Control of fimbrial gene expression in Pseudomonas aeruginosa

描述（由申请人提供）： 铜绿假单胞菌是人类重要的机会致病菌，是囊性纤维化（CF）患者发病和死亡的主要原因。在慢性感染的CF肺中，微生物以生物膜的形式存在，生物膜是包裹在聚合物基质中的表面附着的细菌群落。这种生物膜生长模式增强了铜绿假单胞菌对抗生素的耐药性，并有助于逃避宿主免疫应答。在铜绿假单胞菌的生物膜形成中起重要作用的那些基因中突出的是cupA基因，其编码促进表面附着和宿主定殖的菌毛结构的组分。我们已经确定了三个基因（cgrABC），其产品所需的cupA菌毛基因簇的相位可变（即可逆的开/关）的表达。cgr基因的产物都不类似于任何经典的基因表达正调控因子; cgrA被预测编码腺嘌呤核苷酸水解酶超家族的成员，而cgrB编码一种假定的乙酰化酶，cgrC编码一种与DNA结合蛋白ParB家族同源的蛋白质。在目的1中，我们提出确定Cgr蛋白如何发挥其控制作用。我们将明确测试CgrB功能的假设， 乙酰化CgrA，并且cupA基因的时相可变表达由CgrA乙酰化状态的变化介导。由于许多其他病原菌含有cgr直系同源物，我们预计我们研究的相关性将超出铜绿假单胞菌。最近，我们确定了PrrA，一种新的小调控RNA（sRNA），作为cupA基因的一个额外的正调控因子。初步证据表明，PrrA发挥其作用cupA表达拮抗组蛋白样类核结构蛋白的H-NS家族的成员的沉默效应。在目标2中，我们提出确定PrrA如何作为抗沉默剂发挥作用。我们预计，拟议的研究将使我们能够（i）确定蛋白质乙酰化和sRNA在铜绿假单胞菌中控制相变毒力基因表达的作用，以及（ii）精确确定影响cupA基因表达的局部和全局调控网络如何有效整合。

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.