Serial analysis of gene expression (SAGE) was applied to the malarial parasite Plasmodium falciparum to characterize the comprehensive transcriptional profile of erythrocytic stages. A SAGE library ...of approximately 8335 tags representing 4866 different genes was generated from 3D7 strain parasites. Basic local alignment search tool analysis of high abundance SAGE tags revealed that a majority (88%) corresponded to 3D7 sequence, and despite the low complexity of the genome, 70% of these highly abundant tags matched unique loci. Characterization of these suggested the major metabolic pathways that are used by the organism under normal culture conditions. Furthermore several tags expressed at high abundance (30% of tags matching to unique loci of the 3D7 genome) were derived from previously uncharacterized open reading frames, demonstrating the use of SAGE in genome annotation. The open platform "profiling" nature of SAGE also lead to the important discovery of a novel transcriptional phenomenon in the malarial pathogen: a significant number of highly abundant tags that were derived from annotated genes (17%) corresponded to antisense transcripts. These SAGE data were validated by two independent means, strand specific reverse transcription-polymerase chain reaction and Northern analysis, where antisense messages were detected in both asexual and sexual stages. This finding has implications for transcriptional regulation of Plasmodium gene expression.
The genome of Plasmodium falciparum has one of the most skewed base-pair compositions of any eukaryote, with an AT content of 80–90%. As start and stop codons are AT-rich, the probability of finding ...upstream open reading frames (uORFs) in messenger RNAs (mRNAs) is high and parasite mRNAs have an average of 11 uORFs in their leader sequences. Similar to other eukaryotes, uORFs repress the translation of the downstream open reading frame (dORF) in P. falciparum, yet the parasite translation machinery is able to bypass these uORFs and reach the dORF to initiate translation. This can happen by leaky scanning and/or reinitiation. In this report, we assessed leaky scanning and reinitiation by studying the effect of uORFs on the translation of a dORF, in this case, the luciferase reporter gene, and showed that both mechanisms are employed in the asexual blood stages of P. falciparum. Furthermore, in addition to the codon usage of the uORF, translation of the dORF is governed by the Kozak sequence and length of the uORF, and inter-cistronic distance between the uORF and dORF. Based on these features whole-genome data was analysed to uncover classes of genes that might be regulated by uORFs. This study indicates that leaky scanning and reinitiation appear to be widespread in asexual stages of P. falciparum, which may require modifications of existing factors that are involved in translation initiation in addition to novel, parasite-specific proteins.
Background Information
Like other apicomplexan parasites, Toxoplasma gondii harbours a four‐membraned endosymbiotic organelle – the apicoplast. Apicoplast proteins are nuclear encoded and trafficked ...to the organelle through the endoplasmic reticulum (ER). From the ER to the apicoplast, two distinct protein trafficking pathways can be used. One such pathway is the cell's secretory pathway involving the Golgi, whereas the other is a unique Golgi‐independent pathway. Using different experimental approaches, many apicoplast proteins have been shown to utilize the Golgi‐independent pathway, whereas a handful of reports show that a few proteins use the Golgi‐dependent pathway. This has led to an emphasis towards the unique Golgi‐independent pathway when apicoplast protein trafficking is discussed in the literature. Additionally, the molecular features that drive proteins to each pathway are not known.
Results
In this report, we systematically test eight apicoplast proteins, using a C‐terminal HDEL sequence to assess the role of the Golgi in their transport. We demonstrate that dually localised proteins of the apicoplast and mitochondrion (TgSOD2, TgTPx1/2 and TgACN/IRP) are trafficked through the Golgi, whereas proteins localised exclusively to the apicoplast are trafficked independent of the Golgi. Mutants of the dually localised proteins that localised exclusively to the apicoplast also showed trafficking through the Golgi. Phylogenetic analysis of TgSOD2, TgTPx1/2 and TgACN/IRP suggested that the evolutionary origins of TgSOD2 and TgTPx1/2 lie in the mitochondrion, whereas TgACN/IRP appears to have originated from the apicoplast.
Conclusions and Significance
Collectively, with these results, for the first time, we establish that the driver of the Golgi‐dependent trafficking route to the apicoplast is the dual localisation of the protein to the apicoplast and the mitochondrion.
Research article: Nuclear encoded proteins are known to reach the apicoplast via two exclusive pathways the choice between these pathways was unknown This report clearly establishes that dually localised proteins of the apicoplast and the mitochondria reach the apicoplast through a Golgi dependent pathway Proteins localised exclusively to the apicoplast are trafficked independent of the Golgi.
