Plasmodium berghei crystalloids contain multiple LCCL proteins

Abstract

Malaria crystalloids are unique organelles of unknown function that are present only in the mosquito-specific ookinete and early oocyst stages of the parasite. Recently, crystalloid formation in Plasmodium berghei was linked to the parasite protein PbSR, a member of the Plasmodium LCCL protein family composed of six modular multidomain proteins involved in sporozoite development and infectivity. Here, we show by fluorescent protein tagging that two other LCCL protein family members are targeted to the crystalloids in a similar way to PbSR. These results extend the similarities between the LCCL proteins, and provide strong supporting evidence for the hypothesis that members of this protein family work in concert and are involved in a similar molecular process.

Fig. 1

Generation and molecular analysis of genetically modified parasite lines. (A) Targeting strategy for GFP-tagging PbLAP2 via single crossover homologous recombination. (B) Targeting strategy for GFP-tagging PbLAP3 via double crossover homologous recombination. The pblap genes are indicated with coding sequence (wide bars) and noncoding sequence (narrow bars). Also indicated are the enhanced GFP module (egfp); the human dhfr selectable marker gene cassette (hdhfr); the intron in pblap3 (v-shaped line); the position of key restriction sites (PacI, KpnI, SacII); and primers used for PCR amplification (P1–P10). (C) Diagnostic PCR of genomic DNA from three PbLAP2/EGFP clones (lanes 1–3) and two PbLAP3/EGFP clones (lanes 5 and 6). In lanes 4 and 7 genomic DNA (gDNA) from wild-type parasites was used as template. M = Generuler 1 kb DNA ladder (Fermentas).

1. Primer sequences: P1

2. (ACGAAGTTATCAGTCGACATGAGTCATTACTAGACATAATTACAAGTGAA); P2

3. (ATGAGGGCCCCTAAGCTTTCAGTAATTCCATGAGTTACTTTGC); P3

4. (ACGAAGTTATCAGTCGAGGTACCTAGCGGAAACAACAATGTTC); P4

5. (ATGAGGGCCCCTAAGCTATTTTTAATAATTTGTATCGAAAGTATAGTTG); P5

6. (CCTTCAATTTCGACATATAATGGATTAAAATTTTAGTTCGGT); P6

7. (GCGGCCGCTCTAGCATAGGATTAGAAATACAGTAATAGCAATTTTG); P7

8. (CATCTATACATGCAGGCG); P8

9. (GTGCCCATTAACATCACC); P9

10. (ACAAAGAATTCATGGTTGGTTCGCTAAACT); P10 (CCTCAAGATAGTTACGAATTTAAC).

Fig. 2

Expression and localization of GFP-tagged PbLAP2 and PbLAP3. (A) Confocal images of live gametocytes. (B) Confocal images of live ookinetes. (C) Confocal images of mature oocysts containing sporozoites. (D) Immunogold EM images (with silver enhancement) of purified ookinetes, showing labelling of crystalloids (CR).