Plasmodium falciparum Phospholipase C Hydrolyzing Sphingomyelin and Lysocholinephospholipids Is a Possible Target for Malaria Chemotherapy

Abstract

Sphingomyelinase (SMase) is one of the principal enzymes in sphingomyelin (SM) metabolism. Here, we identified a Plasmodium falciparum gene (PfNSM) encoding a 46-kD protein, the amino acid sequence of which is ∼25% identical to that of bacteria SMases. Biochemical analyses of the recombinant protein GST-PfNSM, a fusion protein of the PfNSM product with glutathione-S-transferase, reveal that this enzyme retained similar characteristics in various aspects to SMase detected in P. falciparum–infected erythrocytes and isolated parasites. In addition, the recombinant protein retains hydrolyzing activity not only of SM but also of lysocholinephospholipids (LCPL) including lysophosphatidylcholine and lysoplatelet-activating factor, indicating that PfNSM encodes SM/LCPL-phospholipase C (PLC). Scyphostatin inhibited SM/LCPL-PLC activities of the PfNSM product as well as the intraerythrocytic proliferation of P. falciparum in a dose-dependent manner with ID50 values for SM/LCPL-PLC activities and the parasite growth at 3–5 μM and ∼7 μM, respectively. Morphological analysis demonstrated most severe impairment in the intraerythrocytic development with the addition of scyphostatin at trophozoite stage than at ring or schizont stages, suggesting its effect specifically on the stage progression from trophozoite to schizont, coinciding with the active transcription of PfNSM gene.

Involvement of ceramide in hyperosmotic shock-induced death of erythrocytes

“…With the exception of the description of a P. falciparum SMase during infection, 40 no data are available about the role of ceramide in erythrocyte (patho-) physiology. In the present study, five lines of evidence are provided that ceramide is involved in erythrocyte death signaling following osmotic shock: (i) accumulation of ceramide is observed after osmotic shock, (ii) osmotic shock induces breakdown of SM, (iii) ceramide induces annexin binding even in the absence of osmotic shock, (iv) osmotic shock-induced annexin binding is inhibited by 3,4-dichloroisocoumarin, which is known to efficiently prevent activation of SMase 23 and (v) knockout of aSMase impairs phosphatidylserine exposure after osmotic shock.…”

Serine Repeat Antigen (SERA5) Is Predominantly Expressed among the SERA Multigene Family of Plasmodium falciparum, and the Acquired Antibody Titers Correlate with Serum Inhibition of the Parasite Growth

“…Parasites-FCR3 (25), Honduras-1 (26), K1 (generous gift from Dr. Masatsugu Kimura), 3D7 (27), and Dd2 (28) strains of P. falciparum were maintained in culture according to the methods mainly by Trager and Jansen (29) and modified by Mitamura and co-workers (30,31). Cultures were maintained in 5% O 2 and 5% CO 2 atmosphere with 3% type O erythrocyte (v/v) in the culture medium containing 10% heatinactivated human serum.…”

Raft ceramide in molecular medicine

“…Genetic deficiency of ASM or pharmacologic inhibition of the enzyme reduces or even prevents infection of mammalian cells with these pathogens. P. falciparum seems to have developed another strategy to circumvent a requirement for endogenous ASM, that is, the pathogen expresses its own sphingomyelinase that appears to be required for the infection of erythrocytes with P. falciparum (Hanada et al, 2002). Targeting endogenous ASM or the pathogen's sphingomyelinase might serve as a novel strategy to alter the course of these infections.…”