The mannan-binding lectin (MBL) is a serum protein, which is involved in the immune defence against viruses, bacteria and parasites. Children who have mutations in the MBL gene that lead to a MBL deficiency are more susceptible to infectious diseases and are more likely to suffer from severe malaria. In this report we investigate the interaction between MBL and the proteins of red blood cells infected with the parasite Plasmodium falciparum. Protein extracts were separated on MBL-sepharose columns. After the elution of bound material, the proteins were detected either by Western blot with human antibodies, or radioactive labelling with 35S-methionine or 3H-glucosamine. MBL recognises proteins of P. falciparum-infected erythrocytes that are immunogenic in humans, parasite-derived and glycosylated. Whether the proteins identified in the different assays are identical remains to be explored. MBL added to in vitro cultures of P. falciparum, however, does not inhibit parasite growth. The positive effect of MBL in the blood of malaria patients could be caused by detoxification of parasite products.

Background Trypanosoma cruzi is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease.Methodology/Principal Findings In silico analysis of the T. cruzi genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in T. cruzi epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. T. cruzi genes TcDPM1, TcGPI10 and TcGPI12 complement conditional yeast mutants in GPI biosynthesis. Attempts to generate T. cruzi knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding TcGPI8, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene.Conclusions/SignificanceAnalyses of T. cruzi sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these T. cruzi genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this parasite.

Toxoplasma gondii is a ubiquitous parasitic protozoan that invades nucleated cells in a process thought to be in part due to several surface glycosylphosphatidylinositol (GPI)-anchored proteins, like the major surface antigen SAG1 (P30), which dominates the plasma membrane. The serine protease inhibitors phenylmethylsulfonyl fluoride and diisopropyl fluoride were found to have a profound effect on the T. gondii GPI biosynthetic pathway, leading to the observation and characterization of novel inositol-acylated mannosylated GPI intermediates. This inositol acylation is acyl-CoA-dependent and takes place before mannosylation, but uniquely for this class of inositol-acyltransferase, it is inhibited by phenylmethylsulfonyl fluoride. The subsequent inositol deacylation of fully mannosylated GPI intermediates is inhibited by both phenylmethylsulfonyl fluoride and diisopropyl fluoride. The use of these serine protease inhibitors allows observations as to the timing of inositol acylation and subsequent inositol deacylation of the GPI intermediates. Inositol acylation of the non-mannosylated GPI intermediate D-GlcN␣1-6-D-myo-inositol-1-HPO 4 -sn-lipid precedes mannosylation. Inositol deacylation of the fully mannosylated GPI intermediate allows further processing, i.e. addition of GalNAc side chain to the first mannose. Characterization of the phosphatidylinositol moieties present on both free GPIs and GPI-anchored proteins shows the presence of a diacylglycerol lipid, whose sn-2 position contains almost exclusively an C18:1 acyl chain. The data presented here identify key novel inositol-acylated mannosylated intermediates, allowing the formulation of an updated T. gondii GPI biosynthetic pathway along with identification of the putative genes involved.

No abstract

Using hypotonically permeabilized