BLADDER GLYCOSPHINGOLIPIDS IN THE PATHOGENESIS OF URINARY TRACT INFECTION

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Although the bladder is by far the most common site of UTI, little is known about the molecules to which infecting bacteria attach in this tissue as compared with the kidney, in which the role of globoseries GSLs as bacterial attachment sites for Escherichia coli has been well studied. Since the carbohydrate portion of GSLs varies in different tissues, often under host genetic control, these molecules serve as determinants of both patient susceptibility to infection and tissue tropism for pathogenic bacteria. The overall goal of this proposal is to utilize primary cell cultures from the bladder as a model system to define the role and regulation of bladder GSLs as attachment sites for uropathogenic bacteria, focusing on the two most common causes of cystitis in women, E. coli and S. saprophyticus, in the following proposed studies: (1) To evaluate the hypothesis that globoseries (Gb) and ganglioseries (Gg) GSLs are present in primary cultures of bladder transitional epithelium and bind E. coli and S. saprophyticus, respectively, we will purify GSLs from the cells, identify E. coli-and S. saprophyticus-binding GSLs, and confirm the identities of the GSLs using specific monoclonal antibodies (MAbs) and formal carbohydrate structural analysis; (2) To evaluate the hypothesis that the GSLs identified in Specific Aim 1 are surface exposed in primary bladder cell cultures and are functionally relevant in E. coli and S. saprophyticus attachment, we will study bacterial adherence before and after pre-treatment of the cell cultures with an saprophyticus attachment, we will study bacterial adherence before and after pre-treatment of the cell cultures with an inhibitor of GSL synthesis or MAbs directed against relevant GSLs; (3) To evaluate the hypothesis that the secretor and ABO genes influence the expression of Gb and Gb GSLs in the bladder and thus the capacity for E. coli binding, we will establish primary bladder cultures from patients of known secretor/ABO status and determine whether the predicted glycosyltransferases and GSL products are present in the cultured cells; and (4) To evaluate the hypothesis that cytokines are release from primary uroepithelial cell cultures and modulate the expression of E.coli-and S. saprophyticus-binding GSLs in the bladder, we will to examine the effects of adherence by E. coli and S. saprophyticus on cytokine release from the cells and the effects of tumor necrosis factor and IL-1, IL-6 and IL-8 on the expression of relevant GSLs in primary urothelial cell cultures and on bacterial adherence to these cells. These studies on the expression and regulation of bladder GSLs are relevant to the pathogenesis of infection, inflammation and differentiation in this organ and could eventually lead to novel approaches to prevention of bacterial attachment and infection.