Host-associated regulation of P. aeruginosa colonization and virulence

描述(申请人提供)：铜绿假单胞菌是最危险的革兰氏阴性条件致病菌之一。在受损的肺部，铜绿假单胞菌是呼吸机相关性肺炎的常见原因，慢性阻塞性肺疾病患者的病情恶化，以及遗传性疾病囊性纤维化患者的慢性致命感染。慢性和急性铜绿假单胞菌肺部感染都很难治疗，而且死亡率很高，因此需要新的战略来抗击这种细菌。一个重要的铜绿假单胞菌毒力因子是一种分泌型磷脂酶C，它能降解含有胆碱的磷脂，如磷脂酰胆碱(PC)，这些磷脂在肺中含量很高。PlcH具有细胞毒性、免疫调节作用，并导致肺功能下降，我们已经发现了一种已被批准用于人类的药物，该药物可以抑制PlcH的活性，并防止PlcH在急性小鼠肺炎模型中的影响。我们的发现还表明，铜绿假单胞菌PlcH介导的宿主磷脂的降解为细菌提供了产生甘氨酸甜菜碱(GB)所需的前体。通过基因芯片的研究，我们发现GB除了是一种营养物质和渗透保护剂外，还在ANR通常不活跃的有氧环境中作为一种新型的中央转录调节因子。我们的数据显示了ANR在生物膜形成和宿主细胞定植中的新作用，并且GB对寄主定植的刺激是依赖于ANR的。因此，我们的中心假设是，PlcH不仅会对宿主造成损害，还会释放促进铜绿假单胞菌侵袭宿主的产物。因此，抑制PlcH可能直接降低铜绿假单胞菌的毒力，并通过减少耐药生物膜来降低其毒力。在这项提议中，我们旨在检验GB直接增加有氧环境中ANR活性的假设并确定其发生的机制(目标1)，并检验ANR通过增加铜绿假单胞菌菌毛的产生而促进铜绿假单胞菌生物被膜形成和宿主定植的假设(目标2)。在目标2中，我们还将确定ANR对CUPA基因表达的调节是否对铜绿假单胞菌在小鼠肺部的定植起重要作用，以及是否有证据表明在人类急性和慢性铜绿假单胞菌感染中存在GB合成、ANR活性和CUPA表达。基于我们的初步数据，我们预测现有药物对PlcH的抑制将降低铜绿假单胞菌GB水平、生物被膜形成和抗生素耐药性，我们将在目标3中验证这一假设。这些研究的完成将揭示宿主衍生分子影响铜绿假单胞菌毒力相关基因表达的新机制，并可能为其他细菌病原体中ANR同源物的调控提供依据。我们将探索一种新颖而令人兴奋的可能性，即使用一种现有的抑制PlcH活性的药物既可以减轻PlcH对肺部的急性影响，又可以减少铜绿假单胞菌对呼吸道的定植，使其更容易受到现有抗菌治疗的影响。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is among the most dangerous Gram-negative opportunistic pathogens. In the compromised lung, P. aeruginosa is a common cause of ventilator-associated pneumonia, exacerbations in patients with chronic obstructive pulmonary disease, and chronic, lethal infections in individuals with the genetic disease cystic fibrosis. Both chronic and acute P. aeruginosa lung infections are difficult to treat and are associated with high rates of mortality, thus new strategies are needed to combat this bacterium. One important P. aeruginosa virulence factor is a secreted phospholipase C, PlcH, which degrades choline- containing phospholipids, such as phosphatidylcholine (PC), that are highly abundant in the lung. PlcH is cytotoxic, immunomodulatory and causes decreased lung function, and we have discovered a drug, already approved for use in humans, that inhibits PlcH activity, and prevents the effects of PlcH in an acute mouse pneumonia model. Our findings also indicate that P. aeruginosa PlcH-mediated degradation of host phopholipids provides the bacteria with precursors necessary to derive glycine betaine (GB). Through microarray studies, we have found that GB, in addition to being a nutrient and an osmoprotectant, also serves as a novel inducer of Anr, a central transcriptional regulator, in oxic environments where Anr is normally inactive. Our data show a novel role for Anr in biofilm formation and host cell colonization, and that GB stimulation of host colonization is Anr-dependent. Thus, our central hypothesis states that PlcH not only causes host damage, but also releases products that promote P. aeruginosa colonization of the host. Thus, inhibition of PlcH may decrease P. aeruginosa virulence directly and by decreasing antibiotic-resistant biofilms. In this proposal, we aim to test the hypothesis that GB directly increases Anr activity in oxic environments and determine the mechanism by which this occurs (Aim 1) and test the hypothesis that Anr promotes P. aeruginosa biofilm formation and host colonization by increasing the production of CupA fimbriae (Aim 2). In Aim 2, we will also determine if Anr regulation of cupA gene expression is important for P. aeruginosa colonization of the mouse lung, and if there is evidence for GB synthesis, Anr activity, and cupA expression in acute and chronic P. aeruginosa infections in humans. Based on our preliminary data, we predict that the inhibition of PlcH by an existing drug will decrease P. aeruginosa GB levels, biofilm formation and antibiotic resistance, and we will test this hypothesis in Aim 3. Completion of these studies will unravel a new mechanism by which host-derived molecules impact virulence-related gene expression in P. aeruginosa, and may provide insight into the regulation of Anr homologs in other bacterial pathogens. We will pursue the novel and exciting possibility that the use of an exisitng drug that inhibits PlcH activity will both attenuate acute effects of PlcH on the lung and reduce airway colonization by P. aeruginosa rendering it more susceptible to existing antimicrobial therapies.