The underlying hypothesis for this project is that soluble peptidoglycan (PG) fragments liberated from bacteria modulate the host response during the course of natural infections. The test of this hypothesis exploits two distinct PG-mediated biological reactions. The first system is based on our previous suggestion that PG derived for Neisseria gonorrhoeae contributes to the acute inflammatory reactions, e.g., urethritis and arthritis, characteristic of local and disseminated gonococcal infections, respectively. The second is based on the intriguing revelation that certain PG fragments accumulate in the brains of sleep- deprived animals and function as natural enhancers of slow-wave sleep. Our goal is to define the mechanisms behind these pro- inflammatory and sleep-promoting properties of PG. Toward this end, we will exploit a unique series of physiologically realistic PG fragments, e.g., N-acetylglucosaminlyl-1,6-anhydro-N- acetylmuramyl-alanyl-glutamyl-diaminopimelyl-alanine, which are purified to homogeneity by reversed-phase HPLC, unambiguously defined by fast-atom bombardment-mass spectrometry and, for some purposes, labeled to very high specific activity with tritium. In conjunction with assay systems for quantitating the release of soluble PG by cultured bacteria and for examining the interactions of PG fragments with the intact host and with cultured host cells, the PG library will be employed to test the following integrated series of hypotheses, (1) that endogenous gastrointestinal flora turn over and release PG fragments (ii) that PG fragments in the gastrointestinal tract cross the intact mucosal epithelium and enter the vascular system, (iii) that some PG fragments ultimately localize in potential target tissues, including the joints and the central nervous system, where they might trigger a cascade of events leading to arthritis and enhanced sleep, respectively, (iv) that PG-mediated arthropathic and somnogenic activities are associated with modulation of the release of common mediators, such as interleukin-1, form specific cultured target cells, e.g., synovial fibroblasts and astroglial cells, and (v) that the ability of PG fragments to cause arthritis, enhance sleep and modulate release of mediators is associated with the specific binding of PG determinants to receptors on susceptible target cells. We feel the defining the cellular and molecular events involved in gonococcal PG-arthritis and enhancement of slow-wave sleep may reveal common mechanisms that operate whenever a biologically relevant burden of these versatile effector molecules gain access to specific host tissues.
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