In P. falciparum, antioxidant proteins of the glutathione and thioredoxin systems are compartmentalized. Some subcellular compartments have only a partial complement of these proteins. This lack of ...key anti-oxidant proteins in certain sub-cellular compartments might be compensated by functional complementation between these systems. By assessing the cross-talk between these systems, we show for the first time, that the glutathione system can reduce thioredoxins that are poor substrates for thioredoxin reductase (Thioredoxin-like protein 1 and Thioredoxin 2) and thioredoxins that lack access to thioredoxin reductase (Thioredoxin 2). Our data suggests that crosstalk between the glutathione and thioredoxin systems does exist; this could compensate for the absence of certain antioxidant proteins from key subcellular compartments.
Display omitted
•Crosstalk between the glutathione and thioredoxin systems exists in P. falciparum.•Glutathione reduces thioredoxins that are poor substrates of thioredoxin reductase.•Glutathione can reduce thioredoxins that lack access to thioredoxin reductase.•Lack of certain redox proteins in key organelles is compensated by this cross talk.
Importin α is a nuclear transporter that binds to nuclear localization signals (NLSs), consisting of 7–20 positively charged amino acids found within cargo proteins. In addition to cargo binding, ...intramolecular interactions also occur within the importin α protein due to binding between the importin β-binding (IBB) domain and the NLS-binding sites, a phenomenon called auto-inhibition. The interactions causing auto-inhibition are driven by a stretch of basic residues, similar to an NLS, in the IBB domain. Consistent with this, importin α proteins that do not have some of these basic residues lack auto-inhibition; a naturally occurring example of such a protein is found in the apicomplexan parasite
Plasmodium falciparum
. In this report, we show that importin α from another apicomplexan parasite,
Toxoplasma gondii
, harbors basic residues (KKR) in the IBB domain and exhibits auto-inhibition. This protein has a long, unstructured hinge motif (between the IBB domain and the NLS-binding sites) that does not contribute to auto-inhibition. However, the IBB domain may have a higher propensity to form an α-helical structure, positioning the wild-type KKR motif in an orientation that results in weaker interactions with the NLS-binding site than a KRR mutant. We conclude that the importin α protein from
T. gondii
shows auto-inhibition, exhibiting a different phenotype from that of
P. falciparum
importin α. However, our data indicate that
T. gondii
importin α may have a low strength of auto-inhibition. We hypothesize that low levels of auto-inhibition may confer an advantage to these important human pathogens.
The Plasmodium falciparum genome being AT-rich, the presence of GC-rich regions suggests functional significance. Evolution imposes selection pressure to retain functionally important coding and ...regulatory elements. Hence searching for evolutionarily conserved GC-rich, intergenic regions in an AT-rich genome will help in discovering new coding regions and regulatory elements. We have used elevated GC content in intergenic regions coupled with sequence conservation against P. reichenowi, which is evolutionarily closely related to P. falciparum to identify potential sequences of functional importance. Interestingly, ∼30% of the GC-rich, conserved sequences were associated with antigenic proteins encoded by var and rifin genes. The majority of sequences identified in the 5′ UTR of var genes are represented by short expressed sequence tags (ESTs) in cDNA libraries signifying that they are transcribed in the parasite. Additionally, 19 sequences were located in the 3′ UTR of rifins and 4 also have overlapping ESTs. Further analysis showed that several sequences associated with var genes have the capacity to encode small peptides. A previous report has shown that upstream peptides can regulate the expression of var genes hence we propose that these conserved GC-rich sequences may play roles in regulation of gene expression.
The Plasmodium falciparum genome being AT-rich, the presence of GC-rich regions suggests functional significance. Evolution imposes selection pressure to retain functionally important coding and ...regulatory elements. Hence searching for evolutionarily conserved GC-rich, intergenic regions in an AT-rich genome will help in discovering new coding regions and regulatory elements. We have used elevated GC content in intergenic regions coupled with sequence conservation against P. reichenowi, which is evolutionarily closely related to P. falciparum to identify potential sequences of functional importance. Interestingly, ~30% of the GC-rich, conserved sequences were associated with antigenic proteins encoded by var and rifin genes. The majority of sequences identified in the 5' UTR of var genes are represented by short expressed sequence tags (ESTs) in cDNA libraries signifying that they are transcribed in the parasite. Additionally, 19 sequences were located in the 3' UTR of rifins and 4 also have overlapping ESTs. Further analysis showed that several sequences associated with var genes have the capacity to encode small peptides. A previous report has shown that upstream peptides can regulate the expression of var genes hence we propose that these conserved GC-rich sequences may play roles in regulation of gene expression. Keywords: Plasmodium, regulatory elements, comparative genomics, genome bias, antigenic variation
Upstream open reading frames (uORFs) and upstream AUGs (uAUGs) can regulate the translation of downstream ORFs. The AT rich genome of Plasmodium falciparum, due to the higher AT content of start and ...stop codons, has the potential to give rise to a large number of uORFs and uAUGs that may affect expression of their flanking ORFs.
