fmicb-10-00127 February 27, 2019 Time: 13:2 # 1 ORIGINAL RESEARCH published: 28 February 2019 doi: 10.3389/fmicb.2019.00127 The Selection of a Hepatocyte Cell Line Susceptible to Plasmodium falciparum Sporozoite Invasion That Is Associated With Expression of Glypican-3 Rebecca E. Tweedell1,2, Dingyin Tao2†, Timothy Hamerly1,2†, Tanisha M. Robinson3†, Simon Larsen4, Alexander G. B. Grønning4, Alessandra M. Norris1, Jonas G. King2,5, Henry Chun Hin Law1, Jan Baumbach4,6‡, Elke S. Bergmann-Leitner3‡ and Rhoel R. Dinglasan1,2* 1 Department of Infectious Diseases and Immunology, Emerging Pathogens Institute, University of Florida, Gainesville, FL, Edited by: United States, 2 W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Malaria Celio Geraldo Freire-de-Lima, Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States, 3 Malaria Vaccine Universidade Federal do Rio Branch, Walter Reed Army Institute of Research, Silver Spring, MD, United States, 4 Computational BioMedicine Lab, de Janeiro, Brazil Department of Mathematics and Computer Science, University of Southern Denmark, Odense, Denmark, 5 Department Reviewed by: of Biochemistry, Molecular Biology, Entomology & Plant Pathology, Mississippi State University, Starkville, MS, United States, Rodrigo Andrés López-Muñoz, 6 Chair of Experimental Bioinformatics, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Southern University of Chile, Chile Munich, Germany Miguel Prudêncio, University of Lisbon, Portugal In vitro studies of liver stage (LS) development of the human malaria parasite *Correspondence: Rhoel R. Dinglasan Plasmodium falciparum are technically challenging; therefore, fundamental questions [email protected]fl.edu about hepatocyte receptors for invasion that can be targeted to prevent infection remain †These authors have contributed unanswered. To identify novel receptors and to further understand human hepatocyte equally to this work ‡These authors also contributed susceptibility to P. falciparum sporozoite invasion, we created an optimized in vitro equally to this work 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 Specialty section: This article was submitted to exoerythrocytic forms—a 3- to 5-fold improvement on current in vitro hepatocarcinoma Infectious Diseases, models for P. falciparum invasion. We juxtaposed this invasion-susceptible cell line a section of the journal with an invasion-resistant cell line (HepG2) and performed comparative proteomics Frontiers in Microbiology and RNA-seq analyses to identify host cell surface molecules and pathways important Received: 13 October 2018 Accepted: 21 January 2019 for sporozoite invasion of host cells. We identified and investigated a hepatocyte cell Published: 28 February 2019 surface heparan sulfate proteoglycan, glypican-3, as a putative mediator of sporozoite Citation: invasion. We also noted the involvement of pathways that implicate the importance Tweedell RE, Tao D, Hamerly T, Robinson TM, Larsen S, of the metabolic state of the hepatocyte in supporting LS development. Our study Grønning AGB, Norris AM, King JG, highlights important features of hepatocyte biology, and specifically the potential role of Law HCH, Baumbach J, glypican-3, in mediating P. falciparum sporozoite invasion. Additionally, it establishes a Bergmann-Leitner ES and Dinglasan RR (2019) The Selection simple in vitro system to study the LS with improved invasion efficiency. This work paves of a Hepatocyte Cell Line Susceptible the way for the greater malaria and liver biology communities to explore fundamental to Plasmodium falciparum Sporozoite Invasion That Is Associated With questions of hepatocyte-pathogen interactions and extend the system to other human Expression of Glypican-3. malaria parasite species, like P. vivax. Front. Microbiol. 10:127. doi: 10.3389/fmicb.2019.00127 Keywords: malaria, Plasmodium falciparum, liver stage, in vitro model, omics, glypican-3, hepatocyte Frontiers in Microbiology| www.frontiersin.org 1 February 2019| Volume 10| Article 127 fmicb-10-00127 February 27, 2019 Time: 13:2 # 2 Tweedell et al. Hepatocyte Susceptibility to P. falciparum Invasion INTRODUCTION can be distributed as a shared resource to laboratories all over the world. Malaria is a devastating disease that affects over 200 million Technical limitations of studying the mammalian Plasmodium people each year and causes approximately 445,000 deaths, LS have hampered the identification of proteins involved mainly among young children (WHO, 2017). Plasmodium in sporozoite host cell invasion and infection and left the falciparum is one of the major parasites responsible for morbidity process poorly understood for P. falciparum. Most