A method for rapid genetic integration into Plasmodium falciparum utilizing mycobacteriophage Bxb1 integrase

Abstract

Genetic manipulation of the human malaria parasite Plasmodium falciparum has presented substantial challenges for research efforts aimed at better understanding the complex biology of this highly virulent organism. The development of methods to perform gene disruption, allelic replacement or transgene expression has provided important insights into the function of parasite genes. However, genomic integration studies have been hindered by low transfection and recombination efficiencies, and are complicated by the propensity of this parasite to maintain episomal replicating plasmids. We have developed a fast and efficient site-specific system of integrative recombination into the P. falciparum genome, which is catalyzed by the mycobacteriophage Bxb1 serine integrase. This system has the advantage of providing greater genetic and phenotypic homogeneity within transgenic lines as compared to earlier methods based on episomal replication of plasmids. Herein, we present this methodology.

Figure 1

Generation of homogeneous parasites by integrase-mediated attB × attP recombination. (a) Live cell imaging of Dd2^attB parasites expressing an mRFP-tagged cytosolic protein (PfVps4) encoded on a plasmid integrated at the cg6-attB locus. Bright field (left panel) and merged fluorescence images (right panel) showing mRFP-PfVps4 (red) and Hoechst 33342-stained nuclei (blue). Prior to imaging, infected RBCs were enriched by magnet-activated cell sorting (MACS, Miltenyi Biotec Inc., Auburn, CA) (18). Bar = 10 µm. (b) and (c) Flow cytometry analysis of parasites expressing GFP either from a plasmid integrated at the cg6-attB locus (Dd2^attB/GFP) or from episomes (Dd2/GFP). Parasites were incubated with Hoechst 33342 dye and analyzed at 16- to 20-h post-invasion. Cells were gated for nuclear staining to distinguish infected from uninfected RBCs. (b) GFP expression profiles show a higher percentage of GFP-expressing parasites in the integrant Dd2^attB/GFP population (94%) than in the episomal Dd2/GFP (74%). (c) Dd2^attB/GFP integrants show a unique peak of GFP expression with negligible variance, whereas Dd2 parasites expressing GFP from episomes display multiple peaks with a significantly larger variance. Panels (b) and (c) were derived from Fig. 4 in ref. with the permission of the Nature Publishing Group.

Figure 2

Dual-plasmid approach for integrase-mediated attB × attP recombination. (a) Map of the attP-carrying plasmid pLN-ENR-GFP that can be used as a template for engineering the stable site-specific integration of a gene of interest. Digestion with AvrII and AflII can be performed to replace the enr-gfp fusion by the gene of interest. Alternatively, digestion with AvrII and BsiWI can place the gene of interest in frame with gfp. The plasmid carries the blasticidin S-deaminase (bsd) selectable marker. (b) Map of the integrase-expressing plasmid pINT, which harbors a neomycin selectable marker. The plasmid is usually maintained under G418 selection pressure only for the first 6 days following transfection. (c) Schematic representation of the attB × attP recombination allowing integration of a gene of interest (yfg) at the cg6-attB locus. Dd2^attB or 3D7^attB parasites are co-transfected with pINT and an attP-plasmid carrying the gene of interest, and selected with 2.5 µg/mL BSD, 2.5 nM WR99210 (to select for the human dihydrofolate reductase (hdhfr) marker in the cg6-attB locus) and 125 or 250 µg/mL G418 (for the Dd2^attB and 3D7^attB parasite lines respectively). Integrase expressed from the pINT plasmid catalyzes insertion of the attP-plasmid harboring the gene of interest into the attB site. PCR reactions using p1 + p2 and p3 + p4 primers as well as Southern blots analyzing digested-DNA hybridization to cg6 or yfg probes (designated as open boxes), can be performed to confirm integration of the gene of interest at the cg6-attB locus. Sequences of the primers for PCR screening are as follows: p1: 5′-GAAAATATTATTACAAAGGGTGAGG, p2: 5′-TTAGCTAATTCGCTTGTAAG, p3: 5′-CTCTTCTACTCTTTCGAATTC, p4 is designed based on the sequence of the gene of interest. pBS refers to the pBluescript backbone. UTR: untranslated region. Panel (c) was adapted from Fig. 3 in ref. with the permission of the Nature Publishing Group.