Parasite‐induced ER stress response in hepatocytes facilitates Plasmodium liver stage infection
2015; Springer Nature; Volume: 16; Issue: 8 Linguagem: Inglês
10.15252/embr.201439979
ISSN1469-3178
AutoresPatricia Inácio, Vanessa Zuzarte‐Luís, Margarida TG Ruivo, Brie Falkard, Nagarjuna Nagaraj, Koos Rooijers, Matthias Mann, Gunnar R. Mair, David A. Fidock, Maria M. Mota,
Tópico(s)Hepatitis Viruses Studies and Epidemiology
ResumoScientific Report25 June 2015free access Parasite-induced ER stress response in hepatocytes facilitates Plasmodium liver stage infection Patricia Inácio Patricia Inácio Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Vanessa Zuzarte-Luís Vanessa Zuzarte-Luís Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Margarida TG Ruivo Margarida TG Ruivo Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Brie Falkard Brie Falkard Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Nagarjuna Nagaraj Nagarjuna Nagaraj Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Martinsried, Germany Search for more papers by this author Koos Rooijers Koos Rooijers Division of Gene Regulation, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Matthias Mann Matthias Mann Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Martinsried, Germany Search for more papers by this author Gunnar Mair Gunnar Mair Department of Parasitology, University of Heidelberg, Heidelberg, Germany Search for more papers by this author David A Fidock David A Fidock Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY, USA Division of Infectious Diseases, Department of Medicine, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Maria M Mota Corresponding Author Maria M Mota Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Patricia Inácio Patricia Inácio Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Vanessa Zuzarte-Luís Vanessa Zuzarte-Luís Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Margarida TG Ruivo Margarida TG Ruivo Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Brie Falkard Brie Falkard Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Nagarjuna Nagaraj Nagarjuna Nagaraj Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Martinsried, Germany Search for more papers by this author Koos Rooijers Koos Rooijers Division of Gene Regulation, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Matthias Mann Matthias Mann Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Martinsried, Germany Search for more papers by this author Gunnar Mair Gunnar Mair Department of Parasitology, University of Heidelberg, Heidelberg, Germany Search for more papers by this author David A Fidock David A Fidock Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY, USA Division of Infectious Diseases, Department of Medicine, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Maria M Mota Corresponding Author Maria M Mota Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Author Information Patricia Inácio1, Vanessa Zuzarte-Luís1, Margarida TG Ruivo1, Brie Falkard2, Nagarjuna Nagaraj3, Koos Rooijers4, Matthias Mann3, Gunnar Mair5, David A Fidock2,6 and Maria M Mota 1 1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal 2Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY, USA 3Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Martinsried, Germany 4Division of Gene Regulation, The Netherlands Cancer Institute, Amsterdam, The Netherlands 5Department of Parasitology, University of Heidelberg, Heidelberg, Germany 6Division of Infectious Diseases, Department of Medicine, Columbia University Medical Center, New York, NY, USA *Corresponding author. Tel: +351 217 999 509; E-mail: [email protected] EMBO Reports (2015)16:955-964https://doi.org/10.15252/embr.201439979 See also: A Kaushansky & SHI Kappe (August 2015) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Upon infection of a mammalian host, Plasmodium parasites first replicate inside hepatocytes, generating thousands of new parasites. Although Plasmodium intra-hepatic development represents a substantial metabolic challenge to the host hepatocyte, how infected cells respond to and integrate this stress remains poorly understood. Here, we present proteomic and transcriptomic analyses, revealing that the endoplasmic reticulum (ER)-resident unfolded protein response (UPR) is activated in host hepatocytes upon Plasmodium berghei infection. The expression of XBP1s—the active form of the UPR mediator XBP1—and the liver-specific UPR mediator CREBH is induced by P. berghei infection in vivo. Furthermore, this UPR induction increases parasite liver burden. Altogether, our data suggest that ER stress is a central feature of P. berghei intra-hepatic development, contributing to the success of infection. Synopsis This study is the first to describe the