Robert G Parton

Wiebke Petersen

Werner Stenzel

Olivier Silvie

Judith Blanz

Paul Saftig

Kai Matuschewski

Alyssa Ingmundson
email: alyssa.ingmundson@hu-berlin.de

Sequestration of cholesterol within the host late endocytic pathway restricts liver-stage Plasmodium development

Keywords: membrane protein, LE, late endosome, MβCD, methyl-β-cyclodextrin, NPC, Niemann-Pick type C, PV, parasitophorous vacuole, PVM, parasitophorous vacuole membrane, TVN, tubovesicular network, UIS4

While lysosomes are degradative compartments and one of the defenses against invading pathogens, they are also hubs of metabolic activity. Late endocytic compartments accumulate around Plasmodium berghei liver-stage parasites during development, and whether this is a host defense strategy or active recruitment by the parasites is unknown. In support of the latter hypothesis, we observed that the recruitment of host late endosomes (LEs) and lysosomes is reduced in uis4 -parasites, which lack a parasitophorous vacuole membrane protein and arrest during liver-stage development. Analysis of parasite development in host cells deficient for late endosomal or lysosomal proteins revealed that the Niemann-Pick type C (NPC) proteins, which are involved in cholesterol export from LEs, and the lysosomeassociated membrane proteins (LAMP) 1 and 2 are important for robust liver-stage P. berghei growth. Using the compound U18666A, which leads to cholesterol sequestration in LEs similar to that seen in NPC-and LAMP-deficient cells, we show that the restriction of parasite growth depends on cholesterol sequestration and that targeting this process can reduce parasite burden in vivo. Taken together, these data reveal that proper LE and lysosome function positively contributes to liver-stage Plasmodium development.

INTRODUCTION

Although many intracellular pathogens encounter, or even exploit, the host endocytic pathway upon invasion, apicomplexan parasites avoid the endocytic pathway upon entry through their active invasion of cells [START_REF] Sibley | Invasion and intracellular survival by protozoan parasites[END_REF]. Recent studies of Plasmodium berghei parasites, however, found that late endosomes (LEs) and lysosomes of the host progressively accumulate around the parasitophorous vacuole (PV) and associated tubovesicular network (TVN) during the first 24 h of parasite liver-stage development (Lopes da [START_REF] Da Silva | The host endocytic pathway is essential for Plasmodium berghei late liver stage development[END_REF][START_REF] Grützke | The spatiotemporal dynamics and membranous features of the Plasmodium liver stage tubovesicular network[END_REF]. The mechanisms and consequences of this recruitment are thus far undefined.

After development within hepatocytes, Plasmodium parasites infect red blood cells and cause malaria. Although red blood cells have minimal resources available to the parasites, blood-stage Plasmodium are well adapted to take advantage of their host environment, for example, through the metabolism of host hemoglobin [START_REF] Sigala | The peculiarities and paradoxes of Plasmodium heme metabolism[END_REF]. Hepatocytes as host cells could offer more opportunities for parasites to usurp host processes but could also present a greater chance of encountering cellular defense pathways. Fusion of the liver-stage PV with host lysosomes can act as one of these defense pathways and lead to parasite killing [START_REF] Prado | Longterm live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms[END_REF][START_REF] Boonhok | LAP-like process as an immune mechanism downstream of IFN-gamma in control of the human malaria Plasmodium vivax liver stage[END_REF]; however, for the majority of liver-stage parasites, any fusion of the host late endocytic compartments with the PV membrane (PVM) is minimal because markers of LEs and lysosomes such as CD63 and lysosome-associated membrane protein 1 (LAMP1) do not appear to label the membrane PVM proteins and investigated the influence of late endosomal host proteins on parasite development. We analyzed parasite development within cells deficient for the late endosomal/lysosomal proteins Niemann-Pick type C (NPC) 1 and 2, LAMP1, and LAMP2. The prominent phenotypes of cells deficient for these proteins include defects in autophagic flux and the accumulation of cholesterol in LEs and lysosomes due to inefficient export of free cholesterol from these vesicles [START_REF] Eskelinen | Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/ LAMP-2 double-deficient fibroblasts[END_REF][START_REF] Chang | Niemann-Pick type C disease and intracellular cholesterol trafficking[END_REF][START_REF] Schneede | Role for LAMP-2 in endosomal cholesterol transport[END_REF]. In the present model, endocytosed cholesteryl esters are hydrolyzed upon reaching LEs, and the resulting unesterified cholesterol in the lysosomal lumen is initially bound by NPC2, and subsequently transferred to NPC1 in the limiting LE/lysosomal membrane [START_REF] Wang | Identification of surface residues on Niemann-Pick C2 essential for hydrophobic handoff of cholesterol to NPC1 in lysosomes[END_REF]. Cells deficient for LAMP1 and LAMP2 phenocopy the defects observed in cells deficient for either NPC1 or NPC2, indicating that these two type I transmembrane glycoproteins are also required for proper export of cholesterol out of LEs and lysosomes [START_REF] Schneede | Role for LAMP-2 in endosomal cholesterol transport[END_REF].

Plasmodium is unable to synthesize cholesterol de novo, but the parasite PVM is enriched with cholesterol [START_REF] Bano | Cellular interactions of Plasmodium liver stage with its host mammalian cell[END_REF]. Although Plasmodium can access cholesterol from the host via either the endogenous or exogenous pathways [START_REF] Labaied | Plasmodium salvages cholesterol internalized by LDL and synthesized de novo in the liver[END_REF], neither pathway has been shown to be necessary for parasite growth, and the significance of this cholesterol acquisition for Plasmodium liver-stage development is undetermined.

RESULTS

Efficient recruitment of host late endocytic compartments requires UIS4

The striking recruitment of host LEs and lysosomes to developing P. berghei liver-stage parasites (Lopes da [START_REF] Da Silva | The host endocytic pathway is essential for Plasmodium berghei late liver stage development[END_REF]; Supplemental Figure S1A) and to the liver-stage TVN [START_REF] Grützke | The spatiotemporal dynamics and membranous features of the Plasmodium liver stage tubovesicular network[END_REF] prompted us to investigate whether parasite PVM/TVN proteins play a role in recruitment of these vesicles. We tested recombinant parasite lines deficient for the proteins UIS4 [START_REF] Mueller | Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface[END_REF] and IBIS1 [START_REF] Ingmundson | The exported Plasmodium berghei protein IBIS1 delineates membranous structures in infected red blood cells[END_REF] for their capacity to recruit late endocytic compartments (Figure 1, A andB). Both UIS4 and IBIS1 are transmembrane P. berghei-encoded proteins that localize to the liver-stage PVM/TVN. Although absence of IBIS1 does not appear to affect liver-stage growth [START_REF] Ingmundson | The exported Plasmodium berghei protein IBIS1 delineates membranous structures in infected red blood cells[END_REF]; Supplemental Figure S2A), infection with uis4 -parasites results in reduced parasite burden in the liver and liver-stage arrest [START_REF] Mueller | Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface[END_REF]; Supplemental Figure S2A). In vitro, uis4 -parasites are less likely to successfully develop into liver-stage forms [START_REF] Mueller | Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface[END_REF]; Supplemental Figure S2B), although those that do develop are approximately the same size as wild-type parasites (Supplemental Figure S2C). We confirmed the absence of UIS4 in these parasites with antibody staining of infected cells (Supplemental Figure S2D).

Although ibis1 -parasites recruited LEs at a frequency comparable to wild-type parasites, noticeably fewer uis4 -parasites were surrounded by CD63-positive vacuoles 24 h after infection. By assessing of the PV, and the PV does not have the acidity of lysosomes (Lopes da [START_REF] Da Silva | The host endocytic pathway is essential for Plasmodium berghei late liver stage development[END_REF][START_REF] Grützke | The spatiotemporal dynamics and membranous features of the Plasmodium liver stage tubovesicular network[END_REF][START_REF] Prado | Longterm live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms[END_REF].

The PV interacts directly with the host autophagy pathway, as seen by the recruitment of the autophagy protein LC3 to the developing parasite [START_REF] Grützke | The spatiotemporal dynamics and membranous features of the Plasmodium liver stage tubovesicular network[END_REF][START_REF] Prado | Longterm live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms[END_REF][START_REF] Thieleke-Matos | Host cell autophagy contributes to Plasmodium liver development[END_REF]. Typically, autophagosome maturation results in fusion with lysosomes, and some of the lysosomes associated with the PV also possess the autophagy marker LC3; however, the PV of properly developing parasites does not appear to mature as an autophagosome beyond LC3 recruitment, and further maturation seems detrimental to the parasites [START_REF] Prado | Longterm live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms[END_REF]. Some of the late endocytic vesicles surrounding the PV do possess both LC3 and lysosomal markers, indicating that some of these associated vesicles are maturing amphisomes [START_REF] Thieleke-Matos | Host cell autophagy contributes to Plasmodium liver development[END_REF]. Parasite-encoded proteins within the liver-stage PVM and TVN are at the interface between the parasite and host and likely play a critical role in the interaction with the host cell and its organelles. For instance, the liver stage-specific protein up-regulated in infective sporozoites 4 (UIS4) delineates the PVM, and reverse genetics established the necessity of this PVM protein for parasite maturation inside the liver [START_REF] Mueller | Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface[END_REF], but the underlying molecular mechanism that leads to growth restriction in the absence of UIS4 remains enigmatic.

