Derbyshire, E. | Search bioRxiv

bioRxiv2020

Phosphatidylinositol 3-phosphate and Hsp70 protect Plasmodium falciparum from heat-induced cell death

Phosphatidylinositol 3-phosphate (PI(3)P) levels in Plasmodium falciparum correlate with tolerance to cellular stresses caused by artemisinin, a first-line malaria treatment, and environmental factors. However, the functional role of PI(3)P in the Plasmodium stress response and a possible mechanism of protection were unknown. Here, we used multiple chemical probes including PI3K inhibitors and known antimalarial drugs to examine the importance of PI(3)P under thermal conditions that recapitulate malaria fever. Live cell microscopy using both chemical and genetic reporters revealed that PI(3)P stabilizes the acidic and proteolytic digestive vacuole (DV) under heat stress. We demonstrate that heat-induced DV destabilization in PI(3)P-deficient P. falciparum precedes cell death and is reversible after withdrawal of the stress condition and the PI3K inhibitor. These phenotypes are not observed with an inactive structural analog of the PI3K inhibitor. A chemoproteomic and biochemical approach identified PfHsp70-1 as a parasite PI(3)P-binding protein. Targeting PfHsp70-1 with a small molecule inhibitor phenocopied PI(3)P-deficient parasites under heat shock. Furthermore, tunable knockdown of PfHsp70-1 showed that PfHsp70-1 downregulation causes DV destabilization and hypersensitizes parasites to heat shock and PI3K inhibitors. Our findings underscore a mechanistic link between PI(3)P and PfHsp70-1, and present a novel PI(3)P function in stabilizing the DV compartment during heat stress.

microbiology↗

bioRxiv2019

RNA-Seq Analysis Illuminates the Early Stages of Plasmodium Liver Infection

The apicomplexan parasites Plasmodium spp. are the causative agents of malaria, a disease that poses a significant global health burden. Plasmodium spp. initiate infection of the human host by transforming and replicating within hepatocytes. This liver stage (LS) is poorly understood when compared to other Plasmodium life stages, which has hindered our ability to target these parasites for disease prevention. We conducted an extensive RNA-seq analysis throughout the Plasmodium berghei LS, covering as early as 2 hours post infection (hpi) and extending to 48 hpi. Our data revealed that hundreds of genes are differentially expressed at 2 hpi, and that multiple genes shown to be important for later infection are upregulated as early as 12 hpi. Using hierarchical clustering along with co-expression analysis, we identified clusters functionally enriched for important liver-stage processes such as interactions with the host cell and redox homeostasis. Furthermore, some of these clusters were highly correlated to the expression of ApiAP2 transcription factors, while showing enrichment of mostly uncharacterized DNA binding motifs. This finding presents potential LS targets for these transcription factors, while also hinting at alternative uncharacterized DNA binding motifs and transcription factors during this stage. Our work presents a window into the previously undescribed transcriptome of Plasmodium upon host hepatocyte infection to enable a comprehensive view of the parasites LS. These findings also provide a blueprint for future studies that extend hypotheses concerning LS gene function in P. berghei to human-infective Plasmodium parasites. IMPORTANCEThe LS of Plasmodium infection is an asymptomatic yet necessary stage for producing blood-infective parasites, the causative agents of malaria. Blocking the liver stage of the life cycle can prevent clinical malaria, but relatively less is known about the parasites biology at this stage. Using the rodent model P. berghei, we investigated whole-transcriptome changes occurring as early as 2 hpi of hepatocytes. The transcriptional profiles of early time points (2, 4, 12, and 18 hpi) have not been accessible before due to the technical challenges associated with liver-stage infections. Our data now provides insights into these early parasite fluxes that may facilitate establishment of infection, transformation and replication in the liver.

microbiology↗