Quantitative Proteomic Analysis of Simian Primary Hepatocytes Reveals Candidate Molecular Markers for Permissiveness to Relapsing Malaria Plasmodium cynomolgi

A major obstacle impeding malaria research is the lack of an in vitro system capable of supporting infection through the entire liver stage cycle of the parasite, including that of the dormant forms known as hypnozoites. Primary hepatocytes lose their liver specific functions in long‐term in vitro culture. The malaria parasite Plasmodium initiates infection in hepatocyte. This corresponds to the first step of clinically silent infection and development of malaria parasite Plasmodium in the liver. Thus, the liver stage is an ideal target for development of novel antimalarial interventions and vaccines. However, drug discovery against Plasmodium liver stage is severely hampered by the poor understanding of host–parasite interactions during the liver stage infection and development. In this study, tandem mass tag labeling based quantitative proteomic analysis is performed in simian primary hepatocytes cultured in three different systems of susceptibility to Plasmodium infection. The results display potential candidate molecular markers, including asialoglycoprotein receptor, apolipoproteins, squalene synthase, and scavenger receptor B1 (SR‐BI) that facilitate productive infection and full development in relapsing Plasmodium species. The identification of these candidate proteins required for constructive infection and development of hepatic malaria liver stages paves the way to explore them as therapeutic targets.


Introduction
Malaria was estimated to cause over 219 million infections resulting in approximately 435 000 deaths annually. [1] Global elimination of Plasmodium vivax and Plasmodium ovale is hampered due to several bottlenecks. Most notably, P. vivax and P. ovale produce a quiescent hepatic form of "hypnozoites" that is responsible for malaria relapses. [2] At present, there is no safe available treatment to tackle hypnozoite-induced relapse. The issue of relapse is compounded by the lack of an adequate continuous in vitro cultivation system for the hepatic stage of the parasite, resulting in the lack of basic understanding of the parasite's biology. Currently, primaquine and tafenoquine are the only licensed drugs available in the market that could curtail the hypnozoite reservoir. [3] However, both drugs have serious toxicity liabilities and are unsuitable for widespread use. This is a major hurdle to successful disease elimination. [4] As such, there is a growing www.advancedsciencenews.com www.proteomics-journal.com need to develop new anti-hypnozoitocidal compounds to tackle this reservoir of the parasite and prevent malaria relapses. In the last decade, a number of new compounds have entered the antimalarial pipeline, most of which originated from high throughput screens on the asexual blood stage of Plasmodium falciparum, a malaria parasite that does not form hypnozoites. [5] This is because the development of liver stage screens entails a number of biological and logistical obstacles. The primary hepatocytes rapidly loses their liver specific functions when grown under in vitro conditions on collagen-coated plates [6] and hepatic cell lines lack many cellular functions of their in vivo counter parts that enable P. vivax to complete the liver stage cycle. Furthermore, as hypnozoites do not multiply and infect new cell, the number of infected cells become diluted as non-infected cells continue to double. Physiological and functional features of primary hepatocytes can be retained to some extent when using matrigel, a protein extract from Engelbreth-Holm-Swarm mouse sarcoma cells. [7] Due to the difficulties in culturing hepatocytes in vitro, only few studies have been undertaken to investigate the liver stage biology of relapsing parasites [8][9][10] and host hepatocyteparasite interactions.
A number of host cell receptors associated with Plasmodium liver stage sporozoite infection of hepatocytes have been identified. The tetraspanin CD81 [11] has been shown to be essential in human P. falciparum and rodent P. yoelii sporozoite infection. Likewise, scavenger receptor B1 (SR-BI) enhances hepatocytes permissiveness to Plasmodium infection and is shown to be critical for Plasmodium liver stage development. [12,13] Interaction between heparan sulfate proteoglycans and Plasmodium circumsporozoite protein (CSP) triggers the host invasion signal in Plasmodium sporozoites and promote host cells entry. [14] Overexpression of host receptor liver fatty acid binding protein (L-FABP) has been shown to promote liver stage malaria parasite growth. [15] Recently, the ephrin type-A receptor 2 (EphA2) was shown to be associated with the onset of productive invasion and development by the malaria parasite. [16] However, none of these receptors have been reported to be associated with the infection and development of liver stages of Plasmodium relapsing species.
To better understand host-parasite interactions, we have set out to analyze the host proteome using a tandem mass tag (TMT)-based quantitative proteomic analysis of simian primary hepatocytes cultured under conditions permissive and inhibitory of Plasmodium liver infection and development. In the process, we identified the asialoglycoprotein receptor (AS-GPR), apolipoproteins, and scavenger receptor B1 (SR-BI) as key component for constructive infection of hepatocytes. Several of the up-regulated proteins observed in Plasmodium-permissive culture conditions include previously identified specialized receptors in binding and entry of other pathogens. Additionally, a number of proteins involved in diverse biosynthetic pathways including cholesterol biosynthesis were up-regulated in cells permissive to Plasmodium infection. Taken together, our data displayed that multiple proteins are required for a constructive Plasmodium parasite infection and development to establish. The definitive role of each individual protein for the constructive infection and development of malaria parasite remains to be investigated. However, identification of these important candidate proteins during malaria liver stage infection could help understand the interplay between the host hepatocytes and the

