ARMed and even more dangerous?: Response

I appreciate Dr Waters’ insightful comments on our work. Indeed, the possibility that Plasmodium has developed mechanisms to modulate protein synthesis provides a fascinating perspective. Not only might the parasite be able to kill potentially dangerous cells of the immune surveillance system of the vertebrate host or the vector, but it might also be able to modulate the cellular environment of its nurse cells to its own advantage. It will be exciting to identify the ‘relief mechanism’ that Plasmodium is likely to possess to keep its nurse cells alive. Hepatocytes are normally responsible for the detoxification of many endogenous and exogenous substances and for the secretion of large quantities of serum proteins, but these functions cannot be in the interest of a rapidly growing intracellular schizont – the nutritional requirements of which are currently unknown. Would it be surprising to discover that additional parasitic factors modulate the environment of the host cell to fulfil the needs of the schizont?Concerning the expression of a protein that could potentially block its own translation, there have been no reports of cytoplasmically targeted, full-length circumsporozoite protein (CS) being expressed in eukaryotic cells. In fact, a full-length CS construct by itself does not induce a protective immune response1xLi, S. et al. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5214–5218CrossRef | PubMedSee all References1. Other constructs lack region II-plus and most of the basic amino acids upstream from region I (Ref. 2xBarr, P.J. et al. J. Exp. Med. 1987; 165: 1160–1171CrossRef | PubMed | Scopus (29)See all ReferencesRef. 2) or are directed towards the cell surface3xFasel, N. et al. Gene. 1992; 111: 57–63CrossRef | Scopus (23)See all References3 and thus never enter the cytosol. In contrast, and in agreement with the successful production of CS constructs in Escherichia coli4xStuber, D. et al. Immunol. Methods. 1990; IV: 121–152See all References4, our recent data indicate that prokaryotic ribosomes are CS protein insensitive (U. Frevert, unpublished). I therefore believe that complete cytosolic CS protein does intoxicate mammalian cells.Regarding effective protein and peptide concentrations, new data suggest that the conformation of the CS protein is crucially important for its ribotoxicity (U. Frevert, unpublished). When purified from E. coli under non-denaturing conditions, Plasmodium falciparum CS protein inhibits eukaryotic in vitro translation completely at 1–2 μm and is, therefore, 100–400 times as active as our region I and II-plus peptides. Native sporozoite-derived CS protein might be even more efficient, so the notion that sporozoites can translocate cytotoxic amounts of the protein into mammalian cells appears realistic.I agree with Dr Waters that much work has to be directed towards the characterization of the RNA-binding motifs of the CS protein. Furthermore, the mechanism of membrane translocation remains to be elucidated. However, a recent discovery might shed some light on this unexplained phenomenon. Fusion of a short membrane-translocating sequence (MTS), the hydrophobic region of a signal peptide, to Plasmodium proteins mediates efficient translocation across the cell membrane and import into the cytosol of mammalian cells5xDu, C. et al. J. Peptide Res. 1998; 51: 235–243CrossRef | PubMedSee all References, 6xRojas, M. et al. J. Biol. Chem. 1998; 271: 27456–27461See all References, 7xLin, Y-Z. et al. J. Biol. Chem. 1995; 270: 14255–14258CrossRef | PubMed | Scopus (710)See all References. The MTS resembles the leader sequence and the hydrophobic anchor sequence of the CS protein. As neither cleavage of the signal peptide from sporozoite-derived CS protein nor replacement of the carboxyl terminus by a glycosylphosphatidylinositol anchor have unambiguously been demonstrated, it is conceivable that one or both of these peptides are retained and mediate membrane translocation. We are looking forward to addressing these unanswered questions on the biology of Plasmodium.