The flagellum expressed by pathogenic microorganisms functions at several stages of infection. In addition to their role in motility, some flagella are attachment organelles, others are used for the secretion of virulence factors, and all are likely to be potent stimuli in triggering the innate immune response and inflammation. The single flagellum of P. aeruginosa serves some of these functions in disease and plays a pivotal role in the formation of bacterial biofilms in nature and possibly in lungs. The long-term goal of this proposal is therefore to understand the biogenesis of this organelle in nature and disease. The major regulatory steps in the flagellar biogenesis have been elucidated but little is known about its regulation in disease. Recent findings demonstrate that growth of P. aeruginosa in respiratory mucus results in a block in flagellin synthesis. The purpose of repression of flagellin synthesis may be to avoid production of potent innate immune response activators and perhaps prevent opsonophagocytosis. The hypothesis that derives from this observation is that down-regulation of flagellin may be a prerequisite for chronic colonization of mucus in Cystic Fibrosis.
The specific aims of this proposal are therefore: 1. Identify the regulatory network which controls flagellin expression and hence flagellum assembly in mucus. 2. Identify the signal for flagellin down-regulation in mucus. 3. Engineer strains of P. aeruginosa that constitutively express a flagellum in mucus and assess their virulence in animal models of infection. The regulatory network that directs the repression of flagellin production within the normal biogenesis pathway will be ascertained by examining the role of an inhibitor of flagellin synthesis, FIgM, in this process if any. The action of novel regulatory mechanisms that may function independent of the flagellar biogenesis pathway will also be examined. The source of the signal in mucus, which controls flagellin expression, will be sought by examining whether adhesion to mucins, or another component of mucus provides these environmental signals. Lastly, P. aeruginosa strains lacking suppression of flagellin synthesis in mucus will be engineered and examined in both an acute and a chronic model of P. aeruginosa lung infection. If flagellin synthesis is deleterious to chronic colonization, this may provide an opportunity for therapeutic intervention, where ectopic expression of flagellin in mucus may be driven to allow host defenses to deal with the organism early in the colonization process.
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