Host cell autophagy contributes to Plasmodium liver development

Summary

Autophagy plays an important role in the defence against intracellular pathogens. However, some microorganisms can manipulate this host cell pathway to their advantage. In this study, we addressed the role of host cell autophagy during Plasmodium berghei liver infection. We show that vesicles containing the autophagic marker LC3 surround parasites from early time‐points after invasion and throughout infection and colocalize with the parasitophorous vacuole membrane. Moreover, we show that the LC3‐positive vesicles that surround Plasmodium parasites are amphisomes that converge from the endocytic and autophagic pathways, because they contain markers of both pathways. When the host autophagic pathway was inhibited by silencing several of its key regulators such as LC3, Beclin1, Vps34 or Atg5, we observed a reduction in parasite size. We also found that LC3 surrounds parasites in vivo and that parasite load is diminished in a mouse model deficient for autophagy. Together, these results show the importance of the host autophagic pathway for parasite development during the liver stage of Plasmodium infection.

Introduction

The first obligatory step of the infection by Plasmodium spp. in the mammalian host is the invasion of liver cells. After being injected into the skin of the host, malaria parasites, in the form of sporozoites, travel in the blood circulation. When the sporozoites reach the liver, they infect hepatocytes by inducing an invagination of the host cell membrane (Prudêncio et al., 2006). This results in the formation of a parasitophorous vacuole (PV) surrounded by a host cell‐derived parasitophorous vacuole membrane (PVM). Each sporozoite then replicates into thousands of newly formed liver merozoites that will further infect red blood cells, leading to the onset of the disease symptoms.

Recently, using an in vitro murine model of Plasmodium hepatocyte infection, we have reported the existence of an interaction between parasites and vesicles from the host endocytic pathway that could represent a source of nutrients for the growing parasites (Lopes da Silva et al., 2012; Thieleke‐Matos et al., 2014). These vesicles contain membrane markers of the late endocytic pathway and are acidic in nature. When vesicle acidification was compromised, parasite survival was not affected but a decrease in schizont size was observed. Moreover, using transmission electron microscopy (TEM), we were able to demonstrate the transport of material from the host endocytic pathway towards the interior of the PV. Altogether, these results point to an important role for the endocytic pathway in Plasmodium growth.

Macroautophagy, hereafter referred to as autophagy, is a major catabolic process by which intracellular cytosolic components, including organelles, are sequestered and delivered to lysosomes for degradation (Kroemer and Levine, 2008). Canonical autophagy is conserved from yeast to mammals and is mediated by a special organelle called the autophagosome. Furthermore, the autophagic and endocytic pathways intersect because endosomes can fuse with autophagosomes to form intermediate organelles called amphisomes, before fusion with lysosomes (Gordon and Seglen, 1988). Under physiological conditions, autophagy has several vital roles such as maintenance of the amino acid pool during starvation, prevention of neurodegeneration, anti‐ageing, tumour suppression and regulation of innate and adaptive immunity (Rubinsztein, 2006; Levine and Deretic, 2007; Mizushima et al., 2008; Wang et al., 2009; Deretic et al., 2013). The autophagic machinery is also used in defence against microbes, and as such, numerous pathogens are degraded through this pathway, including the bacteria Mycobacterium tuberculosis, Shigella flexneri and Listeria monocytogenes, and the parasite Toxoplasma gondii (Levine and Deretic, 2007). Despite this role for autophagy in pathogen elimination, some microorganisms have evolved strategies to take advantage of the different compartments of the autophagic pathway and establish replicative niches. Modulation of the balance between anabolic and catabolic processes may affect the outcome of the competition for limiting nutrients between a pathogen and its host cell. In particular, the nutritive function of autophagy could favour pathogen expansion by providing greater access to host cell biomass. Indeed, T. gondii is an example of an intracellular parasite that is able to induce host cell autophagy to exploit the nutritive function of this pathway and enhance its own proliferation (Wang et al., 2009).

It has recently been described that the host cells respond to Plasmodium infection by targeting LC3 to the PVM, in a process resembling selective autophagy that leads to their degradation in lysosomes (Prado et al., 2015). Nevertheless, some of these parasites are able to resist degradation and benefit from host canonical autophagy to grow. However, the mechanism behind this process remains to be determined.

We have investigated the role of host autophagy during the highly replicative stages of Plasmodium liver development, as a possible source of nutrients, using a murine model of malaria. Together with our previous findings, the results described here suggest that in the course of liver infection Plasmodium parasites subvert amphisomes as a source of nutrients, while avoiding destruction by lysosomes.

