An integrated strategy for efficient vector construction and multi-gene expression in Plasmodium falciparum

Abstract

Background: The construction of plasmid vectors for transgene expression in the malaria parasite, Plasmodium falciparum, presents major technical hurdles. Traditional molecular cloning by restriction and ligation often yields deletions and re-arrangements when assembling low-complexity (A + T)-rich parasite DNA. Furthermore, the use of large 5'- and 3'- untranslated regions of DNA sequence (UTRs) to drive transgene transcription limits the number of expression cassettes that can be incorporated into plasmid vectors.

Methods: To address these challenges, two high fidelity cloning strategies, namely yeast homologous recombination and the Gibson assembly method, were evaluated for constructing P. falciparum vectors. Additionally, some general rules for reliably using the viral 2A-like peptide to express multiple proteins from a single expression cassette while preserving their proper trafficking to various subcellular compartments were assessed.

Results: Yeast homologous recombination and Gibson assembly were found to be effective strategies for successfully constructing P. falciparum plasmid vectors. Using these cloning methods, a validated family of expression vectors that provide a flexible starting point for user-specific applications was created. These vectors are also compatible with traditional cloning by restriction and ligation, and contain useful combinations of commonly used features for enhancing plasmid segregation and site-specific integration in P. falciparum. Additionally, application of a 2A-like peptide for the synthesis of multiple proteins from a single expression cassette, and some rules for combinatorially directing proteins to discrete subcellular compartments were established.

Conclusions: A set of freely available, sequence-verified and functionally validated parts that offer greater flexibility for constructing P. falciparum vectors having expanded expression capacity is provided.

Figure 1

Schematic of the homology-based (yeast HR and Gibson assembly) and traditional restriction/ligation cloning strategies selected as part of an integrated framework for the orthogonal assembly of Plasmodium falciparum constructs. Beginning with a common primer set, PCR products and the desired vector backbone (see Figure 2 for details), the identical target plasmid can be assembled using any of these approaches individually or in parallel.

Figure 2

Schematic summary of the new family of plasmid vectors. Plasmids are designated by the pfYC prefix, a series number (1-4) and a number (0-4) defining the resistance marker present in expression cassette A (5′PfCaM/3′PfHsp86 UTRs) or B (5′-PcDT/3′PfHRPII UTRs). The series number is defined by specific utility features included in the plasmid as follows: 1 = yeast CEN/ARS origin to enable plasmid maintenance in S. cerevisiae during yeast HR and a 2 × Rep20 element to improve plasmid segregation in P. falciparum[19]; 2 = same as in 1, but with a 2 × attP element added to enable site-specific chromosomal integration into existing attB⁺ strains [18]; 3 = 2 × attP element is present, but the yeast CEN/ARS origin and 2 × Rep20 elements have been eliminated; and 4 = the pfcen5-1.5 mini-centromere element is included to facilitate plasmid segregation and maintenance at single copy in P. falciparum[20], while the yeast origin, 2 × Rep20 and 2 × attP elements have been eliminated. P. falciparum resistance markers are designated as: 0 = none; 1 = nptII (G-418 resistance); 2 = bsd (Blasticidin S resistance); 3 = hdhfr (WR99210 resistance); and 4 = ydhodh (DSM-1 resistance). A non-resistance gene cloned into the available expression cassette is indicated by a colon followed by the gene name (e g, pfYC110:FL indicates that the nptII and firefly luciferase genes are present in expression cassettes A and B, respectively). Three HindIII sites present on the base plasmid are noted, as they are useful for topologically mapping these vectors and derivatives to screen for potential rearrangements and large insertions or deletions.

Figure 3

Heterologous and native Plasmodium falciparum genes can be successfully assembled into pfYC vectors using all three cloning strategies. (A) The firefly luciferase gene (FL = 1.65 kb) was cloned into the pfYC1 and pfYC3 series (Additional file 3) using either yeast HR or Gibson assembly. Topological mapping with HindIII digestion yields three fragments, as FL and the selection markers do not contain HindIII sites. A 3.9 kb fragment is released from the pfYC1 series whether FL is present or not (Figure 2). The fragments containing cassettes A and B from pfYC10x:FL plasmids are (1.5 kb + FL) = 3.2 kb and (1.7 + selection marker size) kb, respectively. Similarly, the fragments containing cassettes A and B from pfYC1x0:FL plasmids are (1.5 + selection marker size) kb and (1.7 + FL size) = 3.4 kb, respectively. The sizes of the different selection markers are: nptII (0.8 kb); hdhfr (0.6 kb); bsd (0.4 kb) and ydhodh (0.95 kb). (B) Two native P. falciparum genes, ama1 (apical membrane antigen 1; PF3D7_1133400; 1.87 kb) and trxR (thioredoxin reductase; PF3D7_0923800.1; 1.85 kb) were cloned in parallel using restriction/ligation, Gibson assembly and yeast HR, and the same PCR products and digested pfYC120 vector. Successful gene insertion is expected to yield three HindIII digestion products that include: a backbone fragment (denoted as C); cassette B with the ama1 or trxR gene inserted (denoted as B when no insert is present and B′ when containing the proper insert); and cassette A containing the bsd gene (denoted as A). As a reference, the parent pfYC120 plasmid yields products denoted as A, C and B upon HindIII digestion. The asterisk in the yeast HR trxR panel denotes sample degradation that occurred during storage prior to analysis by gel electrophoresis.

Figure 4

The pfYC plasmid family exhibits typical behaviour during Plasmodium falciparum transfection, and can be maintained episomally and chromosomally integrated. (A and B) The entire pfYC1xx:FL plasmid series was either transfected individually (A) or as a single pair (pfYC110:FL + pfYC120:RL) (B) under the appropriate drug selection initiated on day 4 post-transfection (arrow). Firefly and Renilla luciferase levels were monitored to assess parasite population growth kinetics until a parasitaemia ≥1% was attained. (C). The copy number of each plasmid per parasite genome was determined for both the single and double transfections. (D) PCR confirmation of chromosomal integration of pfYC320 and pfYC340 at the cg6 locus in the P. falciparum 3D7-attB strain. The β-actin gene was PCR amplified as a positive control.

Figure 5

The Thosea asigna virus 2A-like peptide (T2A) enables expression of two functional proteins in Plasmodium falciparum from a single expression cassette. (A) Schematic of FL-nptII and control constructs. (B) Both T2A- and T2Am- containing constructs produce active FL. (C) Western blot detection of FL- and nptII- containing proteins. (D) Northern blot analysis of FL-containing transcripts in transfected parasites. 3D7 + FL indicates the inclusion of a synthetic FL mRNA produced by in vitro transcription.

Figure 6

The 2A sequence can be used to successfully and predictably target proteins to distinct subcellular compartments. Various targeting sequences were N-terminally fused to an upstream vYFP and a downstream tdTom reporter separated by T2A. The vYFP and tdTom proteins were localized using direct fluorescence microscopy imaging. Mitochondria were stained with MitoTracker (MT), and nuclei with Hoechst 33342. Legend: ATS = apicoplast targeting sequence; MTS = mitochondrial targeting sequence and PEX = protein export element.