"Plasmodium falciparum var" Gene Expression Dynamics and its Relevance in Malaria Disease in Children from Papua New Guinea

Abstract

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.