To elucidate the function and mechanism of late endocytic organelle recruitment to liver stage parasites, we assessed this recruitment upon infection with parasites deficient for specific parasite LE recruitment to parasites over time from soon after host cell invasion until the beginning of merosome development, we saw that the percentage of uis4 -parasites that recruit LEs was at least 34% lower than that of wild-type parasites throughout infection (Figure 1C). The same pattern was observed in uis4 --infected cells stained with the LE and lysosome marker LAMP2 (Figure 1C).

Other known host components that have been shown to be associated with the liver-stage PVM are cholesterol and the autophagy protein LC3 [START_REF] Labaied | Plasmodium salvages cholesterol internalized by LDL and synthesized de novo in the liver[END_REF][START_REF] Grützke | The spatiotemporal dynamics and membranous features of the Plasmodium liver stage tubovesicular network[END_REF][START_REF] Prado | Longterm live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms[END_REF]. As previously reported, membranes surrounding liver-stage parasites are enriched with cholesterol, as detected by filipin staining [START_REF] Labaied | Plasmodium salvages cholesterol internalized by LDL and synthesized de novo in the liver[END_REF]. When cells are infected with uis4 -parasites, this enrichment of the PVM with cholesterol is detected less frequently (Figure 1, A andC), and when it is detected, the intensity of filipin staining surrounding parasites is typically less than around wild-type parasites (Supplemental Figure S2, E andF).

Furthermore, UIS4-deficient parasites recruit LC3 less efficiently than wild-type parasites when LC3 localization is examined over the course of infection (Figure 1C and Supplemental Figure S2G). Autophagosome maturation is associated with lysosome fusion. Therefore we assessed whether recruitment of lysosomes to the PVM depends on LC3 recruitment by examining recruitment of LAMP2positive vesicles to liver-stage parasites in Atg5 -/-cells, which are not capable of LC3 processing and lipidation [START_REF] Kuma | The role of autophagy during the early neonatal starvation period[END_REF] and do not allow LC3 recruitment to developing parasites [START_REF] Prado | Longterm live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms[END_REF]. Lysosome accumulation around liver-stage parasites does not depend on LC3 recruitment: no significant difference in LAMP2-vesicle association to parasites was seen in Atg5 -/-cells compared with wild-type cells (Figure 1D). Therefore absence of LC3 around uis4 -parasites is not directly responsible for the lack of recruited late endocytic vesicles. These results demonstrate that the absence of UIS4 on the PVM negatively affects the recruitment of host lysosomes and host cholesterol during liver-stage development. Although this result might be an indirect consequence of incomplete vacuole development of uis4 -parasites rather than a direct interaction of UIS4 with the host endocytic pathway, the correlation between arrested growth and reduced LE and lysosome recruitment in uis4 -parasites indicates that the recruitment of host late endocytic compartments signifies proper liver-stage development and might be of benefit to developing parasites.

Cells deficient for LAMP or NPC proteins fail to support efficient liver-stage parasite development

To begin to ascertain which host lyososomal functions could contribute to liver-stage parasite development, we infected embryonic fibroblasts (MEFs) derived from knockout mouse lines lacking specific lysosomal proteins [START_REF] Scimeca | The gene encoding the mouse homologue of the human osteoclast-specific 116-kDa V-ATPase subunit bears a deletion in osteosclerotic (oc/oc) mutants[END_REF][START_REF] Eskelinen | Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/ LAMP-2 double-deficient fibroblasts[END_REF][START_REF] Huang | Potential pitfalls and solutions for use of fluorescent fusion proteins to study the lysosome[END_REF][START_REF] Kissing | Vacuolar ATPase in phagosome-lysosome fusion[END_REF]. Because mouse embryonic fibroblasts are not a natural target cell for Plasmodium sporozoite infection, we wanted to ensure that these cells support normal parasite development. A comparison of parasite growth in hepatoma (Huh7) and wild-type MEFs showed that, although parasite numbers are in general lower in MEFs (unpublished data), once sporozoites establish infection, they develop into similarly sized liver-stage forms as in Huh7 cells (Supplemental Figure S3A). These data, together with successful merosome production (see Figure 3B later in this article), suggest that MEFs are a suitable model cell line for Plasmodium infection and development. MEFs deficient for the vacuolar ATPase subunit V 0 A3 [START_REF] Scimeca | The gene encoding the mouse homologue of the human osteoclast-specific 116-kDa V-ATPase subunit bears a deletion in osteosclerotic (oc/oc) mutants[END_REF] or V-ATPase accessory protein ATP6AP2 [START_REF] Kissing | Vacuolar ATPase in phagosome-lysosome fusion[END_REF] support P. berghei liver-stage growth as well as the comparable wild-type cell lines (Figure 2, A andB). These findings indicate that these proteins are dispensable embryos, they are prone to clonal differences. To further support our findings, we tested P. berghei growth in a second, independently generated LAMP1/LAMP2-deficient cell line. Parasite growth was similarly restricted in this second clone in comparison to the congenic wild-type line (Supplemental Figure S3, B andC).

Of note, the percentage of infected cells differed between wild-type and knockout MEF in the two LAMP1/LAMP2-deficient cell lines. Lamp1 -/-Lamp2 -/-cells of clone 1, but not clone 2, harbored more parasites after infection than wild-type control cell lines (unpublished data), indicating that infection rates can differ between MEF lines but cannot be attributed to loss of LAMP1 and LAMP2.

The impaired parasite growth in Lamp1 -/- Lamp2 -/-cells led to reduced production of merosomes (Figure 3B), the final stage of liver development, in which parasites are released [START_REF] Sturm | Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids[END_REF]. Merosome numbers were reduced by >60% in knockout cells compared with wild-type MEFs. Because of the higher number of infected cells in Lamp1 -/-Lamp2 -/-cells, these results indicate that formation of first-generation merozoites is severely impaired in cells lacking LAMP1 and LAMP2.

To confirm independently the data we generated in the Lamp1 -/-Lamp2 -/-MEFs and address whether both LAMP proteins are similarly important for parasite development, we used small interfering RNA (siRNA) to target LAMP1 and LAMP2 expression individually. Gene expression was targeted with at least three distinct siRNA sequences (A-C) against either Lamp1 or Lamp2, and each siRNA was transfected individually into hepatoma cells. Using antibodies against LAMP1 or LAMP2, we confirmed efficient knockdown for all three siRNAs targeting Lamp1 and for siRNA A and B but not siRNA C targeting Lamp2 (Figure 3C). When the cells were infected, only siRNAs that reduced protein expression of LAMP2 caused significant reductions in parasite size (Figure 3D). Despite efficient knockdown of LAMP1, no effect on parasite development was observed. When LAMP2 knockdown was not achieved (siRNA C), parasite size was comparable to that for infected control cells. Combining LAMP1 knockdown with LAMP2 knockdown did not further enhance the phenotype (unpublished data). We conclude that LAMP2 expression is crucial for efficient parasite liver-stage development.

Sequestration of cholesterol in late endocytic vesicles restricts liver-stage growth

To validate the parasite growth phenotype observed in NPC-and LAMP-deficient cells, we used the amphipathic steroid 3-b-[2-(diethylamine)eth-oxy]androst-5-en-17-one (U18666A), which is well established to mimic the cellular phenotype caused by Npc mutations [START_REF] Cenedella | Cholesterol synthesis inhibitor U18666A and the role of sterol metabolism and trafficking in numerous pathophysiological processes[END_REF][START_REF] Lu | Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection[END_REF]. U18666A blocks the exit of low-density lipoprotein (LDL)-derived free cholesterol from for liver-stage maturation. In contrast, parasites developing within cells deficient for the lysosomal glycoproteins LAMP1 and LAMP2 were significantly smaller 24 h after sporozoite infection than those growing in wild-type cells (Figure 2C). Parasite growth was similarly restricted in cells lacking the NPC1 and NPC2; Figure 2D).

One phenotype shared by both cell lines in which reduced parasite growth was observed is the accumulation of cholesterol in LEs [START_REF] Liscum | Niemann-Pick type C mutations cause lipid traffic jam[END_REF][START_REF] Eskelinen | Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/ LAMP-2 double-deficient fibroblasts[END_REF]. One important function of late endocytic organelles lies in the processing and sorting of exogenously derived cholesterol, and deletion of Npc1, Npc2, or Lamp1 and Lamp2 blocks the export of free cholesterol from LEs [START_REF] Brasaemle | Rapid intracellular transport of LDL-derived cholesterol to the plasma membrane in cultured fibroblasts[END_REF][START_REF] Liscum | Niemann-Pick type C mutations cause lipid traffic jam[END_REF][START_REF] Eskelinen | Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/ LAMP-2 double-deficient fibroblasts[END_REF].

To determine the effect of cholesterol retention in LEs on infection outcome, we followed infection of Lamp1 -/-Lamp2 -/-cells until parasite egress. At 48 h after infection, the difference in parasite size between control and knockout cell lines was even more severe than at 24 h (Figure 3A). Parasites in control cells increased in size by more than sevenfold from 24 to 48 h, whereas parasites in LAMP1/ LAMP2-deficient cells increased by only approximately fourfold. Because MEF cell lines are derived from individual embyronic day 12.5 detected by filipin, accumulates in enlarged intracellular vacuoles (Figure 5A and Supplemental Figure S4C). With the addition of 1 mM MβCD, lysosomal morphology was restored, and filipin labeled the plasma membrane, similar to control cells. Addition of MβCD also rescued the effect on parasite size caused by U18666A (Figure 5B). Parasites developing in cells treated with both U18666A and MβCD were significantly larger than those growing in cells treated with U18666A alone. MβCD treatment alone does not alter parasite growth, indicating that the drug at this concentration has no effect on parasite development.

Together these data demonstrate that cholesterol accumulation in host LEs and lysosomes impairs parasite growth and indicate that the export of cholesterol from the late endocytic compartments is needed for normal P. berghei development.

