Host-associated regulation of P. aeruginosa colonization and virulence

描述（由申请方提供）：铜绿假单胞菌是最危险的革兰氏阴性机会致病菌之一。在受损的肺中，铜绿假单胞菌是呼吸机相关性肺炎、慢性阻塞性肺疾病患者病情加重以及遗传性疾病囊性纤维化患者慢性致死性感染的常见原因。慢性和急性铜绿假单胞菌肺部感染都难以治疗，并且与高死亡率相关，因此需要新的策略来对抗这种细菌。一种重要的铜绿假单胞菌毒力因子是分泌的磷脂酶C，PlcH，其降解在肺中高度丰富的含胆碱磷脂，例如磷脂酰胆碱（PC）。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药物，该药物已被批准用于人类，可抑制PlcH活性，并防止PlcH在急性小鼠肺炎模型中的作用。我们的研究结果还表明，铜绿假单胞菌PlcH介导的宿主磷脂的降解提供了细菌的前体必需的衍生甘氨酸甜菜碱（GB）。通过微阵列研究，我们发现，GB，除了作为一种营养素和一种抗氧化剂的保护剂，也作为一种新的诱导剂的Anr，一个中央转录调节因子，在有氧环境中，Anr通常是不活跃的。我们的数据表明，一个新的作用，为Anr在生物膜形成和宿主细胞定植，GB刺激宿主定植是Anr依赖。因此，我们的中心假设指出，PlcH不仅导致宿主损伤，而且还释放促进铜绿假单胞菌在宿主中定殖的产物。因此，抑制PlcH可以直接降低铜绿假单胞菌的毒力，并通过减少耐药生物膜。 在该提案中，我们的目的是检验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（目的1），并检验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（目的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中的定殖重要，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、生物膜形成和抗生素耐药性，我们将在目标3中检验这一假设。这些研究的完成将揭示宿主衍生分子影响铜绿假单胞菌中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同系物的调控提供见解。我们将追求新的和令人兴奋的可能性，即使用抑制PlcH活性的药物既可以减弱PlcH对肺的急性作用，又可以减少铜绿假单胞菌的气道定植，使其对现有的抗微生物治疗更敏感。

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.