activation of the unfolded protein response (UPR) in Plasmodium-infected hepatocytes. UPR induction increases parasite liver burden in vivo, in an example of modulation of host pathways to favour infection. Plasmodium berghei infection induces ER stress and UPR activation in hepatocytes. In vivo UPR activation by tunicamycin strongly increases P. berghei exoerythrocytic form (EEF) numbers. Liver-specific Xbp1 deletion impairs P. berghei liver infection. CREBH knockdown in hepatocytes significantly decreases P. berghei liver infection. Introduction Plasmodium spp., the causative agents of malaria, are obligate intracellular parasites with a complex life cycle involving both a mosquito and a mammalian host. In mammals, Plasmodium life cycle is initiated when a motile sporozoite is injected with the bite of an infected Anopheles mosquito. Sporozoites travel, through the bloodstream, to the liver and infect hepatocytes, where each sporozoite develops and multiplies into thousands of merozoites 1. Once released into the bloodstream, each merozoite infects a red blood cell (RBC) where a new replication cycle occurs, culminating in the production of new merozoites. The continuous cycle of invasion, intracellular development and proliferation and release of merozoites from RBCs is central to malaria-associated pathology 2. Plasmodium, as an obligate intracellular parasite, depends on host cell resources to support its development. This feature is particularly important during the liver stage as the Plasmodium replication rate in the liver is exceedingly higher than that in the blood. Hepatocytes are responsible for a myriad of metabolic processes, including protein synthesis and metabolism of lipids, carbohydrates and other nutrients and micronutrients (such as iron) 3. The key players of these pathways exist, entirely or partially, in the lumen or membrane domains of the endoplasmic reticulum (ER). As a consequence, hepatocytes are unusually rich in both smooth and rough ER. In hepatocytes, the ability of the ER to respond to metabolic demands is crucial for cell homeostasis. The unfolded protein response (UPR) of the ER is an elaborate stress signalling pathway activated upon conditions that challenge ER function, in particular the accumulation of unfolded proteins in the lumen owing to an increased demand on its synthesis capacity that the cell cannot cope with, termed ER stress 4. In eukaryotic cells, three ER transmembrane proteins mediate the canonical UPR: the two kinases, IRE1 (inositol requiring enzyme 1) and PERK (PKR-like eukaryotic initiation factor 2α kinase), and the transcription factor precursor ATF6 (activating transcription factor 6). IRE1, the most conserved signalling branch of the UPR response, functions by activating the transcription factor XBP1 (X-box binding protein 1). XBP1 mRNA is activated by an IRE1-dependent unconventional splicing and generates a mature (spliced) XBP1 transcription factor with a potent transactivation domain 5. Together, they activate signalling pathways that restore the folding capacity of the ER. Several recent studies have revealed that ER stress and UPR activation regulatory actions are broad-acting and intersect at certain metabolic pathways including lipid and glucose metabolism, particularly in the liver 6. The metabolic role of the UPR in the liver is further enhanced by the existence of a hepatocyte-specific UPR branch, mediated by the ER transcription factor CREBH (cAMP responsive element-binding protein, hepatocyte specific). This UPR branch does not activate protein folding transcriptional programmes but rather regulates liver metabolic pathways 789. As such, ER stress and UPR are activated in several metabolic syndromes, including obesity and type II diabetes, as well as in specific liver diseases including fatty liver disease and viral hepatitis 10. Plasmodium infection leads to alterations in the hepatocyte transcriptome, in particular by activating metabolic processes 11. Plasmodium development thus represents a metabolic challenge to the host cell. How hepatocytes cope with a developing parasite and how intracellular host signalling events shape the infection outcome remain unclear. Here, we hypothesized that Plasmodium development inside hepatocytes impacts ER function and modulates UPR signalling pathways. We now show that Plasmodium infection induces ER stress and that activation of the UPR strongly increases Plasmodium liver infection through both the XBP1 and CREBH pathways. Results and Discussion Plasmodium hepatocyte infection induces ER stress and UPR activation Our recent transcriptomic analysis of P. berghei sporozoite-infected mouse Hepa 1.6 cells at different time points 11 identified transcriptional upregulation of several key