Distinct and stage specific nuclear factors regulate the expression of falcipains, Plasmodium falciparum cysteine proteases
© Sunil et al; licensee BioMed Central Ltd. 2008
Received: 23 July 2007
Accepted: 14 May 2008
Published: 14 May 2008
Plasmodium falciparum cysteine proteases (falcipains) play indispensable roles in parasite infection and development, especially in the process of host erythrocyte rupture/invasion and hemoglobin degradation. No detailed molecular analysis of transcriptional regulation of parasite proteases especially cysteine proteases has yet been reported. In this study, using a combination of transient transfection assays and electrophoretic mobility shift assays (EMSA), we demonstrate the presence of stage specific nuclear factors that bind to unique sequence elements in the 5'upstream regions of the falcipains and probably modulate the expression of cysteine proteases.
Falcipains differ in their timing of expression and exhibit ability to compensate each other's functions at asexual blood stages of the parasite. Present study was undertaken to study the transcriptional regulation of falcipains. Transient transfection assay employing firefly luciferase as a reporter revealed that a ~1 kb sequence upstream of translational start site is sufficient for the functional transcriptional activity of falcipain-1 gene, while falcipain-2, -2' and -3 genes that exist within 12 kb stretch on chromosome 11 require ~2 kb upstream sequences for the expression of reporter luciferase activity. EMSA analysis elucidated binding of distinct nuclear factors to specific sequences within the 5'upstream regions of falcipain genes. Analysis of falcipains' 5'upstream regulatory regions did not reveal the presence of sequences known to bind general eukaryotic factors. However, we did find parasite specific sequence elements such as poly(dA) poly(dT) tracts, CCAAT boxes and a single 7 bp-G rich sequence, (A/G)NGGGG(C/A) in the 5' upstream regulatory regions of these genes, thereby suggesting the role(s) of Plasmodium specific transcriptional factors in the regulation of falcipain genes.
Taken together, these results suggest that expression of Plasmodium cysteine proteases is regulated at the transcriptional level and parasite specific factors regulate the expression of falcipain genes. These findings open new venues for further studies in identification of parasite specific transcription factors.
Plasmodium falciparum, a human malaria parasite has a complex life cycle that encompasses three major developmental stages; the mosquito, liver and blood stages. During the complex cycle of P. falciparum, the intracellular development of the different asexual and sexual stages proceeds through a dynamic and multistep process for which the parasite has evolved complex molecular strategies . P. falciparum proteome data show a considerable level of regulation of gene expression throughout its life cycle; only 6% of the proteins identified are expressed at all the four stages. The transcriptome analysis of Plasmodium falciparum has revealed that the parasite has evolved an extremely specialized mode of transcriptional regulation at asexual blood stages that produces a continuous cascade of gene expression, beginning with genes corresponding to general cellular processes and ending with genes with specialized functions, such as genes involved in erythrocyte invasion . The transcriptome of intraerythrocytic developmental cycle (IDC) of PIasmodium falciparum thus resembles a "just-in-time" manufacturing process whereby transcripts are essentially produced when required. This concept has also been referred as "transcripts to go model" . For example, merozoite surface proteins that are required for erythrocyte invasion are expressed mainly at late schizont and merozoite stages [2, 4].
Transcriptional regulation and post-transcriptional gene silencing through translational repression of messenger RNA have been shown to be the major regulatory mechanisms in P. falciparum [4–6]. Malaria parasites express structurally distinct sets of rRNA genes in a stage specific manner that supposedly alter the properties of the ribosomes and thus modify patterns of cell growth and development . Transcriptional regulation is the major regulatory mechanism that determines the expression of one var gene and silencing of other var genes in a single parasite. . Regulatory regions within the var gene promoters have been shown to determine their silencing; their introns act as transcriptional silencing elements that help to control antigenic variations [9, 10]. Post-transcriptional regulation has been shown for P28, a gametocyte specific protein that remains in a state of translational repression in developing and mature gametocytes . Based on these studies, it has been proposed that the developmental stages of malaria parasite require coordinated modulation of expression of distinct sets of genes, which could be achieved by transcriptional and/or post-transcriptional control. In Plasmodium species, as in all eukaryotes, gene expression is governed at the level of transcription by the interaction of elements within promoters acting in cis (DNA regulatory elements) and/or trans whose availability is modulated during P. falciparum development . However, little is currently known about these elements in Plasmodium species. Insights into the regulatory pathways of malaria parasites will lead to better understanding of Plasmodium biology and development of new chemotherapeutics and vaccine strategies.