Results

Plasmodium parasites are surrounded by amphisomes throughout liver stage infection

To investigate the relationship between Plasmodium liver infection and host cell autophagic, we started by analyzing the distribution of LC3‐positive vesicles in host cells infected with Plasmodium parasites, by immunofluorescence. The LC3 protein marks autophagic vesicles throughout the autophagic pathway and is commonly used for its monitorization (Klionsky et al., 2012). Hepa 1‐6 cells were infected with freshly‐dissected Plasmodium berghei green fluorescent protein (GFP) sporozoites, fixed at different times post‐infection and stained for the Plasmodium PVM protein UIS4, as well as for autophagic vesicles using an anti‐LC3 antibody. We found that from early time points and throughout infection [1–44 h post‐infection (hpi)], 80–100% of the parasites with a clear UIS4 staining are surrounded by LC3‐positive vesicles (Fig. 1A and B). We then performed the same experiment but this time transducing Hepa 1‐6 cells with adenoviruses encoding GFP‐LC3 and observed that GFP‐LC3 is also recruited to the neighbourhood of the PVM (Fig. S1). Moreover, the results show that LC3 is not only recruited to the neighbourhood of the PVM but it also strongly co‐localizes with it, suggesting that LC3 associates with the PVM. This co‐localization was also observed when we used a different PVM marker, EXP1, which is only expressed at later stages of parasite development (Fig. S2). Furthermore, an increase in LC3 fluorescence intensity was observed in infected cells but not in neighbouring uninfected cells. To quantify this observation, at least 50 Hepa 1‐6 infected and 50 non‐infected cells were analysed and LC3 mean fluorescence intensity per µm² of cell area measured at 16 hpi (early/pre‐replicative stage) or 44 hpi (late/replicative stage). In both cases, the mean fluorescence intensity of LC3 in infected cells is significantly higher than in non‐infected cells, suggesting that there is an increase in LC3 recruitment to membranes in infected cells (Figs 1C and S3A). To further address the physiological relevance of these findings, we performed the same study in isolated murine primary hepatocytes infected with P. berghei sporozoites and obtained a similar result (Figs 1D and S3B). Furthermore, we investigated the presence and distribution of autophagic vesicles in Hepa 1‐6 cells infected with a closely related parasite, P. yoelii, and also found that LC3‐positive vesicles surround these parasites (Figs 1E and S3C). Thus, these results suggest that the increase and accumulation of LC3‐labelled vesicles around the Plasmodium PV is a common process in vitro and ex vivo and is not parasite species‐restricted.

Figure 1

Plasmodium berghei liver forms are surrounded by autophagic vesicles. A. Hepa 1‐6 cells were infected with GFP‐P. berghei sporozoites. Infection was stopped at various times, and the analysis was performed by immunofluorescence microscopy using antibodies for parasite UIS4, in green, and LC3, in red. DAPI was used to stain nuclei (in blue). Representative images of parasites surrounded by LC3 at different times post‐infection are shown. Plot profiles showing colocalization between UIS4 and LC3 are shown for each time point. B. Approximately 50 parasites with clear UIS4 staining for each time‐point were scored positive or negative for LC3 aggregation, depending on the presence or absence of LC3 within 2 µm from the PV. The results are presented as the percentage of parasites surrounded by LC3. C. The mean fluorescence intensity of LC3‐positive vesicles in Hepa 1‐6 cells was quantified per area of infected or non‐infected cells at 16 and 44 h post‐infection (hpi). D. Isolated murine primary hepatocytes were infected with GFP‐P. berghei sporozoites. Infection was stopped at 16 or 44 hpi. Cells were stained for Hsp70 (2E6), in green, LC3, in red, and with DAPI for nuclei detection (in blue). E. Hepa 1‐6 cells were infected with P. yoelii sporozoites. Infection was stopped at 16 or 44 hpi. Cells were stained with antibodies for parasite Hsp70 (2E6), in green, anti‐LC3, in red, and with DAPI for nuclei detection (in blue). Scale bars, 10 µm.

