The ability of the gonococcus to gain access to the intracellular environment appears to be important for its ability to initiate and sustain infection. As part of its intracellular existence, the organism acquires the substrate, cytidine monophospate acetyl neuraminic acid (CMP-NANA) in order to sialylate it lipooligosaccharide (LOS). This is necessary since the gonococcus lacks the enzyme CMP-NANA synthase, Sialylation of the LOS has been shown to confer a number of advantages to the organism in the extracellular environment including inhibition of antibody and complement binding to gonococcal outer membrane structures. CMP-NANA is available to the organism within only two intracellular compartments, the Golgi complex and the nucleus. In order for the LOS to become sialylated, the gonococcus must traffic to these locations in the cell in which CMP-NANA acquisition is unlikely because organisms not be seen within the nuclear membrane in infected human tissues or cell culture infections. This would indicate that the Golgi is the site responsible for providing the CMP-NANA. Trafficking within eukaryotic cells is an organized process, dependent upon a variety of specific signals. One mechanism for trafficking are specific motifs on the cytoplasmic tails of receptors which are involved in clathrin dependent receptor-mediated endocytosis. We have demonstrate the presence of such a receptor, the asialoglycoprotein receptor (ASGP-R), in human urethral epithelial cells and have shown that it is up-regulated during infection. Lipids such as ceramide, preliminary studies indicate that the urethral epithelial cells can produce the cytokines IL-6, IL-8, TNFa. And IL-1b independent of CD14. The hypothesis upon which this project is based is that the gonococcus moves in a directed fashion within the human urethral epithelial cell and that cytokines are induced from urethral epithelial cells in a CD14 independent fashion. These hypothesis will be resolved by the following specific aims. 1. Studies will be done to define the intracellular trafficking pathway of Neisseria gonorrhoeae within primary human urethral epithelial cells. 2. Studies will be done to define the role of the asialoglycoprotein receptor and LOS in trafficking of the N. gonorrhoeae in primary human urethral epithelial cell cultures. 3. Studies will be done to examine LOS signal transduction in primary urethral epithelial cells.
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