Expression was confirmed using an anti-His tag antibody (red arrows in the second panel) and anti-CSP antibody (green arrow in the third panel). as phospholipid scramblase). Further, we show that HP1 interacts with a ~160?kDa hepatocyte membrane putative receptor (identified as carbamoyl-phosphate synthetase 1). Importantly, immunization of mice with the HP1 peptide partially protects them from infection by the rodent parasite infection of human hepatocytes in culture. The sporozoite ligand for hepatocyte invasion is a potential novel pre-erythrocytic vaccine candidate. Subject terms: Parasite biology, Pathogens After transmission of Plasmodium sporozoites from infected mosquitoes, parasites first infect hepatocytes. Here, Cha et al. identify a sporozoite ligand (phospholipid scramblase) and the hepatocytic receptor (carbamoyl-phosphate synthetase 1) as relevant for hepatocyte invasion and show that an?antibody to?hepatocyte-binding peptide 1 (HP1), which structurally mimics the sporozoite ligand, partially protects mice from infection. Introduction Malaria infection is initiated by the release from an infected mosquito of 50C100 sporozoites into the skin of a vertebrate host1. Thereafter sporozoites migrate through dermal tissues in search of a blood vessel, which they traverse to enter the circulation. Circulating sporozoites must exit in the liver, where they infect hepatocytes, each of which produces thousands Lumefantrine of merozoites that are released into the blood circulation and cause disease symptoms2. This complex cycle requires specific parasiteChost recognition at each stage. Binding of the sporozoite surface circumsporozoite protein (CSP) to liver-specific and highly sulfated glycosaminoglycans (GAGs) protruding into the sinusoidal vessels is the first step for liver recognition and invasion3,4. Next, sporozoites glide along the vessel wall and preferentially traverse Kupffer cells via recognition of its Lumefantrine CD68 surface receptor5C8. After crossing the sinusoid lining, sporozoites specifically invade hepatocytes, no other cell types such as Stellate cells or adipocytes. Thus, we Lumefantrine hypothesize that infection requires recognition via interaction of a sporozoite ligand with a hepatocyte receptor. No sporozoite ligand for hepatocyte recognition has been reported. On the other hand, CD81, the scavenger receptor BI (SR-BI), and EphA2 have been proposed as possible hepatocyte receptors for sporozoite invasion9C11. The 6-cysteine domain protein P36 was proposed to be involved in the SR-BI-dependent pathway12. Subsequently, it was determined that CD81 and SR-BI are not sporozoite receptors but rather, are involved in parasitophorous vacuole membrane formation and organization10. Furthermore, a recent report shows that EphA2 is not an obligatory receptor for sporozoite hepatocyte interaction13. Hepatocyte Aquaporin-9 was recently Lumefantrine identified as an important host cell membrane protein for sporozoite permissiveness, however was not characterized as a receptor14. Hence, the molecular basis for specific sporozoiteChepatocyte interaction remains unknown. The most advanced available RTS/S malaria pre-erythrocytic vaccine that uses CSP as an antigen only shows moderate protection (~40%)15. Identification of a sporozoite ligand for hepatocyte invasion may MYCC identify new pre-erythrocytic malaria vaccine targets. Here we show that a peptideHP1identified by the use of a phage display library, specifically binds to hepatocytes and by doing so, inhibits sporozoite infection. Further, HP1 is a structural mimic of the sporozoite ligand phospholipid scramblase (PLS) that for infection, interacts with the hepatocyte receptor carbamoyl-phosphate synthetase 1 (CPS1). Results The hepatocyte-binding peptide HP1 inhibits sporozoiteChepatocyte interaction We used a two-step strategy to investigate the molecular basis for sporozoiteChepatocyte interaction: (1) use a phage peptide display library to select peptides that strongly bind to the hepatocyte surface and (2) determine whether such peptide competitively inhibits sporozoiteChepatocyte interaction. Should competitive inhibition by a small peptide be observed, this would constitute preliminary evidence that the selected peptide binds to a hepatocyte surface molecule that serves as a sporozoite receptor. We screened a phage library displaying 1.5??109 different peptides16 for peptides with high affinity to primary mouse hepatocytes Lumefantrine (Fig.?1a). Of 39 successfully sequenced phages, 17 (43.6%) displayed conserved amino acid sequences. Out of these, five displayed an identical peptide that was named HP1 (Fig.?1b, Supplementary Fig. 1). The recombinant HP1 phage, and the wild-type phage that has no peptide insert as control, were tested for competitive inhibition of sporozoiteChepatocyte.
Expression was confirmed using an anti-His tag antibody (red arrows in the second panel) and anti-CSP antibody (green arrow in the third panel)