Previous data from our laboratory showed that vesicles derived from the late endocytic pathway are essential for Plasmodium parasite development in the liver (Lopes da Silva et al., 2012). Because the endocytic and autophagic pathways converge and intersect originating intermediate organelles called amphisomes, we decided to characterize the autophagic vesicles accumulating in the vicinity of the parasite and their co‐localization with specific markers of the endocytic pathway. For this, Hepa 1‐6 cells were transduced with adenoviruses encoding GFP‐LC3 prior to infection with P. berghei red fluorescent protein (RFP) parasites, fixed at 16 or 44 hpi and stained for late endosome/lysosome markers, using anti‐LAMP2 and anti‐CD63 antibodies. We observed that both endocytic markers partially colocalize with GFP‐LC3 (Fig. 2A and B), suggesting that the vesicles surrounding the parasite converge from both the endocytic and autophagic pathways and thus can be identified as amphisomes. Amphisomes can be distinguished from autolysosomes because they are not highly degradative or highly acidic. The acidification and degradation capacity of autophagic organelles can be monitored by using a tandem fluorescently tagged mCherry‐GFP‐LC3, which displays green and red fluorescence before fusion with lysosomes, but only red fluorescence afterwards, since GFP, but not mCherry loses its fluorescence when in contact with the highly acidic environment of the lysosome (Kimura et al., 2007). Consequently, when acidification is inhibited, the green fluorescent signal becomes predominant (Fig. S4). Using this tool, we performed immunofluorescence and live cell imaging studies on Hepa 1‐6 cells infected with P. berghei parasites and observed that the vesicles that surround the parasites are not highly acidic, because the fluorescence from both mCherry and GFP fluorescence can be detected, indicating that they are not autolysosomes but rather amphisomes (Figs 2C and S5 and Videos 1 and 2).

Figure 2

P. berghei liver forms are surrounded by amphisomes. A, B. Hepa 1‐6 cells were transduced with GFP‐LC3, infected with RFP‐P. berghei sporozoites and stained with antibodies against LAMP2 (A) or CD63 (B), followed by Alexa 647‐conjugated secondary antibodies, at 16 or 44 hpi. In both cases parasites were stained with anti‐Hsp70 antibody (2E6), followed by Alexa 568‐conjugated secondary antibody, because the RFP signal is faint and becomes undetectable. In the merged images, LAMP2 (A) and CD63 (B) are false‐coloured in red; GFP‐LC3 is shown in green and nuclei in blue, after staining with DAPI. Plot profiles show colocalization between (A) LAMP2 and GFP‐LC3 and (B) CD63 and GFP‐LC3. With the exception of the nuclei, merged images are shown separately, in black and white. C. Hepa 1‐6 cells were transduced with GFP‐mCherry‐LC3, infected with GFP‐P. berghei sporozoites and analysed at 16 and 40 hpi. Parasites were stained for Hsp70 (2E6), followed by Alexa 647‐conjugated secondary antibody, because the GFP signal bleaches quickly and becomes undetectable. Both the GFP and the mCherry from the LC3 are shown separately, in black and white. In the merged images the GFP‐LC3 is shown in green, mCherry‐LC3 in red and nuclei in blue, after staining with DAPI. Plot profiles show colocalization between GFP‐ and mCherry‐LC3. Scale bars, 10 µm. D. Electron micrograph showing a parasite cross‐section where the parasite membrane (arrows), the PVM (open arrowheads) and various host vesicles surrounding the PVM are depicted. Insets (2 and 3) show two serial sections of the areas highlighted in 1, where a membrane contact site (filled arrowhead) between a host autophagic vesicle (asterisk) and the PVM can be seen. n, parasite nucleus. Scale bars, 1 µm and 200 nm in insets.

To further investigate the nature of the vesicles that surround parasites, P. berghei‐infected Hepa 1‐6 cells were prepared for TEM at 24 hpi. Interestingly, we found several vesicles in close proximity of the PV (Fig. 2D). Moreover, we could observe that these vesicles are bound by a single membrane and are full of intraluminal vesicles, which is consistent with them being late endosomes/multivesicular bodies or amphisomes. Taking into account our immunofluorescence studies, it is likely that the vesicles seen in the neighbourhood of the PV are amphisomes, after degradation of the inner membrane of the autophagosomes (Klionsky et al., 2012). Furthermore, membrane contact sites between the PVM and the membrane of these vesicles were often observed (Fig. 2D, arrowhead), indicating a close interaction between the two organelles.

Blockade of host autophagy reduces parasite size in the replicative stage of infection

In order to determine the role of autophagy during Plasmodium liver infection, we transduced Hepa 1‐6 cells with lentiviruses encoding LC3‐targeting shRNA, achieving an ~50% reduction in LC3 mRNA and protein expression, as measured by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot, respectively (Fig. S6A, B and C). These cells were then infected with freshly‐dissected P. berghei parasites, and the infection was allowed to proceed for approximately 44 h. Parasite cytoplasm was stained with anti‐Hsp70 antibody, and the size of parasites was determined by microscopy analysis. Strikingly, parasite size in LC3‐silenced cells is significantly decreased when compared with shRNA control‐transduced cells (Fig. 3A and B). When measured by qRT‐PCR, a reduction in parasite load was observed in LC3‐silenced cells (Fig. 3C). To investigate if the decrease in parasite size is specifically because of the lack of LC3, we performed a rescue experiment. For this, both LC3‐deficient and shRNA control‐transduced cells were transduced with GFP‐LC3^wt or a mutant LC3 form, GFP‐LC3^G120A that is not able to be lipidated and thus is unable to form autophagosomes (Kabeya et al., 2000). Importantly, the exogenous expression of GFP‐LC3 is able to restore the levels of LC3 in cells treated with LC3‐targeting shRNA (Fig. S6D and E). These cells were then infected with freshly dissected P. berghei sporozoites, and parasite size was measured by microscopy. The results show that parasite size recovers to the level of control cells when GFP‐LC3^wt is overexpressed in cells silenced for LC3 but not when the non‐lipidated form (GFP‐LC3^G120A) is used, showing the specific requirement for lipidated LC3 in parasite development (Fig. 3A and B). In addition, we could observe that in shRNA control‐transduced cells the overexpression of GFP‐LC3^G120A mutant form leads to slightly smaller parasites, when compared with GFP‐LC3^wt, showing that although not acting as a complete dominant negative, this construct could be partially competing with the endogenous LC3 protein and blocking autophagic flux.