The defect in autophagic flux in U18666A-treated or LAMP1/LAMP2-deficient cells is not likely responsible for restricted parasite development

In addition to their effect on cholesterol export from LEs and lysosomes, Npc mutations, U18666A treatment, or LAMP1/LAMP2-deficiencies cause an increase in the steady-state number of LC3-II-positive autophagosomes due to a defect in amphisome formation [START_REF] Tanaka | Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice[END_REF][START_REF] Eskelinen | Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/ LAMP-2 double-deficient fibroblasts[END_REF][START_REF] Ishibashi | Association of autophagy with cholesterol-accumulated compartments in Niemann-Pick disease type C cells[END_REF][START_REF] Sarkar | Impaired autophagy in the lipid-storage disorder Niemann-Pick type C1 disease[END_REF][START_REF] Maetzel | Genetic and chemical correction of cholesterol accumulation and impaired autophagy in hepatic and neural cells derived from Niemann-Pick Type C patient-specific iPS cells[END_REF]. A similar accumulation of LC3-positive membranes can be observed in cells treated with 1 mM MβCD [START_REF] Cheng | Cholesterol depletion induces autophagy[END_REF]Supplemental Figure S4B). P. berghei liver-stage parasites growing in cells treated with MβCD showed no defect in development, suggesting that accumulation of autophagic bodies alone does not disturb parasite growth. Recently it was shown that LC3 is recruited to developing liver-stage parasites [START_REF] Grützke | The spatiotemporal dynamics and membranous features of the Plasmodium liver stage tubovesicular network[END_REF][START_REF] Prado | Longterm live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms[END_REF][START_REF] Boonhok | LAP-like process as an immune mechanism downstream of IFN-gamma in control of the human malaria Plasmodium vivax liver stage[END_REF][START_REF] Thieleke-Matos | Host cell autophagy contributes to Plasmodium liver development[END_REF]. Published data also indicated that early recruitment of autophagy markers promotes successful liver-stage development [START_REF] Prado | Longterm live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms[END_REF]. To examine further whether the impaired autophagic flux seen in cells with impaired cholesterol transport could influence parasite growth, we monitored the recruitment of LC3 to developing liver-stage parasites over time in cells treated with U18666A. We saw no change in the recruitment of LC3 to liver-stage parasitophorous vacuoles in U18666A-treated Huh7 cells and only a very small difference in LAMP1/LAMP2-deficient MEFs compared with control cells (Supplemental Figure S4, D-F). Thus we have no evidence that the accumulation of autophagic bodies in the knockout and drug-treated cells alters the manner in which the parasite vacuole interacts with the host autophagy pathway or is responsible for the observed impairment of parasite growth.

Block of cholesterol export from late endocytic organelles affects liver-stage development and time to blood-stage patency in vivo

We sought to validate the in vitro data indicating the detrimental effect of cholesterol sequestration in LEs and lysosomes on P. berghei infection in vivo. To this end, we administered U18666A to C57BL/6 mice daily for 15 d, and after drug treatment on day 15, we intravenously infected the mice with P. berghei sporozoites (Figure 6A). Blood-stage parasites were detected 3 d postinfection LEs and causes its accumulation in these compartments [START_REF] Liscum | The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one[END_REF][START_REF] Roff | Type C Niemann-Pick disease: use of hydrophobic amines to study defective cholesterol transport[END_REF]. P. berghei growing in U18666Atreated Huh7 cells reached ∼50% the size of parasites growing in control cells at 24 h postinfection (Figure 4A). This phenotype was confirmed in primary murine hepatocytes treated with U18666A (Figure 4A). To test potential activity of the drug against the parasite, we pretreated sporozoites with U18666A under multiple conditions before infection (Supplemental Figure S4A). This treatment caused no difference in liver-stage development compared with development of phosphate-buffered saline (PBS)-treated control sporozoites, which is consistent with the assumption that the effect of the drug on infection is due to disruption specifically of the host cell physiology.

We predict that the defects in parasite development in the LAMP1/LAMP2-deficient cells and the U18666A-treated cells is due to inhibition of the same host cellular pathways in both conditions. If this assumption is true, combining these two conditions through treatment of cells deficient in LAMP1 and LAMP2 with U18666A should not have an additive effect and lead to further reduced parasite growth. When this was tested, indeed, U18666A treatment of Lamp1 -/-Lamp2 -/-cells resulted in parasite growth that was similar to that seen in untreated Lamp1 -/-L amp2 -/-cells or U18666A-treated wild-type cells (Figure 4B). These results are consistent with the hypothesis that LAMP deficiency and U18666A treatment disrupt a common cellular pathway important for parasite development, which further confirms that the effect of U18666A on infection is due to modulation of the host cell.

If the defect in parasite development upon U18666A treatment is due to the sequestration of cholesterol in LEs and lysosomes, we could predict, on the basis of previous studies [START_REF] Rosenbaum | Endocytosis of beta-cyclodextrins is responsible for cholesterol reduction in Niemann-Pick type C mutant cells[END_REF], that low concentrations of methyl-β-cyclodextrin (MβCD) should release the accumulated cholesterol and therefore restore parasite growth. Cyclodextrins are water-soluble compounds that can release cholesterol from membranes [START_REF] Yancey | Cellular cholesterol efflux mediated by cyclodextrins. Demonstration of kinetic pools and mechanism of efflux[END_REF][START_REF] Atger | Cyclodextrins as catalysts for the removal of cholesterol from macrophage foam cells[END_REF], and MβCD efficiently reduces cholesterol accumulation in late endocytic compartments of cells with Npc mutations [START_REF] Camargo | Cyclodextrins in the treatment of a mouse model of Niemann-Pick C disease[END_REF][START_REF] Lope-Piedrafita | MRI detects therapeutic effects in weanling Niemann-Pick type C mice[END_REF][START_REF] Liu | Reversal of defective lysosomal transport in NPC disease ameliorates liver dysfunction and neurodegeneration in the npc1-/-mouse[END_REF]. In cells treated with U18666A alone, LAMP2-positive compartments are enlarged (Supplemental Figure S4B), and cholesterol, as drug-treated animals showed a strong filipin signal and a clear increase in cholesterol accumulation in liver tissue. These data demonstrate that functional cholesterol transport in host cells is required for efficient liver-stage growth of P. berghei also in vivo.

DISCUSSION

LEs and lysosomes fulfill many different functions in the endocytic network. The highly acidic vesicles are part of the cells' antimicrobial defense mechanism, and their fusion with the PVM can lead to parasite death [START_REF] Prado | Longterm live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms[END_REF][START_REF] Boonhok | LAP-like process as an immune mechanism downstream of IFN-gamma in control of the human malaria Plasmodium vivax liver stage[END_REF]. However, late endocytic vesicles are also a distribution point of substances taken up by the cells and could therefore serve as a nutrient source. During their development, liver-stage Plasmodium parasites undergo a striking size increase, for which they require large amounts of nutrients, which they likely acquire from the nutrient-rich environment in the hepatocyte; however, our knowledge of the mechanisms by which the parasites acquire these resources from host cells is limited.

When we began our studies, the potential benefit of lysosomal recruitment to liver-stage parasites as a source of nutrients had been proposed (Lopes da [START_REF] Da Silva | The host endocytic pathway is essential for Plasmodium berghei late liver stage development[END_REF]. However, this suggestion was based on the use of chemical agents that deacidify the lysosomes of not only the host cell, but also of the parasite, making interpretation of these data difficult. Recently it was shown that generation of phosphatidylinositol 3,5-bisphosphate by PIKfyve at endosomal membranes is required for efficient liver-stage parasite growth, which demonstrated that functions occurring at the host late endocytic compartments are important for parasite growth [START_REF] Thieleke-Matos | Host PI(3,5)P2 activity is required for Plasmodium berghei growth during liver stage infection[END_REF]. Here we found that LEs/lysosomes are not efficiently recruited to liver-stage-arrested uis4 -parasites and that perturbing host LE/lysosome function reduced parasite burden. Together these results further support the hypothesis that LE/lysosome recruitment indicates proper PV maturation and may support parasite development.

What drives the association between late endocytic compartments and the liver-stage PV is unknown. The presence of the autophagy protein LC3 in the PVM may contribute to lysosomal recruitment; however, because we showed that lysosomes are still recruited to the PV in ATG5-deficient cells, the interaction with the host autophagic pathway cannot be the sole factor responsible for lysosomal recruitment. Reduced recruitment of LEs/lysosomes to PVs containing uis4 -parasites could be due to direct interaction between UIS4 and LE/lysosome membranes or proteins, or the cause could be indirect and instead due to improper maturation of the vacuole in the absence of UIS4. In either case, poor liver-stage growth, which has been demonstrated for uis4 -parasites [START_REF] Mueller | Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface[END_REF], coincides with reduced recruitment of host LC3, LEs and lysosomes, and cholesterol. Although several PVM proteins have been shown to be important for parasite development, to our knowledge, this is the first example in which the absence of a parasite protein is shown to alter the interaction between the PV and host cell. These results demonstrate that perturbing the protein composition of the PVM negatively affects the recruitment of host LEs during liver-stage development and suggest that accumulation of these organelles is a normal part of PV maturation.