ER stress markers, as early as 6 h after infection. These include Atf4 and Chop, Atf6 and Atf3, Cebpb, Herpud1 and Trib3 (Fig 1A), all with a well-established role in ER stress 12. These results were independently validated by analysing the expression of 4 of those transcripts (Atf4, Chop, Atf3 and Herpud1) by quantitative real-time PCR (qRT–PCR) of purified Hepa 1.6 GFP-expressing P. berghei ANKA-infected cells infected with GFP-expressing P. berghei ANKA sporozoites 13 at 6 and 24 h after infection (Fig 1B). Uninfected cells (Hepa 1.6 GFP-negative cells), from both time points, were used to control for basal mRNA expression. Additionally, comparative quantitative proteomic analysis of infected and uninfected cells (V. Zuzarte-Luís and M. Mota, unpublished data) identified 59 ER proteins that were differentially expressed (DE) within 12 h of Huh7 human hepatoma cells being infected with GFP-expressing P. berghei ANKA sporozoites (Fig 1C). Among these, gene ontology (GO) analysis identified an enrichment of proteins involved in protein folding processes, as demonstrated by the induced expression of calnexin (Canx) and calreticulin (Calr). These factors play important roles in the ER quality control apparatus through retention of incorrectly folded proteins 1415: the ER resident protein 29 (Erp29) 16 and ERp44 (Erp44) 17, both important to correct protein folding, together with ERp19 (Txndc12) and ERp46 (Txndc5), members of the thioredoxin family of ER proteins that are highly expressed in the liver 18, and protein-modifying enzymes, including dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunits 1 and 2 (Rpn1/Rpn2) and mannosyl-oligosaccharide glucosidase (Mogs) 19 (Fig 1C). Proteins involved in the ER-associated protein degradation (ERAD) pathway were also overrepresented. These include endoplasmin (Hsp90b1), both components of the ERLIN1/ERLIN2 complex, and the UBX domain-containing protein 4 (Ubxn4, also known as Ubxd2) 20 (Fig 1C). The upregulation of the protein folding capacity together with ERAD activation is consistent with the induction of the unfolded protein response (UPR), in response to ER stress. In fact, ERp44 and UBX domain-containing protein 4 are induced, at the protein level, with ER stress 1721. Moreover, the major ER stress marker, the 78 kDa glucose-regulated protein also known as BiP (Hspa5) and protein disulphide-isomerase (PDI, P4hb) 22, were clearly upregulated with infection (Fig 1C). Notably, transcriptomic data from hepatocytes infected with a different rodent model, P. yoelii, show a similar alteration in ER response 11. Altogether, our data analysis on both the transcriptional and ER protein responses to infection suggests that Plasmodium liver stage parasites induce a clear signature of UPR activation in response to ER stress. Figure 1. Plasmodium sporozoite infection induces ER stress and activates the UPR in hepatocytes Heatmap of DE transcripts involved in ER stress/UPR pathways in parasitized hepatocytes at 6, 12, 18 and 24 h after infection. Each row of the plot is a gene and was colour-coded according to the log base 2 of the expression fold changes for each transcript, with red meaning upregulation and blue meaning downregulation. Original data from Albuquerque et al 11. Quantitative real-time PCR (qRT–PCR) analysis of Atf4, Chop, Atf3 and Herpud1 mRNA in sorted Hepa 1.6 cells infected with P. berghei at 6 and 24 h after infection relative to its GFP-negative control (dashed line), normalized to hypoxanthine-guanine phosphoribosyltransferase (Hprt). *P < 0.05, one-sample t-test. Results are expressed as means ± SEM (n = 3 independent experiments). Heatmap of DE proteins identified as ER proteins by gene ontology (GO) (GO_0044432). Each row of the plot is a protein and was colour-coded according to row-normalized log intensity (z-score) with red meaning upregulation and blue meaning downregulation. Each row is identified with gene name and UniProt accession number. Each column represents a replicate. Highlighted in blue are all the proteins mentioned in the text. Data are available via ProteomeXchange with identifier PXD002269. Download figure Download PowerPoint In vivo UPR activation strongly increases exoerythrocytic forms (EEFs) in the liver We next sought to determine how UPR activation impacts Plasmodium hepatocyte infection. To that end, we have used a well-described pharmacological ER stress inducer, tunicamycin (TM) 712. We performed a dose–response analysis of TM impact on P. berghei ANKA liver stage infection 13. Mice were intraperitoneally treated with different TM concentrations (or DMSO as control) 8 h prior to intravenous (i.v.) injection of 50,000 P. berghei ANKA sporozoites. Liver infection was quantified by qRT–PCR and ER stress induction