Aniweh, Y. | Search bioRxiv

bioRxiv2020

Plasmodium falciparum Replication factor C subunit 1 is involved in genotoxic stress response

About half the worlds population is at risk of malaria, with Plasmodium falciparum malaria being responsible for the most malaria related deaths globally. Antimalarial drugs such as chloroquine and artemisinin are directed towards the proliferating intra-erythrocytic stages of the parasite, which is responsible for all the clinical symptoms of the disease. These antimalarial drugs have been reported to function via multiple pathways, one of which induces DNA damage via the generation of free radicals and reactive oxygen species. An urgent need to understand the mechanistic details of drug response and resistance is highlighted by the decreasing clinical efficacy of the front line drug, Artemisinin. The replication factor C subunit 1 protein is an important component of the DNA replication machinery and DNA damage response mechanism. Here we show the translocation of PfRFC1 from an intranuclear localization to the nuclear periphery indicating an orchestrated progression of distinct patterns of replication in the developing parasites. PfRFC1 responds to genotoxic stress via elevated protein levels in soluble and chromatin bound fractions. Reduction of PfRFC1 protein levels upon treatment with antimalarials suggests an interplay of replication and DNA repair pathways leading to cell death. Additionally, mislocalization of the endogenously tagged protein confirmed its essential role in parasites replication and DNA repair. This study provides key insights into DNA replication, DNA damage response and cell death in plasmodium falciparum. ImportanceFrontline drugs have been found to induce DNA damage in the human malaria parasite Plasmodium falciparum. The genotoxic stress response in Plasmodium and the interplay between DNA damage repair, replication and activation of programmed cell death pathways remains largely undescribed. This study shows a distinct pattern of localization of PfRFC1 during replication and DNA repair. PfRFC1 responds to genotoxic stress with an increase in protein expression. Interfering with the RFC complex formation or mislocalization of PfRFC1 is associated with disrupted genotoxic stress response. Additionally, a reduction of PfRFC1 protein levels is observed upon treatment with antimalarial drugs or under apoptosis like conditions, highlighting the role of DEVD/G like motif in mediating programmed cell death in these parasites. This study sheds light on the role of PfRFC1 in differentially responding to replication, genotoxic stress and programmed cell death in Plasmodium parasites.

cell biology↗

bioRxiv2020

Blood donor variability is a modulatory factor for P. falciparum invasion phenotyping assays

Human erythrocytes are indispensable for Plasmodium falciparum development. Unlike other eukaryotic cells, there is no existing erythroid cell line capable of supporting long-term P. falciparum in vitro experiments. Consequently, invasion phenotyping experiments rely on erythrocytes of different backgrounds. However, the contribution of the erythrocytes variation in influencing invasion rates remains unknown, which presents a challenge for conducting large-scale comparative studies. Here, we used erythrocytes of different blood groups harboring different hemoglobin genotypes to assess the relative contribution of blood donor variability in P. falciparum invasion phenotyping assays. For each donor, we investigated the relationship between parasite invasion phenotypes and erythrocyte phenotypic characteristics, including; the expression levels of surface receptors (e.g. the human glycophorins A and C, the complement receptor 1 and decay accelerating factor), blood groups (e.g. ABO/Rh system), and hemoglobin genotypes (e.g. AA, AS and AC). Across all donors, there were significant differences in invasion efficiency following treatment with either neuraminidase, trypsin or chymotrypsin relative to the control erythrocytes. Primarily, we showed that the levels of key erythrocyte surface receptors and their sensitivity to enzyme treatment, significantly differed across donors. However, invasion efficiency correlated neither with susceptibility to enzyme treatment nor with the levels of the selected erythrocyte surface receptors. Upon further analysis, we found no relationship between P. falciparum invasion phenotype and blood group or hemoglobin genotype. ImportanceAssays to decipher P. falciparum invasion phenotypes are of great importance in the quest for an efficient malaria vaccine. Malaria associated mortality is mainly attributed to the blood stage of the parasites life cycle, a major focus of vaccine development strategies. Further, testing and validating blood stage vaccines necessitates conducting large-scale studies in endemic countries. However, comparing results from such studies is challenged by the lack of standard assays. As human erythrocytes play a pivotal role in P. falciparum invasion assays, the need to investigate the effect of blood donor variability in the outcome of such assays is apparent. The significance of our study is in reporting the absence of relationship between P. falciparum invasion efficiency and commonly shared erythrocyte features across different erythrocyte donors, therefore emphasizing the need to consider erythrocyte donor uniformity and to anticipate challenges associated to blood donor variability in early stages of large-scale study design.

microbiology↗