To investigate whether host lysosomal functions contribute to liver-stage Plasmodium development, we assessed parasite growth in MEFs deficient for specific LE/lysosome proteins. Whereas ATP6V0A3 and ATP6AP2 are associated with the vacuolar-ATPase, which is responsible for lysosomal acidification, lysosomes remain acidified in both of these knockout lines [START_REF] Kissing | Vacuolar ATPase in phagosome-lysosome fusion[END_REF]. Therefore, on the basis of our observation that cells deficient for in most control animals. However, U18666A-treated mice displayed a 2-to 3-d delay in patency, with blood-stage infection becoming apparent in most animals 5-6 d postinfection. This delay is unlikely to be due to inhibition of blood-stage parasite replication because subsequent blood-stage replication proceeded normally in both groups (Supplemental Figure S5). To ensure that the delay in patency was due to liver-stage development, we assessed the parasite burden in the livers of infected animals with a quantitative-PCR-based assay 42 h after infection (Figure 6B). Thus administration of U18666A resulted in a significant reduction of liver-stage parasite load and increased time to the appearance of P. berghei in the blood.

To confirm that U18666A influenced cholesterol transport in livers of treated mice, we visualized cholesterol in liver sections by filipin staining and fluorescence microscopy (Figure 6C). Whereas in control livers, filipin intensities were weak and only a few areas with cholesterol accumulation could be detected, liver sections from Coxiella burnetii-containing phagosomes [START_REF] Schulze-Luehrmann | LAMP proteins account for the maturation delay during the establishment of the Coxiella burnetii-containing vacuole[END_REF], and are less permissive to Trypanosoma cruzi invasion [START_REF] Albertti | Role of host lysosomal associated membrane protein (LAMP) in Trypanosoma cruzi invasion and intracellular development[END_REF].

Autophagic flux is altered in cells deficient for the LAMPs or NPC proteins and in cells treated with U18666A [START_REF] Tanaka | Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice[END_REF][START_REF] Eskelinen | Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/ LAMP-2 double-deficient fibroblasts[END_REF][START_REF] Ishibashi | Association of autophagy with cholesterol-accumulated compartments in Niemann-Pick disease type C cells[END_REF][START_REF] Sarkar | Impaired autophagy in the lipid-storage disorder Niemann-Pick type C1 disease[END_REF][START_REF] Maetzel | Genetic and chemical correction of cholesterol accumulation and impaired autophagy in hepatic and neural cells derived from Niemann-Pick Type C patient-specific iPS cells[END_REF]. These cells display a defect in amphisome formation, which increases the number of steady-state autophagic bodies. Because the liver-stage PV is known to interact with the autophagy pathway [START_REF] Grützke | The spatiotemporal dynamics and membranous features of the Plasmodium liver stage tubovesicular network[END_REF][START_REF] Prado | Longterm live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms[END_REF][START_REF] Thieleke-Matos | Host cell autophagy contributes to Plasmodium liver development[END_REF], we initially hypothesized that defects in parasite growth in these conditions relate to changes in host autophagosome maturation. However, parasites grow robustly in cells treated with 1 mM MβCD, which leads to a similar accumulation of autophagic bodies as seen in the aforementioned conditions. Furthermore, LC3 recruitment to liver-stage parasites is unchanged in U18666A-treated cells and barely reduced in LAMP-deficient cells. Thus, although we cannot rule out the possibility that alterations in autophagic maturation contribute to the parasite growth phenotypes we describe here, we have no evidence that autophagy is responsible for the P. berghei growth phenotypes.

The other shared phenotype caused by Npc mutations, Lamp mutations, and U18666A treatment is the sequestration of cholesterol in late endocytic compartments. Cells can acquire cholesterol via two pathways: by uptake of LDL, which is hydrolyzed along the endocytic pathway and released from LEs to allow transport to other membranes in the cell, or via synthesis by the mevalonate pathway in the endoplasmic reticulum. In any of the conditions we tested in which cholesterol export from lysosomes is blocked, parasite growth was inhibited. We were able to validate these results in vivo by infecting mice treated with U18666A. Of note, we could rescue the in vitro phenotype with MβCD. Cyclodextrins bind cholesterol and can extract it from membranes [START_REF] Christian | Use of cyclodextrins for manipulating cellular cholesterol content[END_REF], and MβCD reduces cholesterol accumulation in late endocytic compartments of NPC-deficient cells and U18666A-treated cells and rescues the NPC-associated phenotype [START_REF] Camargo | Cyclodextrins in the treatment of a mouse model of Niemann-Pick C disease[END_REF][START_REF] Lope-Piedrafita | MRI detects therapeutic effects in weanling Niemann-Pick type C mice[END_REF][START_REF] Liu | Reversal of defective lysosomal transport in NPC disease ameliorates liver dysfunction and neurodegeneration in the npc1-/-mouse[END_REF]. These rescue experiments are performed using 1 mM MβCD, which is 5-10 times less than the concentration typically used to deplete cholesterol from membranes. MβCD rescues the U18666A-induced phenotype by acting from within LEs [START_REF] Abi-Mosleh | Cyclodextrin overcomes deficient lysosome-to-endoplasmic reticulum transport of cholesterol in Niemann-Pick type C cells[END_REF][START_REF] Rosenbaum | Endocytosis of beta-cyclodextrins is responsible for cholesterol reduction in Niemann-Pick type C mutant cells[END_REF], and an increase in esterified cholesterol has been detected in this context, indicating that this concentration of MβCD does not just remove cholesterol but makes it available for transport to the ER, the site of cholesterol esterification [START_REF] Rosenbaum | Endocytosis of beta-cyclodextrins is responsible for cholesterol reduction in Niemann-Pick type C mutant cells[END_REF]. It is therefore tempting to speculate that liver-stage Plasmodium grows poorly when cholesterol is sequestered in LEs because the parasite has reduced access to exogenously acquired cholesterol. Hepatocytes can compensate for deficits in cholesterol uptake through cholesterol synthesis [START_REF] Rudney | Regulation of cholesterol biosynthesis[END_REF]), but it is not sufficient to rescue the parasite growth phenotype.

Previous studies showed that host cholesterol labels the liverstage PVM and that both exogenous and endogenous sources contribute to the cholesterol in these membranes [START_REF] Labaied | Plasmodium salvages cholesterol internalized by LDL and synthesized de novo in the liver[END_REF].

ATP6V0A3 or ATP6AP2 support efficient parasite growth, we cannot claim that acidification of host lysosomes is dispensable for parasite development, but only that these proteins are not required for parasite development. In contrast, cells deficient for NPC1, NPC2, LAMP1, or LAMP2 did not support proficient P. berghei growth in comparison to wild-type cells.

NPC2 is a small soluble protein in the LE/lysosome lumen, and NPC1 is a large membrane glycoprotein in the outer LE/lysosome membrane [START_REF] Higgins | Niemann-Pick C1 is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network[END_REF][START_REF] Naureckiene | Identification of HE1 as the second gene of Niemann-Pick C disease[END_REF]. The NPC proteins actively participate in cholesterol transport [START_REF] Vanier | Structure and function of the NPC2 protein[END_REF], and mutation in either of these two genes leads to the lysosomal storage disease Niemann-Pick disease type C [START_REF] Sokol | Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol[END_REF]. Other lipids, for example, sphingolipids, also accumulate in LEs/lysosomes of cells containing Npc mutations, but this is likely a consequence of the cholesterol sequestration (reviewed in [START_REF] Vance | Niemann-Pick C disease and mobilization of lysosomal cholesterol by cyclodextrin[END_REF]. Further, whereas sphingolipid levels are elevated in cells with Npc mutations [START_REF] Chang | Niemann-Pick type C disease and intracellular cholesterol trafficking[END_REF], sphingolipid levels in LAMP1/LAMP2 doubly deficient cells appear comparable to those in wild-type cells [START_REF] Eskelinen | Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/ LAMP-2 double-deficient fibroblasts[END_REF].

In infection studies, host NPC function has been found to be crucial for the filoviruses Marburg and Ebola [START_REF] Carette | Ebola virus entry requires the cholesterol transporter Niemann-Pick C1[END_REF], as well as for HIV-1 infectivity and production [START_REF] Tang | Deficiency of niemann-pick type C-1 protein impairs release of human immunodeficiency virus type 1 and results in Gag accumulation in late endosomal/ lysosomal compartments[END_REF][START_REF] Coleman | Loss of Niemann Pick type C proteins 1 and 2 greatly enhances HIV infectivity and is associated with accumulation of HIV Gag and cholesterol in late endosomes/lysosomes[END_REF]. Furthermore, the replication rate of Toxoplasma gondii is reduced in NPC-deficient cells [START_REF] Coppens | Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition[END_REF].

LAMP1 and LAMP2 are the most abundant proteins in mammalian lysosomes. The exact function of these proteins is unknown, but characterization of cells and animals deficient for these proteins reveals their necessity for proper autophagosome maturation [START_REF] Tanaka | Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice[END_REF], phagosome maturation [START_REF] Huynh | LAMP proteins are required for fusion of lysosomes with phagosomes[END_REF], and cholesterol egress from late endocytic compartments [START_REF] Eskelinen | Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/ LAMP-2 double-deficient fibroblasts[END_REF]. These studies found that deletion of Lamp1 alone is compensated for by increased expression of LAMP2 and therefore results in no severe phenotypes. In contrast, LAMP1 is not able to fully compensate functionally for the loss of LAMP2. Accordingly, knockdown of Lamp1 did not affect Plasmodium infection, whereas knockdown of Lamp2 resulted in smaller developing parasites.

