DESCRIPTION (provided by applicant): Coccidian parasites are important human pathogens that cause severe diarrhea in infants (Cryptosporidium parvum) and disseminated infections in fetuses (Toxoplasma gondii). Both Cryptosporidium and Toxoplasma cause chronic infections in immunosuppressed adults. Oocysts are infectious stages of coccidia, which are spread by the fecal-oral route. While numerous proteins have been identified in oocyst walls (e.g. Cryptosporidium oocyst wall proteins (COWPs)), there are no models for how oocyst walls Toxoplasma and Cryptosporidium are assembled. Here we show that the inner layer of the oocyst wall of Toxoplasma is a porous scaffold of fibrils of ß-1,3-glucan, a sugar polymer present in fungal walls. While glucan synthase inhibitors (echinocandins) kill fungi, these drugs arrest the development of oocyst walls of Eimeria, a chicken parasite that closely models Toxoplasma oocysts. The outer layer of the oocyst wall of Toxoplasma contains acid-fast lipids like those that cover the surface of mycobacteria. Organic solvents release oocyst wall lipids, which are triglycerides with polyhydroxy fatty acyl chain like those of the waxy cuticle of plants. The oocyst wall of Cryptosporidium contains acid-fast lipids but does not contain glucan fibrils. Instead the inner layer of the Cryptosporidium wall contains fibrils of a novel sugar polymer. Proposed studies in Aim 1 test a two-layer model of the Cryptosporidium oocyst wall. We will identify glycoproteins in the outer rigid bilayer of wall that also contains acid-fast lipids. We wll determine the chemical composition of fibrils in the inner layer of the oocyst wall and determine whether COWPS are lectins that bind these sugar fibrils. Proposed studies in Aim 2 focus on the structure and development of the oocyst wall of Toxoplasma. We will identify oocyst wall proteins and determine whether those in the outer, acid-fast layer have lipid-binding properties, while those in the inner layer are lectins that bind to fibrils of ß-1,3-glucan. We will test the dea that ß-1,3-glucan, acid-fast lipids, and wall proteins are made in separate secretory compartments during development of the oocyst wall, and we will identify cytoskeletal elements important for secretion and for determining oocyst wall shape. Proposed studies in Specific Aim 3 focus on the structure and development of the sporocyst wall of Toxoplasma. We will test the hypothesis that sporocyst wall of Toxoplasma is structural similar to the Cryptosporidium oocyst wall, which is also acid-fast, is missing fibrils of ß-1,3-glucan, and contains sporozoites. We tet the idea that sporulation (development of the sporocyst wall and sporozoites) differs markedly from vegetative growth by tachyzoites, which is well-characterized. In particular, nuclear division is uncoupled from cell division; sporoblast formation is by binary fission rather than budding (endodyogeny); and each sporozoite develops synchronously but independently from others, using an IMC complex similar to that of tachyzoites.

PUBLIC HEALTH RELEVANCE: Coccidian parasites cause life threatening diarrhea in infants (Cryptosporidium parvum) and disseminated infections in fetuses and immunosuppressed adults (Toxoplasma gondii). The proposed studies, which characterize the walls of infectious stages (oocysts) of the parasites, should lead to a better understanding of how these important human pathogens cause disease. The information gained concerning the structure of the oocyst wall may also be used in future studies to develop methods to decrease transmission of Cryptosporidium and Toxoplasma (e.g. by determining best methods for hand sanitization).