P. aeruginosa biofilm-specific proteins and regulators

描述（由申请人提供）：囊性纤维化（CF）是欧洲人后裔中最常见的致命遗传疾病之一，在美国影响30,000人。人们认为慢性CF肺部感染是由表面相关的抗微生物耐药性微生物群落引起的，称为生物膜，铜绿假单胞菌是主要病原体之一。目前CF感染的治疗策略，包括频繁的抗生素治疗和胸部物理治疗，都不能清除这些感染，尽管宿主免疫防御完好，但肺中的生物膜细菌仍然存在。最近，有人建议针对生物膜的治疗策略可能成功治疗CF肺部感染。

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is one of the most common lethal genetic diseases among people of European descent, affecting 30,000 individuals in the United States. It is believed that chronic CF lung infections are caused by surface-associated, antimicrobial-resistant communities of microorganisms called biofilms with Pseudomonas aeruginosa being one of the principal pathogens. Current treatment strategies for CF infections, including frequent antibiotic treatment and chest physiotherapy, fail to clear these infections and biofilm bacteria persist in the lung despite intact host immune defenses. Recently, it has been suggested that therapeutic strategies directed towards biofilms may be successful in treating CF lung infections. Our research goal proposed herein is designed to elucidate the nature and identity of proteins that are unique to the biofilm mode of growth for the development of therapeutic strategies directed towards biofilms. Previous work in our laboratories has demonstrated that P. aeruginosa PAO1 undergoes a major shift in its cellular protein profile during biofilm development. This shift is most profound in biofilms grown for 3 and 6 days (maturation-I and maturation-II stage, respectively). We hypothesize that we will identify biofilm-specific proteins - important regulatory, virulence and resistance proteins - that are unique to the maturation-I and maturation-II biofilm stages. We expect that many of the biofilm-specific proteins are post-translational modified and have regulatory functions involved in signal transduction. Our goal will be accomplished by utilizing two-dimensional gel electrophoresis (2D/PAGE) combined with 2D-image analysis and protein identification. Biofilm-specific proteins will be identified by peptide mass fingerprinting using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS). Upon protein identification, functional proteomics will be used to provide an insight in signal transduction cascades: phosphorylated proteins will be immunoprecipitated and separated by 2D/PAGE. Comparative 2D-image analysis will reveal proteins that are uniquely phosphorylated in the protein patterns of biofilms grown to the maturation-I and -II biofilm stages. Uniquely phosphorylated, biofilm-specific proteins will then be analyzed by peptide mass fingerprinting and MALDI-ToF MS.