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Robert Pinches

Publications and source records attributed to Robert Pinches.

8 recordsLinked to original sources
PubMed2004

Variable var transition rates underlie antigenic variation in malaria.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is expressed on the surface of infected erythrocytes where it plays a central role in both infected erythrocytes cytoadhesion and immune evasion. Switches in clonal expression of PfEMP1 result in antigenic variation that facilitates long-term chronic infection of the host. The var gene family encodes PfEMP1 variants, with transcriptional switching between different var variants providing the molecular basis for antigenic variation. Despite the importance of var transcriptional switching in the evasion of the immune response, little is known about the way in which this process is regulated. Here we report the measurement of transition on and off rates for a series of var gene variants. We find (i) that on and off rates for a given variant are dissimilar, (ii) that these rates vary dramatically among different variants, and (iii) that in isogenic clones expressing the same var gene, both on and off rates are constant and appear to be an intrinsic property of that particular gene. These data would suggest that the information that determines the probability of the activation or silencing of var genes is present in their surrounding DNA. Furthermore, some transitions appear to be disallowed depending on the recent variant antigen expression history of the parasite clone. These findings have important implications for both the underlying molecular mechanisms of antigenic variation and the processes that promote chronicity of infection in vivo.

ABO Blood-Group System↗
PubMed2004

Transcription of subtelomerically located var gene variant in Plasmodium falciparum appears to require the truncation of an adjacent var gene.

The Plasmodium falciparum R29 clone preferentially transcribes the R29var gene variant on rosette selection, unlike other isogenic clones from the same parasite lineage. Characterisation of the R29var gene locus revealed that this gene lies internal to, and is in a tail-to-tail orientation with, a second var gene variant (A4var) at one end of chromosome 13. In the R29 clone, a spontaneous deletion event between these two var variants deletes all of the A4var gene and the subtelomeric repetitive sequence arrays. We have previously shown that a simple disruption of the A4var gene is not sufficient to preferentially activate the R29var gene in rosette-selected parasites. We therefore hypothesised that the truncation of the chromosome end may be a key factor in predisposing the R29var variant to transcription under rosette selection conditions. Here, we have generated a panel of isogenic parasite clones with both intact and truncated A4var-R29var loci, and show that R29var transcription is only detected in rosette-selected clones with a truncated locus. Furthermore, we present provisional data describing the relative frequency with which this spontaneous deletion event occurs. These data have implications in our understanding of how spontaneous deletion events within subtelomeric var loci may affect transcription of these var gene variants.

Animals↗
PubMed2003

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.

Animals↗
PubMed2003

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 Maurer's 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 Maurer's 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 Maurer's 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.

Animals↗
PubMed2002

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 degrees C, all ring-infected red blood cells adhered to CD36, and some isolates adhered to ICAM-1, whereas controls incubated at 37 degrees C did not. Heating to 40 degrees 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 degrees 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.

Animals↗
PubMed2002

Stage-specific promoter activity from stably maintained episomes in Plasmodium falciparum.

Genomic DNA is organised at its simplest level within phased arrays of nucleosomes, a structure key to the correct transcriptional regulation of the encoded genes. Here we studied chromatin formation on DNA transfected into Plasmodium falciparum either as an episomal plasmid or following integration by homologous recombination. We show that stably maintained and replicated plasmid assembles phased arrays of nucleosomes and that a reporter gene is transcribed in an appropriate temporal manner. These data provide a key observation for the future investigation of promoter structure and function with transfected DNA in Plasmodium spp.

Animals↗
PubMed2002

Effect of var gene disruption on switching in Plasmodium falciparum.

The molecular mechanisms underpinning switching of variant antigens on the surface of Plasmodium falciparum-infected erythrocytes are poorly understood. We tested the hypothesis that insertional disruption of the A4var gene, one of two var genes located within the subtelomeric region of one end of chromosome 13, would result in a preferential switch in transcription to the adjacent R29var gene upon rosette selection. In this way, we aimed to mimic the preferential transcription of R29var in rosetting R29 parasites, a parasite line in which the A4var gene is deleted through a chromosome end truncation. Initial analysis of the knock-out parasite lines shows that the insertional disruption of the A4var gene prevents A4 PfEMP1 expression, but that switching transcription to other var gene variants is unaffected. Furthermore, analysis of var transcription in the knock-out parasite line during rosette selection shows that, rather than facilitating a switch to R29var gene transcription, this event was suppressed in the transfectants. These data, and the implications for epigenetic transcriptional control of var genes, are discussed.

Animals↗