Of the various toxins and virulence-related factors produced by Bordetella pertussis, only one has been demonstrated to reproduce the specific respiratory tract cytopathology of the pertussis syndrome. That molecule is tracheal cytotoxin (TCT),a 921 dalton peptidoglycan fragment released by Bordetella pertussis during normal growth. During the past 3 years of funding by this NIH grant, most of the research effort has centered on understanding TCT structure-function relationships, defining TCT target cells, and elucidating TCT mechanism of action. This renewal application is focused on experiments to describe further the TCT toxicity pathway, target cell specificity, and the molecular basis for TCT production. Five years are requested to explore the following specific aims: I. Define more precisely the role of interleukin-1 (IL-1) and nitric oxide (NO ) in the mechanism of TCT action. Experiments will evaluate whether IL-1 is indeed an essential step in the TCT toxicity pathway and will define the specific respiratory epithelial cells that respond to TCT (and Bordetella pertussis infection) by synthesizing IL-1 and/or NO ; similar studies will be designed to examine the molecular basis for species-specific responsiveness to TCT. II. Compare TCT's toxicity for neutrophils to the known biochemistry and biology of TCT's respiratory epithelial effects. The potent effects of TCT on neutrophils will be examined in experiments that parallel our previous work with respiratory epithelial cells: structure-activity relationships, evidence for binding to a surface receptor, the potential involvement of IL-1 and NO , and the possibility of synergy with endotoxin. III. Identify the genetic and biological basis of TCT release by Bordetella pertussis. These experiments will test the hypothesis that Bordetella pertussis release of TCT is largely due to a defective or missing membrane transport protein. AmpG, that is critical for peptidoglycan recycling. In addition, a novel mutant screen will be used to identify other gene(s) that may be involved in production of TCT.
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