P. falciparum expresses four cysteine proteases namely, falcipain-1 (FP-1), falcipain-2 and -2' (FP-2A and FP-2B) and falcipain-3 (FP-3) at asexual blood stages of the parasite. These proteases perform multiple functions such as hemoglobin hydrolysis, erythrocyte rupture and erythrocyte invasion and differ in their timing of expression. Falcipain-1 is active at the invasive merozoite stage while falcipain-2/-2' and -3 are expressed mainly at early and late trophozoite stages respectively [13–15]. A recent falcipain-2 knockout study has suggested interplay between different cysteine proteases . In the present study, we evaluated the mechanism of gene regulation of falcipains. We identified specific promoters regions of the four falcipains and observed that distinct nuclear factors bind to these falcipains promoters in a stage specific manner. We also observed stage specific expression of these nuclear factors, thereby suggesting a gene specific transcriptional regulation of falcipains.
Identification of transcription start sites for the falcipain genes
List of Primers and PCR conditions used for 5' RACE
Primer sequences 5'-3'
Identification of promoter regions of falcipain genes
To identify the functional promoter sequences for falcipain-2, -2' and -3 genes, we next cloned ~2 kb 5'-untranslated regions of these genes in pPf86 plasmid. In falcipain 2 and 3, regions spanning -1882 bps, -1903 bps respectively from the translational start site were cloned into pPf86 vector. In case of falcipain-2' gene, 1610 bps of intergenic region between falcipain- 2' and falcipain- 3 was cloned into the luciferase vector. Each plasmid was co-transfected with pPfrluc into P. falciparum ring stage parasites. Fig. 3B shows the reporter gene expression derived from 1.6/2 kb 5' upstream regulatory regions of three falcipain genes. A high level of luciferase activity was observed for the three falcipains promoters. The 5'upstream regulatory region of falcipain-2' gene exhibited 2–3 fold higher luciferase activity than that exhibited by the corresponding sequences of falcipains- 2 and 3 genes. Together, our data suggests that approximately 2 kb long 5'upstream sequences are required for functional promoter activity of falcipain s -2, -2' and -3.
Analysis of falcipains promoter activities at different asexual blood stages
Specific nuclear factors bind to falcipains 5' upstream regulatory sequences
Nuclear proteins interact stage-specifically with falcipains promoters
Transcriptional and post-transcriptional regulatory processes have been shown to regulate the expression of parasite proteins and transcriptional regulation appears to be the major regulatory mechanism operating in malaria parasites . It appears that the molecular mechanisms that regulate gene expression in malaria parasite may differ from that of other eukaryotes [1, 4]. However, a recent study using bioinformatics approaches has predicted the existence of parasite encoded general transcription factors such as TFIIA, TFIIH, TFIIE α unit, and several subunits of TFIID in P. falciparum genome. Another study has identified divergent version of the AP2-integrase DNA binding domain that is present in numerous plant transcription factors in the Apicomplexans . A highly conserved transcription factor of Myb family (PfMyb1) that regulates the expression of a number of parasite proteins has also been described in Plasmodium falciparum [12, 18].
In malaria parasites, multiple cysteine and aspartic proteases have been reported and they show functional redundancy; these proteases show ability to compensate each other's functions. For example, falcipain-2' takes over the function of falcipain-2 in falcipain-2 knockout P. falciparum parasites . The ability to compensate for the individual protein function among these proteases can also be explained by close similarity in their structure and active site residues. These proteases are responsible for hemoglobin degradation in malaria parasite, an important metabolic process central to growth and maturation of parasites [13, 19]. However, nothing is known about the transcriptional regulation of these proteases in P. falciparum. The current study was performed to identify regulatory regions of four falcipains expressed during the asexual blood stages of the parasite.
