Plasmodium falciparum: hrp3 promoter region is associated with stage-specificity and episomal recombination

Abstract

The asexual blood stage of Plasmodium falciparum in the human host is comprised of morphologically distinct ring, trophozoite and schizont stages, each of which possesses a distinct pattern of gene expression. Episomal promoter recombination has been recently reported in malaria parasites. We aim to investigate the nature of this process, and its relationship with promoter activity by employing a series of nested deletions of the ring-specific hrp3 promoter. Our results showed a discrete promoter region that is preferentially used for recombination. The P. falciparum hrp3 mRNA is only seen in ring-stage parasites but deletion of the recombination region was associated with decreased ring-stage expression and concurrent detection of transcripts in trophozoite-stage parasites. Our results describe a ring-stage specific regulatory region possibly involved in episomal promoter recombination, suggesting that common sequences might mediate both processes.

Figure 1. Construction of plasmids used to transfect P. falciparum parasites

(A) Schematic representation of plasmids containing 1.7-kb promoter (pHRP1.7G), 1.1-kb (pHRP1.1G), 0.6-kb (pHRP10.6G) and 0.5-kb (pHRP0.5G) hrp3 truncated promoters driving the GFP gene. The plasmid pDT.Tg23 plasmid is also shown containing the 1.7-kb hrp3 promoter driving the T. gondii dhfr-ts gene (Kadekoppala, et al., 2001). (B) Stable pyr-resistant parasite cultures cotransfected with pHRP1.7G and pDT.Tg23 plasmids show green fluorescent GFP (left panel) in early trophozoite. Nucleic acid staining is shown in blue in the nucleus (Center panel) and the merge image (right panel).

Figure 2. In vivo episomal recombination in P. falciparum parasites

Parasite cultures were cotransfected with pHRP1.7G, pHRP1.1G, pHRP10.6G or pHRP0.5G hrp3 truncated promoters driving the GFP gene and pDT.Tg23 as shown in figure 1. Parasites were collected at 12, 15, 19, 24 and 33 days post transfection. Green fluorescent parasites were scored and expressed as percent of total.

Figure 3. Heteromultimeric plasmids recovered in E. coli

(A) Schematic representation of heteromultimeric plasmids containing 1pyr^r:1gfp copy of pH1.7G and pDT.Tg23 (type 1); 1pyr^r:2gfp (type 2) or 2pyr^r:1gfp (type 3) (Kadekoppala, et al., 2001). (B). E. coli competent cells were transformed with total DNA from parasite cell lines. Plasmid recovered in E. coli transformed with pDT.Tg23/pH1.7HG (pH1.7HG), pDT.Tg23/pH1.1HG (pH1.1HG), pDT.Tg23/pH0.6HG (pH0.6HG) or pDT.Tg23/pH0.5HG (pH0.5HG) plasmid mixtures were digested with Hpa I (H) and/or Xho I (X) and separated on agarose gel with undigested (U) plasmid as control. DNA molecular weight marker (in kb) is presented. Detected bands did not change size after long-term culture (more than a year). Plasmid backbone is pBluescript (not shown).

Figure 4. Deletion of the hrp3 promoter switched stage specific expression from ring to trophozoite stage

(A) Schematic representation of hrp3 promoter truncations (diagonal lines) driving human DHFR fused to GFP (HDGFP, filled box) in plasmid pH1.7HG containing the 1.7-kb full length promoter as well as the plasmids pH1.1HG, pH0.6HG carrying the truncated promoters of 1.1-kb and 0.6-kb respectively. (B) Total RNA from stable cell lines expressing HDGFP was purified from ring (i, iii) and trophozoite (ii, iv) stages, separated on agarose gels stained with ethidium bromide (iii, iv) and analyzed by northern blot (i, ii) using ³²P-GFP antisense probe.