was confirmed by XBP1 splicing assay. While all tested concentrations induced ER stress response within 8 h after intraperitoneal injection (data not shown), the results show that a sustained ER stress response throughout infection was only observed using 0.75 and 1 mg/kg body weight (bw) of TM. Notably, a clear and significant increase in infection was observed for mice treated with these two concentrations of TM (Fig 2A). To exclude any toxic effect of TM concentrations on mouse livers, we performed histopathological analysis on liver sections of TM-treated and DMSO control mice. No histomorphological differences between control and TM-treated mice were found (Appendix Fig S1A). Additionally, none of these treatments had an impact on liver weights (Appendix Fig S1B). As such, 1 mg/kg bw was chosen for the subsequent experiments. Confirmation of TM impact on infection was obtained by initiating infection either through i.v. injection of P. berghei ANKA sporozoites or mosquito bite (the natural route of infection) and analysing parasite liver load by both in vivo light emission quantification of luciferase-expressing P. berghei liver stage parasites 23 and qRT–PCR (Fig 2B and C). To exclude a direct effect of TM on parasites, P. berghei ANKA sporozoites were incubated with TM (10 μg/ml) prior to in vitro infection. No differences in sporozoites' infectivity were found (Appendix Fig S1C). Figure 2. UPR activation increases EEF numbers in the liver Upper panel: P. berghei liver load quantification on mice treated with DMSO (control) or different tunicamycin (TM) doses 8 h prior to sporozoite injection. qRT–PCR of Plasmodium berghei 18S ribosomal RNA at 42 h after sporozoite infection normalized to hypoxanthine-guanine phosphoribosyltransferase (Hprt) expression. ***P < 0.001 and **P < 0.01, t-test. Results are expressed as means ± SD (n = 5 mice per group, two independent experiments) and TM expressed as fold change compared to DMSO. Lower panel: PCR analysis of XBP1 maturation in infected mouse livers previously treated with DMSO and TM. XBP1 unspliced (U) and spliced (S) mRNA species were resolved by high-density agarose gel (3%) (image representative of two independent experiments with n = 5 mice per group). Plasmodium berghei liver load quantification on mice treated with DMSO or TM 8 h prior to sporozoite delivery by either i.v. injection or mosquito bite. qRT–PCR of parasite 18S ribosomal RNA at 42 h after sporozoite infection normalized to Hprt expression. ****P < 0.0001 and **P < 0.01, t-test. Results are expressed as means ± SD (n = 5 mice per group, six independent experiments, for i.v. injection; n = 5 mice per group, three independent experiments for mosquito bite) and TM expressed as fold change compared to DMSO. Luciferase-expressing P. berghei infection load on DMSO and TM-treated mice at 42 h after infection (image representative of 3 independent experiments). Fluorescent microscopy quantification of EEF density (number) and size (area) on DMSO and TM liver sections at 42 h after P. berghei sporozoite injection. **P < 0.01, t-test. Results are expressed as means ± SD (n = 4 mice per group, three independent experiments). Representative fluorescence images of liver sections of DMSO- and TM-treated mice, 42 h after infection with GFP-expressing P. berghei sporozoites; arrows indicate parasite EEFs, left scale bars 50 μm; parasite in green, DNA stained with Hoechst (blue); and F-actin with phalloidin Alexa 555 (white), right scale bars 11 μm. Plasmodium berghei liver load quantification at 14, 24 and 42 h after i.v. sporozoite injection on mice treated with DMSO and TM for 8 h. Infection measured by qRT–PCR of parasite 18S ribosomal RNA normalized to Hprt. **P < 0.01, t-test. Results are expressed as means ± SD (n = 4 mice per group, two independent experiments). Download figure Download PowerPoint The observed increase in parasite load can result from either a higher number of parasites that manage to reach and establish a successful liver infection or an increase in parasite replication while the number of infected cells remains constant. To determine the cause of this increase, we isolated liver sections from TM- and DMSO-treated mice and quantified GFP-positive parasite numbers and growth by fluorescence microscopy. Microscopic examination revealed that the increase in infection is due to higher numbers of exoerythrocytic forms (EEFs) as opposed to increased EEF size (Fig 2D and E). Hence, our data suggest that UPR activation potentiates infection by increasing Plasmodium-infected hepatocyte numbers. Importantly, this effect on parasite numbers could not be ascribed to a defect in initial invasion of hepatocytes, as there was no difference in liver infection at 14 h after