Figure 3

Host LC3 function impairment leads to a reduction in parasite size in the replicative phase of P. berghei infection. A. Parasite size in Hepa 1‐6 cells transduced with shRNA targeting LC3 (sh LC3) or shRNA control (control), not overexpressing (non) or overexpressing GFP‐LC3^wt or GFP‐LC3^G120A. The size of schizonts (in µm²) was determined by microscopy analysis, using Image J software (**P = 0.0015, ***P ≤ 0.0001). B. Representative images of parasites for each of the conditions are shown. Control cells were stained for Hsp70 (2E6), in green, and anti‐LC3, in red. Cells transduced with shRNA targeting LC3 (sh LC3) were stained for Hsp70 (2E6), in green. Nuclei were stained with DAPI, in blue. Scale bars, 10 µm. C. Parasite load at 44 hpi measured by qRT‐PCR in Hepa 1‐6 cells deficient for LC3 and infected with P. berghei sporozoites. Plot represents mean ± SEM.

In order to further characterize the role of autophagy in P. berghei infection, we established autophagy‐deficient cell lines for other important autophagy regulators, namely Atg5, Beclin1 and Vps34. The silencing efficiency was measured by qRT‐PCR and varied between 50% and 75% (Fig. S7A, B and C). Importantly, we confirmed that the viability of silenced host cells is not affected (Fig. S7G) and that the levels of basal and starvation‐induced autophagy are decreased in these cells (Fig. S7D–F, H and I). Parasites were stained with anti‐Hsp70 antibody, and their size was determined by microscopy analysis. Strikingly, we found that parasite size is significantly decreased in all autophagy‐deficient cell lines, similar to the LC3‐silenced cell line (Fig. 4A and B). Nevertheless, LC3 is still present around parasites in all cases (Figure 4B), which might reflect the partial reduction, rather than a complete blockade in autophagy activation and flux (Fig. S7D, E and F). Alternatively, the efficient incorporation of LC3 in the PVM recently described (Prado et al., 2015) can also explain the presence of LC3 around parasites despite the silencing of LC3. In all cases, merosomes were observed at 63 hpi, suggesting that parasites continue to grow (Figure S8).

Figure 4

Host autophagy impairment leads to a reduction of parasite size in the replicative phase of P. berghei infection. A. Hepa 1‐6 cells deficient for Atg5, Beclin1 or Vps34 were infected with GFP‐P. berghei sporozoites, and the infection was stopped at 48 hpi. The size of schizonts (in µm²) was determined by microscopy analysis (**P = 0.0084, *P ≤ 0.05). Plot represents mean ± SEM. B. Examples of parasites in each of the cell lines (sh Atg5, sh Beclin1 or sh Vps34). Parasites were stained for Hsp70 (2E6), in green, LC3, in red, and with DAPI to detect nuclei (in blue). Scale bar, 10 µm.

Altogether, these results point towards an important role for host autophagy in parasite liver stage development, because parasites infecting cells defective for autophagy are smaller and do not replicate as well as parasites infecting control cells. These results are also in agreement with our previous studies, in which we observed a reduction in parasite size when the function of late endocytic vesicles was disturbed by interfering with their acidification (Lopes da Silva et al., 2012).

