James Leech

A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum.

To obtain free amino acids for protein synthesis, trophozoite stage malaria parasites feed on the cytoplasm of host erythrocytes and degrade hemoglobin within an acid food vacuole. The food vacuole appears to be analogous to the secondary lysosomes of mammalian cells. To determine the enzymatic mechanism of hemoglobin degradation, we incubated trophozoite-infected erythrocytes with peptide inhibitors of different classes of proteinases. Leupeptin and L-transepoxy-succinyl-leucyl-amido-(4-guanidino)-butane (E-64), two peptide inhibitors of cysteine proteinases, inhibited the proteolysis of globin and caused the accumulation of undegraded erythrocyte cytoplasm in parasite food vacuoles, suggesting that a food vacuole cysteine proteinase is necessary for hemoglobin degradation. Proteinase assays of trophozoites demonstrated cysteine proteinase activity with a pH optimum similar to that of the food vacuole and the substrate specificity of lysosomal cathepsin L. We also identified an Mr 28,000 proteinase that was trophozoite stage-specific and was inhibited by leupeptin and E-64. We conclude that the Mr 28,000 cysteine proteinase has a critical, perhaps rate-limiting, role in hemoglobin degradation within the food vacuole of Plasmodium falciparum. Specific inhibitors of this enzyme might provide new means of antimalarial chemotherapy.

Plasmodium falciparum: inhibitors of lysosomal cysteine proteinases inhibit a trophozoite proteinase and block parasite development.

Trophozoites of Plasmodium falciparum obtain free amino acids for protein synthesis by degrading host erythrocyte hemoglobin in an acidic food vacuole. We previously reported that leupeptin and L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64), two inhibitors of the cysteine class of proteinases, blocked hemoglobin degradation in the trophozoite food vacuole, and we identified a 28-kDa trophozoite cysteine proteinase as a potential food vacuole hemoglobinase. We now report that the biochemical properties of the trophozoite cysteine proteinase closely resembled those of the lysosomal cysteine proteinases cathepsin B and cathepsin L. The trophozoite proteinase had a pH optimum of 5.5-6.0, near that of both lysosomal proteinases, and it was efficiently inhibited by highly specific diazomethylketone and fluoromethylketone inhibitors of cathepsin B and cathepsin L. The trophozoite proteinase preferred peptide substrates with arginine adjacent to hydrophobic amino acids, as does cathepsin L. Micromolar concentrations of the fluoromethylketone inhibitor Z-Phe-Ala-Ch2F blocked the degradation of hemoglobin in the trophozoite food vacuole and prevented parasite multiplication. In previous studies much higher concentrations of the inhibitor were not toxic for mice. Our results provide additional evidence that the 28-kDa trophozoite proteinase is a food vacuole hemoglobinase and suggest that specific inhibitors of the enzyme may have potential as antimalarial drugs.

Plasmodium falciparum malaria. An amelanotic melanoma cell line bears receptors for the knob ligand on infected erythrocytes.

Erythrocytes infected with Plasmodium falciparum trophozoites and schizonts are not seen in the peripheral circulation because they attach to venular endothelium via knoblike structures on the infected erythrocyte membrane. We have recently shown that erythrocytes containing P. falciparum trophozoites and schizonts likewise attach to cultured human venous endothelial cells via knobs. In search of a more practical target cell for large scale binding studies designed to characterize and isolate the knob ligand, we tested various normal cells and continuous cell lines for their ability to bind P. falciparum-infected erythrocytes. Of the 18 cell types tested, binding of infected erythrocytes was observed to a human amelanotic melanoma cell line and amnion epithelial cells as well as to human aortic and umbilical vein endothelial cells. 96-100% of amelanotic melanoma cells bound 17+/-4 (+/-1 SEM) infected erythrocytes per positive cell, whereas fewer endothelial cells (4-59%) and amnion epithelial cells (8-19%) were capable of binding 12+/-5 and 4+/-1 infected erythrocytes per positive cell, respectively. Further studies designed to compare the mechanism of binding to the amelanotic melanoma cell line and endothelial cells showed the following results. First, that adhesion of infected erythrocytes to these two cell types was parasite stage-specific in that only erythrocytes containing late ring forms, trophozoites, and schizonts bound. Erythrocytes containing early ring forms, which do not attach to venular endothelium in vivo, did not bind to either cell type. Second, erythrocytes infected with trophozoites and schizonts of P. vivax or a knobless strain of P. falciparum, both of which continue to circulate in vivo, did not bind to either target cell type. Third, transmission electron microscopy showed that infected erythrocytes attached to the amelanotic melanoma cells via knobs. We conclude that cultured human endothelial cells and an amelanotic melanoma cell line share common determinants on their surface and that the mechanism of binding to these two different cell types is similar. The amelanotic melanoma cell line offers a useful substitute for endothelial cells in binding studies requiring large numbers of target cells.

