Host-associated regulation of P. aeruginosa colonization and virulence

描述（由申请人提供）：铜绿假单胞菌是最危险的革兰氏阴性机会致病菌之一。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加重，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难以治疗，并与高死亡率相关，因此需要新的策略来对抗这种细菌。一个重要的铜绿假单胞菌毒力因子是分泌的磷脂酶C， PlcH，它能降解含胆碱的磷脂，如在肺中大量存在的磷脂酰胆碱（PC）。PlcH具有细胞毒性，具有免疫调节作用，导致肺功能下降，我们已经发现了一种已经批准用于人类的药物，可以抑制PlcH的活性，并在急性小鼠肺炎模型中阻止PlcH的影响。我们的研究结果还表明，P. aeruginosa plch介导的宿主磷脂降解为细菌提供了生成甘氨酸甜菜碱（GB）所需的前体。通过微阵列研究，我们发现GB除了作为营养物质和渗透保护剂外，还可以在Anr通常不活跃的氧化环境中作为中枢转录调节因子Anr的新型诱导剂。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，并且宿主定植的GB刺激是Anr依赖的。因此，我们的中心假设表明，PlcH不仅会导致宿主损伤，而且还会释放促进铜绿假单胞菌在宿主定植的产物。因此，抑制PlcH可能通过减少耐药生物膜直接降低铜绿假单胞菌的毒力。在本提案中，我们旨在验证GB直接增加氧化环境中Anr活性的假设，并确定其发生的机制（aim 1），并验证Anr通过增加CupA菌毛的产生促进铜绿假单胞菌生物膜形成和宿主定植的假设（aim 2）。在Aim 2中，我们还将确定Anr调控cupA基因表达是否对铜绿假单胞菌在小鼠肺中的定植很重要，以及是否有证据表明GB合成、Anr活性和cupA表达与人类急性和慢性铜绿假单胞菌感染有关。根据我们的初步数据，我们预测现有药物对PlcH的抑制会降低P. aeruginosa GB水平、生物膜形成和抗生素耐药性，我们将在Aim 3中验证这一假设。这些研究的完成将揭示宿主源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同源物的调控提供新的见解。我们将探索一种新颖而令人兴奋的可能性，即使用一种抑制PlcH活性的现有药物，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的气道定植，使其对现有的抗菌疗法更敏感。

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.