As falcipains promoter sequences have not been characterized previously, we mapped the transcription initiation sites for the four falcipains by 5'RACE analysis. The positions of transcription initiation sites differ considerably among four falcipains. The start sites were in concordance with the known and predicted transcriptional start sites with all the genes having either A or C as their start sites . A single RNA initiation site was observed for falcipain- 1, -2 and -3, while two initiation sites were noted for falcipain-2'. The significance of two start sites for falcipain - 2' requires further investigation to determine if alternate transcripts exist. Nevertheless, multiple RNA initiation sites have been reported earlier for P. knowlesi circumsporozoite gene, P. falciparum MSP-1 gene and P. yoelii Py230 gene [21–23]. We did not perform primer extension or RNAse protection experiments to confirm the start sites for the falcipain genes, as the focus of the study was to identify regulatory elements for the falcipain genes. Even though 5'RACE experiment was carried out with good quality of mRNA, there is still a possibility for discrepancies concerning the start sites because of mRNA degradation. We next examined the sequences required for functional promoter activity for each falcipain by analyzing the reporter gene activity (luciferase activity) in transient transfection assays. Our data suggests that a 1 kb 5' upstream region from the translational start site is sufficient for the functional promoter activity of falcipain- 1, while 1.6 to 2 kb upstream sequences of falcipain- 2,-2'and -3 are required to drive significant luciferase expression. We did observe basal level of promoter activities when sequences 1 kb upstream of falcipain-2, -2' and -3 were used in the reporter assay. A comparison of transcriptional activity as determined by reporter assays for falcipain genes showed that falcipain- 2' promoter is a strong promoter. Apparently, the falcipain- 2' promoter represents an intergenic region between falcipain- 2' to falcipain- 3. It is possible that this region may also include the 3'UTR sequences for falcipain-3.
As falcipains differ in their timing of expression at asexual blood stages of P. falciparum, their promoter activities were analyzed at the different blood stages; ring, trophozoite and schizonts. Considerable variations were seen in the falcipains promoter activities at the three stages and these variable activities coincided with the expression profiles of these proteins. Falcipain-2 promoter showed maximum activity at the trophozoite stage which is in accordance with its expression profile. Similar was the case for falcipain- 1 and-3. In case of falcipain- 2', a previous study  showed a maximum transcript level for this protein at the schizont stage, while, in the present study, luciferase activity was maximum at the ring stage. Thus, our study suggests that a tight transcriptional regulation governs the stage specific expression of the falcipains.
To further elucidate the transcriptional regulation of falcipains, we studied the interaction of nuclear proteins with different regions of falcipain promoters. Distinct nuclear factors were found to be interacting with specific sequences within each falcipain promoters. For example, probes Fal 2.3 and Fal 3.2 formed higher molecular weight complexes than probe Fal 2a.2.
Having established the distinct nature of complexes, the question arose, were these specific DNA/protein complexes stage specific? To answer this, we performed EMSA assays with nuclear extracts prepared from three asexual blood stages of P. falciparum. Protein-DNA complexes were formed depending upon the developmental stage of the parasite. In case of falcipain- 2, two independent sequences (probes 2.2 and 2.3) formed complexes at different stages. Probe 2.2 formed complexes with all the three stage specific nuclear extracts while probe 2.3 formed complexes only with trophozoite and schizont specific nuclear extracts. We hypothesize that probe 2.2 contain sequences that probably bind to general transcription factors/cis-acting elements, while probe 2.3 contain sequences that are part of a specific transcriptional machinery and are crucial for the stage dependent expression of the protein. A closer look at the sequence of probe 2.3 revealed the presence of a TGCAC motif, present exclusively in proteases (see Additional file 1). Probe 2a.2 of falcipain- 2' formed complexes with nuclear extracts from the ring and trophozoite stage but no complex was observed with the nuclear extract from schizont stage. This is in concordance with the reporter assay that showed maximum activity for falcipain- 2' promoter at the ring stage. Together these results suggest that the malaria parasite expresses transcription factors and trans/cis regulatory factors in a stage dependent manner that specifically binds to falcipains 5' upstream sequences. We also analyzed the role(s) of falcipain proteins in regulating the expression of other falcipain proteins (data not shown). However, neither falcipain-1 nor falcipain-2 proteins were able to bind any of the upstream sequence or to a DNA protein complex in an EMSA assay.