studies and mortality. This parasite is transmitted to humans as a have focused on the rodent Plasmodium species. However, sporozoite through the bite of an infected female anopheline differences in sporozoite host cell tropism and the lack mosquito during blood feeding. From the bite site, the sporozoite of conservation of hepatocyte surface receptors necessary makes its way to the liver, where it infects a hepatocyte for invasion suggest significant differences exist between (Yamauchi et al., 2007). The infection of hepatocytes causes these species and P. falciparum (Kaushansky and Kappe, no clinical symptoms, allowing the parasite to develop and 2015); focusing studies on rodent parasites alone can cause multiply to prepare for the invasion of red blood cells, which essential factors for P. falciparum sporozoite invasion to be results in clinical disease (Phillips and Pasvol, 1992; Vaughan missed or overlooked. Using various model systems, it has et al., 2008). The LS is a crucial step in the parasite’s life been demonstrated that SCARB1 (Rodrigues et al., 2008), cycle, as it establishes vertebrate infection; however, studying SDC2 (Frevert et al., 1993), EphA2 (Kaushansky et al., P. falciparum LS development has been technically challenging. 2015), LRP1 (Shakibaei and Frevert, 1996), CD81 (Silvie Studies carried out using primary human hepatocytes face the et al., 2003), and c-Met (P. berghei only; Kaushansky and obstacles of these cells not propagating in culture, being in Kappe, 2011) can each play a role as hepatocyte receptors short supply, and producing highly variable infection rates (0.13– for sporozoite invasion and infection, but the molecular 2%) (Smith et al., 1984; Mazier et al., 1985; Vaughan et al., invasion mechanism for P. falciparum remains largely unknown. 2008; Roth et al., 2018). While recent work has improved the Additionally, the steps of LS development following sporozoite utility of primary cells, this system requires the screening of invasion are not well defined for P. falciparum. These different lots of primary cells to identify those that support knowledge gaps in LS biology, along with the difficulty of sporozoite invasion and development, limiting widespread use implementing high-throughput screens for this stage, have (Roth et al., 2018). Development of a suitable alternative to using been major roadblocks in identifying much needed drug primary human hepatocytes for the study of the P. falciparum targets and vaccine candidates (Derbyshire et al., 2012; LS is desirable. Longley et al., 2015). P. falciparum and P. vivax sporozoites can infect and develop Herein, we applied comparative proteomics and RNA- in the human hepatocarcinoma cell line HC-04, but infection seq approaches to identify surface molecules and pathways efficiency remains marginal, customarily between 0.13% and 0.7– from P. falciparum sporozoite invasion-susceptible and 1% for P. falciparum (Sattabongkot et al., 2006; Mikolajczak et al., invasion-resistant hepatocarcinoma cell lines that are 2011; Tao et al., 2014). HC-04 is a spontaneously immortalized potentially important for P. falciparum sporozoite invasion. cell line that was isolated from normal human hepatocytes We further investigated GPC3 as a putative receptor mediating (Prachumsri and Yimamnuaychok, 2002). Recent analyses of this sporozoite invasion of hepatocytes using a robust platform for line suggest that, unlike other commonly used hepatocarcinoma P. falciparum sporozoite invasion of and early exoerythrocytic cell lines, like HepG2, HC-04 exhibits more plasticity and a form development in a hepatocarcinoma line. This platform greater propensity to recover its epithelial characteristics (Tao effectively overcomes the cost barrier and high variability of et al., 2014), opening the possibility to create a sporozoite primary human tissue and expands the utility of in vitro culture invasion system based on this line. Such a system would greatly for LS studies. The comparative multi-omics dataset identifies improve the ability to perform high-throughput drug screening other important host cell pathways that may also influence for LS compounds (malERA Refresh Consultative Panel on hepatocyte susceptibility to P. falciparum sporozoite invasion, Basic Science and Enabling Technology, 2017) and study the spurring hypothesis generation and testing by the greater biology of the LS in a homogeneous population of cells that scientific community. Abbreviations: ACN, acetonitrile; ALPK2, alpha kinase 2; BSA, bovine serum MATERIALS AND METHODS albumin; CLDN1, claudin; CM, culture media; CSP, circumsporozoite protein; DCBLD2, discoidin, CUB, and LCCL domain containing protein