Robert Pinches

Switches in expression of plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes

Plasmodium falciparum expresses on the host erythrocyte surface clonally variant antigens and ligands that mediate adherence to endothelial receptors. Both are central to pathogenesis, since they allow chronicity of infection and lead to concentration of infected erythrocytes in cerebral vessels. Here we show that expression of variant antigenic determinants is correlated with expression of individual members of a large, multigene family named var. Each var gene contains copies of a motif that has been previously shown to bind diverse host receptors; expression of a specific var gene correlated with binding to ICAM-1. Thus, our findings are consistent with the involvement of var genes in antigenic variation and binding to endothelium.

Characterization of the pathway for transport of the cytoadherence-mediating protein, PfEMP1, to the host cell surface in malaria parasite-infected erythrocytes

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of antigenically diverse proteins is expressed on the surface of human erythrocytes infected with the malaria parasite P. falciparum, and mediates cytoadherence to the host vascular endothelium. In this report, we show that export of PfEMP1 is slow and inefficient as it takes several hours to traffic newly synthesized proteins to the erythrocyte membrane. Upon removal by trypsin treatment, the surface‐exposed population of PfEMP1 is not replenished during subsequent culture indicating that there is no cycling of PfEMP1 between the erythrocyte surface and an intracellular compartment. The role of Maurers clefts as an intermediate sorting compartment in trafficking of PfEMP1 was investigated using immunoelectron microscopy and proteolytic digestion of streptolysin O‐permeabilized parasitized erythrocytes. We show that PfEMP1 is inserted into the Maurers cleft membrane with the C‐terminal domain exposed to the erythrocyte cytoplasm, whereas the N‐terminal domain is buried inside the cleft. Transfer of PfEMP1 to the erythrocyte surface appears to involve electron‐lucent extensions of the Maurers clefts. Thus, we have delineated some important aspects of the unusual trafficking mechanism for delivery of this critical parasite virulence factor to the erythrocyte surface.

Plasmodium falciparum: The human agglutinating antibody response to the infected red cell surface is predominantly variant specific

There is mounting evidence that an important component of the host-protective immune response to Plasmodium falciparum is the antibody response to the altered surface of the infected erythrocyte. The nature of these surface changes and the responses to them have been difficult to analyse because of the diverse nature of the parasite-derived neoantigens (PDN) expressed, because of the additional presence of modified host determinants, and because of the lack of monospecific reagents. We have studied the reactivity of field isolates and laboratory clones with pooled or individual sera using a novel approach which obviates the need for specific antibody. We see marked diversity in PDN but in contrast to previous studies, we also find that the predominant agglutinating antibody response in humans is variant specific. Antibodies which cross-react between different serotypes are rare and react only with a subset of PDN types. These results have implications for mechanisms underlying the development of acquired immunity to P. falciparum.

A well‐conserved Plasmodium falciparum var gene shows an unusual stage‐specific transcript pattern

The var multicopy gene family encodes Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variant antigens, which, through their ability to adhere to a variety of host receptors, are thought to be important virulence factors. The predominant expression of a single cytoadherent PfEMP1 type on an infected red blood cell, and the switching between different PfEMP1 types to evade host protective antibody responses, are processes thought to be controlled at the transcriptional level. Contradictory data have been published on the timing of var gene transcription. Reverse transcription‐polymerase chain reaction (RT‐PCR) data suggested that transcription of the predominant var gene occurs in the later (pigmented trophozoite) stages, whereas Northern blot data indicated such transcripts only in early (ring) stages. We investigated this discrepancy by Northern blot, with probes covering a diverse var gene repertoire. We confirm that almost all var transcript types were detected only in ring stages. However, one type, the well‐conserved varCSA transcript, was present constitutively in different laboratory parasites and does not appear to undergo antigenic variation. Although varCSA has been shown to encode a chondroitin sulphate A (CSA)‐binding PfEMP1, we find that the presence of full‐length varCSA transcripts does not correlate with the CSA‐binding phenotype.

Molecular mechanisms of sequestration in malaria.

Cell surface molecules have received intense attention in recent years because of the central roles they play at the interface between the external environment and the cellular interior. Their functions include adhesion to other cells or extracellular matrices, protection against hostile physical, chemical and biological agents and the transport of metabolites into and out of the cell. In addition, cell surface molecules transduce signals across the cell membrane, relaying information inwards and presenting altered characteristics to the exterior as the environment changes.

Antigenic Variation in Plasmodium falciparum Malaria Involves a Highly Structured Switching Pattern

Many pathogenic bacteria, fungi, and protozoa achieve chronic infection through an immune evasion strategy known as antigenic variation. In the human malaria parasite Plasmodium falciparum, this involves transcriptional switching among members of the var gene family, causing parasites with different antigenic and phenotypic characteristics to appear at different times within a population. Here we use a genome-wide approach to explore this process in vitro within a set of cloned parasite populations. Our analyses reveal a non-random, highly structured switch pathway where an initially dominant transcript switches via a set of switch-intermediates either to a new dominant transcript, or back to the original. We show that this specific pathway can arise through an evolutionary conflict in which the pathogen has to optimise between safeguarding its limited antigenic repertoire and remaining capable of establishing infections in non-naïve individuals. Our results thus demonstrate a crucial role for structured switching during the early phases of infections and provide a unifying theory of antigenic variation in P. falciparum malaria as a balanced process of parasite-intrinsic switching and immune-mediated selection.