Significance Statement
Much of our efforts on malaria control and elimination are focused on Plasmodium falciparum, the human parasite associated with over 90% of malaria related death, and to a lesser extent, Plasmodium. vivax; which accounts for the highest level of morbidity. P. vivax, as well as Plasmodium ovale, are associated with the dormant form of the parasite known as hypnozoites.
In the past decade, we witnessed great advances in the development of new antimalarials resulting in several candidatecompounds presently in clinical trials. While many of these new drug candidates are promising, none is known to target the dormant form of the malaria parasite found in P. vivax and P. ovale. Hypnozoites are formed in hepatocytes upon infection and are responsible for relapses occurring weeks, months or years later. The objective of this study is to identify putative receptors and other proteins present in the host hepatocytes, which facilitate the invasion and development of the infecting malaria sporozoites. We report the identification of putative proteins and cell surface receptors involved in invasion and development of the parasite in hepatocytes. Future manipulation of the expression of these proteins may enhance hepatocytes infectivity in in vitro systems and improve assays for liver-stage drug screening.
malaria parasite and could potentially be useful for the discovery and development of novel vaccines and therapeutic candidates against relapsing malaria.

Ethics Statement
Simian hepatocytes were isolated from segments of liver taken from healthy Macaca fascicularis monkeys provided by the institutions, Novartis Laboratory of Large Animal Services, New Jersey, USA, (Novartis-LAS); and SingHealth, Singapore. Macaca fascicularis monkeys were used for sporozoites production in the Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand (AFRIMS). Prior to commencement of the study, Novartis Animal Welfare first evaluated and certified all institutions. The collection and use of materials were performed in accordance with the recommendations of the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) Standards and the Guide for the Care and Use of Laboratory Animals. Efforts were made to minimize discomfort of all animals. All experiments with animal were approved by the "Novartis Institutional Animal Care and Use Committees", prior to the study start. This project was reviewed for compliance with all established regulatory and accreditation standards at AFRIMS. AFRIMS' Institutional Animal care and Use Committee (IACUC) reviewed and approved that the project was in line with the humane animal care and use standards expressed in the Guide for the

Parasites
Plasmodium cynomolgi (B strain) sporozoites were obtained from infected Anopheles dirus. Salivary glands were collected 14-30 days after a blood meal from an infected monkey. Infected salivary glands were removed and sporozoites were recovered as previously reported. [4] All P. cynomolgi infected A. dirus mosquitoes were obtained from USAMD-AFRIMS, Bangkok 10400, Thailand.
The different hepatocyte culture conditions were infected with P. cynomolgi sporozoites ( Figure 1A) for parasite infectivity and development assessment. Uninfected cultures ( Figure 1B) were used to identify potential host proteins that aid in establishing P. cynomolgi infection. Culture condition FMD1 was infected with P. cynomolgi sporozoites at time D1 and ended at time D7, while SGD7 and OMD7 were infected with P. cynomolgi sporozoites at time D7 then ended at time D14 ( Figure 1A). Uninfected cultures were collected at time D1 for FMD1 and at time D7 for SGD7 and OMD7 for proteomic analysis ( Figure 1B).

