Default Pathway of var2csa switching and translational repression in Plasmodium falciparum.

Antigenic variation is a subtle process of fundamental importance to the survival of a microbial pathogen. In Plasmodium falciparum malaria, PfEMP1 is the major variable antigen and adhesin expressed at the surface of the infected erythrocyte, which is encoded for by members of a family of 60 var-genes. Peri-nuclear repositioning and epigenetic mechanisms control their mono-allelic expression. The switching of PfEMP1 depends in part on variable transition rates and short-lived immune responses to shared minor epitopes. Here we show var-genes to switch to a common gene that is highly transcribed, but sparsely translated into PfEMP1 and not expressed at the erythrocyte surface. Highly clonal and adhesive P. falciparum, which expressed distinct var-genes and the corresponding PfEMP1s at onset, were propagated without enrichment or panning. The parasites successively and spontaneously switched to transcribe a shared var-gene (var2csa) matched by the loss of PfEMP1 surface expression and host cell-binding. The var2csa gene repositioned in the peri-nuclear area upon activation, away from the telomeric clusters and heterochromatin to transcribe spliced, full-length RNA. Despite abundant transcripts, the level of intracellular PfEMP1 was low suggesting post-transcriptional mechanisms to partake in protein expression. In vivo, off-switching and translational repression may constitute one pathway, among others, coordinating PfEMP1 expression.

A highly conserved segmental duplication in the subtelomeres of Plasmodium falciparum chromosomes varies in copy number.

BACKGROUND: Segmental duplications (SD) have been found in genomes of various organisms, often accumulated at the ends of chromosomes. It has been assumed that the sequence homology in-between the SDs allow for ectopic interactions that may contribute to the emergence of new genes or gene variants through recombinatorial events. METHODS: In silico analysis of the 3D7 Plasmodium falciparum genome, conducted to investigate the subtelomeric compartments, led to the identification of subtelomeric SDs. Sequence variation and copy number polymorphisms of the SDs were studied by DNA sequencing, real-time quantitative PCR (qPCR) and fluorescent in situ hybridization (FISH). The levels of transcription and the developmental expression of copy number variant genes were investigated by qPCR. RESULTS: A block of six genes of >10 kilobases in size, including var, rif, pfmc-2tm and three hypothetical genes (n-, o- and q-gene), was found duplicated in the subtelomeric regions of chromosomes 1, 2, 3, 6, 7, 10 and 11 (SD1). The number of SD1 per genome was found to vary from 4 to 8 copies in between different parasites. The intragenic regions of SD1 were found to be highly conserved across ten distinct fresh and long-term cultivated P. falciparum. Sequence variation was detected in a approximately 23 amino-acid long hypervariable region of a surface-exposed loop of PFMC-2TM. A hypothetical gene within SD1, the n-gene, encoding a PEXEL/VTS-containing two-transmembrane protein was found expressed in ring stage parasites. The n-gene transcription levels were found to correlate to the number of n-gene copies. Fragments of SD1 harbouring two or three of the SD1-genes (o-gene, pfmc-2tm, q-gene) were also found in the 3D7 genome. In addition a related second SD, SD2, of approximately 55% sequence identity to SD1 was found duplicated in a fresh clinical isolate but was only present in a single copy in 3D7 and in other P. falciparum lines or clones. CONCLUSION: Plasmodium falciparum carries multiple sequence conserved SDs in the otherwise highly variable subtelomeres of its chromosomes. The uniqueness of the SDs amongst plasmodium species, and the conserved nature of the genes within, is intriguing and suggests an important role of the SD to P. falciparum.

Genome wide gene amplifications and deletions in Plasmodium falciparum.

The extent to which duplications and deletions occur in the Plasmodium falciparum genome, outside of the subtelomeres, and their contribution to the virulence of the malaria parasite is not known. Here we show the presence of multiple genome wide copy number polymorphisms (CNPs) covering 82 genes, the most extensive spanning a cumulative size of 110kilobases. CNPs were identified in both laboratory strains and fresh clinical isolates using a 70-mer oligonucleotide microarray in conjunction with fluorescent in situ hybridizations and real-time quantitative PCR. The CNPs were found on all chromosomes except on chromosomes 6 and 8 and involved a total of 50 genes with increased copy numbers and 32 genes with decreased copy numbers relative to the 3D7 parasite. The genes, amplified in up to six copies, encode molecules involved in cell cycle regulation, cell division, drug resistance, erythrocyte invasion, sexual differentiation and unknown functions. These together with previous findings, suggest that the malaria parasite employs gene duplications and deletions as general strategies to enhance its survival and spread. Further analysis of the impact of discovered genetic differences and the underlying mechanisms is likely to generate a better understanding of the biology and the virulence of the malaria parasite.