Previous infection studies in cells deficient for the LAMPs used pathogens that reside within LAMP-positive compartments during their development. LAMP1/LAMP2-deficient cells fail to kill Neisseria gonorrhoeae [START_REF] Binker | Arrested maturation of Neisseria-containing phagosomes in the absence of the lysosome-associated membrane proteins, LAMP-1 and LAMP-2[END_REF], harbor faster-maturing, smaller and hepatocyte wash medium (Life Technologies) and plated on collagen-coated dishes in William's E Medium supplemented with 10% fetal bovine serum and penicillin-streptomycin (Life Technologies). Wild-type P. berghei were either ANKA clone 507 [START_REF] Janse | High efficiency transfection of Plasmodium berghei facilitates novel selection procedures[END_REF] or ANKA Bergreen [START_REF] Kooij | Expansion of experimental genetics approaches for Plasmodium berghei with versatile transfection vectors[END_REF], both of which express green fluorescent protein (GFP). The ibis1 -P. berghei parasite line was described previously [START_REF] Ingmundson | The exported Plasmodium berghei protein IBIS1 delineates membranous structures in infected red blood cells[END_REF]. The uis4 -ANKA P. berghei was generated in clone 507 as described [START_REF] Mueller | Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface[END_REF]. Anopheles stephensi mosquitoes were raised under a 14-h light/10-h dark cycle at 28°C and 80% humidity and were fed daily on 10% sucrose.

In vitro infections with P. berghei Huh7 cells, MEFs, and primary hepatocytes seeded in collagencoated 96-well µclear plates (Greiner) were infected with P. berghei sporozoites as previously described [START_REF] Silvie | A sporozoite asparagine-rich protein controls initiation of Plasmodium liver stage development[END_REF]. When required, cells were treated as indicated in the figure legends with 3 µM U18666A (Enzo Life Sciences; [START_REF] Liscum | The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one[END_REF]) and 1 µM MβCD (Sigma-Aldrich). Because the drugs are reversible [START_REF] Härmälä | Cholesterol transport from plasma membranes to intracellular membranes is inhibited by 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one[END_REF][START_REF] Rodal | Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles[END_REF], they remained in the medium throughout infection. For merosome production, infection proceeded for 70 h; merosomes in the culture medium were harvested and counted in a Neubauer chamber.

Indirect immunofluorescence analysis

At the indicated time points, samples were fixed with 4% paraformaldehyde in PBS and permeabilized with either 100% ice-cold methanol (for the LC3 antibody) or 0.3% Triton X-100 (all other antibodies) and labeled with the following primary antibodies: polyclonal chicken anti-GFP (1:1000; ab13970; Abcam), monoclonal mouse anti-Pb-Hsp70 [START_REF] Potocnjak | Monovalent fragments (Fab) of monoclonal antibodies to a sporozoite surface antigen (Pb44) protect mice against malarial infection[END_REF], polyclonal rabbit anti-UIS4 [START_REF] Müller | The Puf-family RNA-binding protein Puf2 controls sporozoite conversion to liver stages in the malaria parasite[END_REF], monoclonal mouse anti-LC3 (1:100; 5F10; Nanotools), polyclonal rabbit anti-EEA1 (1:500; ab2900; Abcam), monoclonal mouse anti-CD63 (1:100; CBL553; Merck Millipore), and mouse anti-LAMP2 (1:500; H4B4; Developmental Studies Hybridoma Bank) followed by Alexa Fluor-conjugated secondary antibodies (Invitrogen) and Hoechst 33342 (Invitrogen). When required, filipin staining was carried out before permeabilization using 0.025 mg/ml of filipin III (Sigma-Aldrich) in PBS for 1 h at room temperature.

Fluorescence microscopy, parasite quantification, and quantification of LE/lysosome recruitment Fluorescent samples were analyzed using a Zeiss Axio Observer.Z1 microscope (Zeiss) equipped with 10, 40, and 63×/1.2 oil immersion lens, and pictures were collected with an AxioCam MRm (Zeiss) and Zeiss AxioVision software (Zeiss). Images were processed in Fiji [START_REF] Schindelin | Fiji: an opensource platform for biological-image analysis[END_REF] or Adobe Photoshop.

The P. berghei liver-stage size (in micrometers squared) was measured in Fiji by identifying and measuring areas of fluorescence signal using defined threshold levels. The identified fluorescent areas were used to measure parasite area. Quantification of LE/lysosome recruitment was performed visually on stained samples. Parasites were considered to recruit LEs/lysosomes when a continuous portion of the parasite periphery comprising at least 30% of the parasite circumference had closely associated LAMP2-or CD63-positive vesicles. If <30% of the parasite was covered with these signals, the parasite was considered to not recruit these vesicles.

siRNA knockdown

Huh7 cells were reverse transfected with target-specific or control siRNAs (Silencer Select; Life Technologies) according to the manufacturer's instructions. In brief, 24 h after cell seeding, siRNAs diluted Whereas cholesterol levels at the parasite PVM were shown to decrease when progesterone treatment blocked exogenous cholesterol trafficking, parasite development was not investigated in this context [START_REF] Labaied | Plasmodium salvages cholesterol internalized by LDL and synthesized de novo in the liver[END_REF]. Here we see that preventing release of late endocytic cholesterol has a negative effect on parasite development. These results could be due to blocked access to cholesterol by the parasite, which would indicate that blocking transfer of cholesterol out of late endosomes is more problematic for the parasite than temporarily depleting the source of exogenous cholesterol. Alterations in access to other late endocytic lipids could also contribute to the observed phenotypes. Cholesterol is not the only lipid species obtained by the parasite from the host cell. Acquisition of both lipoic acid [START_REF] Deschermeier | Mitochondrial lipoic acid scavenging is essential for Plasmodium berghei liver stage development[END_REF] and phosphatidylcholine [START_REF] Itoe | Host cell phosphatidylcholine is a key mediator of malaria parasite survival during liver stage infection[END_REF] from the host environment is needed for robust liver-stage parasite growth. The latter is present in LE membranes but also in all other cellular membranes, so it seems unlikely that LEs and lysosomes would be the exclusive source of this lipid. However, transfer of this or other lipid species from LEs and lysosomes to the parasite or PVM could be altered when the cholesterol content of LE and lysosomes is increased.

Taken together, our results show that specific targeting of host LE/lysosomal proteins can restrict liver-stage parasite development. Because interfering with the lipid homeostasis in LEs interferes with parasite development, we conclude that proper host LE function actively contributes to successful parasite growth. Although fusion of lysosomes with the PVM can be a successful host defense against the parasite, we and others have provided evidence that these host late endocytic compartments positively contribute to parasite growth and that their close association with the PV and potential fusion with the liver-stage TVN could provide a source of lipids for the parasite during liver-stage development. Successful liver-stage parasites appear to maintain a balance between exploiting these degradative host compartments and avoiding their antiparasitic potential.

MATERIALS AND METHODS

Experimental animals, cells, and parasite lines

Female C57BL/6 and NMRI mice were obtained from Charles River Laboratories and were 6-12 wk of age at the time of experiments. All animal work was conducted in accordance with the German Tierschutzgesetz in der Fassung vom 18. Mai 2006 (BGBl. I S. 1207), which implements the Directive 86/609/EEC from the European Union and the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes. All appropriate measures were taken to reduce the pain or discomfort of the animals, and the protocol was approved by the ethics committee of the Max Planck Institute for Infection Biology and the Berlin state authorities (LAGeSo Reg# G0469/09 and G0294/15). The hepatoma cells used in this study were Huh7 cells cultured in RPMI supplemented with 10% fetal calf serum (FCS), 100 U penicillin, 100 µg/ml streptomycin, 2 mM l-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 1× nonessential amino acids (Life Technologies). MEFs including wild type, Npc -/- [START_REF] Huang | Potential pitfalls and solutions for use of fluorescent fusion proteins to study the lysosome[END_REF] kindly provided by Peter Lobel, Rutgers University, New Brunswick, NJ), Lamp1/2 -/- [START_REF] Eskelinen | Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/ LAMP-2 double-deficient fibroblasts[END_REF], Atg5 -/- [START_REF] Kuma | The role of autophagy during the early neonatal starvation period[END_REF]; kindly provided by Ingo Schmitz, Helmholz Centre for Infection Research, Braunschweig, Germany), Atp6v0a3 -/-(derived from the TCIRG1 oc/oc line; [START_REF] Scimeca | The gene encoding the mouse homologue of the human osteoclast-specific 116-kDa V-ATPase subunit bears a deletion in osteosclerotic (oc/oc) mutants[END_REF][START_REF] Kissing | Vacuolar ATPase in phagosome-lysosome fusion[END_REF]; and Atp6ap2 -/-(derived from the Atp6ap2 conditional deletion line; [START_REF] Kissing | Vacuolar ATPase in phagosome-lysosome fusion[END_REF] were maintained in DMEM supplemented with 10% FCS, 100 U penicillin, and 100 µg/ml streptomycin. Primary murine hepatocytes were isolated from NMRI mice as described [START_REF] Renia | A malaria heat-shock-like determinant expressed on the infected hepatocyte surface is the target of antibody-dependent cell-mediated cytotoxic mechanisms by nonparenchymal liver cells[END_REF] using liver perfusion, liver digest, in Opti-MEM (Life Technologies) and Lipofectamine RNAiMAX transfection solution (Life Technologies) were added to cells. At 48 h after siRNA transfection, either knockdown efficiency was analyzed by Western blot using antibodies against LAMP1 (Abcam) and LAMP2 (Developmental Studies Hybridoma Bank) or cells were infected with P. berghei sporozoites.