Infection and Parasite Quantification
Three million (3 000 000) sporozoites per milliliter of P. cynomolgi were re-suspended in the above complete medium and used to infect hepatocyte cultures seeded in 96-well plates (adjusted to 60 000 sporozoites in 50 µL per well). The infected culture plates were centrifuged for 10 min at 2000 rpm, allowing fast parasite sedimentation, followed by incubation at 37°C with 5% CO 2 . At 3 h post infection, the medium was removed, hepatocytes were washed thrice, and fresh media was added back to the cultures before incubating at 37°C, 5% CO 2 . Matrigel (BD Biosciences) was added as per the manufacturer's recommendations. The medium was refreshed every 48 h. At defined time points shown in Figure 1A, the cultures were fixed with 100% cold methanol for 3 min before the pre-erythrocytic (hypnozoite: Hyp, schizont: Schz, and bursting schizont: Bur Schz) parasites were detected by immunofluorescence. Following fixation, the respective cultures were specifically stained with a rabbit anti-P. cynomolgi HSP70 (Genscript 299349-3, 1:5000) and a mouse anti-P. cynomolgi UIS4 polyclonal antibodies (1:200) for 1 h incubation at 37°C and washed thrice with PBS before addition of (red) Alexa 594-conjugated goat anti-mouse immunoglobulin for UIS4 (ref 11032 Invitrogen) and (green) Alexa 488-conjugated goat anti-rabbit immunoglobulin for HSP70 (ref 11034 Invitrogen). Parasites and cell nuclei were stained with 1 µg/mL of DAPI (Sigma). Parasites were enumerated under a fluorescence microscope with a 40× magnification and images were obtained from NIKON ECLIPSE TS100 microscope. GraphPad Prism software and ANOVA Statistical test were used to compare the infectivity and parasite development of the different cell culture conditions. The populations compared were derived from three independent biological replicates. A p-value of 0.05 or less was considered to be statistically significant.

Sample Collection and Protein Extraction
Samples from SGD7, FMD1, and OMD7 cultures were collected at the defined time points shown in Figure 1B. The culture medium was removed and the cells were washed thrice with PBS. The cells were lysed in 100 µL lysis buffer (2% SDS, 100 mm Tris-HCl [pH 7.4], 1 µm dithiothreitol, and protease inhibitor cocktail) by sonication at 40% amplitude for 1 min. Insoluble cell debris were removed by centrifugation at 20 000 × g for 10 min at 4°C. The protein samples were purified by acetone precipitation. The purified protein pellets were reconstituted in 250 µL urea buffer (8 m urea and protease inhibitor cocktail in 100 mm ammonium Overview of study procedures. A) Cultures infected with P. cynomolgi sporozoites and used for evaluation of parasite infectivity and development in FMD1, OMD7, and SGD7 conditions. B) Uninfected cultures used for proteomic study to assess host cell candidate markers required for parasite infectivity and development in FMD1 and SGD7 when compared to OMD7. FMD1 consists of fresh monolayer day 1 cultures; OMD7 is old monolayer day 7 cultures; and SGD7 is sandwich gel overlaid cultures day 7.
bicarbonate buffer [ABB], pH 8). Protein amount of each sample was measured by BCA-based protein quantitation method.

