Glycan structure and biosynthesis in Entamoeba histolytica

Aside from the well-known and deadly infectious diseases malaria, tuberculosis and AIDS, there are less-known but nevertheless lethal infectious agents, in particular in countries with limited resources. The present project addresses the protozoan parasite Entamoeba histolytica. An estimated 50,000 patients annually die from infections with this amoeba, which attacks the human colon and liver. Our project deals with a complex surface molecule, of which every invasive amoeba possesses about 80 million copies. The molecule carries the complex name lipopeptidophosphoglycan (LPPG), and also alternative names. It possesses a core protein molecule anchored in the cell membrane with a structure containing both sugar and lipid. The core protein, which has not been identified, is known to possess a large number of serine residues. Carbohydrate chains are linked to these serines indirectly via a bridging phosphate group. We encountered LPPG when we raised monoclonal antibodies from mice immunised with amoeba surface preparations. The antibody EH5 binds to LPPG and is able to protect amoeba-infected mice from liver abscesses. Later we showed that the antibody recognises a defined short amino acid sequence. Subsequently, when the data from the E. histolytica genome project became available, we discovered a gene coding for a protein containing the short amino acid sequence, which would therefore be a candidate for the LPPG core protein. One aim of our project is to demonstrate if this gene product is the core protein of LPPG. Furthermore, we will study the biosynthesis of the side chains of the LPPG. These consist primarily of glucose, the most abundant sugar molecule, which is linked similarly as the material on the surface of caries bacteria. A further question is how the amoeba generates the basis of these chains. In the structure of LPPG it is striking also to observe that galactose is incorporated in several positions. Galactose differs from glucose by a single group pointing in another direction. The enzyme GalE named UDP-glucose 4-epimerase is able to convert UDP-glucose to UDP-galactose, thereby generating the activated form of galactose. We plan to disrupt the GalE gene in the amoebae to examine the consequences for the LPPG and in general for the amoebae. In addition, we are looking for the transferases which could specifically incorporate galactose. We searched the genome databases and established a preliminary list of candidate enzymes for carbohydrate biosynthesis. An analysis in more depth is planned in collaboration with a colleague in France. Taken together, we expect that our cooperation between the Molecular Parasitology of the Medical University of Vienna and the Carbohydrate Biochemistry of the University of Natural Resources and Life Sciences (BOKU) will significantly contribute to the understanding of the the surface of pathogenic entamoebae.

The project addressed the human protist parasite Entamoeba histolytica, which is responsible for amoebic dysentery and liver abscesses. Worldwide it causes around 50,000 deaths annually, but in Austria the availability of clean and safe water and food prevents the spread of amoebiasis. We had studied the parasite for many years but in the present project we focussed on the glycan biochemistry. In one part of the work we studied the enzyme UDP-glucose 4-epimerase (GalE). This enzyme with the complicated name carries out a very special chemical reaction, essentially it interconverts the sugars glucose and galactose. We found that the enzyme helps to convert modified galactose which the parasite cleaves from the mucus in the human intestine to modified glucose that the parasite can use for its own purposes to synthesize its cyst wall. In the second part of our project, we addressed a complex surface molecule of the parasite called lipopeptidophosphoglycan (LPPG). It covers quite a large part of the amoebic surface and contains protein, sugars and phosphate. It has an unknown core protein with dextran side chains which are chemically similar to the dextran produced by dental caries bacteria. Previously we had produced a monoclonal antibody against LPPG which was able to protect mice from amoebic liver abscess. In this project, the antibody was used to test for the LPPG antigen. In this way we were able to find an improved method to isolate it from the amoebae. We then tried to analyse the core protein of the LPPG, this with limited success. However, various cleavage experiments showed that dextranase destroyed the target of our monoclonal antibody. We then found that our antibody really binds to dextran. This is interesting as dental bacteria and invasive amoebae use similar methods to protect themselves from the attack of the host immune system. Taken together, our studies show the intensive biochemical interactions between the parasite and its human host, and that the parasite uses similar methods as pathogenic bacteria to protect itself from the host immune system.