Abstract
In vitro studies of liver stage (LS) development of the human malaria parasite Plasmodium falciparum are technically challenging; therefore, fundamental questions about hepatocyte receptors for invasion that can be targeted to prevent infection remain unanswered. To identify novel receptors and to further understand human hepatocyte susceptibility to P. falciparum sporozoite invasion, we created an optimized in vitro system by mimicking in vivo liver conditions and using the subcloned HC-04.J7 cell line that supports mean infection rates of 3–5% and early development of P. falciparum exoerythrocytic forms—a 3- to 5-fold improvement on current in vitro hepatocarcinoma models for P. falciparum invasion. We juxtaposed this invasion-susceptible cell line with an invasion-resistant cell line (HepG2) and performed comparative proteomics and RNA-seq analyses to identify host cell surface molecules and pathways important for sporozoite invasion of host cells. We identified and investigated a hepatocyte cell surface heparan sulfate proteoglycan, glypican-3, as a putative mediator of sporozoite invasion. We also noted the involvement of pathways that implicate the importance of the metabolic state of the hepatocyte in supporting LS development. Our study highlights important features of hepatocyte biology, and specifically the potential role of glypican-3, in mediating P. falciparum sporozoite invasion. Additionally, it establishes a simple in vitro system to study the LS with improved invasion efficiency. This work paves the way for the greater malaria and liver biology communities to explore fundamental questions of hepatocyte-pathogen interactions and extend the system to other human malaria parasite species, like P. vivax.
Highlights
Malaria is a devastating disease that affects over 200 million people each year and causes approximately 445,000 deaths, mainly among young children (WHO, 2017)
Identifying hepatocyte receptors and pathways for P. falciparum sporozoite invasion first required the establishment of an in vitro platform that could be used as the basis for comparing invasion susceptible and non-susceptible cell lines (Figure 1)
When we cultured HC-04 in Dulbecco’s Modified Eagle Medium (DMEM)-NoGlc in an attempt to reduce the Warburg effect typically seen in cancer cells (Warburg et al, 1924), we observed differences in cellular morphology (Supplementary Figure S1A) and expression levels for proteins involved in oxidative phosphorylation and proteins found in the mitochondria (Supplementary Figures S1B–E and Supplementary Tables S1–S3)
Summary
Malaria is a devastating disease that affects over 200 million people each year and causes approximately 445,000 deaths, mainly among young children (WHO, 2017). Plasmodium falciparum is one of the major parasites responsible for morbidity and mortality. This parasite is transmitted to humans as a sporozoite through the bite of an infected female anopheline mosquito during blood feeding. The infection of hepatocytes causes no clinical symptoms, allowing the parasite to develop and multiply to prepare for the invasion of red blood cells, which results in clinical disease (Phillips and Pasvol, 1992; Vaughan et al, 2008). Studies carried out using primary human hepatocytes face the obstacles of these cells not propagating in culture, being in short supply, and producing highly variable infection rates (0.13– 2%) (Smith et al, 1984; Mazier et al, 1985; Vaughan et al, 2008; Roth et al, 2018). Development of a suitable alternative to using primary human hepatocytes for the study of the P. falciparum LS is desirable
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