In-Solution Protein Digestion and TMT Labeling
One-hundred microgram equivalent amount of protein from each sample was taken and used for subsequent proteomic experiment. The urea concentration of the samples was reduced to <1 m by diluting with 100 mM ABB and reduced with 10 mM tris 2-carboxyethyl phosphine hydrochloride (TCEP) at 37°C for 2 h. Reduced samples were then alkylated with 20 mM iodoacetamide at room temperature for 45 min in dark. Protein samples were subsequently digested with trypsin at a trypsin-protein ratio of 1:40 overnight at 37°C. The reaction was quenched by adding 10% TFA (final concentration of 0.4%). Tryptic peptides were desalted by using Sep-Pak C18 cartridges (Waters, Mil-ford, MA) and dried in a SpeedVac (Eppendrop, USA). TMT labeling was performed using TMT 10-plex kits according to the manufacturer's protocol (Thermo scientific, USA). The samples were labeled accordingly with the following tags as listed in the Table 1. The TMT-labeled peptides were pooled together and desalted.
TMT-labeled peptides were fractionated by reverse phase chromatography at high pH (approx. pH 8). Briefly, labeled peptides were reconstituted in buffer A (0.02% NH 4 OH in water) and fractionated using X-Bridge C18 column (4.6 × 200 mm, 5 µm particle size, 130 Å pore size) (Waters, Milford, MA) on an HPLC unit (Prominence, Shimadzu, Kyoto, Japan) at a flow rate of 1 mL/min. The HPLC gradient comprised 100-95% buffer A for 3 min, 5-35% buffer B (80% ACN and 0.02% NH 4 OH) for 40 min, then 35-70% buffer B for 12 min, followed by 70% buffer B for 5 min. The chromatograms were recorded at 280 nm. A total of 60 fractions were collected within 65 min time period. The collected fractions were combined into 15 fractions by using concatenated pooling strategy and concentrated to dryness using vacuum centrifugation.

Liquid Chromatograph Coupled to Tandem Mass Spectrometry and Data Analysis
The liquid chromatograph coupled to tandem mass spectrometry (LC-MS/MS) analysis was performed using a Q Exactive mass spectrometer coupled with online nano-HPLC system (Thermo Scientific, USA). Data acquisition was performed using Xcalibur 2.2 software (Thermo Scientific, USA). Protein identification and quantification were performed using Proteome Discoverer 1.4.1.14 (Thermo Fisher, MA) together with the Sequest search engine (parameters included cysteine alkylation with MMTS as static modification, methionine oxidation and asparagine and glutamine deamidation as dynamic modifications, full trypsin digestion and maximum two missed and/or non-specific cleavages as digestion parameter, 10 ppm precursor mass, and 0.02 Da fragment mass tolerance). The NCBI proteins of Macaca fascicularis proteins (downloaded on December 2016, including 73 374 sequences and 58 200 443 residues) was used as a search database with a cut-off false discovery rate (FDR) of <0.05% and <0.01% at the peptide and protein level accordingly. The resulting data set was automatically bias-corrected to eliminate potential variation due to unequal mixing when combining samples. All LC-MS/MS analysis was performed in triplicate. All statistical calculations were performed using the biological triplet. Cluster analysis of the identified proteins was performed using the online bioinformatics tool Gene Pattern (http://genepattern.broadinstitute.org) followed by hierarchical clustering and Pearson correlation.

Statistical Analysis
The quantitative proteomic results were first examined for quality using a Pearson correlation that showed a satisfactory linear correlation among all three biological replicates (n = 3) (Figure 3A,B). The proteomic data distribution patterns and cut-off levels of statistical significance were determined using Student's t-test and volcano plots (Figure 4A-C). Distribu-tion of the Log2 TMT ratio was used to determine the cut-off for the differentially regulated hits ( Figure 4A). The OMD7 samples were used as denominator for the relative quantification conditions given that they had the least infections. Proteins with ratios <0.76 (Log2Ratio less than −0.4) were classified as down-regulated hits, whereas proteins with ratios >1.32 (Log2Ratio >0.4) were considered as up-regulated hits ( Figure 4A).

Availability of Data and Material
The mass spectrometry proteomics raw data files along with Proteome Discoverer search data file (including protein summary) have been deposited to the ProteomeXchange Consortium (http: //proteomecentral.proteomexchange.org) via the PRIDE partner repository. [33] ProteomeXchange provides globally coordinated proteomics data submission and dissemination with the dataset identifier PXD013310. The protein and peptide information from Proteome Discoverer search are available in datasets S1 and S2, Supporting Information.

