Identification and characterization of a liver stage-specific promoter region of the malaria parasite Plasmodium

Abstract

During the blood meal of a Plasmodium-infected mosquito, 10 to 100 parasites are inoculated into the skin and a proportion of these migrate via the bloodstream to the liver where they infect hepatocytes. The Plasmodium liver stage, despite its clinical silence, represents a highly promising target for antimalarial drug and vaccine approaches. Successfully invaded parasites undergo a massive proliferation in hepatocytes, producing thousands of merozoites that are transported into a blood vessel to infect red blood cells. To successfully develop from the liver stage into infective merozoites, a tight regulation of gene expression is needed. Although this is a very interesting aspect in the biology of Plasmodium, little is known about gene regulation in Plasmodium parasites in general and in the liver stage in particular. We have functionally analyzed a novel promoter region of the rodent parasite Plasmodium berghei that is exclusively active during the liver stage of the parasite. To prove stage-specific activity of the promoter, GFP and luciferase reporter assays have been successfully established, allowing both qualitative and accurate quantitative analysis. To further characterize the promoter region, the transcription start site was mapped by rapid amplification of cDNA ends (5'-RACE). Using promoter truncation experiments and site-directed mutagenesis within potential transcription factor binding sites, we suggest that the minimal promoter contains more than one binding site for the recently identified parasite-specific ApiAP2 transcription factors. The identification of a liver stage-specific promoter in P. berghei confirms that the parasite is able to tightly regulate gene expression during its life cycle. The identified promoter region might now be used to study the biology of the Plasmodium liver stage, which has thus far proven problematic on a molecular level. Stage-specific expression of dominant-negative mutant proteins and overexpression of proteins normally active in other life cycle stages will help to understand the function of the proteins investigated.

Figure 1. Liver stage-specific gene expression.

(A) Results of RT-PCR analysis of PB103464.00.0 mRNA expression, comparing blood stage (BS), oocysts (Oo) and in vitro liver stage (LS) 48 hpi. Total RNA was extracted, and RT-PCR reactions were performed with (+) or without (−) reverse transcriptase (negative control). RT-PCR analysis of tubulin mRNA expression served as a control.

Figure 2. Promoter-dependent GFP expression.

(A) The vector pGFP₁₀₃₄₆₄ with GFP under the control of the promoter region 103464 and the vector pGFP_ef1α with GFP under control of the ef1α promoter were generated and their transfection resulted in the parasite lines PbGFP₁₀₃₄₆₄ and PbGFP_ef1α. (B) Live imaging of PbGFP_ef1α and PbGFP₁₀₃₄₆₄ parasites at different life cycle stages. HepG2 cells were infected with transgenic P. berghei sporozoites and analyzed at different time points after infection (hpi, hours post-infection). GFP expresion was monitored by fluorescent microscopy. DNA was stained with Hoechst 33342. Arrows indicate young liver stage parasites. (iRBC: infected red blood cell; LS: liver stage) Scale bars: 10 µm.

Figure 3. Generation of plasmids and transgenic P. berghei parasites for use in dual-luciferase assays.

(A) The vector pFL₁₀₃₄₆₄RL_ef1α with FL under the control of the promoter region 103464 and RL under the control of the ef1α promoter was generated and its transfection resulted in the parasite line PbFL₁₀₃₄₆₄RL_ef1α. The plasmid pFL_ef1αRL_ef1α with FL and RL under the control of the ef1α promoter was used to obtain control parasites PbFL_ef1αRL_ef1α. Diamonds represent 3′UTRs, which in all cases were from the pbdhfr/ts gene. In the upper plasmid, the selection marker (tgdhfr/ts) is displayed but for simplicity in all other plasmid diagrams only those genes and features directly related to the experiments described are displayed. (B) Comparison of the luciferase activity (FL expression relative to RL expression) of transgenic parasites during the blood stage (BS), in oocysts (Oo), in salivary gland sporozoites (Sp) and in vitro in the liver stage (LS), 48 hours post-infection (hpi) of hepatoma cells. Standard deviation values (shown as error bars) were determined from three different measurements. Statistical analysis was performed using two-tailed unpaired t-tests (*P<0.05; ***P<0.001).

Figure 4. Comparison of the activities of promoter regions of the genes PB000869.01.0 and PB103464.00.0 during different life cycle stages.

(A) Plasmid map of pFL₈₆₉RL_ef1α, used to generate the parasites PbFL₈₆₉RL_ef1α, which express Renilla (RL) and firefly (FL) luciferases under the control of the pbeef1αa and the PB000869.01.0 promoter region, respectively. The diamond represents the 3′UTR of the pbdhfr/ts gene. (B) PB000869.01.0 promoter activity in respect to the constitutive promoter pbeef1αa, which was set to 100%. PbFL₈₆₉RL_ef1α and PbFL_ef1αRL_ef1α parasites were harvested at different life cycle stages (BS; blood stage, Oo; oocysts, Sp; salivary gland sporozoites, LS 24 hpi; liver stage 24 hours post-infection in vitro), the ratio of FL to RL expression was determined and the percentage of luciferase activity of PbFL₈₆₉RL_ef1α parasites was calculated relative to that of PbFL_ef1αRL_ef1α parasites. (C) Comparison of luciferase activity in the parasites PbFL₁₀₃₄₆₄RL_ef1α and PbFL₈₆₉RL_ef1α during the liver stage, as determined by the ratio of FL to RL expression. Standard deviation values (shown as error bars) were determined from three different measurements.

Figure 5. Identification of the transcription start site (TSS) of the promoter region of PB103464.00.0.

(A) Schematic representation of the promoter region. A flag at position −318 shows the TSS. The primers used for the 5′-RACE are indicated. (B) The RACE product, obtained from the second PCR amplification, was analyzed by gel electrophoresis. (C) The 5′ UTR of PB103464.00.0 with the start codon, shown in bold. The arrow indicates the TSS of the promoter region.

Figure 6. Promoter deletion affects expression of FL.

Parasites transfected with the plasmid pFL₁₀₃₄₆₄RL_ef1α carrying the complete promoter region (−989/+1), promoter deletions (PbFL_{103464(−775)}RL_ef1a (−775/+1) and PbFL_{103464(−318)}RL_ef1a (−318/+1)) or a promoter mutation (PbFL_103464(*825)RL_ef1amut-825/−818)were passed through mosquitoes and used for HepG2 cell infections. 48 hpi cells were harvested and dual-luciferase assays of the cell extracts were performed. The FL/RL ratio obtained from extracts of parasites transfected with the plasmid pFL₁₀₃₄₆₄RL_ef1α was set to 100% and the FL/RL ratio obtained from the parasite strains transfected the other constructs was calculated.