Role of Plasmodium berghei cGMP-dependent protein kinase in late liver stage development

Abstract

The liver is the first organ infected by Plasmodium sporozoites during malaria infection. In the infected hepatocytes, sporozoites undergo a complex developmental program to eventually generate hepatic merozoites that are released into the bloodstream in membrane-bound vesicles termed merosomes. Parasites blocked at an early developmental stage inside hepatocytes elicit a protective host immune response, making them attractive targets in the effort to develop a pre-erythrocytic stage vaccine. Here, we generated parasites blocked at a late developmental stage inside hepatocytes by conditionally disrupting the Plasmodium berghei cGMP-dependent protein kinase in sporozoites. Mutant sporozoites are able to invade hepatocytes and undergo intracellular development. However, they remain blocked as late liver stages that do not release merosomes into the medium. These late arrested liver stages induce protection in immunized animals. This suggests that, similar to the well studied early liver stages, late stage liver stages too can confer protection from sporozoite challenge.

FIGURE 1.

RT-PCR detection of PbPKG expression in infected HepG2 cells. mRNAs of PbPKG (top panel) and mammalian glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected in HepG2 cells infected with P. berghei sporozoites 24, 48, and 72 h post-infection. Reverse transcription reactions were carried in the presence (+) or absence (−) of RT. P. berghei genomic DNA (gDNA) was used as a positive control.

FIGURE 2.

Conditional mutagenesis of PbPKG. The PbPKG open reading frame (ORF) consists of 5 exons (open rectangles). A, the targeting plasmid for generating the conditional knock-out (pKO) parasites contains two FRT sites (gray arrowheads) flanking exons 4–5 and an expression cassette for human dihydrofolate reductase (hDHFR). Activation of the FlpL activity in sporozoites leads to the loss of exons 4–5 in cKO parasites. Black arrowheads indicate the position of primers used in diagnostic PCRs. B, the targeting plasmid for generating the control (pCON) parasites contains a single FRT site. pKO and pCON were transfected into TRAP/FlpL erythrocytic stage parasites. C, a Southern hybridization is shown to confirm integration of pKO into the PbPKG locus. SpeI-digested genomic DNA from parental TRAP/FlpL parasites and the cKO clone was probed with labeled exon 5. Integration of the construct at the wild type locus is revealed by the presence of a 7.8-kb fragment. D, shown is PCR amplification using primers 1 and 2 to probe the PbPKG locus in cKO erythrocytic stages (lane 1), cKO sporozoites (lane 2), CON erythrocytic stages (lane 3), CON sporozoites (lane 4), and wild type (WT) erythrocytic stages (lane 5). Excision (E) of exons 4–5 in cKO sporozoites generated a 0.6-kb product. The 1.5-kb product is a result of nonspecific amplification. The non-excised (NE) locus generated a 5.5-kb product in cKO erythrocytic stages, CON erythrocytic stages, and CON sporozoites and a 3.5-kb product in wild type parasites.

FIGURE 3.

Infectivity of the cKO sporozoite population as determined by pre-patent period. A, groups of 5 animals were infected with cKO or CON sporozoites (10,000 sporozoites/animal). Animals were checked daily for the appearance of erythrocytic stages. The number of animals patent on each day post-infection is shown. B, groups of C57Bl/6 mice were infected with different doses of cKO or CON sporozoites via intravenous injection to determine the pre-patent period of infection (±S.D.). The number of animals in each group that were positive for erythrocytic stages is shown. Asterisks indicate that analysis of genomic DNA from parasites obtained from these animals revealed the presence only PbPKG+ parasites, which develop when cKO sporozoites do not undergo the expected DNA excision (Fig. 4).

FIGURE 4.

Analysis of erythrocytic stage parasites obtained after infection by the cKO sporozoite population. A, the PbPKG loci in cKO parasites before DNA excision and CON parasites are differentiated by the presence of an XhoI site in the 5′ FRT site of cKO parasites. Amplification of the PbPKG locus using primers 1 (P1) and 2 (P2) (arrowheads) generates a product of ∼5.2 kb from the non-excised cKO and the CON loci and 0.6 kb from the excised cKO locus. Digestion of the ∼ 5.2-kb product (P1+P2) with XhoI produces 3 fragments in cKO and 2 fragments in CON parasites. B, shown is a PCR analysis of the PbPKG locus present in parasites recovered from the mouse made patent by an infection with the cKO sporozoite population (lane 1), the cKO clone used for mosquito feeding (lane 2), and the CON clone (lane 3). PCR detected the presence only of non-excised PbPKG loci (PbPKG+) in lane 1. C, the PCR products (P1+P2) were digested with XhoI to confirm that the amplified product in lane 1 was from the cKO non-excised locus.

FIGURE 5.

Liver stage development in cKO parasites. A, real-time PCR was used to quantify the infectivity of cKO sporozoites in vivo. The average parasite load (±S.D.) in the livers of cKO or CON-infected mice is expressed as equivalent to plasmid copies of the P. berghei 18S rRNA gene. B, LS that developed 40 and 72 h post-infection of HepG2 cells with either cKO or CON sporozoites were detected using immunofluorescence assays (n = 2 or 3). C, merosomes released into the medium of cKO or CON-infected HepG2 cells were collected 65 h post-infection and quantified. Results from a typical experiment are shown. D, shown are representative images of CON and cKO LS in HepG2 cells, 65 h post-infection. LSs were stained with an anti-parasite Hsp70 antibody. Cells were examined at 100× magnification using fluorescence and differential interference contrast (DIC) microscopy.