P. cynomolgi Liver Stage Productive Infection and Full Development Depend On Host-Cell Culture Conditions In Vitro
We assessed the hepatocyte permissiveness to P. cynomolgi sporozoites infection through proteomic analysis of cells cultured under different conditions of permissiveness ( Figure 1). These conditions included FMD1, OMD7, and SGD7 described in Section 2 and shown in Figure 1. Development of hepatic stages were analyzed using antibodies raised against Plasmodium proteins HSP70 (heat shock protein 70) and UIS4 (up-regulated in infectious sporozoite-4, a parasitophorous vacuole membrane [PVM] marker). [17] At 7 days post infection, P. cynomolgi (Pc) infectivity, measured with anti-PcHSP70 antibody (green), was similar in both FMD1 and SGD7 cultures (Figure 2A,B). In contrast, infection of the OMD7cultured hepatocytes was significantly lowered as compared to that in FMD1 (p = 0.0383) and SGD7 (p = 0.0006) cultures (Figure 2A,B). The diameters of the developing parasites (schizonts) were noticeably larger in FMD1 (p = 0.0024) and SGD7 (p = 0.0002) cultures as compared to that of OMD7 ( Figure 2C). All the parasite stages, including hypnozoites and schizonts were observed in all three conditions except for the presence of mature bursting schizonts in OMD7 ( Figure 2D). The rupture of the mature bursting schizonts and release of the pre-erythrocytic merozoites could be clearly discerned using the anti-PcUIS4 antibody (red) displaying UIS4 rupture in SGD7 and FMD1 cultures ( Figure 2D). Our results suggest that significant productive sporozoite infection of the hepatocytes were only possible in the fresh (FMD1) and matrigel overlaid (SGD7) hepatocyte culture conditions (Figure 2A,B). Having demonstrated that the culture conditions of the host cells significantly pre-determine their permissiveness to Plasmodium sporozoite infection and parasite development, we set out to identify the required host molecular markers involved.

