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. This laboratory has a long-standing interest in characterizing antigenic variation in Giardia lamblia including an understanding of the process, the biological advantage to the parasite and the consequences to the host. The surface of the trophozoite is covered by one of a family of related proteins called varying specific surface protein (VSPs) that change. Even though these proteins have certain common motifs such as an absolutely conserved cytoplasmic tail, CRGKA, their external residing portion is antigenically distinct and they have individual biochemical differences. As a consesquence both biological as well as immunological selection has been documented in vivo and in vitro. Prior studies showed that the cysteine of the CRGKA is post translationally modified by addition of a palmitate residue and this modification altered membrane location and altered the effects of antigenic variation. The present study indicates that the R in the tail is also modified to citrulline by arginine deiminase (gADI), an enzyme previously shown in Giardia lamblia and most other prokaryotes to be part of a enegy supplying pathway under anaerobic conditions. In contrast eukaryotes employ peptidyl-arginine deiminases to post translationally modify arginine to citrulline. The biological purpose of citrullination is not well defined in humans although responses to citrullinated proteins are felt to play a role in the pathogenesis of autoimmune diseases in humans. Our studies show that arginine deiminase binds to the CRGKA tail of the VSPs and unexpectedly functions as a peptidyl- arginine deiminase modifying the arginine in the tail to citrulline. While antibodies that bind to the extracellular portion of specific VSPs normally result in cytotoxicity to the trophozoites expressing the antigenic VSP, the response to the same citrullinated VSP fails to confer cytotoxicity. Citrullination therefore is one of the posttranslational processes that control the effects of host responses to the parasite. In addition, other studies indicate gADI has multiple biological functions including in encystation. In Giardia extracts, gADI is present in multiple forms including a 85kDa form that reacts with antibodies to SUM0-1 indicating control of gADIs function and localization by sumoylation, a finding enhanced by the presence of a known SUMO-l binding motif. These studies define a biological system that appears to play a crucial role in the biology of Giardia lamblia. Giardia is a eukaryotic protozoal parasite with unusual characteristics,such as the absence of a morphologically evident Golgi apparatus. Although both constitutive and regulated pathways for protein secretion are evident in Giardia, little is known about the mechanisms involved in vesicular docking and fusion. In higher eukaryotes, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of the vesicle-associated membrane protein and syntaxin families play essential roles in these processes. In this work we identified and characterized genes for 17 SNAREs in Giardia to define the minimal set of subcellular organelles present during growth and encystation, in particular the presence or not of a Golgi apparatus. Expression and localization of all Giardia SNAREs demonstrate their presence in distinct subcellular compartments, which may represent the extent of the endomembrane system in eukaryotes. Remarkably, Giardia SNAREs, homologous to Golgi SNAREs from other organisms, do not allow the detection of a typical Golgi apparatus in either proliferating or differentiating trophozoites. However, some features of the Golgi, such as the packaging and sorting function, seem to be performed by the endoplasmic reticulum and/or the nuclear envelope. Moreover, depletion of individual genes demonstrated that several SNAREs are essential for viability, whereas others are dispensable. Thus, Giardia requires a smaller number of SNAREs compared with other eukaryotes to accomplish all of the vesicle trafficking events that are critical for the growth and differentiation of this important human pathogen.
