Loss-of-function analyses defines vital and redundant functions of the Plasmodium rhomboid protease family.

Rhomboid-like proteases cleave membrane-anchored proteins within their transmembrane domains. In apicomplexan parasites substrates include molecules that function in parasite motility and host cell invasion. While two Plasmodium rhomboids, ROM1 and ROM4, have been examined, the roles of the remaining six rhomboids during the malaria parasite's life cycle are unknown. We present systematic gene deletion analyses of all eight Plasmodium rhomboid-like proteins as a means to discover stage-specific phenotypes and potential functions in the rodent malaria model, P. berghei. Four rhomboids (ROM4, 6, 7 and 8) are refractory to gene deletion, suggesting an essential role during asexual blood stage development. In contrast ROM1, 3, 9 and 10 were dispensable for blood stage development and exhibited no, subtle or severe defects in mosquito or liver development. Parasites lacking ROM9 and ROM10 showed no major phenotypic defects. Parasites lacking ROM1 presented a delay in blood stage patency following liver infection, but in contrast to a previous study blood stage parasites had similar growth and virulence characteristics as wild type parasites. Parasites lacking ROM3 in mosquitoes readily established oocysts but failed to produce sporozoites. ROM3 is the first apicomplexan rhomboid identified to play a vital role in sporogony.

The exported Plasmodium berghei protein IBIS1 delineates membranous structures in infected red blood cells.

The importance of pathogen-induced host cell remodelling has been well established for red blood cell infection by the human malaria parasite Plasmodium falciparum. Exported parasite-encoded proteins, which often possess a signature motif, termed Plasmodium export element (PEXEL) or host-targeting (HT) signal, are critical for the extensive red blood cell modifications. To what extent remodelling of erythrocyte membranes also occurs in non-primate hosts and whether it is in fact a hallmark of all mammalian Plasmodium parasites remains elusive. Here we characterize a novel Plasmodium berghei PEXEL/HT-containing protein, which we term IBIS1. Temporal expression and spatial localization determined by fluorescent tagging revealed the presence of IBIS1 at the parasite/host interface during both liver and blood stages of infection. Targeted deletion of the IBIS1 protein revealed a mild impairment of intra-erythrocytic growth indicating a role for these structures in the rapid expansion of the parasite population in the blood in vivo. In red blood cells, the protein localizes to dynamic, punctate structures external to the parasite. Biochemical and microscopic data revealed that these intra-erythrocytic P. berghei-induced structures (IBIS) are membranous indicating that P. berghei, like P. falciparum, creates an intracellular membranous network in infected red blood cells.

Critical role for a stage-specific actin in male exflagellation of the malaria parasite.

Male gametogenesis occurs directly after uptake of malaria parasites by the mosquito vector and leads to the release of eight nucleated flagellar gametes. Here, we report that one of the two parasite actin isoforms, named actin II, is essential for this process. Disruption of actin II in Plasmodium berghei resulted in viable asexual blood stages, but male gametogenesis was specifically inhibited. Upon activation, male gametocyte DNA was replicated normally and axonemes assembled, but egress from the host cell was inhibited, and axoneme motility abolished. The major actin isoform, actin I, displayed dual localization to the cytoplasm and the nucleus in male gametocytes. After activation actin I was found to be restricted to the cytoplasm. In actII(-) mutant parasites, this re-localization was abolished and actin I remained in both cellular compartments. These findings reveal vital and pleiotropic functions for the actin II isoform in male gametogenesis of the malaria parasite.

Expression profiling of Plasmodium berghei HSP70 genes for generation of bright red fluorescent parasites.

Live cell imaging of recombinant malarial parasites encoding fluorescent probes provides critical insights into parasite-host interactions and life cycle progression. In this study, we generated a red fluorescent line of the murine malarial parasite Plasmodium berghei. To allow constitutive and abundant expression of the mCherry protein we profiled expression of all members of the P. berghei heat shock protein 70 (HSP70) family. We identified PbHSP70/1, an invariant ortholog of Plasmodium falciparum HSP70-1, as the protein with the highest expression levels during Plasmodium blood, mosquito, and liver infection. Stable allelic insertion of a mCherry expression cassette into the PbHsp70/1 locus created constitutive red fluorescent P. berghei lines, termed Pbred. We show that these parasites can be used for live imaging of infected host cells and organs, including hepatocytes, erythrocytes, and whole Anopheles mosquitoes. Quantification of the fluorescence intensity of several Pbred parasite stages revealed significantly enhanced signal intensities in comparison to GFP expressed under the control of the constitutive EF1alpha promoter. We propose that systematic transcript profiling permits generation of reporter parasites, such as the Pbred lines described herein.