infection between TM- and DMSO-treated mice (Fig 2F). The increase in infection in TM-treated mice only became apparent 24 h after sporozoite delivery (Fig 2F). Both XBP1 and CREBH pathways contribute to Plasmodium infection Our results so far showed that infection led to an UPR activation in the liver and that exogenous induction of ER stress led to an increase in the number of productively infected cells. We thus hypothesized that UPR activation during infection may therefore play a role in the maintenance of a successful infection. Owing to the complexity of UPR signalling, the study of its individual pathways is crucial to understand how liver UPR pathways and Plasmodium EEFs crosstalk to determine in vivo liver infection. In the liver, the XBP1 242526 and CREBH 789 pathways have been shown to regulate aspects of hepatic metabolism. We first sought to determine whether Plasmodium infection activated splicing of Xbp1 mRNA. We detected maturation of Xbp1 to its active form, Xbp1s (Fig 3A and B), together with an increased expression of its target gene Dnajb9, in P. berghei-infected cells (Fig 3A). We next investigated the relevance of our findings to Plasmodium liver infection. We infected mice with an inducible, conditional disruption of the Xbp1 gene in the liver (Xbp1∆), as previously described 24 (Fig 3C). We observed a significant decrease in parasite liver load in Xbp1∆ mice compared to their wild-type (WT) littermates (Fig 3D). In agreement with our finding of UPR activation by TM, we observed that the effect in P. berghei liver load is due to an alteration in numbers of EEFs rather than in their development. Our results show that Xbp1∆ livers exhibit a reduction in the number of infected cells (Fig 3E), suggesting a direct effect of XBP1 regulated pathways on P. berghei liver stage infection. During this stage, a bulk of lipids is required to support the generation of thousands of merozoites. Plasmodium possesses a type II fatty acid biosynthetic pathway 27; however, several studies have shown that in the liver the parasite relies on its own synthesis pathway (in addition to scavenging host fatty acids) only at the transitional stage from the liver to the blood 282930. Therefore, during the majority of the liver stage, we hypothesize that the host must fulfil the bulk of the parasite's lipid needs. The ER has a central role in the regulation of lipid metabolism, and in the liver, XBP1s was shown to regulate the expression of genes involved in fatty acid synthesis 24. Thus, it is tempting to hypothesize a functional link between XBP1-mediated regulation of de novo hepatic fatty acid synthesis and infection. On the other hand, XBP1s overexpression was shown to induce the synthesis of phospholipids, mainly phosphatidylcholine (PC), the primary phospholipid of eukaryotic membranes in general, and the ER membrane in particular 31. Importantly, we have now shown that Plasmodium relies on the abundance of PC within hepatocytes to support infection 32. Whether XBP1 role in infection is mediated by its impact in PC levels remains to be explored. Figure 3. Liver Xbp1 deletion inhibits Plasmodium liver infection qRT–PCR analysis of Xbp1 maturated form, Xbp1s, and its target Dnajb9 mRNA in sorted Hepa 1.6 cells infected with P. berghei at 6 and 24 h after infection relative to its GFP-negative control (dashed line), normalized to hypoxanthine-guanine phosphoribosyltransferase (Hprt) and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) expression. *P < 0.05, one-sample t-test. Results are expressed as means ± SEM (n = 3 independent experiments). Representative PCR analysis of XBP1 maturation in 8 h TM-treated Hepa 1.6 cells (positive control) and in sorted Hepa 1.6 sporozoite-infected (GFP+) and uninfected (GFP−) cells at 6 and 24 h after infection. XBP1 unspliced (U) and spliced mRNA species were resolved by high-density agarose gel (3%). Protein expression analysis of XBP1s protein in Xbp1Δ mice and CTRL mice with tubulin protein expression as a loading control. Plasmodium berghei liver load quantification on Xbp1∆ and CTRL mice by qRT–PCR of parasite 18S ribosomal RNA at 42 h after sporozoite infection, normalized to Hprt. **P < 0.01, t-test. Results are expressed as means ± SD (n ≥ 4 per group, three independent experiments). EEF density (numbers) and size (EEF area) quantified on Xbp1Δ mice and wild-type (WT) control mice by fluorescent microscopy of GFP-expressing P. berghei parasites at 42 h after infection, *P < 0.05, t-test (n ≥ 3 per group, three independent experiments). Results are expressed as means ± SD. Download figure Download PowerPoint CREBH is an ER-bound transcription factor that is specifically and highly expressed in the liver and small intestine 933 and is activated upon ER