Short communication

Deletion analysis of the 5′ flanking sequence of the Plasmodium gallinaceum sexual stage specific gene pgs28 suggests a bipartite arrangement of cis-control elements☆

Little is known about the structure of malaria parasite gene promoters and how their activity is regulated during parasite development. We here report results of a functional study of the genomic flanking regions of the Plasmodium falciparum gametocyte-specific gene pfg27, whose promoter is inactive in asexual parasites and is specifically switched on at the onset of gametocytogenesis. Promoter deletion analysis with plasmids containing the green fluorescent protein reporter, conducted on asexual and sexual stage parasites of clone 3D7, showed that 140 bp immediately preceding the pfg27 transcription start sites are sufficient to achieve timing and specificity of gene expression comparable to those of the endogenous pfg27 gene. Observations that the promoter sequences were functioning on episomal plasmids, and our failure to detect dramatic alterations in DNaseI hypersensitivity in the pfg27 chromosomal upstream region upon activation of the endogenous promoter, suggest that chromatin conformation may not have a major influence on the developmental regulation of the pfg27 promoter. Further deletion analysis of the 140 bp promoter led to identification of sequences involved in the repression of the pfg27 promoter in a sub-population of asexual parasites, and of sequences required for the efficient gametocyte-specific transcription of the gene, the latter characterised by the presence of long homopolymeric dA and dT tracts. These results together indicate that in P. falciparum short sequences of apparent minimal complexity nevertheless contain sufficient genetic information to act as a promoter responsive to developmental regulation. In this analysis, the functional role of the downstream flanking region of pfg27 was also investigated, revealing that the gene 3′ untranslated region contributes to stability of the pfg27 transcript in the maturation of P. falciparum gametocytes.

In this review, we bring together some of the approaches toward understanding the cellular and molecular biology of Plasmodium species and their interaction with their host red blood cells. Considerable impetus has come from the development of new methods of molecular genetics and bioinformatics, and it is important to evaluate the wealth of these novel data in the context of basic cell biology. We describe how these approaches are gaining valuable insights into the parasite–host cell interaction, including (1) the multistep process of red blood cell invasion by the merozoite; (2) the mechanisms by which the intracellular parasite feeds on the red blood cell and exports parasite proteins to modify its cytoadherent properties; (3) the modulation of the cell cycle by sensing the environmental tryptophan-related molecules; (4) the mechanism used to survive in a low Ca²⁺ concentration inside red blood cells; (5) the activation of signal transduction machinery and the regulation of intracellular calcium; (6) transfection technology; and (7) transcriptional regulation and genome-wide mRNA studies in Plasmodium falciparum.

Control of gene expression is poorly understood in the Plasmodium system, where relatively few homologues to known eukaryotic transcription factors have been uncovered. Recent evidence suggests that the parasite may utilize a combinatorial mode of gene regulation, with multiple cis-acting sequences contributing to overall activity at individual promoters [1]. To further probe this mechanism of control, we first searched for over-represented sequence motifs among gene clusters sharing similar expression profiles in Plasmodium falciparum. More specifically, we applied bioinformatic tools to a previously characterized micro-array data set from drug-treated asexual stage cultures (Gunasekera et al., submitted). Cluster analysis of 600 drug responsive genes identified only a single 5′ motif, GAGAGAA. Two additional 5′ motifs, ACTATAAAGA and TGCAC, were also shared among loci displaying patterns of coordinate expression across varying asexual growth stages. Secondly and most importantly, the functional relevance of each motif was tested in two independent assays—transient transfection and gel-retardation experiments. The GAGAGAA and TGCAC motifs were both active in the former. The GAGAGAA and ACTATAAAGA elements formed specific RNA-protein, but not DNA-protein complexes in gel shift assays, suggesting a key level of control at the RNA level. This is the first report of functionally characterized motifs in P. falciparum that were uncovered following clustering analysis of its asexual stage transcriptome. Together, both the bioinformatic and functional data reported here imply that multiple forms of gene regulation, including post-transcriptional control, may be important in the malarial system.

