Localisation and timing of expression of putative Plasmodium berghei rhoptry proteins in merozoites and sporozoites

Invasive forms of apicomplexan parasites contain secretory organelles (which may include micronemes, rhoptries or dense granules), the contents of which mediate invasion of host cells. Only few rhoptry proteins have been identified in Plasmodium and then only in merozoites and none in the sporozoite or ookinete. Epitope-tagged proteins (with either green fluorescent protein or C-MYC) were used to analyse the expression and cellular localisation of a known Plasmodium rhoptry protein (RAP2/3) and putative homologues of two Toxoplasma rhoptry proteins (rhoptry neck protein 2, RON2 and putative rhoptry protein 2, PRP2) at different stages across the life cycle. This analysis showed correct targeting to the merozoite rhoptries of GFP-tagged RAP2/3 and, for the first time, a distinct apical fluorescence pattern in sporozoites indicating a rhoptry location. In addition, tagged PBRON2 and PBPRP2 also show a merozoite rhoptry localisation similar to that of RAP2/3. RON2 is expressed in sporozoites and has the same timing of expression and location as RAP2/3. While PRP2 is also expressed in sporozoites, both its pattern of expression and location differ from RON2 and RAP2/3. None of the tagged proteins were detected in ookinetes, which is in agreement with the proposed lack of rhoptries in this third invasive form of Plasmodium. The analysis of tagged rhoptry proteins reveals new insights into the role of these proteins in host-cell invasion in different malarial ‘zoites’ and will facilitate more detailed studies into the role of rhoptries in establishing an infection of not only red blood cell but also the hepatocytes.