"Plasmodium falciparum var" gene expression dynamics and its relevance in malaria disease in children from Papua New Guinea

Malaria is a tremendous global public health problem. While especially hitting the poorest
countries in the world, malaria elicits each year 300 million febrile illnesses and up to 1
million deaths. Widespread drug resistances, climatic changes, but also disintegrated health
services and armed conflicts have contributed to a global increase of malaria while a
vaccine will not be at hand for many more years to come.
Malaria is caused by the protozoan parasite Plasmodium and transmitted by the female
Anopheles mosquito. Of 4 Plasmodium species infecting humans, Plasmodium falciparum
is by far the most harmful parasite responsible for nearly all mortality. The increased
virulency of P. falciparum can be ascribed to special immune evasion strategies inherent of
this species. This mainly refers to a process called cytoadherence, the sequestration and
adhesion of infected erythrocytes (IE) to endothelial cells of the microcapillary system. To
evade spleen dependent killing, cytoadherence is a benefit for the parasite, but detrimental
to the host by leading to poorly diffused tissues and hypoxia in the upstream segments and
thus, contributing substantially to severe manifestations.
Related to sequestration is a process called rosetting, the binding of IE to uninfected
erythrocytes. This leads to erythrocyte clusters impeding local blood flow and accordingly,
rosette formation was also associated with severe disease.
On the surface of IE, the parasite derived protein family Plasmodium falciparum
erythrocyte membrane protein 1 (PfEMP1) is thought to be the key mediator for
sequestration and rosetting.
However, by exposing a parasite derived antigen on the surface of IE, the parasite gets
vulnerable to immune attack. Therefore, P. falciparum evades the immune system by a
process called antigenic variation, the switching of the expression between different
members of PfEMP1.
PfEMP1 is encoded by approximately 60 var genes per haploid genome. The highly
diverse and large var genes are structured into several adhesive, semi-conserved domains.
Most var genes can be subgrouped into var group A, B and C according to their diverse,
but within one group highly conserved untranslated regions.
Despite of the substantial contribution of PfEMP1 to malaria pathogenesis and parasite
survival, few studies on var genes and PfEMP1 have been carried out in vivo. This is
mainly due to their immense diversity interfering with most study designs. We conducted 2 studies on var gene expression in naturally infected children from Papua
New Guinea.
In a longitudinal study over a 4-month period in older, semi-immune children, we studied
antigenic variation of var genes, namely the dynamics and distribution of var transcripts
over time. Diversity and patterns of full-length var transcripts were evaluated by magnetic
bead-anchored reverse-transcription polymerase chain reaction (RT-PCR), cloning and
sequencing. We identified a highly dynamic picture of var expression with mostly new var
transcripts at a 2-weeks interval but with some var transcripts recurring for up to 10 weeks.
The number of detected var transcripts correlated with the number of infecting P.
falciparum strains. On average, 1.7 different var transcripts were detected per child and
infecting strain. The analysis of 286 different sequences of selected var gene domains
confirmed the recombinogenic nature of var genes.
In a malaria case-control study on children from Papua New Guinea, we quantitatively
compared the distribution of var transcripts among var groups A, B and C in children with
severe malaria, with mild malaria and in asymptomatic children. The sub-division of var
genes into these var groups raises questions about the biological or clinical significance of
these structural differences. Upon expression, different var groups might have different
pathological implications on the host leading to distinct virulences and different clinical
outcomes. By using real-time quantitative PCR, we found a major expression difference
between parasites causing clinical attack and parasites leading to asymptomatic infections.
A significant up-regulation of var group B transcripts was evident in children with clinical
malaria (mild and severe) while var group C genes were mainly switched on in
asymptomatic children. No change in the distribution of var transcripts was detected
between mild and severe disease. Finally, we found a significant up-regulation of var
group A genes in parasites conferring the formation of rosettes.
Together, these studies on var gene expression are the first of its kind, conducted in
naturally infected children in an endemic area. They are a step towards the comprehension
of the dynamics and impacts of var gene expression in vivo. Together with previous
studies, our data emphasize the substantial implications of PfEMP1 in malaria morbidity.