P. aeruginosa biofilm specific proteins and regulators

描述（由申请方提供）：铜绿假单胞菌是与囊性纤维化（CF）肺部感染相关的主要病原体之一，导致这些患者健康状况下降和预后不良。一旦建立，铜绿假单胞菌在生物膜中的生长使得很难通过抗微生物处理来根除生物体。最近的研究结果表明，生物膜的发展与Ser/Thr磷酸化模式的显着变化相一致，生物膜的发育过程是由一系列磷酸化的调节蛋白控制。此外，我们的数据表明，生物膜特异性调节蛋白的失活损害或逮捕的生物膜发育过程。拟议研究的目标是鉴定参与调节铜绿假单胞菌生物膜形成的磷酸化蛋白质，这些蛋白质一旦失活，就会损害或阻止体外生物膜发育的进展。我们假设铜绿假单胞菌生物膜的形成是由一系列磷酸化的调节蛋白控制的，这些调节蛋白的失活不仅会损害生物膜的形成，而且会导致生物膜比成熟生物膜更容易受到抗菌剂的影响。我们将首先确定磷酸化的调节蛋白，这是独特的过渡从非稳态生长阶段的成熟-1和-2生物膜发育阶段。这将通过金属氧化物亲和色谱法（MOAC）纯化磷酸化的调节蛋白，然后通过2D/PAGE和随后的质谱法使用肽质量指纹识别来实现。为了阐明生物膜形成的调控过程，我们将利用插入突变的调节蛋白，并检查是否这些监管机构的失活损害初始附着和/或生物膜的发展在流动条件下，以及如何失活的调节蛋白影响蛋白磷酸化和蛋白生产模式。此外，将检测在生物膜发育进程中受损的突变菌株对抗菌剂的敏感性。将通过RT-PCR进一步分析对抗菌剂更敏感的突变体，以通过确定mexF、mexC和转录调节因子PA 4878的转录丰度来进一步了解抗菌剂敏感性的机制。来自对铜绿假单胞菌生物膜发育过程的调节的详细研究的发现预期将导致基于生物膜形成的抑制或调节的创新且更有效的治疗策略，以治疗和控制生物膜感染。生物膜被认为是许多持续性和慢性细菌感染的根源，这些细菌感染对抗生素治疗无效，几乎不受宿主防御的影响。这项研究的结果旨在为治疗此类生物膜感染提供新的更有效的方法。

DESCRIPTION (provided by applicant): P. aeruginosa is one of the principal pathogens associated with Cystic fibrosis (CF) pulmonary infection and is responsible for a decline in health and poor prognosis for these patients. Once established, growth of P. aeruginosa in biofilms makes it very difficult to eradicate the organisms by antimicrobial treatment. Recent findings indicate that the progression of biofilm development coincides with marked changes in the Ser/Thr phosphorylation patterns and that the biofilm developmental process is controlled by a series of phosphorylated regulatory proteins. Furthermore, our data indicate that inactivation of biofilm-specific regulatory proteins impaired or arrested the biofilm developmental process. The goal of the proposed studies is to identify phosphorylated proteins involved in the regulation of P. aeruginosa biofilm formation that once inactivated, impair or arrest the progression of biofilm development in vitro. We hypothesize that P. aeruginosa biofilm development is controlled by a series of phosphorylated regulatory proteins and that inactivation of these regulatory proteins will not only impair the progression of biofilm development but also result in biofilms that are more susceptible to antimicrobial agents than mature biofilms. We will first identify phosphorylated regulatory proteins that are unique to the transition from planktonic growth stage to maturation-1 and -2 biofilm developmental stages. This will be achieved by purifying phosphorylated regulatory proteins from planktonic and biofilm growth stages by metaloxide affinity chromatography (MOAC), followed by 2D/PAGE and subsequent identification by mass spectrometry using peptide mass fingerprinting. To elucidate the regulatory process of biofilm formation, we will make use of insertional mutation of regulatory proteins and examine whether inactivation of these regulators impairs initial attachment and/or biofilm development under flowing conditions and how inactivation of regulatory proteins affects the protein phosphorylation and protein production patterns. Furthermore, mutant strains impaired in the progression of biofilm developmental will be tested for susceptibility to antimicrobial agents. Mutants that are more susceptible to antimicrobial agents will be further analyzed by RT-PCR to gain further insight into the mechanism of antimicrobial susceptibility by determining the transcript abundance of mexF, mexC and the transcriptional regulator PA4878. Findings from this detailed investigation of the regulation of the P. aeruginosa biofilm developmental process are expected to lead to innovative and more effective treatment strategies based on inhibition or regulation of biofilm formation to treat and control biofilm infections. Biofilms are considered the root of many persistent and chronic bacterial infections, which are both refractory to antibiotic therapy and barely affected by host defenses. Findings from this research are intended to lead to novel and more effective approaches for the treatment of such biofilm infections.