Gene inactivation of Pf11-1 of Plasmodium falciparum by chromosome breakage and healing: identification of a gametocyte-specific protein with a potential role in gametogenesis.

We report the identification of the product of the Plasmodium falciparum Pf11‐1 gene and demonstrate that it is a gametocyte‐specific protein that has a potential role in the rupture of the host erythrocyte and emergence of the gametes (gametogenesis). The Pf11‐1 gene is a large locus (30 kb) whose sequence predicts a glutamic acid‐rich polypeptide. Our identification of the Pf11‐1 gene product as gametocyte specific was greatly facilitated by the isolation of a mutant parasite clone in which greater than 90% of the Pf11‐1 gene was deleted. Molecular analysis of the mutant locus suggests that the underlying genetic mechanism is chromosome breakage and subsequent healing by the addition of telomere repeats. PCR‐based analysis showed that similar DNA rearrangements occur commonly in small subpopulations of most laboratory strains, suggesting that the Pf11‐1 locus represents a fragile chromosome region. Northern blot analysis demonstrates that a large Pf11‐1 gene‐specific transcript (much greater than 10 kb) is present in gametocytes but not in asexual blood stage parasites. The Pf11‐1 protein was localized by electron microscopy to granules in the cytoplasm of gametocytes adjacent to the membrane of the parasitophorous vacuole. Following in vitro stimulation of gametogenesis, the Pf11‐1 protein was found in the membrane of lysed erythrocytes, suggesting a role for Pf11‐1 in erythrocyte rupture within the mosquito gut.

Rectal Leishmaniasis in a patient with acquired immunodeficiency syndrome

A severe rectal lesion due to Leishmania infection is described in an American-born homosexual man with the acquired immunodeficiency syndrome. The infection, which may have been venereally transmitted, responded to treatment with amphotericin B. There was no evidence of visceral leishmaniasis. The contribution of the patients immunodeficiency to the development of the atypical cutaneous leishmanial lesion is unclear. The case may foretell increasing problems with protozoan infections in AIDS as the epidemic spreads to areas with endemic protozoan diseases.

The mature erythrocyte surface antigen of Plasmodium falciparum is not required for knobs or cytoadherence

Intraerythrocytic Plasmodium falciparum parasites at the trophozoite and schizont stages synthesize a greater than 200-kDa protein, the mature erythrocyte surface antigen (MESA), that is localized at the membrane of infected red blood cells and manifests size polymorphism and antigenic diversity among parasite isolates. Because MESA is localized in the host cell membrane, we examined parasites with differing knob and cytoadherence phenotypes to determine whether MESA expression correlated with knob formation and cytoadherence. A cloned line of P. falciparum that was cultured with repeated selection for the knobbed and cytoadherent phenotypes did not express MESA, due to at least partial deletion of the single-copy MESA gene. In contrast, parasites from the same clone that were cultured without this selection lost the knobbed and cytoadherent phenotypes, but continued to express MESA. These results indicate that MESA is apparently not required for differentiation and multiplication of erythrocyte stage P. falciparum parasites in vitro, or for knob formation and cytoadherence. We speculate that MESA may have a role in evasion of the host immune response by P. falciparum.