The Harry M. Zweig Memorial Fund for Equine Research


Endoproteases of Sarcocystis Neurona

Dr. Stephen C. Barr

Sarcocystis neurona is a protozoan parasite that enters the brain and spinal cord of horses causing Equine Protozoal Myeloencephalitis. The disease has probably existed for a long time but it was not until the 1970s that a protozoan was associated with the disease, and it was not until the early 90s that the parasite was isolated from horse tissues.

Opossums are the definitive host for the parasite, but birds also become infected. No one really knows how or why horses become infected. We assume the horse becomes infected by eating the parasite in the feces of opossums (spread on pasture), then the parasite makes its way from the horse's gastrointestinal tract to the brain and spinal cord. The life stages of the parasite found in the brain are merozoites. The cell damage merozoites cause in the brain and spinal cord resulting in ataxic (horse becomes wobbly on its feet) making the horse impossible to ride or work. There are no drugs currently known that will kill the parasite, so once the horse has the disease, it is very difficult to treat. There are no vaccines.

One way of finding new drugs to attack a parasite is to find molecules in the parasite that have important functions essential for its survival and the progression of its life cycle. Thus, if the function of the particular enzyme, for example, is removed or inhibited by the action of drugs or vaccine, the parasite would die. The aim of this project is to find out if certain molecules exist in merozoites, which, as mentioned, is the life stage of S neurona that is responsible for the damage to the brain. One group of important molecules present in protozoan parasites, and in fact a wide range of other organisms, are endoproteases. These enzymes have very important functions in the lives of most organisms. For example, a protease of schistosome cercariae facilitates the parasite's penetration of people's skin to initiate infection. An aspartic protease (a class of endoproteases) of HIV that causes AIDS in man, has been shown to be very important in helping the virus enter cells. Recently, drugs have been made to inhibit this protease and are the next generation of effective chemotherapy for HIV-infected people. Thus, because the structure of many of these proteases is well known, we can use this knowledge to help find similar proteases in S. neurona. Because the proteases of each endoprotease class from each organism have some areas of them that are very similar (conserved regions), we can use the DNA sequence of the conserved regions as a starting point (a probe) to find the rest of the gene of the protease in other organisms. Once we have the entire gene, we can determine how the protease will look and act and make large amounts. Once made, we can use the protease to design drugs that inhibit it, or as a basis for vaccine production.

Using funding from last years Zweig grant, we determined that merozoites of S neurona contain a gene which makes an aspartic protease. We have also determined that merozoites contain aspartic protease activity - this may be the product of the aspartic protease gene we have isolated. In this specific project we aim to put the aspartic protease gene we have isolated into a specific bacterium which is designed to produce the aspartic protease enzyme in reasonably pure and large amounts. We can also inject the aspartic protease into a rabbit to produce antibodies against the enzyme. These antibodies can be used in a number of future studies (e.g. to examine where the enzyme is produced in the organism) which are not specifically part of this grant application. However, as part of this application, we can use the pure aspartic protease enzyme to see if serum from horses that have had EPM reacts against the protease. If it does, this suggests that the protease may form the basis for a vaccine against the disease. There is a lot of information already about the effectiveness of drug treatment against proteases of various parasites and viruses such as HIV. Also, there are several vaccines being tested in animals against certain parasite proteases, including the blood worm of sheep and cattle (Haemonchus contortus), cattle ticks (Boophilus microplus), and liver fluke (Fasciola hepatica). Our long term objective is to create an effective treatment or vaccine against a protease of S. neurona.