Role of host autophagy in vivo

To analyse the role of autophagy in Plasmodium liver infection in vivo, we challenged GFP‐LC3 mice (Mizushima et al., 2004), where autophagy can be monitored by GFP fluorescence, with P. berghei RFP parasites. Immunofluorescence analysis of Plasmodium‐infected liver sections at both 16 and 44 hpi showed an accumulation of LC3‐positive vesicles around parasites, consistent with the in vitro data (Fig. 5). To investigate the physiological importance of autophagy in vivo, we challenged Atg5‐deficient mice (Hara et al., 2006) with P. berghei GFP sporozoites. Because Atg5^−/− mice die immediately after birth from developmental defects, we used Atg5^flox/flox mice, which have a 60% reduction in expression of Atg5, as measured by qRT‐PCR (Fig. 6A). Moreover, these mice have an impaired response to autophagic stimuli, because primary hepatocytes isolated from their livers present low levels of lipidated LC3 after starvation, when compared with primary hepatocytes isolated from wild‐type animals (Fig. 6B and C). This suggests that the Atg5^flox/flox mice are hypomorphs and are deficient in the expression of Atg5 and autophagy induction. Therefore, we used these mice to investigate the role of autophagy in malaria infection in vivo. After infection with P. berghei, we used qRT‐PCR of parasite 18S rRNA to assess parasite load. Strikingly, a potent and significant reduction in P. berghei load was observed in mice deficient for autophagy when compared with wild‐type mice (Figure 6D), confirming in vivo the importance of autophagy in the liver stage of infection.

Figure 5

GFP‐LC3 surrounds P. berghei parasites in vivo. Representative images of an infected hepatocyte from 50 µm liver slices of GFP‐LC3^wt mice infected with RFP‐P. berghei sporozoites, fixed at (A) 16 or (B) 44 hpi and analysed by immunofluorescence microscopy. The liver slices were stained with anti‐GFP in order to enhance the GFP signal from the LC3, in green, phalloidin to mark the cortical actin meshwork that is immediately underneath the plasma membrane and DAPI to detect nuclei. Pictures of sequential Z‐planes are shown. Scale bars, 10 µm.

Figure 6

Role of host autophagy in in vivo infection. A. Relative Atg5 expression levels in the liver of control and Atg5^flox/flox mice infected for 44 hpi with 5 × 10⁴ GFP‐P. berghei sporozoites, measured by qRT‐PCR. B. Immunoblot of primary hepatocytes isolated from wild‐type or Atg5^flox/flox mice treated with EBSS (starvation media) to induce autophagy or with EBSS together with NH₄Cl to induce autophagy and block LC3 degradation, simultaneously. Blots were incubated with anti‐LC3 and anti‐Calnexin, used as a loading control. C. Quantification of LC3‐II immunoblot. Results are presented as the ratio of integrated density of LC3‐II normalized to Calnexin. D. Parasite liver load was assessed at 44 hpi after infection with 5 × 10⁴ of P. berghei ANKA‐GFP sporozoites in control or Atg5^flox/flox mice, measured by qRT‐PCR.

Discussion

To date, the cellular and molecular events that occur within host liver cells infected with Plasmodium spp. remain poorly characterized. We found evidence that P. berghei and P. yoelii parasites are surrounded by vesicles marked by LC3, a widely used autophagic marker, in a hepatocyte cell line, in primary hepatocytes and in vivo. In addition, and consistent with our previous observations (Lopes da Silva et al., 2012; Thieleke‐Matos et al., 2014), vesicles containing markers of late endosomes, such as LAMP2 and CD63, partially colocalize with LC3 around the PV until at least 48 hpi. This is in line with two recent studies, which show by live cell imaging and super‐resolution microscopy that host LC3 colocalizes with UIS4 and marks vesicles surrounding the PV, during the highly replicative stages of infection (Grützke et al., 2014; Prado et al., 2015). Furthermore, we show that by depleting host cells of autophagy regulators such as LC3, Atg5, Beclin1 or Vps34, partially inhibiting the autophagic flux, parasite size is decreased. Importantly, this phenotype is rescued when LC3‐depleted cells are complemented with exogenous LC3. Thus, our studies suggest that during the highly replicative phase of malaria liver infection, parasites are able to subvert autophagy‐derived vesicles, which we determined to be amphisomes, and use them as a possible source of nutrients for growth.

An important but still unanswered question is how parasites recruit the host autophagic pathway and how are nutrients taken‐up by the parasite. Our previous studies showed that when the host cell endocytic pathway was loaded with BSA‐gold, gold particles were found within the PV, indicating the transport of intravesicular material from the host endocytic pathway to the interior of the PV (Lopes da Silva et al., 2012). Moreover, we have recently described a role for host PIKfyve and TRPML1 in the fusion of late endocytic vesicles with the PVM (Thieleke‐Matos et al., 2014). It is known that, once inside hepatocytes, the majority of malaria parasites localize close to the nucleus, where they replicate (Bano et al., 2007). Possibly not a coincidence, it is in the perinuclear area that most autophagic vesicles fuse with late endosomes/ lysosomes (Kimura et al., 2008; Mackeh et al., 2013). Therefore, we suggest that by localizing to the perinuclear area, parasites position themselves to interact and fuse with several types of host vesicles. Nevertheless, we cannot rule out that direct incorporation of LC3 into the PVM occurs. Indeed, this was recently shown by the Heussler group (Prado et al., 2015) and also fits with our results. Noteworthy, a parasite export system that has recently been described in liver stage Plasmodium parasites and is used to secrete vesicles to the host cytoplasm (Orito et al., 2013) could potentially be used to secrete factors that recruit host molecules necessary for the subversion of host autophagy by parasites.

