Giardia lamblia is the most common disease-causing parasite in the United States responsible for an estimated 3 million cases a year. Besides causing problems in day care centers, travelers, backpackers, and homosexuals, it not uncommonly contaminates water supplies and has been responsible for massive epidemics. The organism lives in the small intestines and causes diarrhea, abdominal pain, nausea and vomiting. Symptoms are commonly intermittent and long lasting. An environmentally resistant cyst form is passed in the feces, and, because large numbers of cysts are excreted and only a few can initiate infection, infections are common.During the past year, a system for stably placing DNA into Giardia was improved (stable transfection) so that enough protein could be expressed to make it easily detected in the parasite. This allows the expression of any protein in the organism. For instance, a foreign protein, or more of a protein normally present, or a protein defective in some way that would interfere with normal function could be made. We are presently using this system to study a number of questions.Giardia has the ability to change its surface relatively frequently, a process called antigenic variation. Usually only one surface antigen or VSP can be seen on its surface. How only one VSP among many is expressed on the surface of the parasite, how VSPs are made and transported to the surface, and what is their biologic role are of major interest. Using the transfection system we have been able to express a novel VSP that is continually expressed on its surface along with a native VSP. Changes in the protein were then introduced into the VSP to determine the importance of particular regions and whether they could be secreted and placed onto the surface of the parasite. These studies showed that certain changes to conserved regions of the VSP resulted in inability of the parasite to process the VSP and place it on its surface. Substitution of an unrelated sequence in one conserved area unexpectedly showed no effects One question is how Giardia controls antigenic variation. The ability of the parasite to express two VSPs at once indicates that control of VSP expression does not reside in the coding region of the protein. In other studies, a foreign model protein with known and characterized secretion and processing in more developed cells was introduced into Giardia. We are comparing how Giardia handles this protein compared to mammalian cells.In order to survive and infect others, Giardia forms a cyst wall. Two constituents of the cyst wall were previously defined in this laboratory. Both proteins are not normally made or found in the growing form of the parasite but are induced or made when the growing form of the parasite (trophozoite) is cultured under the right conditions. We are presently studying how Giardia controls the production of cyst wall proteins and found that the controlling elements lie immediately upstream to the cyst wall protein gene. The two cyst wall protein genes are induced in exactly the same manner although their upstream regions are not identical. Exactly which small DNA pieces are important is currently being studied. Studies on Microsporidium were begun. This is a parasite that causes diarrhea and other problems in AIDS but recently has been found to cause diarrhea in normal persons as well. The parasite is only found inside the cell it infects. Using very special techniques we have been able to define proteins that are important for the parasite in general, important for infecting the host, and proteins that are specifically seen in the host cell when infected with this parasite. Understanding what proteins are important can lead to a better way to treat or prevent infection. - Giardia lamblia. Microsporidium, transfection, antigenic variation
