Identification of an AP2-family protein that is critical for malaria liver stage development

Abstract

Liver-stage malaria parasites are a promising target for drugs and vaccines against malaria infection. However, little is currently known about gene regulation in this stage. In this study, we used the rodent malaria parasite Plasmodium berghei and showed that an AP2-family transcription factor, designated AP2-L, plays a critical role in the liver-stage development of the parasite. AP2-L-depleted parasites proliferated normally in blood and in mosquitoes. However, the ability of these parasites to infect the liver was approximately 10,000 times lower than that of wild-type parasites. In vitro assays showed that the sporozoites of these parasites invaded hepatocytes normally but that their development stopped in the middle of the liver schizont stage. Expression profiling using transgenic P. berghei showed that fluorescent protein-tagged AP2-L increased rapidly during the liver schizont stage but suddenly disappeared with the formation of the mature liver schizont. DNA microarray analysis showed that the expression of several genes, including those of parasitophorous vacuole membrane proteins, was significantly decreased in the early liver stage of AP2-L-depleted parasites. Investigation of the targets of this transcription factor should greatly promote the exploration of liver-stage antigens and the elucidation of the mechanisms of hepatocyte infection by malaria parasites.

Figure 1. AP2-L and related proteins in apicomplexan parasites.

A. Schematic diagrams of P. berghei AP2-L and related proteins are shown. The paralogs in P. berghei are indicated by their gene identifiers in PlasmoDB. The related proteins in other apicomplexan parasites are indicated by the species name and their GenBank accession number. AP2 domains (blue) and another conserved sequence (red) are highlighted by rectangles. Bars indicate the regions shown in B and C. B. Amino acid sequences of a region containing two A domains (highlighted with rectangles) are compared in the AP2-Ls of four Plasmodium species: P. berghei (Pb), P. falciparum (Pf), P. vivax (Pv), and P. knowlesi (Pk). The amino acids conserved across all four species are indicated by asterisks. This region is indicated in A by the left bar. C. Amino acid sequences of another region conserved among the AP2-Ls. This region is indicated in A by the right bar. The amino acids conserved across all four species are indicated by asterisks.

Figure 2. Deletion of the AP2-L gene does not affect the ability of sporozoites to invade hepatocytes.

A. Schematic diagram of targeting P. berghei AP2-L by double cross-over homologous recombination. A Southern blot analysis of wild-type (WT) and two independent AP2-L knockout populations (AP2-L(−) 1 and AP2-L(−) 2) is shown on the right. B. Replication of asexual blood stages in mice. Mice were intravenously injected with blood infected with AP2-L(−) or wild-type (WT) parasites (∼1×10⁵ parasites per mouse). The parasitemia of each mouse was checked each day. The values are the mean ± standard error of the mean (SEM) for four measurements. C. Cell-traversal assay in HepG2 cells. Sporozoites (4×10⁴) were inoculated into a culture of HepG2 cells and incubated at 37°C for 30 min. The number of injured cells that incorporated FITC-labeled dextran was counted. The values are the mean ± SEM of four measurements. D. Double staining assay. Sporozoites (5×10⁴) were inoculated into a culture of HepG2 cells and incubated at 37°C for 1 h. The sporozoites outside (stained both red and green) and inside (stained only red) the HepG2 cells were counted independently. The results are shown as the percentage of sporozoites inside the cell out of the total. The values are the mean ± SEM of eight measurements. E. Fluorescence images of AP2-L(−) 1 and wild-type parasites inside HepG2 cells 6 h after sporozoite inoculation. The parasites were stained with anti-CSP (red) and anti-UIS4 (green) antibodies. The scale bars represent 10 µm.

Figure 3. LS development is arrested during the schizont stage in AP2-L(−) parasites.

A. Sporozoites (2×10⁴) were inoculated into a culture of HepG2 cells and incubated at 37°C. The number of LS parasites was counted 48 hpi after staining with anti-CSP antibody. The data are the mean ± SEM of four measurements. B. The diameters of LS AP2-L(−) 1 and wild-type parasites were compared. When the parasites were not round, the average of the longest and the shortest axis was used as the diameter. The horizontal axis indicates the number of hours post-sporozoite inoculation. The data are the mean ± SEM of 200 measurements. C. Fluorescence images of AP2-L(−) 1 and wild-type (WT) LS parasites at 24 hpi and 48 hpi. The parasites were stained with an anti-CSP antibody. The arrows indicate densely stained portions that were specifically observed in AP2-L(−) LS parasites. D. Representative image of wild-type and AP2L(−) 1 LS parasites at 53 hpi with nuclear staining by DAPI. The scale bars in C and D represent 10 µm. E. Parasite burden in the rat liver was compared between the wild-type and AP2-L(−) parasites. Wild-type or AP2-L(−) salivary gland sporozoites were intravenously injected into rats, and the liver was dissected after perfusion at 24 hpi or 48 hpi. Parasite loads in the liver were determined by measuring P. berghei 18S rRNA using RT-PCR. The rat glyceraldehyde 3-phosphate dehydrogenase gene was used as an internal control to normalize the mean cycle threshold value of the rat 18S rRNA gene. Four rats were used for each condition.

Figure 4. The expression profile of AP2-L in P. berghei pre-erythrocytic stages.

A. A schematic diagram showing the preparation of transgenic P. berghei expressing GFP-tagged AP2-L. The expression construct was introduced into the 3′ portion of the endogenous AP2-L gene by double cross-over homologous recombination. The results of the Southern blot analysis of wild-type (WT) and transgenic AP2-L parasites (AP2-L ::GFP (−)) are shown on the left. B. Fluorescence images of a salivary gland sporozoite (SG; expressing GFP-tagged AP2-L, extreme left column) and LS parasites (expressing mCherry-tagged AP2-L, other columns). LS parasites were harvested at 12-h intervals following sporozoite inoculation. The LS parasites expressed GFP constitutively in the cytoplasm (upper images). The nuclei were stained with Hoechst 21486 (in sporozoites) or DAPI (in LS parasites). The scale bars represent 10 µm.