Novel insights into telomere biology and virulence gene expression in plasmodium falciparum

Plasmodium falciparum malaria is still one of the most preeminent and deadliest infectious diseases worldwide, imposing a tremendous health and economic burden on endemic countries. The high virulence of P. falciparum is mostly attributable to the expression of P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells. PfEMP1 mediates intravascular parasite sequestration in vital organs, which contributes substantially to severe disease and death. Mutually exclusive transcription of the 60 var genes (encoding PfEMP1) and switching to formerly silenced variants results in antigenic variation and allows the parasite to efficiently evade host immune responses and to establish chronic infection. Members of the var multigene family are predominantly positioned close to chromosome ends. Characteristically, these regions are transcriptionally inert and demarcated by the repressive histone mark H3K9me3 and the evolutionary conserved silencing factor P. falciparum heterochromatin protein 1 (PfHP1). It is believed that this specialised environment at chromosome ends generates a structural framework for the epigenetic control of var gene expression. Moreover, telomeres play a crucial role in preserving genome integrity by protecting chromosome ends from inappropriate fusion and recombination events, as well as in regulating telomere length. However, we still lack a detailed functional understanding of the underlying molecular mechanisms that regulate Plasmodium chromosome end biology. During my PhD thesis, I tackled chromosome end biology from three different angles to improve our understanding of how virulence gene expression is regulated and how genome integrity is preserved. In a first project I performed an in-depth functional analysis of the epigenetic silencing factor PfHP1 by generating an inducible loss-of-function mutant. We showed that upon PfHP1 depletion parasites display a complete breakdown of mutually exclusive var expression and antigenic variation. Intriguingly, we also found that over 50% of PfHP1-deprived parasites represented viable gametocytes that complete sexual development up to stage V maturity. This high conversion rate was linked to the targeted de-repression of the ap2-g locus that codes for the ApiAP2 transcription factor AP2-G, which is essential for gametocyte conversion. Thus, our data unveiled PfHP1 not only as a master regulator of variegated expression of exported virulence factors, but also as a crucial factor in the regulation of sexual cell differentiation. In a second project I aimed at the functional characterisation of the chromosome-end associated protein PfSIP2, which was shown to specifically interact with SPE2 elements in subtelomeric regions. In-depth analysis of the expression profile of endogenous PfSIP2 revealed that this protein is only expressed during a very narrow time window of approximately 10hrs in late stage parasites, which coincides with intra-erythrocytic schizogony. Genome-wide ChIP-Seq experiments confirmed the exclusive binding of endogenous PfSIP2 to subtelomeric SPE2 landmarks in upsB var promoter regions and subtelomeric non-coding regions. Surprisingly, however, neither phenotypic changes nor differential gene expression were observed in a conditional PfSIP2-loss-of-function mutant and hence this approach didn’t uncover novel insights into the function of this ApiAP2 factor. In a third project I aimed at the identification of the telomere repeat-binding factor (TRF) in P. falciparum. Although TRFs are highly conserved and play essential roles in preserving chromosome integrity and regulating chromosome length in model eukaryotes, so far no TRF homologue has been found in the malaria parasites. My work reports about the successful de novo identification of the P. falciparum telomere repeat-binding protein (PfTRF). Intriguingly, this protein appears to be evolutionary distinct from TRFs in other eukaryotes as it binds to telomere repeat DNA via a C-terminal C2H2-type zinc finger domain instead of a MYB domain. Genome-wide mapping by ChIP-Seq experiments not only confirmed that PfTRF indeed binds to all chromosome termini in vivo, but as well revealed an unexpected second binding hotspot at telomere repeat-like sequences found in subtelomeric var gene promoters. A comprehensive characterisation of PfTRF using a conditional loss-of-function mutant identified essential roles for this protein in mitotic cell cycle progression and telomere length regulation. Hence, our findings provide important new insight into mechanisms underlying genome maintenance and possibly virulence gene silencing in P. falciparum. They further suggest that malaria parasites employ an evolutionary divergent molecular complex to preserve telomere function. In summary, my results provide important new and detailed understanding of the molecular processes involved in genome maintenance, virulence gene expression and sexual conversion in P. falciparum, processes that are highly relevant for malaria pathogenesis, parasite viability and malaria transmission. I am confident that these findings have important implications for the development of intervention strategies targeting parasite propagation and transmission.

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