The Selection of a Hepatocyte Cell Line Susceptible to Plasmodium falciparum Sporozoite Invasion That Is Associated With Expression of Glypican-3

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.

Keywords: Plasmodium falciparum; glypican-3; hepatocyte; in vitro model; liver stage; malaria; omics.

FIGURE 1

Schematic of the overall experimental approach utilized in this study. ILSDA, inhibition of liver stage development assay.

FIGURE 2

Establishment of an optimized in vitro system for Plasmodium falciparum sporozoite invasion. (A) Inside-outside staining of P. falciparum sporozoites in HC-04 cells after 24 h. Red staining denotes sporozoites inside cells; yellow staining denotes sporozoites outside cells. Scale bars = 200 μm. (B,C) The invasion efficiency of P. falciparum NF54 sporozoites in HC-04 (parental) cells grown under normoxic conditions (5% CO₂) (B) or in 5% oxygen (C). The dashed red line at 2% indicates the highest invasion percentages reported in the literature using primary human hepatocyte monoculture. AA: amino acids (arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, valine). Lipid: arachidonic, linoleic, linolenic, myristic, oleic, palmitic, stearic, cholesterol, Tween-80, tocopherol acetate, and Pluronic F-68. Glc: 15 mM D-glucose was added at the time of sporozoite addition. (D) The invasion efficiency of P. falciparum NF54 sporozoites isolated from the same pool of mosquitoes in parental HC-04 and the HC-04.J7 subclone grown in DMEM-NoGlc in three biological replicate (R1–R3) assays. Mean ± SEM is shown. P-values were calculated using a using a two-tailed Student’s t-test. ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 3

Multi-omics analysis of HC-04, HC-04.J7, and HepG2 cell lines. (A) Volcano plot of the quantifiable surface-enriched proteome comparing HC-04.J7 protein levels to HepG2 protein levels when both cell lines are grown in IMDM. (B) Immunofluorescence staining of HC-04, HC-04.J7, and HepG2 cells grown in DMEM-NoGlc or IMDM with anti-GPC3 antibody. (C) An inhibition of liver stage development assay with anti-GPC3 antibody. Results from two representative experiments carried out using the same methodology with two different sporozoite pools with mean ± SEM are shown. A two-way ANOVA showed a significant effect of the antibody on sporozoite invasion in HC-04.J7 (P < 0.001).

FIGURE 4

Transcriptomic mining reveals pathways and genes that partition hepatocarcinoma lines that are (i) highly susceptible, HC-04.J7, (ii) susceptible, HC-04 (parental), or (iii) non-susceptible, HepG2, to P. falciparum sporozoite infection. (A) Heatmap displaying the relative transcript reads of various transcripts comparing HC-04.J7 (highly susceptible, HS) and HepG2 (non-susceptible, NS; left); HC-04 (susceptible, S) and HepG2 (center); and HC-04.J7 and HC-04 (right). (B) Boxplot analyses for a subset of genes through the transcriptomic profiling of the three cell lines. Read counts are indicated along the y-axes for each comparison. P-values were calculated using a two-sided Mann–Whitney U test. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 5

Hepatocyte receptors and pathways involved in P. falciparum sporozoite invasion. (A) The glypican-3 (GPC3) STRING protein–protein interaction network. Proteins on the right side of the hepatocyte include GPC3 and its known interactors; proteins on the left side are other hepatocyte receptors implicated in sporozoite invasion. [GPC, glypican; NDST, N-deacetylase/N-sulfotransferase (heparan glucosaminyl); IGFBP, Insulin-like growth factor binding protein 4; IGF, insulin-like growth factor; IGF1R, Insulin-like growth factor 1 receptor; PTCH, patched; HHP, Hedgehog interacting protein; GLI2, GLI family zinc finger 2; purple lines—experimentally determined interactors; light blue lines—curated database-determined interactors; light green lines—text mining-determined interactors; black lines—co-expression-determined interactors]. (B) Characteristics of a more-susceptible hepatocyte for P. falciparum invasion based on proteomic and transcriptomic analyses.