stress 33. We tested whether Crebh mRNA is induced during Plasmodium infection. Results showed that Crebh mRNA expression was highly up-regulated at 6 and 24 h after infection in infected Hepa 1.6 cells (Fig 4A). To understand the functional significance of Crebh upregulation, we infected mouse primary hepatocytes with adenovirus expressing a small hairpin RNA for Crebh (Ad-CREBHi) prior to sporozoite infection. A significant decrease in infection was observed upon Crebh knock-down (Fig 4B and C), without affecting cell viability (Appendix Fig S1D). Additionally, administration of Ad-CREBHi in vivo, which led to a significant decrease in expression of both mRNA and protein, resulted in a marked decrease in liver infection (Fig 4D–F). Crebh has been shown to regulate the expression of the major regulator of mammalian iron homeostasis, hepcidin (encoded by Hamp) 7, which we have shown to influence liver stage infection 34. Iron is an essential nutrient that has been shown to be required for Plasmodium liver infection 343536 and infected hepatoma cells significantly increase the iron importer divalent metal transporter-1 (DMT1), while ferroportin expression is highly reduced 11. It is thus tempting to postulate that CREBH role in infection might be mediated by modulation of iron levels and availability. Future research will establish whether that is the case or not. Figure 4. CREBH knock-down, ex vivo and in vivo, restricts Plasmodium liver infection qRT–PCR analysis of hepatic Crebh mRNA in sorted Hepa 1.6 cells infected with P. berghei sporozoites and analysed at 6 and 24 h after infection relative to its GFP-negative control (dashed line) normalized to hypoxanthine-guanine phosphoribosyltransferase (Hprt). *P < 0.05 and **P < 0.01, t-test. Results are expressed as means ± SEM (n = 5 independent experiments). Plasmodium berghei infection quantification on mouse primary hepatocytes transduced ex vivo with adenovirus expressing a short hairpin RNA for Crebh (Ad-CREBH RNAi) and control adenovirus (Ad-US) by qRT–PCR for parasite 18S ribosomal RNA at 44 h after sporozoite delivery normalized to Hprt expression. Results are expressed as means ± SD, ***P < 0.001, t-test (n = 3 independent experiments). qRT–PCR analysis of Crebh mRNA in primary hepatocytes after Ad-US and Ad-CREBHi transduction normalized to Hprt expression. Results are expressed as means ± SD, ***P < 0.001, t-test (n = 3 independent experiments). Plasmodium berghei liver load quantification at 44 h after sporozoite delivery in mice transduced with Ad-US and Ad-CREBHi 48 h prior to infection by qRT–PCR for parasite 18S ribosomal RNA normalized to Hprt, ***P < 0.001, t-test. Results are expressed as means ± SD (n ≥ 4 mice per group, two independent experiments). qRT–PCR analysis of Crebh mRNA livers of mice transduced with Ad-US and Ad-CREBHi transduction normalized to Hprt expression. Results are expressed as means ± SD, ****P < 0.0001, t-test (n ≥ 4 mice per group, two independent experiments). Western blot analysis of CREBH precursor (CREBH-full) and the processed nuclear form (CREBH-N) of whole-liver lysates showing the complete absence of the precursor and a significant decrease in the processed form upon adenovirus-mediated CREBH knock-down (Ad-CREBHi). Gp96 served as a loading control. Download figure Download PowerPoint Altogether, our data show that Plasmodium infection induces ER stress in hepatocytes via both XBP1 and CREBH pathways being activated and contributing significantly to infection. This raises the question of what is the nature of disrupted ER homeostasis mechanisms in the context of malaria liver infection? The liver, a highly metabolically active organ, has a protein synthesis rate of ~13 million secretory proteins per minute 37. Thus, in hepatocytes, even subtle perturbations to protein synthesis can lead to ER stress. Moreover, alterations to ER fatty acid/lipid composition can also induce ER stress and activate the UPR 638. We have recently reported a global analysis of the total lipid repertoire of Plasmodium-infected hepatocytes, which revealed an enrichment of neutral lipids as well as the major membrane phospholipid, PC 32. Whether these alterations are the underlying cause of the ER stress during Plasmodium liver stage infection remains to be elucidated. Many pathogens induce ER stress and use diverse strategies to activate or modulate it. Viral replication, for example, co-opts ER functions to produce viral glycoproteins, which leads to induction of the UPR 39. On the other hand, several bacteria seem to induce this response via the secretion of virulence factors into the host cell 40414243. Interestingly, an active Plasmodium export machinery may be ope
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