期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2020
卷号：117
期号：50
页码：32098-32104
DOI：10.1073/pnas.2016775117
出版社：The National Academy of Sciences of the United States of America
摘要：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. The symptoms of malaria occur as Plasmodium parasites replicate within blood. This is a rich environment, replete with the nutrients required for growth and providing the opportunity for transmission by blood-sucking insects. However, blood also contains much of the machinery of the host immune defense. To survive under immune attack, Plasmodium parasites have evolved to replicate while hidden within host cells. Only a few parasite proteins are exposed on host cell surfaces, and these have mostly diversified into large protein families, allowing a population survival strategy based on antigenic variation ( 1 , 2 ). The best understood of the infected-erythrocyte surface protein families is the PfEMP1, members of which interact with human endothelial receptors, causing infected erythrocytes to adhere within the vasculature away from splenic clearance ( 1 , 3 , 4 ). However, PfEMP1 are found only in Plasmodium falciparum and the closely related Laverania . More ubiquitous across Plasmodium species are families of small variant surface antigens (VSAs) ( 1 ). These include the CIRs of Plasmodium chabaudi ( 5 ) and the VIRs of Plasmodium vivax ( 6 ), often known as the “ Plasmodium -interspersed repeats” (PIRs) ( 7 ). The PIRs can be very abundant, with thousands of members in some genomes ( 8 ). However, whether some families of small VSAs in the Laverania and more distantly related Plasmodium species are part of the PIR superfamily is unclear. For example, the RIFINs and STEVORs of P. falciparum ( 9 ⇓ – 11 ) were proposed to be PIRs due to their sizes, cellular locations, and the presence of shared sequence elements within intron regions of their genes ( 12 ). However, differences in gene structure and a low protein sequence identity make this assignment uncertain ( 7 ). Are the small VSAs part of a larger superfamily with related functions, or are they different protein families with different roles? Also uncertain is whether the small VSAs are universally found on infected erythrocyte surfaces. Studies have located PIRs on or close to the surfaces of blood cells infected with P. vivax ( 6 ), Plasmodium yoelii ( 13 ), and Plasmodium berghei ( 14 ). Similarly, RIFINs and STEVORs have been located to surfaces of P. falciparum -infected erythrocytes ( 10 , 15 ⇓ – 17 ). Indeed, natural infection with P. vivax induces antibodies that target VIRs ( 18 ), while unusual RIFIN-targeting antibodies found in adults in malaria endemic regions of Africa also recognize infected erythrocytes ( 19 ⇓ – 21 ). These studies suggest that the small VSAs are molecules of infected blood cell surfaces and indicate that family expansion and diversification have occurred to allow them to undergo antigenic variation. However, other studies have cast doubt on the universality of this model, indicating an intracellular location for some small VSAs ( 22 ⇓ – 24 ) or showing them be expressed in other life cycle stages of the parasite, including merozoites or gametocytes ( 23 , 25 ⇓ – 27 ). Has diversification of these small VSA families led to their use at different life cycle stages and different cellular locations during infection? A number of recent studies indicate that small VSAs have important functions. First, P. chabaudi introduced into mice through mosquito bite are less virulent than those introduced by direct injection of infected blood ( 28 ). The major differences in gene expression in these parasites are in the cir gene repertoires, with a broader range of cir genes expressed on mosquito transmission ( 28 ). Different cir genes are also transcribed during the chronic phase of a mosquito-transmitted P. chabaudi infection compared with those expressed during the acute phase ( 29 ). When passaged in naïve mice, these chronic parasites are more virulent than those from acute stages of infection. Indeed, a more virulent P. chabaudi strain, PcCB, expresses more of the pir genes associated with chronic infection than a less virulent strain, PcAS ( 30 ). These findings combine to suggest an as-yet unknown role for PIRs in modulating the virulence of infection. One possible mechanism in some Plasmodium species might be through causing adhesion of infected cells within the vasculature, allowing avoidance of splenic clearance. Indeed, VIRs from P. vivax are proposed to cause infected-reticulocytes to adhere to endothelial receptors including ICAM-1 ( 22 , 31 ), while RIFINs and STEVORs can cause infected erythrocytes to adhere to uninfected erythrocytes, by interacting with blood group antigens or glycophorin C ( 16 , 32 ). Alternatively, subgroups of RIFINs have been shown to interact with human inhibitory immune receptors, such as LILRB1 and LAIR1 ( 17 ). The LILRB1-binding RIFINs mimic the natural ligand of LILRB1, MHC class I, allowing the RIFIN to inhibit markers of natural killer cell activation, most likely reducing parasite clearance ( 33 ). These studies imply wide-ranging roles for the small VSAs in both mammalian and insect hosts. The important roles emerging for different small VSAs highlights the need to understand these mysterious parasite protein families in greater detail. In particular, are they members of the same Plasmodium superfamily, evolved to perform similar functions during infection, or do the small VSAs represent different protein families with different roles? To explore this question, we determined the structure of the extracellular domain of a CIR protein from P. chabaudi . We compared this structure with that of the variable domain of a LILRB1-binding RIFIN ( 33 ) and used structure-guided sequence analysis to predict which small VSAs are part of the PIR superfamily. This provides a framework for understanding PIR protein function and evolution. Results The Structure of a P. chabaudi PIR Protein Ectodomain. To determine the structure of a member of the PIR family, we focused on proteins from P. chabaudi, often known as CIRs. These consist of an N-terminal extracellular domain ranging in size from 28 kDa (236 residues) to 133 kDa (1,331 residues) in the AS strain. This is followed by a predicted transmembrane helix and small intracellular peptide. We assessed the expression of a panel of seven of the smaller CIR ectodomains ( SI Appendix , Fig. S1 ). Five of these were expressed in HEK293F cells and were subjected to crystallization trials. Crystals formed for PCHAS_1200500 and contained three copies in the asymmetric unit. A complete dataset was collected to 2.15 Å, and the structure was determined by Sulfur-SAD phasing, using anomalous scattering from the sulfur atoms found in the five disulfide bonds and six methionine residues in each monomer ( Fig. 1 A and SI Appendix , Table S1 ). A complete model was built for residues 5 to 242. Another 20 residues, which connect the ectodomain to the predicted transmembrane helix, are missing from the C terminus, suggesting that this domain is connected to the membrane through a flexible, disordered linker. Indeed, residues 242 to 258 are predicted to be disordered ( Fig. 1 B ).