Sequence alignment of the nuclear factors binding sequences of falcipain s did not reveal a common element among these promoters, nor were homologies seen with many known binding sites of higher eukaryotic transcription factors. However, we did find parasite specific sequence elements in the upstream regions of falcipains. Long poly(dA) poly(dT) tracts that have been previously shown to regulate Pfcam activity were present in all falcipains 5'upstream regulatory sequences . The length and density of these tracts varied amongst the four falcipains. These poly(dA) poly(dT) tracts have been shown to influence the transcriptional activity in yeast, Giardia lamblia and Dictyostelium discoideum [25–27]. We also identified a CCAAT box  in upstream sequences of falcipain-3 gene; however, the significance of this element in falcipain- 3 needs further investigation. A single G-rich sequence, (A/G)NGGGG(C/A) that is typical of 5'UTR of heat shock genes in Plasmodium  was present in upstream regions of falcipain- 2 & -3. In falcipain 2, this sequence is present within the EMSA probe (see Additional file 1), while in falcipain 3, this motif is present upstream to the EMSA probe. A motif, TGCAC previously identified in a class of functionally related protease was also seen in upstream region of falcipain-2 . Thus, sequence analysis and alignment data of the falcipains 5'upstream regulatory regions suggest that cis-acting factors/transcription factor(s) distinct from that of human host are involved in regulating the expression of falcipains. Though, our initial attempts to identify the DNA binding proteins failed, isolation and characterization of parasite's cis-regulatory elements and transcription factors, which are apparently highly divergent from those of other eukaryotes due to the high A+T-richness of P. falciparum genome will be valuable to gain insight into the gene regulatory processes in P. falciparum.
In conclusion, we have identified sequences that are essential for functional promoter activity for each of the four falcipains. We have also shown that distinct nuclear factors bind to specific sequences of each falcipain promoter that in turn may determine the developmental stage specific patterns of falcipains expression. Characterization of parasite specific regulatory elements may provide new insights into the biology and pathogenicity of malaria parasite that can reveal new opportunities for intervention.
Parasite culture and transfection
P. falciparum 3D7 strain was continuously cultured in vitro with human erythrocytes (4% hematocrit) in complete RPMI 1640 media (Invitrogen) supplemented with 10% human sera following the protocol described previously . Parasites were synchronized by rounds of Percoll-enrichment of late-stage schizonts, followed by sorbitol treatment using a protocol described by Lambros and co-workers. . Ring stage parasites (parasitemia 5–7%) were transfected as described by Wu and co workers . Transfections were carried out with 75 μg of maxiprep (Qiagen) purified DNA. Renilla luciferase vector (pPfrluc) was used as an internal control for all the transfection experiments. Vector pGL2 with the luciferase gene but no promoter (Promega) served as a negative control for all the luciferase assays. Parasites were harvested at different time points, 48 h, 75 h and 90 h post transfection for the analysis of luciferase activity. All the experiments were done in triplicates.
5'Rapid Amplification of cDNA Ends Assay (5'RACE)
Total RNA was extracted from 3D7 parasite culture having parasitemia 8–12% using TRIzol reagent. mRNA was extracted from the total RNA using biotinylated oligo dT primers. Following mRNA isolation, the first strand synthesis was done with gene specific primer (GSP1) using SuperScript II Reverse Transcriptase (Invitrogen). TdT tailing was done with column purified cDNA sample and the tailed cDNA was amplified directly by PCR with gene specific primer (GSP2) and abridged anchor primers. In the case of falcipain 2 and 2', GSP1 and GSP2 were the same as the two genes shared 97% homology. The PCR conditions for the amplification of 5'UTR sequences of different falcipains are listed in Table 1. A second round of amplification using the product obtained from first PCR reaction was done with a primer, GSP3. In the second round of PCR reaction, the initial five cycles had the annealing temperature of 48°C with an initial denaturation of 94°C for 1 min and extension at 68°C for 1 min followed by 30 cycles of PCR conditions given in Table 1. Secondary PCR products were ligated into pGEMT-Easy (Promega) vector, and ligations were transformed into competent JM109 cells. Plasmid DNAs were prepared from individual clones using the Qiagen Miniprep Spin kit and the inserts were sequenced using gene-specific primers.
