Hepatocyte growth factor and its receptor are required for malaria infection (original) (raw)
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PLoS Pathogens, 2008
Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites cocultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface proteins involved in hepatocyte invasion, while the other two were predominantly expressed during hepatic parasite development. The genome-wide up-regulation of expression observed supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes validated our approach as one means to determine the repertoire of proteins implicated during the early events of the Plasmodium infection, and in this case that of P. falciparum, the species responsible for the severest forms of malaria.
1 cm x 1 cm strips of skin from the back of a male Balb/C mouse were soaked in penicillin/streptomycin for 3 min before mincing into 1 mm x 1 mm pieces under sterile conditions. Skin pieces were then incubated in Liberase III (Roche Applied Sciences) for 1 hr at 37°C with agitation followed by grinding with PBS/0.2% BSA and centrifugation for 10 min at 100 x g. The tissue was then filtered through 70 µm mesh, centrifuged, resuspended in DMEM/FCS and transferred to a 25-cm 2 culture flask. Disaccharide Analysis. Cell lysates were treated overnight with 2 mg/ml Pronase followed by purification of the glycopeptides by anion exchange chromatography using DEAE Sephacel. Columns were washed with 0.25 M NaCl in 20 mM sodium acetate, pH 6.0 and eluted with 2 M NaCl. Glycans were released from peptides by β-elimination and repurified by anion exchange chromatography. Uronic acid concentration was determined by carbazole assay (Bitter and Muir, 1962) and HS composition was determined as follows: Chains were digested with heparin lyases I, II, and III (Ibex, Montreal, Canada), and the liberated disaccharides were resolved by anion exchange HPLC with postcolumn derivatization and fluorescent detection (Toyoda et al., 1997). Confocal imaging of sporozoites in tissue explants. Female Swiss Webster mice were anesthetized and liver or ear removed and immediately rinsed in cold PBS. Ear tissue was splayed into two leaflets and cut into 0.5 cm squares which were placed hair-side down into an 8-well LabTek chamber. Thin liver slices were cut by hand and placed into LabTek chambers in a similar manner. P. berghei-GFP sporozoites that had been preincubated on ice ± 10 µM E-64d for 30 min, were spun onto tissue sections at 300 x g for 4 min at 12°C then immediately placed at 37°C for 3 min and fixed in 4% PFA/PBS for 20 min. Tissue sections were washed in PBS and blocked in 1% BSA/PBS for 1 hr 15 min. Sporozoites on the tissue sections were stained with antiserum that recognizes full length CSP, washed, incubated with anti-rabbit Ig conjugated to Texas Red, washed,
Nomadic or sessile: can Kupffer cells function as portals for malaria sporozoites to the liver
Cellular Microbiology, 2006
The initial site of replication for Plasmodium parasites in mammalian hosts are hepatocytes, cells that offer unique advantages for the extensive parasite replication occurring prior to the erythrocytic phase of the life cycle. The liver is the metabolic centre of the body and has an unusual relationship to the immune system. However, to reach hepatocytes, sporozoites must cross the sinusoidal barrier, composed of specialized endothelia and Kupffer cells, the resident macrophages of the liver. Mounting evidence suggests that, instead of taking what would seem a safer route through endothelia, the parasites traverse Kupffer cells yet suffer no harm. Kupffer cells have a broad range of responses towards incoming microorganisms, toxins and antigens which depend on the nature of the intruder, the experimental conditions and the environmental circumstances. Kupffer cells may become activated or remain anergic, produce pro- or anti-inflammatory mediators. Consequently, outcomes are diverse and include development of immunity or tolerance, parenchymal necrosis or regeneration, chronic cirrhotic transformation or acute liver failure. Here we review data concerning the unique structural and functional characteristics of Kupffer cells and their interactions with Plasmodium sporozoites in the context of a model in which these hepatic macrophages function as the sporozoite gate to the liver.
Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity
Nature Medicine, 2002
Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and first invade the liver of the mammalian host, as an obligatory step of the life cycle of the malaria parasite. Within hepatocytes, Plasmodium sporozoites reside in a membrane-bound vacuole, where they differentiate into exoerythrocytic forms and merozoites that subsequently infect erythrocytes and cause the malaria disease. Plasmodium sporozoite targeting to the liver is mediated by the specific binding of major sporozoite surface proteins, the circumsporozoite protein and the thrombospondin-related anonymous protein, to glycosaminoglycans on the hepatocyte surface 1 . Still, the molecular mechanisms underlying sporozoite entry and differentiation within hepatocytes are largely unknown. Here we show that the tetraspanin CD81, a putative receptor for hepatitis C virus 2 , is required on hepatocytes for human Plasmodium falciparum and rodent Plasmodium yoelii sporozoite infectivity. P. yoelii sporozoites fail to infect CD81-deficient mouse hepatocytes, in vivo and in vitro, and antibodies against mouse and human CD81 inhibit in vitro the hepatic development of P. yoelii and P. falciparum, respectively. We further demonstrate that the requirement for CD81 is linked to sporozoite entry into hepatocytes by formation of a parasitophorous vacuole, which is essential for parasite differentiation into exoerythrocytic forms.
The Journal of Immunology, 2008
Plasmodium sporozoites traverse several host cells before infecting hepatocytes. In the process, the plasma membranes of the cells are ruptured, resulting in the release of cytosolic factors into the microenvironment. This released endogenous material is highly stimulatory/immunogenic and can serve as a danger signal initiating distinct responses in various cells. Thus, our study aimed at characterizing the effect of cell material leakage during Plasmodium infection on cultured mouse primary hepatocytes and HepG2 cells. We observed that wounded cell-derived cytosolic factors activate NF-B, a main regulator of host inflammatory responses, in cells bordering wounded cells, which are potential host cells for final parasite infection. This activation of NF-B occurred shortly after infection and led to a reduction of infection load in a time-dependent manner in vitro and in vivo, an effect that could be reverted by addition of the specific NF-B inhibitor BAY11-7082. Furthermore, no NF-B activation was observed when Spect ؊/؊ parasites, which are devoid of hepatocyte traversing properties, were used.