A bioinformatics approach was used to detect uATGs associated with different genes in the parasite. To study the effect of some of these uAUGs on the expression of the downstream ORF, promoters and 5' leaders containing uAUGs and uORFs were cloned upstream of a luciferase reporter gene. Luciferase assays were carried out in transient transfection experiments to assess the effects of uAUGs and mutations on reporter expression.
The average number of uATGs and uORFs seen in P. falciparum coding sequences (CDS) is expectedly high compared to other less biased genomes. Certain genes, including the var gene family contain the maximum number of uATGs and uORFs in the parasite. They possess ~5 times more uORFs and ~4.5 times more uAUGs within 100 bases upstream of the start codons than other CDS of the parasite. A 60 bp upstream region containing three ORFs and five ATGs from var gene PF3D7_0400100 and a gene of unknown function (PF3D7_0517100) when cloned upstream of the luciferase start codon, driven by the hsp86 promoter, resulted in loss of luciferase activity. This was restored when all the ATGs present in the -60 bp were mutated to TTGs. Point mutations in the ATGs showed that even one AUG was sufficient to repress the luciferase gene.
Overall, this work indicates that the P. falciparum genome has a large number of uATGs and uORFs that can repress the expression of flanking ORFs. The role of AUGs in translation initiation suggests that this repression is mediated by preventing the translation initiation complex from reaching the main AUG of the downstream ORF. How the P. falciparum ribosome is able to bypass these uAUGs and uORFs for highly expressed genes remains a question for future research.
FORS-D is a measure of the contribution of base order to the stem loop potential of a nucleic acid sequence and can also give information on evolutionary pressures on sequences to move away from ...secondary structure. Negative FORS-D values in a gene are associated with exons and nucleotide substitutions such as SNPs. An analysis of P. falciparum genes under selection pressure shows a correlation between negative FORS-D values and SNP density for genes that drug targets but not for drug transporters or antigenic variation genes. Analysis of the dhfr gene shows that a majority of rare mutations that associate with drug resistance also fall into regions with negative FORS-D values. These data suggest that FORS-D values might be predictors for drug target genes and drug resistance mutations in these genes.
On day 6, blood smears and staining for parasites from control animals (no treatment) showed a high parasitemia and many parasites in the ring stages (A). But for animals treated with 40mg/kg body ...weight of curcuminoids-loaded liposomes, the parasitemia was lower and interestingly red blood cells showed fewer ring stage parasites (B). Display omitted
► Liposomal deliver system was used to enhance antimalarial activity of curcuminoids. ► Plasmodium berghei infected mice was used to test the efficacy of the delivery system. ► Curcuminoids loaded liposomes treated animals shown marginal increase in survivality. ► Curcuminoids-loaded liposomes and α/β arteether combination cured infected mice. ► Curcuminoids-loaded liposomes and α/β arteether therapy also prevented recrudescence.
Curcuminoids are poorly water-soluble compounds with promising antimalarial activity. To overcome some of the drawbacks of curcuminoids, we explored the potential of liposomes for the intravenous delivery of curcuminoids in a model of mouse malaria. The curcuminoids-loaded liposomes were formulated from phosphatidylcholine (soy PC) by the thin-film hydration method. Antimalarial activity of curcuminoids-loaded liposomes alone and in combination with α/β arteether when administered intravenously, was evaluated in Plasmodium berghei infected mice. Animals treated with curcuminoids-loaded liposomes showed lower parasitemia and higher survival when compared to control group (no treatment). Importantly, the combination therapy of curcuminoids-loaded liposomes (40mg/kg body wt) along with α/β arteether (30mg/kg body wt) was able to not only cure infected mice but also prevented recrudescence. These data suggest that curcuminoids-loaded liposomes may show promise as a formulation for anti-malarial therapy.
PDF