Febrile temperatures induce cytoadherence of ring-stage Plasmodium falciparum-infected erythrocytes

In falciparum malaria, the malaria parasite induces changes at the infected red blood cell surface that lead to adherence to vascular endothelium and other red blood cells. As a result, the more mature stages of Plasmodium falciparum are sequestered in the microvasculature and cause vital organ dysfunction, whereas the ring stages circulate in the blood stream. Malaria is characterized by fever. We have studied the effect of febrile temperatures on the cytoadherence in vitro of P. falciparum-infected erythrocytes. Freshly obtained ring-stage-infected red blood cells from 10 patients with acute falciparum malaria did not adhere to the principle vascular adherence receptors CD36 or intercellular adhesion molecule-1 (ICAM-1). However, after a brief period of heating to 40°C, all ring-infected red blood cells adhered to CD36, and some isolates adhered to ICAM-1, whereas controls incubated at 37°C did not. Heating to 40°C accelerated cytoadherence and doubled the maximum cytoadherence observed (P < 0.01). Erythrocytes infected by ring-stages of the ICAM-1 binding clone A4var also did not cytoadhere at 37°C, but after heating to febrile temperatures bound to both CD36 and ICAM-1. Adherence of red blood cells infected with trophozoites was also increased considerably by brief heating. The factor responsible for heat induced adherence was shown to be the parasite derived variant surface protein PfEMP-1. RNA analysis showed that levels of var mRNA did not differ between heated and unheated ring-stage parasites. Thus fever-induced adherence appeared to involve increased trafficking of PfEMP-1 to the erythrocyte membrane. Fever induced cytoadherence is likely to have important pathological consequences and may explain both clinical deterioration with fever in severe malaria and the effects of antipyretics on parasite clearance.

PfEMP1 expression is reduced on the surface of knobless Plasmodium falciparum infected erythrocytes

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a key virulence factor for this species of human malarial parasite. PfEMP1 is expressed on the surface of infected erythrocytes (IEs) and directly mediates adhesion to a variety of host cells. A number of other parasite-encoded proteins are similarly exported to the IE plasma membrane and play an indirect role in this adhesion process through the modification of the erythrocyte cytoskeleton and the formation of electron dense knobs into which PfEMP1 is anchored. Analysis of the specific contribution of knob-associated proteins to adhesion is difficult due to rapid PfEMP1 switching during in vitro culture. Furthermore, these studies typically assume that the level and distribution of PfEMP1 exposed in knobby (K+) and knobless (K–) IEs is unaltered, an assumption not yet supported with data. We describe here the preparation and characterisation of a panel of isogenic K+ and K– parasite clones that express one of two defined PfEMP1 variants. Analysis of the cytoadhesive properties of these clones shows that both static and flow adhesion is reduced in all the K– clones and, further, that this correlates with an approximately 50% reduction in PfEMP1 displayed on the IE surface. However, despite this reduction, the gross distribution of PfEMP1 in K– IEs appears unaltered. These data impact on our current interpretation of the role of knobs in adhesion and the mechanism of trafficking PfEMP1 to the IE surface.

MORN1 has a conserved role in asexual and sexual development across the apicomplexa.

ABSTRACT The gene encoding the membrane occupation and recognition nexus protein MORN1 is conserved across the Apicomplexa. In Toxoplasma gondii, MORN1 is associated with the spindle poles, the anterior and posterior rings of the inner membrane complex (IMC). The present study examines the localization of MORN1 during the coccidian development of T. gondii and three Eimeria species (in the definitive host) and erythrocytic schizogony of Plasmodium falciparum. During asexual proliferation, MORN1 is associated with the posterior ring of the IMCs of the multiple daughters forming during T. gondii endopolygeny and schizogony in Eimeria and P. falciparum. Furthermore, the expression of P. falciparum MORN1 protein peaked in late schizogony. These data fit a model with a conserved role for MORN1 during IMC assembly in all variations of asexual development. An important new observation is the reactivity of MORN1 antibody with certain sexual stages in T. gondii and Eimeria species. Here MORN1 is organized as a ring-like structure where the microgametes bud from the microgametocyte while in mature microgametes it is present near the flagellar basal bodies and mitochondrion. These observations suggest a conserved role for MORN1 in both asexual and sexual development across the Apicomplexa.

Plasmodium falciparum var gene expression is developmentally controlled at the level of RNA polymerase II-mediated transcription initiation

The Plasmodium falciparum var gene family codes for a major virulence factor in this most lethal of human malaria parasites. A single var protein variant type is expressed on each infected red blood cell, with antigenic variation allowing progeny parasites to escape host immune detection. The control of mutually exclusive var gene expression in the parasite relies on in situ epigenetic changes. Whether control of expression occurs at transcription initiation or post transcription, however, remains to be established. Recent evidence supports existence of a unique var transcription site at the nuclear periphery containing the dominantly expressed var gene, although silent var genes can colocalize to the same region. We demonstrate here that exclusive var gene expression is controlled at the level of transcription initiation during ring stages and that var genes are transcribed by RNA polymerase II. This represents another example where P. falciparum differs from the paradigm for antigenic variation, Trypanosoma brucei.