List of primers and PCR conditions used for making luciferase constructs
Primer sequences 5'-3'
Fal1Luc 1 kb F
Fal1Luc 0.5 kb F
Fal2Luc 2 kb F
Fal2Luc 1 kb F
Fal2Luc 0.5 kb F
Fal2aLuc 2 kb F
Fal2aLuc 1 kb F
Fal2aLuc 0.5 kb F
Fal3Luc 2 kb F
Fal3Luc 1 kb F
Fal3Luc 05.kb F
To perform the luciferase assay, transfected parasites were harvested by pelleting the infected erythrocytes (300 μl of packed cell volume/culture). The parasite pellets were lysed for 10 min on ice with PBS containing 0.15% saponin. The parasites were centrifuged at 7000 rpm for 10 min at 4°C and the pellets were washed twice with 1% PBS to eliminate residual debris. The pellets were then resuspended in 50 μl of passive lysis buffer (Promega) and kept at RT for 10 min to ensure total lysis and centrifuged at 13,000 rpm. Renilla and firefly luciferase activities were measured as described in the Dual Luciferase reporter assay system kit (Promega) using a Berthold luminometer. All experiments were performed at least twice and done in triplicates.
Preparation of parasite nuclear protein extracts
Nuclear protein extracts were prepared as described by Voss and co-workers  with some modifications. About 10 × 1010 parasitized erythrocytes (~10% parasitemia) at the three asexual blood stages, rings, trophozoites or schizonts were lysed in PBS containing 0.15% saponin. The parasites were washed with 1× PBS and resuspended in ice-cold lysis buffer (20 mM HEPES pH 7.8, 10 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.65% NP-40) for 5 min on ice. The lysates were centrifuged at 2500 g for 5 min and the supernatants containing the cytoplasmic proteins were removed. The remaining pellets containing intact nuclei were washed twice with lysis buffer. After washing, the nuclear pellets were resuspended in one pellet volume of extraction buffer (20 mM HEPES pH 7.8, 800 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 3 uM Pepstatin A, 10 uM leupeptin) and the nuclei were extracted with vigorous shaking at 4°C for 30 min. The extracted nuclei were centrifuged at 13000 g for 30 min. The supernatants containing the nuclear proteins were diluted with one volume of dilution buffer (20 mM HEPES pH 7.8, 1 mM EDTA, 1 mM DTT, 30% glycerol) and stored at -80°C until further use.
Electromobility Shift Assay (EMSA)
List of primers for EMSA probes
Fal 1.1 F
Fal 1.1 R
Fal 2.1 F
Fal 2.1 F
Fal 2.2 F
Fal 2.2 R
Fal 2.3 F
Fal 2.3 R
Fal 2a.1 F
Fal 2a.1 R
Fal 2a.2 F
Fal 2a.2 R
Fal 3.1 F
Fal 3.1 R
Fal 3.2 F
Fal 3.2 R
Fal 3.3 F
Fal 3.3 R
EMSA reactions were carried out by incubating 2–5 μg of nuclear proteins with 5 fmol of radiolabeled probe in EMSA buffer (50 mM Tris pH 7.5, 250 mM NaCl, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 20% glycerol) containing 500 ng of poly(dI-dC) as nonspecific competitor in a 20 μl reaction volume for 20 min at room temperature. Binding reactions were analyzed on a 6% native polyacrylamide gel in 0.5% TBE. Gels were dried and exposed for autoradiography. Scanning was performed using Typhoon 9210, variable mode Imager from Amersham Biosciences, USA. For competition experiments, the labeled probe was added 10 min after incubation of competitor DNA.
We thank Manish Sharma for his help in 5'RACE assays. We also thank Reshma Korde for her help. This work was funded in part by the Department of Biotechnology, New Delhi.
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