Differential Trafficking and Expression of PIR Proteins in Acute and Chronic Plasmodium Infections.

Plasmodium multigene families are thought to play important roles in the pathogenesis of malaria. Plasmodium interspersed repeat (pir) genes comprise the largest multigene family in many Plasmodium species. However, their expression pattern and localisation remain to be elucidated. Understanding protein subcellular localisation is fundamental to reveal the functional importance and cell-cell interactions of the PIR proteins. Here, we use the rodent malaria parasite, Plasmodium chabaudi chabaudi, as a model to investigate the localisation pattern of this gene family. We found that most PIR proteins are co-expressed in clusters during acute and chronic infection; members of the S7 clade are predominantly expressed during the acute-phase, whereas members of the L1 clade dominate the chronic-phase of infection. Using peptide antisera specific for S7 or L1 PIRS, we show that these PIRs have different localisations within the infected red blood cells. S7 PIRs are exported into the infected red blood cell cytoplasm where they are co-localised with parasite-induced host cell modifications termed Maurer's clefts, whereas L1 PIRs are localised on or close to the parasitophorous vacuolar membrane. This localisation pattern changes following mosquito transmission and during progression from acute- to chronic-phase of infection. The presence of PIRs in Maurer's clefts, as seen for Plasmodium falciparum RIFIN and STEVOR proteins, might suggest trafficking of the PIRs on the surface of the infected erythrocytes. However, neither S7 nor L1 PIR proteins detected by the peptide antisera are localised on the surface of infected red blood cells, suggesting that they are unlikely to be targets of surface variant-specific antibodies or to be directly involved in adhesion of infected red blood cells to host cells, as described for Plasmodium falciparum VAR proteins. The differences in subcellular localisation of the two major clades of Plasmodium chabaudi PIRs across the blood cycle, and the apparent lack of expression on the red cell surface strongly suggest that the function(s) of this gene family may differ from those of other multigene families of Plasmodium, such as the var genes of Plasmodium falciparum.

Structure of the Plasmodium-interspersed repeat proteins of the malaria parasite.

The deadly symptoms of malaria occur as Plasmodium parasites replicate within blood cells. Members of several variant surface protein families are expressed on infected blood cell surfaces. Of these, the largest and most ubiquitous are the Plasmodium-interspersed repeat (PIR) proteins, with more than 1,000 variants in some genomes. Their functions are mysterious, but differential pir gene expression associates with acute or chronic infection in a mouse malaria model. The membership of the PIR superfamily, and whether the family includes Plasmodium falciparum variant surface proteins, such as RIFINs and STEVORs, is controversial. Here we reveal the structure of the extracellular domain of a PIR from Plasmodium chabaudi We use structure-guided sequence analysis and molecular modeling to show that this fold is found across PIR proteins from mouse- and human-infective malaria parasites. Moreover, we show that RIFINs and STEVORs are not PIRs. This study provides a structure-guided definition of the PIRs and a molecular framework to understand their evolution.

Structural basis for RIFIN-mediated activation of LILRB1 in malaria.

The Plasmodium species that cause malaria are obligate intracellular parasites, and disease symptoms occur when these parasites replicate in human blood. Despite the risk of immune detection, the parasite delivers proteins that bind to host receptors on the cell surfaces of infected erythrocytes. In the causative parasite of the most deadly form of malaria in humans, Plasmodium falciparum, RIFINs form the largest family of surface proteins displayed by erythrocytes1. Some RIFINs can bind to inhibitory immune receptors, and these RIFINs act as targets for unusual antibodies that contain a LAIR1 ectodomain2-4 or as ligands for LILRB15. RIFINs stimulate the activation of and signalling by LILRB15, which could potentially lead to the dampening of human immune responses. Here, to understand how RIFINs activate LILRB1-mediated signalling, we determine the structure of a RIFIN bound to LILRB1. We show that this RIFIN mimics the natural activating ligand of LILRB1, MHC class I, in its LILRB1-binding mode. A single mutation in the RIFIN disrupts the complex, blocks LILRB1 binding of all tested RIFINs and abolishes signalling in a reporter assay. In a supported lipid bilayer system, which mimics the activation of natural killer (NK) cells by antibody-dependent cell-mediated cytotoxicity, both RIFIN and MHC are recruited to the immunological synapse of NK cells and reduce the activation of NK cells, as measured by the mobilization of perforin. Therefore, LILRB1-binding RIFINs mimic the binding mode of the natural ligand of LILRB1 and suppress the function of NK cells.