The Gram-negative bacterium Pseudomonas aeruginosa is responsible for a significant percentage of nosocomial infections and is typically the terminal respiratory pathogen in cystic fibrosis patients. Production of exopolysaccharides (EPS) is important in virulence and is critical for biofilm formation in P. aeruginosa. The long-term objectives of this project are to characterize the interaction of the EPS alginate, Psl, and Pel with several components of the innate immune system and evaluate the role of these EPS in the pathogenesis of infection in acute and chronic animal models. The hypothesis to be tested is that Psl, Pel, and alginate increase resistance to antimicrobial effectors and innate immune cells, and contribute to in vivo pathogenesis.
The first aim will address the role of P. aeruginosa EPS in resistance to antimicrobials and human polymorphonuclear leukocytes (neutrophils, or PMNs). The first subaim will utilize standard survival assays to investigate the effects of EPS on P. aeruginosa killing mediated by reactive oxygen species (ROS) and antimicrobials. The second subaim will utilize chemiluminescence assays to evaluate EPS effects on activation of the PMN respiratory burst. The third subaim will define the mechanism for the observed reduction in PMN response when Psl is expressed. Complement binding assays as well as phagocytosis and phagocytic killing experiments will be utilized.
The second aim will evaluate the effects of P. aeruginosa EPS on virulence in vivo. In the first subaim an acute murine model will be used to compare virulence of wild type and EPS mutants lacking one or more EPS. Colonization will be determined by viable plate counts of recovered bacteria at specific time points. Lung samples will be processed for histology, and morbidity and mortality will be observed in infected mice. The second subaim will use a chronic chinchilla model to evaluate the same strains as in the acute infections over a longer period of time. Virulence will be compared using the same readouts as in the acute infections.
The specific aims presented in this proposal will generate new knowledge of exopolysaccharides in P. aeruginosa pathogenesis. Understanding the role of EPS in the innate immune response and in vivo models could result in new strategies for treatment of P. aeruginosa infections. The estimated 30,000 Americans diagnosed with CF and a significant percentage of patients with nosocomial infections could directly benefit from this research, in accordance with the NIH mission of decreasing the burden of disease.