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

Abstract

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 hrp3 mRNA is only seen in ring-stage parasites, deletion of a specific sequence in the 5' end of the promoter region decreased ring-stage expression of hrp3 and enabled detection of its transcripts in trophozoite-stage 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 demonstrate 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.

Figure 1

Deletion of hrp3 promoter reduced the ring parasite firefly luciferase activity. A) Schematic representation of hrp3 promoter truncations driving firefly luciferase (FFL). B) Transient expression of luciferase gene in ring and trophozoite parasites. FFL activity was normalized by cotransfection using Chloramphenicol acetyltransferase gene (CAT) as described in Material and Methods. Bars are the mean of four independent experiments ± SD.

Figure 2

Ring specific cam-dependent luciferase activity is stimulated by hrp3 region between -1.7 to -1.1. A) Schematic representation of the 1.7 kb hrp3 promoter region depicting the different hrp3 fragments placed upstream of the cam 5’ end region. In addition, the organization of the hybrid promoter is shown. B) Transient luciferase activity was measured in ring and trophozoite parasites and normalized to CAT activity. The control plasmid (pcamSluc) contains cam promoter and firefly luciferase gene (FFL). pAhrpcamSluc, pA1hrpcamSluc, pA2hrpcamSluc represent plasmids with hrp3 A, A1 and A2 fragments placed upstream of the cam promoter respectively. Bars are the mean of three independent experiments ± SD. C) Schematic representation of the 1.7 kb hrp3 promoter region depicting the different hrp3 fragments placed upstream the cam 5’ end region. In addition, the general organization of the hybrid promoter is shown. D) Transient luciferase activity was measured in ring and trophozoite parasites and normalized by cotransfection with a plasmid carrying the Renilla luciferase gene driven by the cam promoter. The control plasmid (pcamSluc) contains the cam promoter driving firefly luciferase gene (FFL). pBhrpcamSluc, pB1hrpcamSluc, pB2hrpcamSluc represent plasmids with hrp3 B, B1 and B2 fragments placed upstream the cam promoter respectively. Bars are the mean of four independent experiments ± SD.

Figure 2

Ring specific cam-dependent luciferase activity is stimulated by hrp3 region between -1.7 to -1.1. A) Schematic representation of the 1.7 kb hrp3 promoter region depicting the different hrp3 fragments placed upstream of the cam 5’ end region. In addition, the organization of the hybrid promoter is shown. B) Transient luciferase activity was measured in ring and trophozoite parasites and normalized to CAT activity. The control plasmid (pcamSluc) contains cam promoter and firefly luciferase gene (FFL). pAhrpcamSluc, pA1hrpcamSluc, pA2hrpcamSluc represent plasmids with hrp3 A, A1 and A2 fragments placed upstream of the cam promoter respectively. Bars are the mean of three independent experiments ± SD. C) Schematic representation of the 1.7 kb hrp3 promoter region depicting the different hrp3 fragments placed upstream the cam 5’ end region. In addition, the general organization of the hybrid promoter is shown. D) Transient luciferase activity was measured in ring and trophozoite parasites and normalized by cotransfection with a plasmid carrying the Renilla luciferase gene driven by the cam promoter. The control plasmid (pcamSluc) contains the cam promoter driving firefly luciferase gene (FFL). pBhrpcamSluc, pB1hrpcamSluc, pB2hrpcamSluc represent plasmids with hrp3 B, B1 and B2 fragments placed upstream the cam promoter respectively. Bars are the mean of four independent experiments ± SD.

Figure 3

A putative multipartite element on hrp3 promoter controls ring specific expression of a reporter gene. Schematic representation of hrp3 deletions (left side of panel). The constructs’ name indicates the promoter size after deletion. hrp3 promoter was deleted by PCR and placed upstream the firefly luciferase gene. Constructs were transfected into P. falciparum. Firefly and Renilla luciferase activities were determined 12–14 hours post-invasion (hpi) (ring) and 32–34 hpi (trophozoite). Bars are the mean of four independent experiments ± SD.

Figure 4

Logo visualization of hrp3 promoter transcription start site. cDNA, from endogenous and episomal located promoter, was cloned in E. coli and recombinant plasmids were purified from 15 independent colonies for each group. Recombinant plasmids were then sequenced and used to determine the 5’ end of cloned cDNA that represents the TSS. The logo sequence shows alignment of 15 different TSS for endogenous and episomal located hrp3 promoter. The first transcribed nucleotide (position 11) was adenosine in most cases. Ten nucleotide upstream and downstream of the TSS in endogenous (A) and episomal (B) were also included. The height of each letter is proportional to the frequency of nucleotides in each position. The total letter height indicates the amount of information (bits) contained in the nucleotides at that position.