences in the contacts between TFIIIA and the 5' border of the control region influence the ability of additional transcription factors to bind to the TFIIIA:5S DNA complex.

Transition mutations within the Xenopus borealis somatic 5S RNA gene can have independent effects on transcription and TFIIIA binding.

Base-pair changes were introduced into the Xenopus borealis somatic 5S RNA gene by treatment with sodium bisulfite. Mutants were screened by sequence determination. The collection of mutants permitted a detailed investigation of the fine structure of the intragenic control region that binds the transcription factor TFIIIA. Selected mutants were recloned in tandem with a somatic 5S RNA maxigene to permit sensitive measurement of their relative transcription activities. The transcription efficiencies of a number of mutations at the borders of the control region were correlated with TFIIIA binding by using DNase I protection (footprinting) assays. Mutations affecting transcription and TFIIIA binding extended from gene residues 46 to 91. The results reinforce a model in which the distal half of the protected region constitutes a tight binding domain for TFIIIA. A number of transitions in the 5' domain led to significant increases or decreases in transcription efficiency, but resulted in barely detectable changes in TFIIIA binding. Two mutants with C----T transitions at gene residues 52 and 53 were transcribed with increased efficiencies (up phenotype). These results suggest that TFIIIA must make appropriate contacts within the 5' domain of the control region to permit subsequent binding of other factors in stable transcription complexes.