Giardia lamblia undergoes surface antigenic variation where only one of a set of variable specific surface antigens (VSPs) is expressed on the surface of each trophozoite at any one time. For reasons that are not known, one VSP is periodically exchanged for another. Although it is commonly taught that antigenic variation exists solely as an immune escape mechanism, antigenic variation also occurs in the absence of immune selection. A number of types of evidence suggests that biological selection also occurs. Prior experiments indicated that VSPs possess unique or different physical properties that under the appropriate conditions allow specific VSPs to be either positively or negatively selected. Most Giardia lamblia isolates can be adapted to grow in vitro in a complex media. However, Giardia can only be maintained under conditions favorable for mammalian cell culture for only a limited time period, only a few hours. Since Giardia adhere to the intestinal epithelium in vivo, it is important to understand their interactions. Results of studies to date have been limited because of inadequate culture conditions for either Giardia or mammalian cells or both. A system was developed that allowed Giardia to grow and multiply in the presence of epithelial cells for over a year. The WB isolate of Giardia was adapted to grow in a number of epithelial cells lines but multiple attempts to grow the GS isolate in the presence of epithelial cells were unsuccessful. This result lends support to the idea that the GS isolate of Giardia is so different from the WB isolate that it fulfills the criteria for being a new species. Giardia were continuously maintained for over a year through subculture and/or replacement of medium. The system required the presence of viable adhered epithelial cells and Giardia growth required a healthy monolayer. Different epithelial cell types supported growth to variable degrees. The type size, geometry and chemical make up of the culture vessel were important variables. Adapted Giardia grew faster than non-adapted Giardia. Certain VSPs were favored under in vitro conditions, a finding that supports biological selection. This system will allow detailed study of the interaction of Giardia and epithelial cells and why one VSP is favored over others under specific conditions.? ? One of the major problems in understanding antigenic variation in Giardia is the multiple copies of identical or almost identical vsp genes and inability to know which of the family of highly related vsps is being expressed or not expressed. In previous experiments, HA tagged VSPH7 was integrated into the genome of its own host, GS, and underwent antigenic variation identical to native VSPH7. Analysis indicated that integration occurred into a site that differed from vspH7 but nevertheless was virtually identical up to about 3kb. Despite the similar upstream regions, vspH7 and vspH7HA were expressed independently in the absence of gene movement, definitively proving that epigenetic control of antigenic variation in Giardia. Chromosomal precipitin assays of the immediate upstream region of VSPH7 HA were performed using trophozoites expressing or not expressing the tagged VSP. An anti-acetylated lysine Mab was able to precipitate immediate upstream chromatin from expressing but not from non-expressing trophozoites confirming epigenetic control of antigenic variation. Because of technical reasons we were unable to compare other nearby regions but nevertheless the differences between expressing and non-expressing organisms implicate the 5? UTR of VSPs as regions that control expression. However, it remains unknown how two identical upstream regions are controlled independently. The downstream. 3? UTR regions of vspH7 HA and vspH7 were sequenced and found to be identical for at least 1 kb downstream confirming the virtual identical immediate surrounding sequences for vspH7 and vspH7HA.
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