Quantification of liver parasite load by quantitative PCR

Quantification of parasite burden in mouse liver after sporozoite infection into U18666A-or PBS-treated animals was performed as described [START_REF] Friesen | Natural immunization against malaria: causal prophylaxis with antibiotics[END_REF]. Briefly, C57Bl/6 mice were injected intraperitoneally with 10 mg/kg U18666A in PBS or PBS alone daily for 15 d. On day 15, animals were injected with 10 4 sporozoites intravenously. At 42 h after infection, livers of infected animals were harvested and homogenized in TRIzol (Life Technologies), total RNA was extracted, and cDNA was prepared using the RETROscript reverse transcription kit (Life Technologies). Relative parasite levels were determined with quantitative PCR by comparing the mean Ct value of the P. berghei 18s ribosomal subunit to the mean Ct value of the Mus musculus GAPDH. To assess time to patency and parasitemia, Giemsa-stained blood smears were examined microscopically daily starting at day 3 after infection.

Staining of liver tissue sections

Liver tissue sections were generated from U18666A-treated and control animals. At 42 h postinfection, mice were killed and perfused with PBS. Livers were removed and frozen in liquid nitrogen immediately. Thin liver sections (7 µm) were stained with filipin as described [START_REF] Vanier | Laboratory diagnosis of Niemann-Pick disease type C: the filipin staining test[END_REF]. Briefly, after fixing for 15 min in Formalin, sections were washed with PBS, and 0.05 mg/ml filipin was added to the sections for 1 h. Slides were mounted using ProLong Gold Antifade Mountant (Life Technologies) and examined using an Olympus BX50 microscope. Images were processed in Fiji [START_REF] Schindelin | Fiji: an opensource platform for biological-image analysis[END_REF].

Statistical analysis

Statistics for parasite size analysis are based on the average values of three wells, not on the sizes of the individual parasites. All experiments were carried out at least three times, with the exception of those in primary hepatocytes and in mice, which were both performed twice. In each figure, one representative experiment is shown. All statistical analyses were performed using Prism 5.0 for Mac OS X (GraphPad Software, San Diego, CA). p < 0.05 was considered to indicate statistical significance.

FIGURE 1 :

 1 FIGURE 1: UIS4 is required for maximal host LE and cholesterol recruitment to developing liver-stage P. berghei parasites. (A-C) Huh7 cells infected with GFP-expressing wild-type (WT), uis4 -, or ibis1 -parasites were fixed 24 h postinfection and stained with filipin (blue) and antibodies against CD63 (red) and GFP (green). Scale bars, 10 μm. (D) Huh7 cells were infected with P. berghei WT or mutant sporozoites, and infection was left to proceed until the indicated time points. Samples were stained with anti-GFP (parasite) and either anti-CD63 (LE/lysosomes; (top), anti-LAMP2 (LE/lysosomes; middle), or anti-LC3 (autophagic bodies; bottom). (E) WT and Atg5 -/-MEFs infected with P. berghei sporozoites were fixed 24 h postinfection and stained with antibodies against PbHsp70 (parasite) and LAMP2 (LE/lysosomes). At least 50 parasites per well were scored for host marker recruitment. Results are shown as a mean percentage of total parasites analyzed per well (±SD). One representative experiment of three performed is shown. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 (Fisher's exact test).

FIGURE 2 :

 2 FIGURE 2: Host proteins involved in lipid export from late endocytic vacuoles are important for P. berghei development. (A-D) MEF cell lines deficient for late endocytic/lysosomal host proteins and corresponding WT MEFs were infected with P. berghei sporozoites, and parasite size was assessed by fluorescence microscopy 24 h postinfection. In all experiments, parasites were visualized by staining with anti-PbHsp70 antibody. One representative experiment of at least three performed is shown. ns, not significant; **p < 0.01; ***p < 0.001 (Student's unpaired t test, n = 3).

FIGURE 3 :

 3 FIGURE 3: Host LAMP1 and LAMP2 are required for efficient P. berghei liver-stage development. (A, B) WT and Lamp1 -/-Lamp2 -/-MEFs were infected with P. berghei sporozoites. Parasite size was assessed 48 h postinfection (A; right, representative images of mean-sized parasites), and the total number of merosomes was counted 70 h postinfection (B). (C) Protein levels of LAMP1 or LAMP2 in Huh7 cells transfected with siRNAs targeting either Lamp1 or Lamp2 were evaluated by Western blotting 48 h after transfection. The effect of three independent siRNAs (A-C) targeting Lamp1 or Lamp2 were compared with a nonspecific negative control siRNA (neg) using antibodies against LAMP1 or LAMP2, respectively. (D) siRNA-transfected cells were infected with P. berghei sporozoites 48 h posttransfection, and parasite size was evaluated 48 h postinfection. Parasites were visualized with anti-PbHsp70 antibody. One representative of at least three independent experiments is shown. ns, not significant; * p < 0.05; ** p < 0.01 (Student's unpaired t test, n = 3).

FIGURE 4 :

 4 FIGURE 4: P. berghei development is impaired when host cell cholesterol export from late endocytic vacuoles is blocked. (A) Hepatoma cells and mouse primary hepatocytes or (B) WT and LAMP1/LAMP2-deficient MEFs were treated with 3 μM U18666A 12 h before and throughout infection with P. berghei sporozoites. Parasite size was assessed 24 h postinfection by fluorescence microscopy. Parasites were visualized by staining with anti-PbHsp70 antibody. One representative of at least three independent experiments is shown, with the exception of the experiment in primary hepatocytes, in which the combined results of two experiments are shown. ns, not significant; * p < 0.05, ** p < 0.01 (Student's unpaired t test, n = 3).

FIGURE 5 :

 5 FIGURE 5: Release of cholesterol from LE/lysosomes rescues P. berghei development in U18666A-treated cells. Huh7 cells were treated with 3 μM U18666A or 1 mM MβCD 36 h before infection. Where indicated, MβCD was added to U18666A-treated cells 12 h before infection. Cells were either fixed and stained with filipin (A; scale bars, 30 μm) or infected with P. berghei sporozoites and kept under continual presence of the drug until fixation 24 h postinfection (B). Parasites were visualized by staining with an anti-PbHsp70 antibody. Size was assessed, and the area of each measured parasite was plotted individually. One representative of three independent experiments is shown. * p < 0.05; ** p < 0.01; *** p < 0.001 (Student's unpaired t test, n = 3).

FIGURE 6 :

 6 FIGURE 6: Blocking cholesterol export from late endocytic organelles delays time to bloodstage patency and reduces liver-stage parasite burden in vivo. Mice were treated daily with 10 mg/kg U18666A (intraperitoneally) for 15 d before receiving an intravenous dose of 10,000 P. berghei sporozoites. (A) The prepatent period was followed by microscopic examination of Giemsa-stained blood smears. (B) Parasite burden in infected mouse livers at 42 h postinfection was quantified by quantitative PCR. Relative expression levels of the Pb18S gene were normalized to expression levels of the mouse GAPDH gene. Data from two biological replicates (n = 3 and 5) are displayed. *p < 0,05 (Student's unpaired t test; n = 8). (C) After treatment with U18666A for 15 d, mouse livers were removed and immediately frozen in liquid nitrogen. Sections (7 μm) were Formalin fixed, stained with filipin (0.05 mg/ml), and imaged under identical conditions.

ACKNOWLEDGMENTS

We thank Peter Lobel, Noboru Mizushima, Ingo Schmitz, Meryem Senkara, and Sandra Kissing for providing cell lines and reagents and Özlem Günay-Esiyok, Nishith Gupta, and Katja Müller for experimental assistance and advice. We also thank Carolin Rauch, Manuel Rauch, and Petra Matylewski for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SPP 1580; IN 182/1-1) and in part by the Max Planck Society and the European Commission through the EviMalaR network (#34).


domain: spi phys

text:
Maria-Elisavet Chatzitheodoridi
email: maria-elisavet.chatzitheodoridi@onera.fr

Abigael Taylor
email: abigael.taylor@onera.fr

Hélène Oriot
email: helene.oriot@onera.fr

Olivier Rabaste
email: olivier.rabaste@onera.fr

A Joint SAR-Communication System using Continuous Phase Frequency Shift Keying Codes and Mismatched Filtering Compression

The continuous growth of communication applications, in combination with the limited possibilities of bandwidth allocation, congests the electromagnetic spectrum. Joint radar-communication systems are thus attracting attention as they can alleviate the frequency spectrum by using the same bandwidth to perform both applications. To design such a system, continuous phase frequency-shift keying (CPFSK) codes are chosen, in order to transmit information and generate high resolution images from an airborne radar. However, the use of CPFSK codes requires sidelobe energy mitigation which is achieved by mismatched filters. The re-synthesized SAR images are created using a reconstruction algorithm from the aforementioned CPFSK codes and the mismatched filters, as well as from matched filters for the sake of analysis. These outputs are then compared using different evaluation criteria. Results of which show that the use of mismatched filtering and different messages embedded in the code phase, that maximize the data rate, provide enhanced image performance.

Continuous Phase Frequency-Shift

Keying (CPFSK) Codes

Introduction

The on-going growth of radio frequency applications gradually congests the electromagnetic spectrum. Concurrently, the need of alleviating the bandwidth occupation, adds up to the spectrum congestion problem [START_REF] Griffiths | Challenge problems in spectrum engineering and waveform diversity[END_REF]. To deal with this problem, two different applications of the electromagnetic spectrum can be combined together, such as radar and communications. Three different categories of joint radar-communications systems are presented in the literature; co-existence, cooperation and co-design [START_REF] Feng | Joint radar and communication: A survey[END_REF][START_REF] Ma | Joint radar-communication strategies for autonomous vehicles: Combining two key automotive technologies[END_REF]. Co-existence takes place when the radio-frequency applications are functioning autonomously and they consider the other system as interference that needs to be mitigated, for example in [START_REF] Han | Analysis for the BER of LTE system with the interference from radar[END_REF]. Cooperation occurs when there is a mutual operation between the two systems. For example, either using different signals for each application but optimizing their combined performance [START_REF] Chalise | Performance tradeoff in a unified passive radar and communications system[END_REF], or using communication signals to perform radar applications [START_REF] He | Performance gains from cooperative MIMO radar and MIMO communication systems[END_REF] or vice versa [START_REF] Sahin | A novel approach for embedding communication symbols into physical radar waveforms[END_REF]. Co-design consists in designing dual-function waveforms [START_REF] Liu | Toward dual-functional radarcommunication systems: Optimal waveform design[END_REF], that maximizes simultaneously the performance of both radio-frequency applications.