Quantitative Proteomic Analysis of Simian Primary Hepatocytes Revealed Candidate Molecular Markers for Permissiveness to Plasmodium cynomolgi Infection
Protein samples of the three cultures conditions from three monkey donors ( Figure 1B) were analyzed simultaneously using TMT quantitative proteomic analysis by liquid chromatograph coupled to LC-MS/MS. The quantitative proteomic results were first examined for quality using a Pearson correlation that showed a satisfactory linear correlation among all the three biological replicates ( Figure 3A,B). The combined three biological replicates' proteomic data distribution patterns and cut-off levels of statistical significance were determined using Student's t-test and volcano plots ( Figure 4A-C). A total of 5763 proteins were identified with a FDR <1%, including 4588 proteins that incorporated more than one peptide. All the proteins and peptides identified are listed in datasets S1 and S2, Supporting Information. Out of the 5763 proteins identified, 1627 were found to be non-annotated proteins. In FMD1 or OMD7 condition, a total of 5163 proteins follow a similar trend with less than 20% deviation among the three biological replicates. Under SGD7 conditions, 5012 proteins were identified. Further analysis focused on 2187 proteins that were significantly enriched (p-value <0.05) ( Figure 4A-C and S1, Supporting Information). Distribution of the Log2 TMT ratio was used to determine the cut-off for the differentially regulated hits ( Figure 4A). The OMD7 samples were used as denominator for the relative quantification conditions, given that they had the least infections and would not highly express required proteins for successful infection. Proteins with ratios <0.76 (Log2Ratio less than −0.4) were classified as down-regulated hits, whereas proteins with ratios >1.32 (Log2Ratio >0.4) were considered as up-regulated hits ( Figure 4A). These criteria resulted in a total of 477 down-regulated and 574 up-regulated hits in FMD1 condition, while 89 down-regulated and 50 up-regulated in the SGD7 culture as compared to OMD7 ( Figure 4A). Of the hits identified in FMD1 and SGD7, there was an overlap Figure 4. A) Frequency distribution plot of Log2TMT ratio. B,C) Volcano plot highlighting significantly differentially regulated proteins for SGD7 and FMD1 samples. Every data point in the volcano plot serves as fold change difference between FMD1 and OMD7 (B) and between SGD7 and OMD7 (C) plotted against their respective p-values. The x-axis represents the log2 fold change, whereas the y-axis is the −log10 p-values. D) Venn diagram showing the proteins that were differentially expressed in FMD1 and SGD7 samples; up-regulated hits (red) and down-regulated hits (blue). There are 117 proteins that are commonly regulated in both FMD1 and SGD7 samples. This is from combined three biological replicates' proteomic data. of 28 up-regulated and 89 down-regulated proteins, respectively ( Figure 4D).
We focused on the 28 proteins that were commonly upregulated in FMD1 and SGD7 samples (Figure 5; Tables 2-4). This cluster consists of proteins involved in biosynthesis of isoprenoid, cholesterol and fatty acid; signal transduction; glycoprotein surface receptors and proteins involved in the general metabolism of hepatocytes ( Figure 5; Tables 2-4). Notably, from the up-regulated protein dataset, we identified the AS-GPR in both FMD1 and SGD7, which is known to interact with other human pathogens during infection. These pathogens included Mycobacterium tuberculosis, Helicobacter pylori, Candida albicans, Leishmania spp., hepatitis C, and HIV-1. [18] In addition, apolipoproteins were identified in this screen that have previously been reported to interact with the malaria parasite P. falciparum PFE1590w/ETRAMP [19] and P. berghei parasitophorous vacuole membrane exported protein 1 (EXP-1) during liver stage development. [20] Squalene synthase, an essential host hepatocyte enzyme required for sterol production [21] was also significantly up-regulated in both FMD1 and SGD7. Previously, it has been shown that squalestatin, a squalene synthase inhibitor kills the blood stage Plasmodium parasites. [22] In future studies, it would be interesting to test its inhibitory effect on the dormant liver stage, hypnozoite stage.
Given that a single protein may not be required for the infection but rather a collection of proteins involved in a specific pathway(s) or in a mechanism to facilitate infection and development, we sought to identify key pathways that allow hepatocytes to be permissive to infection. A pathway enrichment analysis was performed on FMD1 and SGD7 (Tables 2 and 3) using GO and KEGG pathways. Proteins in several pathways were uniquely up-regulated in FMD1 samples but not in SGD7. These include hepatocyte developmental and metabolism-related pathway, mitochondrial-related pathways, peroxisome, and endoplasmic reticulum ( Table 2). In contrast, there were only few pathways in which proteins were up-regulated in SGD7 samples. www.advancedsciencenews.com www.proteomics-journal.com Figure 5. Hierarchical clustering of up-and down-regulated genes in FMD1 and SGD7 as compared to OMD7 for three biological replicates is shown. Red ones are up-regulated genes, white are non-affected genes, and green are down-regulated genes. Nine hundred and five up-regulated genes of FMD1 samples were analyzed and significantly enriched pathways were highlighted using GO and KEGG database. Selected pathways related to liver stage development and metabolism are shown here. Number of genes and the receptive p-value for each pathway in our dataset is also included (p-value < 0.05). *Only pathway up-regulated in both FMD1 and SGD7 culture conditions. These comprised pathways related to extracellular spaces of hepatocytes. The only pathway in which proteins were commonly upregulated in both FMD1 and SGD7 was the extracellular exosome secretion pathway. Noteworthy is the observation that most of the documented host surface receptors required for P. falciparum sporozoite infection were not significantly altered in either FMD1 or SGD7 samples (Table 4) when compared to OMD7. Here, we only identified scavenger receptor class B member 1 (SR-B1) to be significantly up-regulated in simian FMD1 samples, while the liver fatty acid binding protein L-FABP was significantly downregulated in both FMD1 and SGD7 samples. Further studies are required to test the role of SR-BI in sporozoite infection for relapsing malaria species.

