Expression of "Plasmodium falciparum var" genes in naturally infected children from Tanzania

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.