Plasmodium Cysteine Repeat Modular Proteins 3 and 4 are essential for malaria parasite transmission from the mosquito to the host

Abstract

Background: The Plasmodium Cysteine Repeat Modular Proteins (PCRMP) are a family of four conserved proteins of malaria parasites, that contain a number of motifs implicated in host-parasite interactions. Analysis of mutants of the rodent parasite Plasmodium berghei lacking expression of PCRMP1 or 2 showed that these proteins are essential for targeting of P. berghei sporozoites to the mosquito salivary gland and, hence, for transmission from the mosquito to the mouse.

Methods: In this work, the role of the remaining PCRMP family members, PCRMP3 and 4, has been investigated throughout the Plasmodium life cycle by generation and analysis of P. berghei gene deletion mutants, Δpcrmp3 and Δpcrmp4. The role of PCRMP members during the transmission and hepatic stages of the Plasmodium lifecycle has been evaluated by light- and electron microscopy and by analysis of liver stage development in HEPG2 cells in vitro and by infecting mice with mutant sporozoites. In addition, mice were immunized with live Δpcrmp3 and Δpcrmp4 sporozoites to evaluate their immunization potential as a genetically-attenuated parasite-based vaccine.

Results: Disruption of pcrmp3 and pcrmp4 in P. berghei revealed that they are also essential for transmission of the parasite through the mosquito vector, although acting in a distinct way to pbcrmp1 and 2. Mutants lacking expression of PCRMP3 or PCRMP4 show normal blood stage development and oocyst formation in the mosquito and develop into morphologically normal sporozoites, but these have a defect in egress from oocysts and do not enter the salivary glands. Sporozoites extracted from oocysts perform gliding motility and invade and infect hepatocytes but do not undergo further development and proliferation. Furthermore, the study shows that immunization with Δcrmp3 and Δcrmp4 sporozoites does not confer protective immunity upon subsequent challenge.

Conclusions: PCRMP3 and 4 play multiple roles during the Plasmodium life cycle; they are essential for the establishment of sporozoite infection in the mosquito salivary gland, and subsequently for development in hepatocytes. However, although Δpcrmp3 and Δpcrmp4 parasites are completely growth-impaired in the liver, immunization with live sporozoites does not induce the protective immune responses that have been shown for other genetically-attenuated parasites.

Figure 1

Generation of Δpcrmp3 and Δpcrmp4 parasites. A. Transfection of P. berghei parasites with constructs PL1082 or PL1083 containing the tgdhfr gene flanked by target sequences for homologous recombination within the pbcrmb3 or 4 loci generated Δpcrmp3a and b and Δpcrmp4a and b respectively. Fragment sizes produced by restriction enzyme digestion are shown in kb. X, Xho1; K, Kpn1; H, HindIII; S, StuI; E, EcoR1, B, BamH1. B. Southern blots of genomic DNA from WT and Δpcrmp3 or 4 clones digested with enzymes indicated were hybridized with specific probes within the 5' target region of pcrmp3 or 4. C. Chromosome analysis of Δpcrmp3 and 4 parasite clones. Chromosomes were hybridized to probes specific for the P. berghei dhfr/ts 3'UTR region or for tgdhfr within the selection cassette, as indicated. The pbdhfr/ts probe hybridizes to the P. berghei dhfr/ts gene on chromosome 7. Both probes hybridize to the site of integration of constructs PL1082 in the pbcrmp3 locus on chromosome 6 in Δpcrmp3 or the site of integration of PL1083 in the pbcrmp4 locus on chromosome 13 in Δpcrmp4.

Figure 2

Δpcrmp3 and Δpcrmp4 sporozoites develop normally but remain within mature oocysts. 1) At day 18 post infection, WT oocysts (A) contain few sporozoites and show areas of degradation (arrowed), whilst sporozoites within Δpcrmp3 oocysts (B) are radially-aligned and are retained within an intact cell wall. Scale = 100 mm. 2) WT (A), Δpcrmp3 (B) and Δpcrmp4 (C) oocysts are found in mosquito guts 10 days post infection, developing between the mosquito epithelium (mo) and haemolymph (h). Several nuclei (n) in developing sporozoites are visible. No morphological differences are observed. At day 18 post infection, WT oocysts contain few nuclei (D) whilst most Δpcrmp3 (E) and Δpcrmp4 (F) mature oocysts are full of sporozoites (sp). Scale = 5 μm.

Figure 3

Δpcrmp3 and Δpcrmp4 sporozoites migrate through and infect HepG2 cells. 1) HepG2 cells were incubated with WT salivary gland sporozoites or with Δpcrmp3 or Δpcrmp4 oocyst-derived sporozoites in the presence of rhodamine dextran. Migration rates are expressed as the percentage of uninfected traversed (rhodamine dextran +ve) cells ± the standard deviation. 2) HepG2 cells were incubated with WT salivary gland sporozoites or with Δpcrmp3 or Δpcrmp4 oocyst-derived sporozoites and the development of EEFs was quantified at 24 hours. Infected cells are expressed as the mean ± the standard deviation for each coverslip. 3) WT parasites show normal development in EEFs at 24 hours post-infection and develop into trophozites at 48 hours and schizonts at 56 hours. Δpcrmp3 and Δpcrmp4 parasites establish infection at 24 hours but remain small with irregular shapes. Fluorescence pictures of EEF (green) and nuclei (blue). Scale = 5 μm.

Figure 4

C57BL/6 mice immunized with Δpcrmp3 (black square) or Δpcrmp4 (black triangle) sporozoites develop a patent infection with the same kinetics as unimmunized mice (black diamond) upon subsequent infection with WT sporozoites.