The ability of Giardia lamblia to differentiate in response to external stimuli is central to its pathogenesis. The Giardia cytoskeleton undergoes drastic changes during encystation and excystation. However, the pathways mediating these key transformations are poorly understood. The overall hypothesis unifying this revised renewal is that the centrosomes are cellular """"""""control centers"""""""", while cytoskeletal elements unique to Giardia have specific effector functions that are reflected in their protein compositions. We will test our hypothesis with these Specific Aims: During encystation, the motility apparatus is internalized.
In Specific Aim 1 A, we test the hypothesis that encystation entails quantitative and qualitative changes in the protein composition of Giardia centrosomes/basal bodies that point to new levels of regulation. We will compare the proteomes of centrosomes from growing and encysting trophozoites by Multidimensional Protein Identification Technology (MudPIT).
In Specific Aim 1 B, we will epitope-tag and express selected proteins in Giardia to validate their localization and for functional analyses in Aims 2 and 3. A major goal of cell biology research is to relate changes in protein localization to physiologic stimuli of differentiation. Most centrosomal proteins identified so far also localize to cytoskeletal elements that are unique to Giardia.
Specific Aim 2 is to test the hypothesis that the localization of certain proteins to centrosomes or Giardia-specific structures is responsive to physiologic signals.
Specific Aim 3 is to analyze the biological roles of selected centrosomal signaling proteins and proteins unique to Giardia.
In Aim 3 A, we will identify proteins that form complexes with signaling proteins to test the hypothesis that they localize and function by association with specific targeting or regulatory subunits.
In Aim 3 B, we will use post-transcriptional gene silencing (PTGS) and over- expression to further test this hypothesis by defining the functions of selected signaling and unique proteins in growth and differentiation. Relevance to public health: Giardia lamblia is a major cause of water-borne diarrheal disease in the US and worldwide and a Class B bioterrorism agent. These studies will greatly advance our understanding of the biology of G. lamblia, whose pathogenesis depends on its ability to respond rapidly to changing signals from its environment by encysting or excysting. Giardia is a valuable model for other parasites whose life cycles have not been completed. ? ? ?
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