MGH-PGA Genomic Analysis of Stress and Inflammation: Pseudomonas aeruginosa Infections of CF Mouse Models Nicole T. Liberati, Dan G. Lee, Jacinto M. Villanueva and Frederick M. Ausubel Department of Molecular Biology Massachusetts General Hospital Carolyn Cannon, Fadie Coleman, Mike Kowalski Jeff Lyczak, Martin Lee, Gloria Meluleni, and Gerald Pier Channing Laboratory Brigham and Women’s Hospital

Use of Transgenic CF Mice for Establishing Chronic Oropharyngeal Colonization Difficult to establish chronic P. aeruginosa infections in CF KO mice. Maintain mice 5-7 days on water containing antibiotics. 107 CFU P. aeruginosa/ml in drinking water for 5 days. Sterile water for 1 week followed by water containing antibiotics. Monitor mice by throat culture every 1-2 weeks.

P. aeruginosa PA14 Chronically Colonizes Oropharynx of CF Mice 100 Percent Positive Throat Cultures 80 60 C57sBl/6 WT mice SPC-CFTR* *Over-express CFTR via lung-specific surfactant protein C promoter CF mice 40 20 1 3 6 8 10 12 15 17 19 21 23 26 29 Weeks After Infection with P. aeruginosa PA14

Percentage probability Summary of Results from Seven CF Mouse Colonization Studies Probability of Colonization Negatively Correlates with CFTR Expression 100 P<.001 75 P=.02 Percentage probability of colonization 50 25 CF mice Wild-type mice SPC-CFTR mice Mouse strain

PA14 algD (alginate) and gacA (global accessory regulator) Mutants Fail to Colonize CF Mice 100 Percent Positive Throat Cultures 80 PA14 Wild Type PA14 algD 60 PA14 gacA 40 20 1 3 7 11 15 17 19 21 23 Weeks after Infection with P. aeruginosa

PA14 algD (alginate) Mutant Fails to Chronically Colonize the Oropharynx of CF Mice PA14 algD in transgenic CF mice 100 Percent Positive Throat Cultures 80 C57Bl/6 WT-mice 60 CF mice 40 20 1 3 7 11 15 17 19 21 23 Weeks after Infection with P. aeruginosa algD

Weeks After Infection with P. aeruginosa gacA PA14 gacA (global accessory regulator) Mutant Fails to Chronically Colonize the Oropharynx of CF Mice PA14 gacA in transgenic CF mice 100 Percent Positive Throat Cultures 80 C57Bl/6 WT-mice CF mice 60 40 20 1 3 5 7 11 15 19 21 23 25 27 Weeks After Infection with P. aeruginosa gacA