Comparative transcriptomal analysis of isogenic Plasmodium falciparum clones of distinct antigenic and adhesive phenotypes.

Antigenic variation is a survival mechanism developed by the malaria parasite Plasmodium falciparum in order to allow for the establishment of a chronic infection. Here we have studied clonal differences in the transcriptomes of two isogenic P. falciparum clones (3D7S8.4 and 3D7AH1S2) of distinct adhesive and antigenic phenotypes employing a P. falciparum 70-mer oligonucleotide microarray. Fifteen transcripts were highly differentially expressed (greater than a 5-fold change) with five transcripts upregulated in 3D7AH1S2 compared to 3D7S8.4, and ten downregulated. Identified genes encode apical organellar (Gbph2, GBP-related antigen), cell cycle and DNA/RNA processing (SERA-5, RNA-methylase), cell-rescue, defense/virulence (RESA-2, RIFIN, PfEMP1) and hypothetical proteins (PFB0115w, PFI1445w, MAL13P1.121). A number of short and full-length var transcripts were differentially expressed between the clones but one full-length transcript was dominant in both rings and trophozoites (PFD0630c versus PFF0845c). Distinct members of two other variant gene families (phist-a and rif-like), scattered over the subtelomeric areas of the 14 chromosomes, were also found to be clonally and developmentally expressed. Three sibling-clones of 3D7AH1S2 (3D7AH1S1, -S3, -S4) were further studied for the expression of transcripts upregulated in 3D7AH1S2 compared to 3D7S8.4. Individual var and phist-a genes were found expressed in all of the clones while the expression of a rif-like gene and gbph2 varied in-between the clones. The present data provides evidence for complex transcriptional differences between closely related isogenic P. falciparum of distinct adhesive and antigenic characteristics.

Whole-body imaging of sequestration of Plasmodium falciparum in the rat.

The occlusion of vessels by packed Plasmodium falciparum-infected (iRBC) and uninfected erythrocytes is a characteristic postmortem finding in the microvasculature of patients with severe malaria. Here we have employed immunocompetent Sprague-Dawley rats to establish sequestration in vivo. Human iRBC cultivated in vitro and purified in a single step over a magnet were labeled with 99mtechnetium, injected into the tail vein of the rat, and monitored dynamically for adhesion in the microvasculature using whole-body imaging or imaging of the lungs subsequent to surgical removal. iRBC of different lines and clones sequester avidly in vivo while uninfected erythrocytes did not. Histological examination revealed that a multiadhesive parasite adhered in the larger microvasculature, inducing extensive intravascular changes while CD36- and chondroitin sulfate A-specific parasites predominantly sequester in capillaries, inducing no or minor pathology. Removal of the adhesive ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), preincubation of the iRBC with sera to PfEMP1 or preincubation with soluble PfEMP1-receptors prior to injection significantly reduced the sequestration. The specificity of iRBC binding to the heterologous murine receptors was confirmed in vitro, using primary rat lung endothelial cells and rat lung cryosections. In offering flow dynamics, nonmanipulated endothelial cells, and an intact immune system, we believe this syngeneic animal model to be an important complement to existing in vitro systems for the screening of vaccines and adjunct therapies aiming at the prevention and treatment of severe malaria.

Sp1 site is crucial for the mouse claudin-19 gene expression in the kidney cells.

Members of the claudin family play important roles in the formation of tight junctions in the kidneys, liver and intestine. Claudin-19 (Cldn19), a newly identified member of this family, is highly expressed in the kidney of the mouse. To have a better understanding on mouse claudin-19 gene expression, a 0.9-kb DNA fragment containing the 5'-flanking region of the Cldn19 gene was isolated. DNA sequence comparison between the mouse and human Cldn19 promoter regions exhibited little homology. One transcription initiation site was located at 104 nucleotides upstream of the start codon (ATG) of the Cldn19 gene. The mouse claudin-19 promoter lacked typical CAAT or GC-box. Deletion constructs of the 0.9-kb DNA fragment were generated and fused to a promoterless luciferase (Luc) reporter plasmid. Transfection studies using various kidney cell lines (MDCK, mIMCD3 and HEK293) revealed that the minimal promoter fragment resided in the -39 to -108 region, which contained a number of binding sites for transcription factors including Sp1. Site-directed mutagenesis using specific oligo probes confirmed that Sp1 was crucial for Cldn19 transactivation in the three cell lines studied. Electromobility shift assay confirmed that the nuclear extracts of these cells bound to the Sp1 oligo derived from Cldn19 promoter, but not to the mutated Sp1 oligo probe. Moreover, this DNA-protein complex would be recognized by Sp1 antibody, indicating specific Sp1 binding. Collectively, our data suggest that Sp1 binds to the claudin-19 promoter region and is responsible for its expression in the kidney cell lines in vitro.