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Expression of "Plasmodium Falciparum Var" Genes in Naturally Infected Children from Tanzania

Mugasa, J. P. (2008) Expression of "Plasmodium Falciparum Var" Genes in Naturally Infected Children from Tanzania. Doctoral thesis, University of Basel.

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Abstract

Plasmodium falciparum is the most pathogenic malarial parasite and a major cause of morbidity and mortality among young children in sub-Saharan Africa. The virulence of P. falciparum has been linked to its expression of variant surface antigens (VSAs) on the surface of infected red blood cells. These VSAs subvert acquisition of protective immunity and mediate cytoadherence of infected erythrocytes to the microvasculature lining of various endothelial cell receptors. It causes sequestration of infected erythrocytes in post capillary venules of the vital organs such as the brain or placenta. Cytoadherence causes retention and accumulation of the infected erythrocytes to endothelial membranes of deep postvenous capillaries leading to occlusion of micro-vessels. This result in obstruction of free blood flow with serious pathological consequences associated with severe malaria. Sequestration facilitates parasite multiplication and enables the parasites to avoid the passage of infected erythrocytes through the spleen, where deformed erythrocytes are removed from blood circulations. This cytoadherence is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 is a VSA family encoded by ~ 60 highly polymorphic var genes per haploid genome, expressed on the surface of infected red blood cells. PfEMP1 is expressed in a mutually exclusive manner, and switching the expression creates extensive antigenic variation and the potential for multiple adhesion profile. Antigenic variation is a strategy employed by P. falciparum to avoid antibody-mediated destruction by alternating expression of individual var genes each of which encodes an antigenically distinct form of PfEMP1. Sequence analysis of the var gene repertoire of the 3D7 clone revealed genetic structuring in which var genes fall into 3 distinct groups (A, B, and C) and two intermediate groups (B/A and B/C) based on chromosomal location, gene orientation and the 5' flanking sequences. It has been postulated that this genetic organization helps to restrict recombination within a specific group of genes and leads to their structural and functional specialization for binding to different endothelial receptors. The sequences of var genes vary substantially within and between the parasites genome. This has been clearly indicated by the fact that there is minimal overlap in the var gene repertoire between isolates due to high inter-genic and intra-genic recombination within the var gene family. Despite the complex nature of this molecule, the var gene still remains the best defined factor contributing to malaria pathogenesis. Different research groups have attempted to define the repertoire of var gene from different isolates, and reported vast global var gene diversity. Only a tip of iceberg of the var genes diversity is currently in view. The big challenge to date is to understand how the var gene diversity and selection pressure influence malaria pathogenesis in order to device a control strategy based on interference with PfEMP1 expression. Clinical and sero-epidemiological studies have suggested that severe disease is attributed by the parasite expressing a restricted and antigenically conserved subset of VSAs which are frequently recognized by sera from semi-immune individuals, proposing that expression of a particular VSA may be associated with disease manifestation. Pregnancy associated malaria (PAM) is well understood and has often been linked with the expression of a var gene called var2csa which is unusually conserved across parasite isolates and binds a low sulfated form of chondroitin sulfate A (CSA) in the placenta. Different studies have attempted to link a particular var gene expression with a disease phenotype. It is becoming evident that var group A and B/A are involved in severe childhood malaria. Protective immunity to severe malaria develops earlier in childhood after only few severe episodes pointing to a relatively conserved target antigen. This phenomenon makes it theoretically possible to protect non immune children against severe and complicated malaria by accelerating acquisition of PfEMP1 specific immunity. Given the proposed importance of immunity to PfEMP1 in protection against malaria, it is essential that we gain a better understanding of var gene expression during infection. Despite substantial contribution of var genes to malaria pathogenesis and parasites survival, few studies on var gene transcription during natural infections have been carried out in field isolates. This is mainly attributed to technical difficulties, and the complexity and immense diversity interfering with most study design. For this thesis, two studies on var gene expression in naturally infected children with severe P. falciparum malaria from Tanzania were conducted. In the first study, the transcription levels of var gene groups were compared in children with severe, uncomplicated and asymptomatic malaria by using quantitative real-time PCR. Transcripts of var group A and B genes were up-regulated in children with severe malaria compared to patients with uncomplicated malaria. In general, the transcript abundances of var group A and B genes were higher for children with clinical malaria than for children with asymptomatic infections. var group C was not linked with any disease phenotype. In the second study, the genetic diversity of expressed P. falciparum var genes in children with severe malaria from Tanzania was determined. The var transcripts isolated from children with severe malaria (Blantyre score ≤ 3) were compared with isolates from children with asymptomatic malaria. Diversity patterns of dominant full-length var transcripts were determined by isolation of mRNA followed by magnetic bead capture through an ATS-anchor and reversetranscription into var cDNA. The different PCR amplified expressed sequence tags were cloned and sequenced. Large sequence diversity of the amplified var DBL-1α and the 5’ non-coding regions was observed and minimal overlapping was evident among the isolates providing strong evidence that the transcribed var gene repertoire is immense. var DBL-1α sequences isolated from AM were more diverse with more singletons (P<0.05) compared with DBL-1α sequences from SM. Unique var sequences that were exclusively expressed with P. falciparum isolated from children with SM were found. Despite the fact that var gene diversity is unlimited, transcripts from SM isolates were more restricted, supporting the hypothesis that certain PfEMP1 repertoires are involved in triggering severe infections.

Item Type: Thesis (Doctoral)
Keywords: Plasmodium Falciparum, Malarial, Morbidity and Mortality, Blood Cells, Immunity, Tanzania
Subjects: Malaria > Diagnosis & treatment
Malaria > Vaccines
Divisions: Other
Depositing User: Mr Joseph Madata
Date Deposited: 15 Feb 2013 07:08
Last Modified: 15 Feb 2013 07:08
URI: http://ihi.eprints.org/id/eprint/1135

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