The role of metacaspase 1 in Plasmodium berghei development and apoptosis

Abstract

The malaria parasite encodes a wide range of proteases necessary to facilitate its many developmental transitions in vertebrate and insect hosts. Amongst these is a predicted cysteine protease structurally related to caspases, named Plasmodium metacaspase 1 (PxMC1). We have generated Plasmodium berghei parasites in which the PbMC1coding sequence is removed and replaced with a green fluorescent reporter gene to investigate the expression of PbMC1, its contribution to parasite development, and its involvement in previously reported apoptosis-like cell death of P. berghei ookinetes. Our results show that the pbmc1 gene is expressed in female gametocytes and all downstream mosquito stages including sporozoites, but not in asexual blood stages. We failed to detect an apparent loss-of-function phenotype, suggesting that PbMC1 constitutes a functionally redundant gene. We discuss these findings in the context of two other putative Plasmodium metacaspases that we describe here.

Fig. 1

Structure of PxMC1, PxMC2 and PxMC3. (A) Schematic diagram of the three proteins in P. falciparum with their predicted sizes in amino acids shown in parentheses. The conserved domains are the Peptidase_C14/Caspase domain (rectangle) and the C2 domain (diamond). (B) ClustalW multiple alignment (default parameters) of the conserved part of the Peptidase_C14/Caspase domains of PxMC1, PxMC2 and PxMC3 from P. berghei (Pb), P. falciparum (Pf) and P. vivax (Pv), and that of yeast YCA1. Amino acid identity and similarity shared by at least five of the aligned sequences are shaded. Numbers in parentheses denote numbers of residues not shown. Asterisks marks the position of predicted catalytic dyad histidine and cysteine residues. Sequences are based on PlasmoDB gene IDs PB001074 (PbMC1), PB000485 (PbMC2), PB000564 (PbMC3), PF13_0289 (PfMC1), PF14_0363 (PfMC2), PF14_0160 (PfMC3), Pv114725 (PvMC1), Pv118575 (PvMC2) and Pv08564 (PvMC3).

Fig. 2

Construction of PbMC1-KO parasites. (A) Schematic diagram of the gene-disruption strategy. HincII sites are indicated by arrows, with predicted restriction fragment sizes given. Probes used in Southern blot are indicated by hatched bars. (B) Southern blot analysis of HincII-digested WT and PbMC1-KO parasite genomic DNA. Fragment sizes (in kb) are indicated.

Fig. 3

EGFP fluorescence in PbMC1-KO parasites. (A) Female gametocyte. (B) Ookinete. (C) Day 15 oocysts containing sporozoites. DIC, differential interference contrast. (D) Sporozoites inside an infected mosquito salivary gland.

Fig. 4

Diagram of proportion of Annexin V-FITC (green) and propidium iodide (red) labelled in vitro cultured P. berghei ookinetes. Circles shows values for individual experiments, bars represent average values across 13 experiments.