Expression of the Plasmodium falciparum calmodulin gene (pfcam) is developmentally regulated throughout the blood-stage cycle. The promoter lies within ∼1 kb of intergenic sequence that separates the pfcam open reading frame (ORF) from an upstream inverted ORF encoding a product homologous to the co-chaperone STI1. Using the oligo-capping method, which selectively reverse-transcribes cDNA from only full-length, capped transcript, we have mapped multiple transcription-initiation sites for both genes. Transcription of the pfSTI1 gene initiates over a 150 bp region centred ∼350 bp upstream of the ORF. The pfcam transcription start sites cluster into four ∼30 bp regions lying within 180 bp upstream of the pfcam ORF, generating transcripts with 5′ untranslated regions (UTR) of 3–173 nucleotides in length. Remarkably, splicing was found to be related to UTR length, with apparent preferential splicing of longer transcripts. Activity of the pfcam promoter diminished in a linear fashion to undetectable levels upon step-wise removal of sequence between 625 and 230 bp upstream of the start ATG. Electromobility-shift assays demonstrated nuclear factor binding to eight oligonucleotide probes spanning 657 bp of the pfcam ORF proximal upstream sequence. The degree of binding correlated with the density of poly(dA)poly(dT) tracts within the probes, and in all cases could be inhibited by excess synthetic poly(dA)poly(dT), but not by poly(dAdT)poly(dAdT). The multiple transcription-initiation sites of both pfSTI1 and pfcam genes lie just downstream of 25 bp-long poly(dA)poly(dT) tracts, and the intergenic region contains over 20 poly(dA)poly(dT) tracts of 4 bp or more. Our results suggest that the basal pfcam promoter is situated between approximately −300 and −230 bp upstream of the pfcam ORF and that the P. falciparum transcription-initiation complex has a low degree of sequence-specificity for the sites of initiation but preferentially acts downstream of long poly(dA)poly(dT) tracts.

There is little information regarding regulatory sequences in the newly sequenced genome of the malaria parasite, Plasmodium falciparum. Thus, for the first time, a bioinformatic strategy was utilized to identify regulatory elements in this genome using the P. falciparum heat shock protein (hsp) gene family as a model system. Our analysis indicates that the P. falciparum hsp genes do not contain standard eukaryotic regulatory elements. However, a novel G-rich regulatory element named the G-box was identified upstream of several P. falciparum hsp genes and the P. yoelii yoelii, P. berghei, and P. vivax hsp86 genes. Remarkably, the Plasmodium sp. G-boxes were required for maximal reporter gene expression in transient transfection assays. The G-box is not homologous to known eukaryotic elements, and is the best-defined functional element elucidated from Plasmodium sp. Our analysis also revealed several other elements necessary for reporter gene expression including an upstream sequence element, the region surrounding the transcription start site, and the 5′ and 3′ untranslated regions. These data demonstrate that unique regulatory elements are conserved in the genomes of Plasmodium sp., and demonstrate the feasibility of bioinformatic approaches for their identification.

All Plasmodium species produce a brown birefringent crystal known as malarial pigment or hemozoin. This work compares the morphology of hemozoin from P. falciparum, P. vivax, P. ovale, P. malariae, P. knowlesi, P. brasilianum, P. yoelii and P. gallinaceum. The human, primate and mouse hemozoins have a regular, flat-faced cuboidal morphology with modest size differences in contrast to the larger, regularly irregular barrel shape with a waffle surface of the avian, P. gallinaceum, pigment. Hydrogen peroxide (H₂O₂), as a biochemical test reagent, can distinguish the hemozoins by different concentrations to degrade half of the crystals. A surface area to volume ratio explains both the appearance and susceptibility to H₂O₂ degradation. The hemozoin from each species is able to be a template for hemozoin extension inhibitable by the quinolines. P. gallinaceum hemozoin more closely resembles the hemozoin from another avian apicomplexan, Haemoproteus, rather than the hemozoin from the mammalian malaria species. These distinct morphological characteristics between mammalian and avian crystals suggest different biochemical environments that affect morphology.