Control of fimbrial gene expression in Pseudomonas aeruginosa

描述(由申请人提供)： 铜绿假单胞菌是一种重要的人类条件致病菌，是囊性纤维化患者发病和死亡的主要原因。在慢性感染的CF肺中，有机体以生物膜的形式存在--一个包裹在聚合物基质中的表面附着的细菌群落。这种生物被膜的生长模式增加了铜绿假单胞菌对抗生素的抵抗力，并有助于逃避宿主的免疫反应。在这些在铜绿假单胞菌生物被膜形成中起重要作用的基因中，突出的是CUPA基因，它编码有利于表面附着和宿主定植的菌毛结构的组成部分。我们已经鉴定了三个基因(CgrABC)，它们的产物是CUPA菌毛基因簇的相位可变(即可逆的开/关)表达所必需的。CGR基因的产物没有一个类似于任何经典的正向基因表达调控因子；CGRA被预测编码腺嘌呤核苷酸水解酶超家族的成员，而cgrB编码一个假定的乙酰酶，cgrC编码一个与DNA结合蛋白PARB家族同源的蛋白质。在目标1中，我们建议确定CGR蛋白如何发挥其控制作用。我们将明确测试CgrB通过以下方式发挥作用的假设 CGRA的乙酰化和CUPA基因的相变表达是由CGRA乙酰化状态的变化所介导的。由于许多其他致病细菌含有CGR同源基因，我们预计我们的研究将延伸到铜绿假单胞菌以外。最近，我们发现PrrA是一种新的小调节RNA(SRNA)，是CUPA基因的另一个正调控因子。初步证据表明，PrrA通过拮抗组蛋白样核结构蛋白H-NS家族成员的沉默效应而影响CUPA的表达。在目标2中，我们建议确定PrrA是如何发挥抗沉默作用的。我们预计，这些研究将使我们能够(I)确定蛋白质乙酰化和sRNA在控制铜绿假单胞菌毒力基因表达中的作用，以及(Ii)准确地确定影响CUPA基因表达的局部和全球调控网络如何变得有效整合。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is an important opportunistic pathogen of humans that is notorious for being the principal cause of morbidity and mortality in Cystic Fibrosis (CF) patients. In the chronically infected CF lung the organism persists as a biofilm-a surface attached community of bacteria encased in a polymeric matrix. This biofilm mode of growth augments the resistance of P. aeruginosa to antibiotics and facilitates evasion of the host immune response. Prominent amongst those genes that play an important role in biofilm formation in P. aeruginosa are the cupA genes, which encode components of a fimbrial structure that facilitates surface- attachment and host colonization. We have identified three genes (cgrABC), whose products are required for phase-variable (i.e. reversible ON/OFF) expression of the cupA fimbrial gene cluster. None of the products of the cgr genes resembles any classical positive regulator of gene expression; cgrA is predicted to encode a member of the adenine nucleotide ¿-hydrolase superfamily, whereas cgrB encodes a putative acetylase, and cgrC encodes a protein with homology to the ParB family of DNA-binding proteins. In Aim 1 we propose to determine how the Cgr proteins exert their control. We will explicitly test the hypotheses that CgrB functions by acetylating CgrA and that phase-variable expression of the cupA genes is mediated by changes in the acetylation state of CgrA. Because many other pathogenic bacteria contain cgr orthologs, we expect that the relevance of our studies will extend beyond P. aeruginosa. Recently, we identified PrrA, a novel small regulatory RNA (sRNA), as an additional positive regulator of the cupA genes. Preliminary evidence suggests that PrrA exerts its effects on cupA expression by antagonizing the silencing effects of a member of the H-NS family of histone-like nucleoid structuring proteins. In Aim 2 we propose to determine how PrrA functions as an anti-silencer. We anticipate that the proposed studies will enable us to (i) define roles for protein acetylation and an sRNA in the control of phase-variable virulence gene expression in P. aeruginosa and (ii) determine precisely how the local and global regulatory networks that influence cupA gene expression become effectively integrated.