Pseudomonas aeruginosa conversion to mucoid phenotype plays a critical role in the establishment of chronic respiratory infections in cystic fibrosis (CF) which are responsible for the high morbidity and mortality in this disease. Conversion to mucoidy is controlled by the algU mucABCD locus. The algU gene is a positive regulator of mucoidy and encodes an equivalent of the extreme that shock sigma factor RpoE from enteric bacteria. This strongly suggests a connection between the extreme stress response and conversion of P. aeruginosa to mucoidy. The goal is of this proposal are: i) to delineate the molecular mechanism of conversion to mucoidy in P. aeruginosa; ii) to investigate the role of the stress system that controls mucoidy and possibly additional aspects of P. aeruginosa virulence; and iii) to assess the contributions of mucoidy and co-regulated systems to the pathogenic processes during chronic respiratory infections in CF. The following studies will be carried out: i) AlgU-dependent transcription of the critical biosynthetic and regulatory genes necessary for the production of and oxidative stress response will be established; ii) MucA (or MucB) activity as an anti-IgMa factor or inhibitor of AlgU activity will be investigated and additional mutations and genes causing conversion to mucoidy will be characterized; iii) the proteins and genes that are controlled by AlgU and induced by stress or overexpressed in muc mutants will be analyzed by metabolic labeling, 2D gel analysis, and expression technology based on green fluorescent protein; iv) the role of AlgU-dependent systems in P. aeruginosa pathogenesis in acute and chronic infections will be investigated by: (a) comparing the isogeneic algU+ and algU null strains for their susceptibility to killing with reactive oxygen intermediates and by phagocytic cells; (b) determining virulence of isogeneic exposed to P. aeruginosa aerosols; and (c) analyzing AlgU-dependent immunoreactive proteins using sera from CF patients. These studies will provide a complete model of the molecular mechanisms which govern conversion to mucoidy and develop a comprehensive view of the role that the extreme stress sigma factor AlgU plays in P. aeruginosa virulence and pathogenesis in CF with implications for pharmacological or immunological intervention.
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