| Microsporidia are obligate intracellular pathogens of invertebrate and vertebrate animals. These parasites cause disease in several economically important species of insects, fish, and mammals. Infections in humans have become prevalent with the advent of HIV and AIDS. Despite their pervasiveness in the animal kingdom, relatively little is known about the cellular and molecular biology of microsporidia. Microsporidia posses a polar tube, a unique preformed structure that functions as an invasion organelle. Extrusion of this tube from the spore, penetration across a host cell membrane, and subsequent deposition of the parasite DNA into the host cytoplasm initiates microsporidial infection. The signals and mechanics involved in initiating polar tube extrusion are not known. Work by others suggested that water channels, also known as aquaporins, may be involved in polar tube extrusion. The first chapter of this dissertation reviews aquaporin biochemistry and use of genetically altered mice to investigate the function of these water permeable, integral membrane proteins. Review of the literature revealed that most mouse aquaporin mutants are genetically heterogeneous and therefore conclusions regarding the observed phenotype may not be correct. The second chapter describes a quantitative molecular assay developed to determine the life cycle and effects of various tissue culture conditions on in vitro replication of Encephalitozoon intestinalis, a frequently diagnosed microsporidial parasite of humans. Results indicated that E. intestinalis invades host cells, replicates, and releases mature spore forms in 72 hours. Addition of albendazole, a microtubule inhibitor, to the culture media suppressed---but did not inhibit---parasite replication. The third chapter describes the identification and cloning of an aquaporin-like gene from E. cuniculi, E. hellem and E. intestinalis. These parasite proteins are 80% similar to each other, and 22% similar to human aquaporin-1. Infection with E. intestinalis in a tissue culture system was inhibited upon pre-exposure of parasite spores to mercuric chloride, a common aquaporin channel inhibitor. Infectivity was restored upon counter treatment with mercaptoethanol. Expression of these proteins in a heterologous expression assay confirmed their function as aquaporins. Lastly, chapter four is a review of murine protozoa, including the flagellates, the amebae, the apicomplexans, the ciliates, and microsporidia. |