There is evidence suggesting that autophagy helps ‘feed’ intracellular parasites such as T. gondii by promoting intracellular proliferation of parasites in nutrient‐limiting conditions. Consequently, impaired T. gondii growth is observed in Atg5‐deficient mouse embryonic fibroblasts (MEFs) (Levine and Deretic, 2007). In the case of malaria parasites, we show that in mice deficient for Atg5, P. berghei infection is decreased, confirming in vivo the importance of autophagy during the liver stage of infection. This result is also in accordance with recently published data showing that Plasmodium parasites developing in Atg5^−/− MEFs are significantly smaller than parasites developing in wild‐type MEFs (Prado et al., 2015). These authors reported that a process resembling selective autophagy is induced in Plasmodium‐infected cells during the early stage of infection and that the percentage of live parasites decreases until 24 hpi. We have also shown previously that the number of parasites dramatically decreases until around 16 hpi (Lopes da Silva et al., 2012). Thus, we propose that during the initial stages of infection (until 16–24 hpi) parasites are targeted for destruction by a process resembling selective autophagy but the parasites that are able to survive this host defence mechanism are able to replicate and ultimately subvert the host autophagic pathway in order to access nutrients for their fast growth. Interestingly, a similar strategy has recently been described for Yersinia pseudotuberculosis (Ligeon et al., 2015).

In conclusion, we present evidence supporting a specific role for host autophagy during the replicative phase of Plasmodium liver infection. We speculate that growing parasites have evolved to subvert this host pathway for their advantage and utilize it as a source of nutrients, essential for parasite growth during this phase of the life cycle.

Experimental procedures

Cell culture and stable cell line production

Mouse Hepa 1‐6 hepatoma cells (ATTC) were cultured in Dulbecco's Modified Eagle Medium (Gibco/Invitrogen) supplemented with 10% foetal calf serum (FCS) (Gibco/Invitrogen), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco/Invitrogen). Cells were maintained in a humidified cabinet at 37°C and 10% CO₂. Primary mouse hepatocytes were isolated from female C57BL/6 mice (8–10 weeks) according to the protocol described elsewhere (Goncalves et al., 2007). Stable mouse Hepa 1‐6 cell lines depleted for Atg5 (NM_053069.5), Vps34 (NM_181414.5), Beclin1 (NM_019584.3) or LC3 (NM_026160.4) were generated by transduction with MISSION® shRNA lentiviral particles (Sigma), according to the manufacturer's instructions. Between 3 and 5 shRNA sequences for each gene were tested (Table S1), and the one that gave the best silencing level was used. Puromycin selection (1 mg/ml) was performed to generate the stable cell lines (Table S1).

Parasite strains and culture

Plasmodium yoelli 17X, GFP (parasite line 259cl2) or RFP (parasite line 733 cl1) expressing Plasmodium berghei ANKA salivary gland sporozoites were collected from infected female Anopheles stephensi mosquitoes. These were bred in the insectarium of the Instituto de Medicina Molecular, Lisbon. Infected mosquitoes were dissected between 20 and 24 days after the infectious blood meal. Mean number of sporozoites were determined using a hemocytometer.

Mice and in vivo infections

GFP‐LC3 mice (Riken GFP‐LC3#53) and Atg5^flox/flox mice (RIKEN B6.129S‐Atg5<tm1Myok>) were maintained under specific pathogen‐free conditions. GFP‐LC3 mice were challenged with 2 × 10⁵ sporozoites by intravenous injection of freshly dissected sporozoites. For liver sections, mice were sacrificed at the indicated time points and livers removed and fixed for 4 h in 4% paraformaldehyde (Electron Microscopy Sciences). Afterwards, 50 µm thick liver lobule sections were made using a Leica VT1000 S Vibrating Blade Microtome (Leica Microsystems©). For in vivo infections, Atg5^flox/flox and control C57BL/6 mice were challenged with 5 × 10⁴ sporozoites by intravenous injection of freshly dissected sporozoites. Livers were collected and homogenized in denaturing buffer (4 M guanidine thiocyanate; 25 mM sodium citrate pH = 7; 0.5% N‐Lauroylsarcosineand; 0.7% ®‐ Mercaptoethanol in DEPC‐treated water). Total RNA was extracted using RNeasy Mini kit (Qiagen). Infection load in the liver was determined by qRT‐PCR.