In this paper, a cooperative joint radar communications system is introduced, that uses communication codes, in order to perform an airborne radar application. A variety of communication codes exists, from which a broadly used code is the Orthogonal Frequency-Division Multiplexing (OFDM), due to their carrier orthogonality [START_REF] Ma | Joint radar-communication strategies for autonomous vehicles: Combining two key automotive technologies[END_REF]. Nevertheless, these codes have amplitude modulation, and the peakto-average power ratio (PAPR) is high [START_REF] Paredes | The problem of peak-to-average power ratio in OFDM systems[END_REF], which is costly for the radar hardware [START_REF] Ma | Joint radar-communication strategies for autonomous vehicles: Combining two key automotive technologies[END_REF]. In this paper, to tackle this issue, signals that embed information in their phase are considered. Phase modulated schemes, like phase shift keying or quadrature amplitude modulation, are avoided due to their brutal phase transitions that lead to out-band spectral energy. Thus, the waveform choice in this paper is Continuous Phase Modulated (CPM) codes, which have simultaneously constant envelop and better spectral efficiency thanks to phase continuity.

The radar framework of this paper is Synthetic Aperture Radar (SAR). In such framework, linear frequency modulated signals are mainly used due to their favorable properties and the matched filter is performed for range compression. However, when using other signals, the sidelobe level after compression is high. Hence, in order to assure good SAR image quality, mismatched filtering [START_REF] Rabaste | Mismatched filter optimization for radar applications using quadratically constrained quadratic programs[END_REF][START_REF] De | Design of radar receive filters optimized according to L p -norm based criteria[END_REF][START_REF] Stoica | Transmit codes and receive filters for radar[END_REF] is used for range compression, which minimizes the level of sidelobes.

In order to provide realistic results, we will present resynthesized SAR images in this article. A real-data SAR image is taken into account, on which the impulse response of the communication codes is applied along with their associated matched or mismatched filters. Different evaluation criteria are presented in order to compare the results. This paper is organized in 4 sections. The introduction of the paper is presented in Section 1. Section 2 provides the definitions of the continuous phase frequency-shift keying codes and of the mismatched filter. These definitions are fundamental for Section 3, where the re-synthesized SAR images are presented, along with their reconstruction algorithm and their evaluation criteria. Finally, the conclusion of the paper is presented in Section 4.

Signal and Range Compression Technique Choice

In this section, we present the continuous phase modulated frequency-shift keying codes, along with the mismatched filter formulation for range compression.

In this subsection, we present a special category of continuous phase modulated signal, which has constant envelop and encodes the desired information continuously in the phase, to prevent spreading out of the spectral band. They are called continuous-phase frequency-shift keying (CPFSK) [START_REF] John B Anderson | Digital phase modulation[END_REF]. The embedded symbols I = [I 1 , I 2 , . . .] belong to the set {±1, ±3, ..., ±(M -1)}, with M the number of different possible symbols in the constellation. The constellation determines the number k of bits per symbol through the expression 2 k = M . The CPFSK signal can be formulated as s(t; I) = e jφ(t;I) , with the phase φ expressed as [START_REF] John B Anderson | Digital phase modulation[END_REF],

φ(t; I) = πh i<n I i + πh T (t-nT )I n , nT < t ≤ (n+1)T.
(1) The particularities of CPFSK signals are the constant modulation index h over the different symbols, and a rectangular phase smoothing function [START_REF] John B Anderson | Digital phase modulation[END_REF]. In the following, s is a N -length sampled CPFSK signal expressed as s = s 1 s 2 . . . s N T , where and . T is the transpose operator and s i = s(iT s ; I) with T s the sampling period.

Mismatched Filter

Range compression is essential for radar processing and it is usually performed by a matched filter. However, such a filter combined with CPFSK signals creates high sidelobes which can create unacceptable image contrast. Hence, the mismatched filter is presented, which is mostly defined as a solution to an optimization problem subject to certain constraints, and usually convex [START_REF] Rabaste | Mismatched filter optimization for radar applications using quadratically constrained quadratic programs[END_REF][START_REF] De | Design of radar receive filters optimized according to L p -norm based criteria[END_REF][START_REF] Stoica | Transmit codes and receive filters for radar[END_REF]. In our framework, the filter is designed to minimize the sidelobe energy of the range-compressed CPFSK codes for the SAR image generation by using the Integrated Sidelobe Level (ISL) criterion.

The output signal after range compression can be expressed as [START_REF] Rabaste | Mismatched filter optimization for radar applications using quadratically constrained quadratic programs[END_REF]

y = Λ * K (s) q, (2)
where . * is the complex conjugate, q = q 1 q 2 . . . q K T ∈ C K the mismatched filter, and Λ K is a correlation matrix of size (K + N -1) × K [START_REF] Rabaste | Mismatched filter optimization for radar applications using quadratically constrained quadratic programs[END_REF]. The length K of q is K ≥ N . If the filter is chosen as q = s, then the classical matched filter is obtained. Extensive literature is available [START_REF] Rabaste | Mismatched filter optimization for radar applications using quadratically constrained quadratic programs[END_REF][START_REF] De | Design of radar receive filters optimized according to L p -norm based criteria[END_REF][START_REF] Stoica | Transmit codes and receive filters for radar[END_REF][START_REF] Jm Baden | Optimal peak sidelobe filters for biphase pulse compression[END_REF] when optimizing the mismatched filter with respect to the Integrated Sidelobe Level. It has been shown that the corresponding optimization problem can be expressed as a L 2 -norm minimization problem, in order to consider the sidelobe energy [START_REF] Rabaste | Mismatched filter optimization for radar applications using quadratically constrained quadratic programs[END_REF]:

min q ||F y|| 2 2 , s.t. s H q = s H s q H q ≤ 10 β 10 s H s (3)
where F is a diagonal matrix of ones except for some zero values which correspond to the indices of the mainlobe position, and β is a positive constant in dB that expresses the loss-in-processing gain.

As mentioned in [START_REF] Rabaste | Mismatched filter optimization for radar applications using quadratically constrained quadratic programs[END_REF], the first constraint allows to discard the trivial null solution and is convex because of its linearity and the second one enables to constraint the lossin-processing gain and is also convex as it is a positive semi-definite quadratic constraint. Globally, the optimization problem is thus convex, as the L 2 -norm is convex, with convex constraints. As a consequence, any local optimum of the optimization problem is necessarily a global optimum. We shown in article [START_REF] Chatzitheodoridi | Fast computation of the optimal integrated sidelobe level filter for large-scaled problems[END_REF] that the problem (3) can be solved very fast by considering the dual problem.

SAR Image Generation and Different Evaluation Criteria

This section is dedicated to the presentation of different SAR images generated with different signals and by a reconstruction algorithm, as well as to the evaluation criteria applied on them in order to compare the outputs. These criteria consider different configurations for both high and low energy points.

SAR Reconstruction Algorithm

A reconstruction algorithm that generates simulated SAR images, using CPSFK impulse responses, is applied in order to provide an insight of how actual CPFSK SAR images would be like. The SAR image reconstruction algorithm takes as input a real-data high resolution SAR image generated with classical chirp pulses, acquired using the SETHI airborne radar system [START_REF] Henrion | SETHI : Digital radar system for signal generation and acquisition[END_REF], and the impulse response of the CPFSK codes compressed in range and azimuth directions. The resolution of the generated image is degraded in comparison to the reference image, so that this latter roughly gives the ground reflectivity and prevents boundary spectral problems. Once the scene reflectivity is obtained, it is spectrally multiplied by the CPFSK impulse response spectrum, in order to create the output SAR im-age using these communication codes. The input impulse response is created by matched or mismatched filtering the CPFSK codes in the range direction, and then by performing an inverse Fourier transform for the azimuth direction.

Generated SAR Images

Different examples of re-synthesized images are generated using the reconstruction algorithm presented in subsection 3.1. Firstly, a synthesized chirp SAR image compressed with the matched filter is generated in order to provide a reference image, whose resolution is smaller than the realdata one. The rest of the images are created using CPFSK communication codes, either with matched (MF) or mismatched (MMF) filtering for range compression. Two different configurations are considered. Firstly, the impact of transmitting the same CPFSK code for all the pulses in the pulse train is analyzed, instead of one chirp. Afterwards, in order to maximize information transmission, CPFSK signals with different messages embedded in the phase are transmitted at each pulse.

The impulse response and the SAR image of the chirp pulse are illustrated in Figure 3. This reference case is compared with the CPFSK impulse responses and their associated generated SAR images, displayed in Figure 4.

The impulse response provides various information on the waveform, especially on its sidelobes which are evaluated in the sub-section 3.3. For the sake of clarity, for the simulated displayed impulse responses, no squint angle is applied, so that the sidelobes of the azimuth direction are not inclined over a different direction than the horizontal one, and the evaluation criteria presented in sub-section 3.3, are accurate. For the SAR images displayed in Figures 3 &4, the signals undergo a squint angle in order to follow the acquisition characteristics of the airborne platform.

