P. aeruginosa biofilm-specific proteins and regulators

描述(申请人提供)：囊性纤维化(CF)是欧洲人后裔中最常见的致命性遗传病之一，在美国影响30,000人。慢性肺部感染被认为是由表面相关的、耐药微生物群落生物膜引起的，其中铜绿假单胞菌是主要病原体之一。目前对CF感染的治疗策略，包括频繁的抗生素治疗和胸部理疗，无法清除这些感染，尽管宿主免疫防御完好，但生物膜细菌仍留在肺内。最近，有研究表明，针对生物膜的治疗策略可能在治疗CF肺部感染方面取得成功。 我们在这里提出的研究目标是为了阐明生物膜生长模式特有的蛋白质的性质和特性，以便开发针对生物膜的治疗策略。我们实验室以前的工作已经证明，在生物膜发育过程中，铜绿假单胞菌PAO1的细胞蛋白质谱发生了重大变化。这种转变在生长3天和6天(分别为成熟-I期和成熟-II期)的生物膜中最为明显。 我们假设我们将识别生物膜特有的蛋白质--重要的调节蛋白、毒力蛋白和抗性蛋白--它们是成熟-I和成熟-II生物膜阶段特有的。我们预计许多生物膜特异的蛋白质是翻译后修饰的，并具有参与信号转导的调节功能。 我们的目标将通过利用二维凝胶电泳(2D/PAGE)结合2D图像分析和蛋白质鉴定来实现。生物膜特异性蛋白质将通过使用基质辅助激光解吸电离飞行时间质谱仪(MALDI-ToF MS)的多肽质量指纹图谱进行鉴定。在蛋白质鉴定的基础上，功能蛋白质组学将被用来提供对信号转导级联的洞察力：磷酸化的蛋白质将被免疫沉淀并通过2D/PAGE分离。比较2D图像分析将揭示生长到成熟-I和-II生物膜阶段的生物膜的蛋白质模式中独特的磷酸化蛋白质。然后，通过多肽质量指纹图谱和MALDI-ToF MS分析独特的磷酸化的生物膜特异蛋白质。

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.