Discussion
Malarial parasites' initial site of infection and development in the vertebrate host is the liver. However, very little is known about the host molecular markers and pathways required for parasite permissiveness and development. Previously, four host receptors SR-B1, [12] EphA2, [16] CD81, [23] and alpha-V containing integrin [24] were shown to be required for P. falciparum sporozoite infection to hepatocytes. However, none of these receptors are solely essential for P. falciparum sporozoite infection and development, suggesting that they interact in a mechanism that remains to be established. While host receptors required for sporozoite infection and development of the liver stage cycle have been comprehensively characterized in P. falciparum and rodent malaria species, no receptors have been identified for the relapsing form of the parasites found in P. vivax and P. ovale. The current study aims to identify candidate proteins potentially required in relapsing parasitic infection and development. We report that the above known receptors for malaria entry in liver stages did not show significant changes in their expression level in all culture conditions. The exception was SR-BI, which was significantly up-regulated in only FMD1 (p-value = 0.009) as compared to OMD7. Interestingly, SR-BI expression level in FMD1 correlated with sporozoites infection rate and development in our dataset suggesting SR-BI role in P. cynomolgi sporozoite infection and full development. SR-BI is a glycosylated membrane protein present in several cell types, including primary hepatocytes and was not up-regulated in SGD7 hepatocytes. Like FMD1, significant infection rate and parasitic full development was also found in SGD7 samples. In agreement with other observations, [12,13] this result suggests that SR-BI may play a facilitator role in the bidirectional movement of cholesterol, which is required for malaria parasite infection and development. [11] The ASGPR receptor has been identified as a common host factor involved in the entry of several other infections of bacterial, parasitic, and viral origin. [25] Here, we identify ASGPR up-regulation in conditions conducive to permissive infection (FMD1 and SGD7) by P. cynomolgi sporozoite infection. ASGPR is a heterodimeric Ctype lectin receptor expressed on the surface of hepatocytes. [25] ASGPR and SR-BI receptors have been reported together to bind lipoproteins. [26,27] Other studies have shown apolipoprotein as an important ligand for the high-density lipoprotein and SR-BI. [28] Apolipoprotein also interacts with malaria parasite. [19,20] Like AS-GPR, apolipoprotein expression level was markedly high only in both FMD1 and SGD7 but not OMD7, suggesting a potential interaction with malaria-relapsing species. ASGPR, apolipoprotein, and SR-BI could be modulators of entry and development of pathogens such as malaria parasite and hepatitis C virus. We also identified obscurin-like protein and squalene synthase that were significantly up-regulated in both FMD1 and SGD7 samples. The squalene synthase is essential for cholesterol biosynthesis, [22] which is also required for malaria parasite and hepatitis C virus infection and development. [11,22] It has been reported that there is interaction between mosquito titin, an ortholog of human obscurin and dengue NS3 virus. [29] Collectively, our data suggest contribution of a few proteins in a pathway or mechanism required for successful malaria parasite infection and development. A significant release of extracellular exosomes was found in both FMD1 and SGD7 as compared to OMD7. Exosomes are invagination of endosomal membranes forming vesicles that display cell surface lipids and proteins on their exterior face. [30] Released extracellular exosomes are shown to interact with target pathogens or cells through three major mechanisms: receptor-mediated binding, membrane fusion, and nonspecific entry through the endocytic pathway. [30] Production of these vesicles during infection has been correlated with malarial severity in humans and animals. [31] Earlier studies have shown the host exosomes to interact with other pathogens. [30,32] The overall result of this study suggests that the proteins commonly up-regulated in FMD1 and SGD7 hepatic cells that respectively displayed significant parasite count (p = 0.0383 and p = 0.0006) and development (p = 0.0024 and p = 0.0002) when compared to OMD7 are potentially required for productive P. cynomolgi sporozoite infection and development (Figures 2 and 4D). Identifying conditions for human hepatocyte cultures that enable constructive malarial infection brings the field one step closer to establishing an in vitro system that will permit further studies in hypnozoite biology and drug development. However, more investigations are required to determine the potential role of these proteins individually or collectively.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.