In the following, different criteria are provided in order to be able to evaluate the images with quantitative criteria.

Evaluation Criteria on the SAR Images

To evaluate the generated images, different image characteristics are taken into account, such as sidelobe behaviour, image contrast, and image correlation.

Integrated Sidelobe Level Ratio (ISLR) and Peak-to-Sidelobe Level Ratio (PSLR)

The ISLR, defined as the ratio of the energy of all the sidelobes to the energy of the main lobe, and the PSLR, defined as the ratio between the intensity of the highest sidelobe and the intensity of the mainlobe, are two classical SAR image evaluation criteria [START_REF] Lu | Parameter assessment for SAR image quality evaluation system[END_REF].

Table 2 provides the PSLR and ISLR of the simulated impulse response and the corner reflector of the SAR image, in the range direction. The impulse response can be related to an ideal corner reflector, without the sidelobe influence of other scatterers. Thus, the exact sidelobe behaviour after range and azimuth compression is observed. The resulting values over the corner reflector of each SAR image are less satisfying, possibly due to sidelobe summation of neighbouring scatterers and to the undergoing squint angle, but it represents a more realistic configuration.

The images, that use the mismatched filter for the compression (Sub-figures 4c, 4d, 4g, 4h), provide better values for the PSLR and the ISLR compared to the matched filter (Sub-figures 4a, 4b, 4e, 4f). For the ISLR, this remark also validates the criterion of the optimization problem [START_REF] Ma | Joint radar-communication strategies for autonomous vehicles: Combining two key automotive technologies[END_REF] Table 2 Evaluation criteria for the impulse response (IR) and the corner reflector (CR) on each SAR image, both in the range direction.

Pearson Correlation Coefficient

The Pearson correlation coefficients, also called coherence coefficients, of two signals or images provide the degree of resemblance of the inputs. More precisely, when the output values are converging to 1, the inputs have strong resemblance, and when the output values are converging to 0, then the images tend to be very different [START_REF] Hachemi | The interest of coherence from radar sar images in the follow-up of urban extensions of focşani city[END_REF]. In our framework, we want to compare the pixel energy of the generated images in order to provide the best combination of signal transmission and processing procedure. Let consider two images A, B and d the number of pixels of the image patch side, then the coherence coefficients between A and B are defined as follows [START_REF] Asamoah | Measuring the performance of image contrast enhancement technique[END_REF][START_REF] Jacob | Sentinel-1 insar coherence for land cover mapping: A comparison of multiple feature-based classifiers[END_REF],

ρ i,j = E A I -E [A I ] B I -E [B I ] V ar [A I ] V ar [B I ] , (4)
where i, j are the current index positions, I = {i -d : i + d, j -d : j + d} is the set of indices considered for the computation of the pixel (i, j), and d = 5. Figures 1 & 2 illustrate the correlation between the SAR image generated using the chirp pulse, which is considered as the reference image, and the SAR images generated with CPFSK codes in different configurations, respectively for a corner reflector and a building. In both figures, the compression using the mismatched filter provides a high correlation with the reference image. However, the image generated with different CPFSK codes provides slightly better results. More precisely, the high energy pixel areas, have similar energy in both configurations, but when it comes to lower energy points as in the borders, the images generated with multiple signals and MMF compression have slightly higher values. These observations are also validated by Table 3, which displays the coherence coefficient computed over each sub-image. The highlighted purple values show the best results for the coherence which corresponds to the image generated with different CPFSK signals and compressed with mismatched filters, but it is also worth mentioning that even with one CPFSK and the mismatched filter, correlation is high.

Signals

Structural Similarity (SSIM) Index

The authors in [START_REF] Wang | Image quality assessment: from error visibility to structural similarity[END_REF] propose a different comparative error estimator based on various characteristics of the selected images that need to be evaluated. Their motivation stems from the human eye precision and they show that structural information creates a satisfying error estimator when comparing similar images. Let us denote A, B the two images. The SSIM index can be mathematically expressed as [START_REF] Wang | Image quality assessment: from error visibility to structural similarity[END_REF][START_REF] Avanaki | Exact histogram specification optimized for structural similarity[END_REF],

SSIM(A, B) = (2µ A µ B + C 1 )(2σ AB + C 2 ) (µ 2 A + µ 2 B + C 1 )(σ 2 A + σ 2 B + C 2 )
where C 1 , C 2 are close to zero constants, µ • is the mean, σ • is the standard deviation of the image in question and σ AB is the covariance of A and B. The output of this formula is equal to 1 when the input images are identical. This method is compared to the mean square error (MSE) [START_REF] Asamoah | Measuring the performance of image contrast enhancement technique[END_REF][START_REF] James | An introduction to statistical learning[END_REF]. The MSE and the SSIM values are calculated over the two same areas of the image, as in the previous subsection 3.3.2. Table 4 displays the values of MSE and SSIM criteria for the two different zones. For the corner reflector, results closer to the reference image are obtained when transmitting a CPFSK code and compress it with its MMF. This means that the range and azimuth axes are closer to a sinus-cardinal as the chirp image and there is no sidelobe spreading out these axes as shown in its impulse response in figure 4c, contrary to the impulse response of multiple transmitted codes, shown in figure 4g. This spreading, created when using different codes, shown in sub-figures 4e, 4g, comes from the sidelobe summation during azimuth processing of the random messages. Thus, locally using one signal provides satisfying results, but in larger distance, where the sidelobe impact can be observed, as shown in the SAR image 4d, the sidelobes are higher, as shown also from the ISL value, and create a high energy line over the range axis. For the building area, the image created by multiple CPFSK codes and the MMF provides results closer to the chirp image, as shown in Table 4. This area combines high, medium and low energy pixels, so it is a good comparison reference, also mentioned in subsection 3.3.2.

Conclusion

Our paper proposes a joint SAR-communications framework which uses CPFSK codes, a family of communications codes, and mismatched filtering to perform range compression. The use of different CPFSK pulses allows information transmission, as well as the maximization of the data rate, along with the use of the mismatched filters that improves the image contrast. Different criteria are applied on these configurations while using matched or mismatched filtering in order to evaluate the images. It is thus shown, both on a simple simulated impulse response as well as on re-synthesized images from real data, that when transmitting multiple codes and compress them with their mismatched filters, the proposed output provides better performance in terms of contrast and sidelobe energy. Besides, such results and their evaluation criteria resemble to the reference chirp case, for which information transmission is not possible.

Figure 1

 1 Figure 1 Correlation coefficient on the corner reflector area. The reference image is considered the chirp one, which gives the correlation with rest of the images. The first column represents MF compression and the second column MMF compression. The first row illustrates the correlation between the chirp and the images created with one CPFSK signal, and the second row the images obtained using different CPFSK signals.

Figure 2

 2 Figure 2 Correlation coefficient images of the building area in the SAR image. The image order is the same as for Figure 1.

Figure 3

 3 Figure 3 Zero squint impulse response (left sub-figure) and generated SAR image using chirp pulse (right sub-figure).

Figure 4

 4 Figure 4 Zero squint impulse response and generated SAR images using communication codes.

( a )

 a One CPFSK signal with MF (b) One CPFSK signal with MF (c) One CPFSK signal with MMF (d) One CPFSK signal with MMF (e) Different CPFSK signals at each acquisition with MF (f) Different CPFSK signals at each acquisition with MF (g) Different CPFSK signals at each acquisition with MMF (h) Different CPFSK signals at each acquisition with MMF

Table 1

 1 presents different parameters used for the simulation results, associated either with the signal characteristics, the mismatched filtering or the SAR image processing.

	Parameter	Value
	Bandwidth B	305 MHz
	Constellation M	16
	Modulation Index h	0.4
	Pulse duration T p	4.8 µs
	Chip duration T	19.2 ns
	Sampling frequency f s	625 MHz
	Symbol Number	250
	Signal Length	3000
	Mismatched Filter Length	9000
	Allowed loss in processing gain β	2 dB
	Squint Angle	7.28 •
	Pulse Repetition Frequency	5000 Hz
	Number of transmitted signals	6522

Table 1

 1 Signal, mismatched filtering and SAR processing parameters used for the generation of the impulse responses and the SAR image generation displayed in Figures3 & 4.

  , that minimizes the integrated sidelobe energy. Observing table 2, the best PSLR values are given by the images generated with one communication code, even if the images with multiple codes have close PSLR values. The best ISLR values are provided by the images created with several transmitted signals. An important remark is that the transmission of multiple codes favors the ISLR values because of an average over the sidelobe energy due to different compressed signals whose points have different intensity levels, and thus the overall sidelobe level decreases. This effect can be also observed for the image with multiple signals and the matched filters, where the ISLR is better than expected.

	Signals	PSLR	IR	ISLR	PSLR	CR	ISLR
	Chirp-MF	-13.266 -10.05 -11.625 -8.890
	Diff CPFSK-MF	-14.84 -13.13 -13.07		-10.69
	Diff CPFSK-MMF	-15.23 -14.17 -13.484 -11.66
	One CPFSK-MF	-14.87 -2.865 -13.266	-5.97
	One CPFSK-MMF -15.805 -8.77	-14.03 -11.086

Table 3

 3 Pearson correlation coefficient for the corner reflector image and the building image.

		Pearson Coefficient
		Reflector Building
	Diff CPFSK-MF	0.913	0.969
	Diff CPFSK-MMF	0.964	0.974
	One CPFSK-MF	0.897	0.923
	One CPFSK-MMF	0.962	0.969

Table 4

 4 MSE and SSIM values for the corner reflector and the building in the SAR image.

5

Literature