Immunoblotting

Cells were seeded on 6‐well plates and homogenized in 300 µl of lysis buffer (50 mM NaCl, 50 mM Tris‐Cl pH = 7.4, 0.5% deoxycholic acid, 0.1% SDS and 1% NP‐40). The cell debris was removed, and protein quantification was performed using the BCA protein assay (Pierce). Samples were then subjected to SDS‐PAGE and transferred to polyvinylidine fluoride membranes (Immobilon, Millipore). These were blocked in 1% non‐fat dried milk in PBS/0.1% Tween 20 (PBS_T buffer) for 1 h at room temperature. Primary antibodies were diluted as follows: anti‐LC3 (Nanotools, 1:1000) and anti‐Calnexin (Sicgen 0037160412, 1:2000). Membranes were incubated overnight at room temperature, and proteins were visualized using HRP‐conjugated secondary antibodies (Sigma, 1:10,000) and ECL Plus Western Blotting Detection Reagent (GE Healthcare) using a ChemiDoc (BioRad). Western blot analysis and quantification was performed using Image J software (NIH).

Quantitative real‐time PCR

Isolated primary hepatocytes, Hepa 1‐6 cells and autophagy‐deficient cell lines (2 × 10⁴) were seeded on 96‐well plates the day before the addition of freshly dissected sporozoites. Cells were centrifuged at 1600xg for 5 min at 4°C for parasites to come into contact with the cells.

Total RNA was extracted using the RNeasy Mini Kit (Qiagen). One microgram of total RNA was reverse‐transcribed using SuperScriptII RNase H‐reverse transcriptase (Invitrogen, CA) and random hexamer primers (Invitrogen). Reactions were incubated at 65°C for 5 min, 25°C for 10 min, 42°C for 50 min and finally at 70°C for 15 min. qRT‐PCR was performed in ABI Prism 7900HT system using ABI Power SYBR Green PCR Master Mix. qRT‐PCR reactions were performed in triplicate using specific primers (Table S2). For each protein, gene expression was calculated relative to control wells and normalized for GAPDH expression.

Constructs and virus recombination

For production of pENTR‐GFP‐C2‐LC3 construct, LC3 was amplified by qRT‐PCR from total RNA isolated from Hepa 1‐6 cells (Table S3) and cloned into the modified mammalian expression vector pENTR‐GFP‐C2 previously described (Lopes et al., 2007) using EcoRI/SalI. To generate the mammalian expression vector pcDNA‐ENTR‐BP‐V5‐mcherry‐C2‐LC3 with Gateway® technology (Invitrogen), LC3 was excised from pENTR‐GFP‐C2‐LC3 using EcoRI/SalI and inserted into pcDNA‐ENTR‐BP‐V5‐mcherry‐C2. pcDNA‐ENTR‐BP‐V5‐mcherry‐C2 was generated by cloning the V5 tag and the polylinker C2 excised from pENTR‐V5‐C2 (Lopes et al., 2007) into pcDNA‐ENTR‐BP1848. mCherry was amplified using specific primers (Table S3) from pCMV‐myc‐mCherry (Nightingale et al., 2009) and inserted downstream and in frame with V5 tag, using BglII/XhoI. pcDNA‐ENTR‐BP1848 was constructed by removing EmGFP from pcDNA™6.2/C‐EmGFP‐GW (Invitrogen) and cloning the polylinker with DraI/XhoI. The polylinker was generated by oligonucleotide annealing (Table S3). pENTR‐myc‐mcherry‐GFP‐C2‐LC3 was constructed by inserting myc‐mCherry into pENTR‐GFP‐C2‐LC3 upstream of GFP, with AgeI. Myc‐mCherry was amplified from pCMV‐myc‐mCherry (Nightingale et al., 2009) using specific primers. All constructs were confirmed by sequencing. The resulting plasmids were used to generate pAd adenoviral vectors and viral particles by LR recombination, according to the manufacturer's instructions (Invitrogen). Adequate viral titres that transduced 80–90% of cells were used. The rescue experiment was performed using LentiBrite™ GFP‐LC3‐G120A Lentivirus (#17‐10189) and LentiBrite™ GFP‐LC3 wild‐type (catalogue # 17‐10193).

