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 consesquencce 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 tranlationally 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.? ? Class I and class II fructose-1,6-bisphosphate aldolases (FBPA), glycolytic pathway enzymes, exhibit no amino acid sequence homology and utilize two different catalytic mechanisms. The mammalian class I FBPA employs a Schiff base mechanism, whereas the human parasitic protozoan Giardia lamblia class II FBPA is a zinc-dependent enzyme. In this study, we have explored the potential exploitation of the Giardia FBPA as a drug target. First, synthesis of FBPAwas demonstrated in Giardia trophozoites by using an antibody-based fluorescence assay. Second, inhibition of FBPAgene transcription in Giardia trophozoites suggested that the enzyme is necessary for the survival of the organismunder optimal laboratory growth conditions. Third, two crystal structures of FBPA in complex with the transition state analog phosphoglycolohydroxamate (PGH) show that the enzyme is homodimeric and that its active site contains a zinc ion. In one crystal form, each subunit contains PGH, which is coordinated to the zinc ion through the hydroxamic acid hydroxyl and carbonyl oxygen atoms. The second crystal formcontains PGHonly in one subunit and the active site of the second subunit is unoccupied. Inspection of the two states of the enzyme revealed that it undergoes a conformational transition upon ligand binding. The enzyme cleaves D-fructose-1,6-bisphosphate but not D-tagatose-1,6-bisphosphate, which is a tight binding competitive inhibitor. The essential role of the active site residue Asp-83 in catalysis was demonstrated by amino acid replacement. Determinants of catalysis and substrate recognition, derived from comparison of the G. lamblia FBPA structure with Escherichia coli FBPA and with a closely related enzyme, E. coli tagatose-1,6-bisphosphate aldolase (TBPA), are described.
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