Genetic variability in Plasmodium liver stage schizogony

Malaria imposes an immense public health and economic burden on large parts of the world Health Organization. Malaria control and/or elimination requires a better understanding of Plasmodium , the malaria causative agent: its basic biological processes and interaction with its hosts. Malaria control is undermined by mutation. The known library of antimalarial compound is regularly impacted by drug resistance mutations, and vaccine effectiveness is limited due to widespread variation in all candidate antigens. Understanding how malaria parasites generate genetic diversity is critical to control. Plasmodium infection initiates when the vector, the Anopheles mosquito, injects sporozoites spz in the skin of the mammalian host, after which most parasites travel to the liver and infect hepatocytes. Inside the hepatocyte, each spz replicates into thousands of erythrocyte-infectious forms, which are then released into the bloodstream and initiate the symptomatic blood stage of infection Prudêncio et al .. In spite of the critical role that the intra-hepatic phase of parasite development has for the successful establishment of infection and the onset of disease Dondorp et al ., How the parasite achieves such a high replication rate and the consequences of that remain utterly unexplored. Plasmodium cell cycle progression control is mostly unknown and it has many peculiarities. First, it divides by schizogony, a process characterized by nuclear divisions that occur without cytokinesis, originating a multinucleated ‘schizont’ Francia and Striepen. Moreover, throughout replication, Plasmodium cannot salvage pyrimidine bases or nucleosides from the extracellular environment and relies solely on nucleotides synthesized through the de novo biosynthetic pathway Janse et al .. Such peculiarities hamper research on this fundamental aspect of malaria parasite biology. In addition, we lack tools that can be transferred from cell cycle studies in model organisms to Plasmodium.

Recently, a Thymidine kinase TK coding sequence from Herpes simplex virus was introduced into P. falciparum genome Merrick. This allowed, for the first time, the labelling of parasites with BrdU a thymidine analogue and the characterization of DNA replication dynamics throughout intra-erythrocytic schizogony Stanojcic et al .. In the lab the study soughts to characterize Plasmodium schizogony during the liver stage of infection. to that end, we generated a P. berghei-gfp-tk parasite line, allowing to study cell cycle progression throughout a liver stage ls infection. Preliminary data unveiled for the first time: 1) temporal dynamics of DNA replication throughout parasite intra-hepatic development – P. berghei DNA replication starts as early as 12 hours post infection hpi with replication in some but not all nuclei – asynchronous replication. Importantly, from 56 to 60 hpi all nuclei inside a schizont replicate their DNA at the same time, suggesting that, contrary to earlier development, the last round of DNA replication is synchronous. 2) High replication rates are accompanied by DNA damage – The replication observed in the liver at 60 hpi is similar to that previously described for blood stage schizogony, where the last round of DNA replication is synchronous Stanojcic et al . Notably, in the liver stage such high replication rate at 60 hpi is accompanied by an accumulation of phosphorylated histone H2A foci. In mammals, that was recently showed as having the same role in Plasmodium Goyal et al .. The project aims to test if the accumulation of DNA damage in liver stage parasites is a generator of Plasmodium genetic variability prior to an intra-erythrocytic infection. The transformative idea that rapid replication at this early stage of Plasmodium life cycle could generate DNA damage, and hence genetic diversity, to the potential benefit of the parasite, remains utterly unexplored.

Understanding the connection between parasite replication capabilities and genetic diversity, which the malaria parasite must finely balance in order to generate antigenic variation to aid immune escape and also retain the core cellular functions, has certainly a high impact in disease outcome and will provide valuable tools to underpin novel strategies to tackle malaria.

Genetics and Genomics

Jan 2023 — Jun 2024

$55,330

Portugal