Antibodies and Immunofluorescence microscopy

Isolated primary hepatocytes, Hepa 1‐6 cells and autophagy‐deficient cell lines (9 × 10⁴) were seeded on coverslips on 24‐well plates day before the addition of around 4 × 10⁴ freshly dissected sporozoites. Cells were centrifuged at 1600xg for 5 min at 4°C for parasites to come into contact with the cells. At the appropriate times, cells were washed twice with 1× PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in 1× PBS for 15 min at room temperature, or with ice‐cold methanol for 5 min. Cells were blocked and permeabilized with 1% BSA (Bovine Serum Albumin, Sigma), 0.05% saponin (Sigma) and 1% FCS (Gibco/Invitrogen) in PBS for 30 min. Cells were then incubated with primary antibodies in the same buffer for 1 h. After washing in PBS, cells were incubated with secondary antibodies in the same buffer for 30 min. To visualize the nuclei DAPI was added (Invitrogen) for 1 min. Samples were mounted using MOWIOL mounting medium (Calbiochem). For liver section stainings, primary antibodies were incubated overnight at room temperature. Antibodies and dilutions used were as follows: 2E6 (1:2500), anti‐UIS4 (SicGen, 1:100), anti‐EXP1 (1:500), anti‐Lamp2 (Hybridoma Bank, University of Iowa, 1:300), anti‐CD63 (MBL, 1:500) and anti‐LC3 (Sigma, 1:1000). Specific Alexa fluor® labelled secondary antibodies (Invitrogen) were used at 1:400. Images were taken using an inverted Leica SP5 confocal microscope.

Transmission electron microscopy

Hepa 1‐6 cells were infected with P. berghei GFP sporozoites, and sorted at 3–5 hpi to enrich for infected cells using a FACSAria cell sorter (Becton Dickinson). Sorted cells were seeded on Thermanox (Nunc) coverslips and further incubated in growth medium until ready to fix at the indicated times. Cells were fixed in 2% paraformaldehyde (Sigma) and 2% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M sodium cacodylate buffer for 30 min. After washing in 0.1 M sodium cacodylate buffer, cells were post‐fixed in 1.5% potassium ferricyanide (Sigma) and 1% osmium tetroxide (Sigma) for 1 h on ice. Cells were subsequently incubated in 1% tannic acid in 0.05 M sodium cacodylate for 45 min and dehydrated in ethanol (70%, 90% and absolute). Coverslips were then transferred to 1:1 propylene oxide: epon for 1 h, followed by two changes and embedding in epon. Ultra‐thin sections (70 nm) were sectioned and stained with lead citrate before examination on a JEOL 1010 transmission electron microscope (Welwyn Garden City, United Kingdom). Images were taken with a Gatan OriusSC100B charge coupled device camera and analysed with Gatan Digital Micrograph and Image J software (NIH).

Live cell confocal imaging

Hepa 1‐6 cells (4 × 10⁴) were seeded on glass bottom culture dishes (MatTek Corporation) infected with 5 × 10⁴ P. berghei sporozoites and transduced with mCherry‐GFP‐LC3. Samples were visualized using an inverted spinning disc microscope system (Andor technology^TM), fitted with a temperature and CO₂ control chamber.

Image analysis

All the images were processed and analysed using Image J software (NIH). To measure schizont size, images for each condition were acquired using Nikon Eclipse TE2000‐S automated widefield microscope, and schizont area (in µm²) was calculated. Quantification of LC3 intensity in infected and non‐infected cells was performed using the drawing tool of ImageJ and selecting the area of interest to measure the integrated density fluorescence of LC3 staining. Quantification of LC3 in autophagy‐silenced cell lines was performed using the drawing tool of ImageJ and selecting the area of interest using the brightfield image to define the cell edges. The number of LC3 dots in autophagy‐deficient cell lines was counted by applying the Otsu threshold filter to each image. The number of LC3 dots per cell was counted. Cell viability in autophagy‐deficient cell lines was measured by counting the number of nuclei per field in at least 10 fields (each field covers 116 mm²).

Statistics

The mean ± SEM of at least three independent experiments is shown in figures unless otherwise indicated, and P values were calculated using a two‐tailed two‐sample unequal variance Student's t‐test. A P value of less than 0.05 was determined to be statistically significant and marked with an asterisk and a P value < 0.0005 was marked with triple asterisk.

Ethics statement

All experimental procedures were performed according to EU recommendations and approved by the Instituto de Medicina Molecular Animal Care and Ethical Committee (AEC_2010_024_MM_RDT_General_IMM).

Acknowledgements

The authors would like to thank Lígia A. Gonçalves for her help with primary hepatocyte isolation. We also thank Ghislain Cabal and Eliana Real from Maria M. Mota's lab for their help in conducting experiments and critical discussions. We would also like to thank the IGC Imaging Unit. The authors declare that there are no conflict of interests.

Accession numbers

LC3 (NM_026160), Beclin1 (NM_019584), Vps34 (NM_181414), Atg5 (NM_053069), UIS4 (PBANKA 050120), EXP1 (PBANKA 092670), CD63 (NM_177356), LAMP2 (NM_010685).