Stable transgene expression in Plasmodium falciparum

Crabb, Brendan S.; Triglia, Tony; Waterkeyn, Jacqueline G.; Cowman, Alan F.

doi:10.1016/s0166-6851(97)00143-6

Cited by 129 publications

(105 citation statements)

References 21 publications

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“…A dipeptidyl aminopeptidase type I (DPAP1) 1 truncation plasmid (pFA5KO) was made by PCR amplification of bases 928 -1857 of the DPAP1 open reading frame from P. falciparum 3D7 genomic DNA with primers 5Ј-AGTCACTCGAGTAAGTTGTTGATAATGTAATAAATA-AAGG (FA5KO1) and 5Ј-AGTCACTCGAGTTACCAACCTGTTATGTT-ATAGACAC and cloning the product into the XhoI site of pHC1 (26). The orientation of the insert was such that the 3Ј end of the DPAP1 open reading frame was adjacent to the 5Ј end of the HSP86 3Ј-untranslated region.…”

Section: Methodsmentioning

confidence: 99%

A Plasmodium falciparum Dipeptidyl Aminopeptidase I Participates in Vacuolar Hemoglobin Degradation

Klemba

Gluzman

Goldberg

2004

Journal of Biological Chemistry

175

174

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Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum requires the catabolism of large amounts of host cell hemoglobin. Endoproteolytic digestion of hemoglobin to short oligopeptides occurs in an acidic organelle called the food vacuole. How amino acids are generated from these peptides is not well understood. To gain insight into this process, we have studied a plasmodial ortholog of the lysosomal exopeptidase cathepsin C. The plasmodial enzyme dipeptidyl aminopeptidase 1 (DPAP1) was enriched from parasite extract by two different approaches and was shown to possess hydrolytic activity against fluorogenic dipeptide substrates. To localize DPAP1 we created a transgenic parasite line expressing a chromosomally encoded DPAP1-green fluorescent protein fusion. Green fluorescent protein fluorescence was observed in the food vacuole of live transgenic parasites, and anti-DPAP1 antibody labeled the food vacuole in parasite cryosections. Together these data implicate DPAP1 in the generation of dipeptides from hemoglobin-derived oligopeptides. To assess the significance of DPAP1, we attempted to ablate DPAP1 activity from blood stage parasites by truncating the chromosomal DPAP1-coding sequence. The inability to disrupt the coding sequence indicates that DPAP1 is important for asexual proliferation. The proenzyme form of DPAP1 was found to accumulate in the parasitophorous vacuole of mature parasites. This observation suggests a trafficking route for DPAP1 through the parasitophorous vacuole to the food vacuole.

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Section: Methodsmentioning

confidence: 99%

A Plasmodium falciparum Dipeptidyl Aminopeptidase I Participates in Vacuolar Hemoglobin Degradation

Klemba

Gluzman

Goldberg

2004

Journal of Biological Chemistry

175

174

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“…The nucleotide sequence encoding for the first 87 amino acids of PfEMP2 was PCR-amplified from the PfEMP2 open reading in a 3D7 26-h cDNA library (MR4) with primers 5′-CGCTAGAG CTCCATGGAGGTAATTTGTAGAAA-3′ and 5′-GACTAGAATTCGCCTCTAAACTC ATCGGTGGTT-3′ and inserted into SacI/EcoRI-restricted His 6 -GFPmut2 pET-MALcH to place the PEXEL processing site 19 amino acids upstream of a thrombin cleavage site (TCS) and generate PfEMP2(1-87)-TCS-His 6 -GFPmut2 pET-MALcH. The subcloned PfEMP2-GFP construct was PCR-amplified with primers 5′-GGTGGATACTCGAGAT GGAGGTAATTTGTAGAA-3′ and 5′-GATACTCGAGTTATTTGTATAGTTCATCCA TGCCATG-3′ and inserted into the XhoI site in pHC1 (MR4) to obtain PfEMP2-GFP pHC1 [21].…”

Section: Plasmid Construction Transfection and Microscopy Of Pfemp2mentioning

confidence: 99%

“…If the PEXEL of PfEMP2-GFP is fully processed, thrombin cleavage of the processed protein would generate a single peptide at m/z 2095.04. This construct was cloned into the pHC1 Plasmodium expression vector and overexpressed under the parasite cam promoter that is maximally active in the trophozoite stage [21].…”

Section: Pexel Of Pfemp2 Is Cleaved and N-acetylatedmentioning

confidence: 99%

N-terminal processing of proteins exported by malaria parasites

Chang

Falick

Carlton

et al. 2008

Molecular and Biochemical Parasitology

171

177

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Malaria parasites utilize a short N-terminal amino acid motif termed the Plasmodium export element (PEXEL) to export an array of proteins to the host erythrocyte during blood stage infection. Using immunoaffinity chromatography and mass spectrometry, insight into this signalmediated trafficking mechanism was gained by discovering that the PEXEL motif is cleaved and N-acetylated. PfHRPII and PfEMP2 are two soluble proteins exported by Plasmodium falciparum that were demonstrated to undergo PEXEL cleavage and N-acetylation, thus indicating that this Nterminal processing may be general to many exported soluble proteins. It was established that PEXEL processing occurs upstream of the brefeldin A-sensitive trafficking step in the P. falciparum secretory pathway, therefore cleavage and N-acetylation of the PEXEL motif occurs in the endoplasmic reticulum (ER) of the parasite. Furthermore, it was shown that the recognition of the processed N-terminus of exported proteins within the parasitophorous vacuole may be crucial for protein transport to the host erythrocyte. It appears that the PEXEL may be defined as a novel ER peptidase cleavage site and a classical N-acetyltransferase substrate sequence.

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“…While transcriptome and proteome analyses indicate that some genes are regulated post-transcriptionally [8,9], other genes are regulated at the level of transcription. For example, analyses of 5' end upstream regions of the asexual and sexual stages have been shown by transfection studies that they function as promoters by supporting reporter gene expression [10][11][12][13][14][15][16]. Moreover, functional and bioinformatic studies of sexual and asexual Plasmodium promoters have uncovered putative cis-acting regulatory elements possibly involved in gene regulation [17][18][19][20][21][22].…”

Section: Introductionmentioning

confidence: 99%

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

López-Estraño

Gopalakrishnan

Semblat

et al. 2007

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expres

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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 RNA of the P. falciparum hrp3 gene is only seen in ring-stage parasites, deletion of a specific sequensce in the 5' end of the promoter region decreased ring-stage expression of hrp3 and enabled detection of its transcripts in trophozoitestage 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 demonstrates 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.

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Stable transgene expression in Plasmodium falciparum

Cited by 129 publications

References 21 publications

A Plasmodium falciparum Dipeptidyl Aminopeptidase I Participates in Vacuolar Hemoglobin Degradation

A Plasmodium falciparum Dipeptidyl Aminopeptidase I Participates in Vacuolar Hemoglobin Degradation

N-terminal processing of proteins exported by malaria parasites

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

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