Mechanism Underlying Nitrite Sensitivity of Mucoid Pseudomonas in COPD

COPD 中粘液假单胞菌亚硝酸盐敏感性的机制

• 批准号: 8196343
• 负责人: DANIEL J. HASSETT
• 金额: --
• 依托单位: CINCINNATI VA MEDICAL CENTER RESEARCH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-10-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8196343
• 关键词: Accounting; Adverse effects; Aerobic; Affect; Alginates; Anabolism; Animal Model; Animals; Bacteria; Bathing; Biochemistry; Biological Models; Blood; Caring; Cell Death; Cells; Cerebrospinal Fluid; Chronic; Chronic Bronchitis; Chronic Obstructive Airway Disease; Clinical Trials; Communities; Complex; Diagnosis; Dyspnea; Expenditure; Exposure to; Genes; Genetic; Genetic Transcription; Goals; Growth; Health Care Costs; Hospitalization; Hospitals; Human; In Vitro; Infection; Journals; Kinetics; Libraries; Liquid substance; Lung; Lung diseases; Mediating; Microbial Biofilms; Modeling; Molecular; Morbidity - disease rate; Mucous body substance; Mus; Mutation; NBL1 gene; Nitric Oxide; Nitrites; Opportunistic Infections; Organism; Paper; Patients; Pattern; Play; Prevalence; Protein S; Proteins; Pseudomonas; Pseudomonas aeruginosa; Publishing; Pulmonary Emphysema; Relative (related person); Resistance; Role; Sigma Factor; Site; Sodium Nitrite; System; Testing; Thick; Urinary tract; airway epithelium; airway surface liquid; base; bronchial epithelium; cystic fibrosis airway; design; improved; in vivo; killings; member; mortality; mouse model; mucoid; mutant; novel; public health relevance; research study; respiratory

DESCRIPTION (provided by applicant): Nearly 40% of Cincinnati VA patients suffer from chronic obstructive pulmonary disease (COPD) that often suffer from airway infection by opportunistic bacteria, the most prevalent of which is Pseudomonas aeruginosa (PA). PA is found at high titers in chronically infected COPD airways and many strains are mucoid, resulting from overproduction of a viscous exopolysaccharide called alginate. The major mechanism of mucoid conversion of PA is via mutations. These mutations occur predominantly (>84-92%) within mucA, encoding an anti-sigma factor. Without MucA, the sigma factor AlgT(U) directs transcription of genes involved in alginate biosynthesis, resulting in mucoidy. In 2006, we published a paper in the Journal of Clinical Investigation, demonstrating that mucoid mucA mutant bacteria are killed during anaerobic exposure to acidified nitrite (A- NO2-). However, inactivation of algT(U) in the mucA background did not relieve sensitivity to acicified nitrite strongly indicating the affects observed were MucA-specific. Provision of mucA in trans restored A-NO2- resistance and subsequent experiments established that nitric oxide (NO) plays a role in cell death. Importantly, no adverse effects were observed when A-NO2- was applied to human airway epithelia. In summary, we have discovered a novel, non-toxic agent that could potentially achieve the translational goal of eradicating mucoid PA from the airways of COPD patients. Three specific aims are proposed and designed to determine (i) the mechanism(s) underlying A-NO2- sensitivity in mucA mutant bacteria, (ii) the role of MucA and members of the anaerobic respiratory cascade in biofilm sensitivity to A-NO2-, and (iii) to test the hypothesis that mucA and double anaerobic regulator mutants will be even more sensitive to A-NO2- in a tried-and-true mouse model of chronic lung infection. Aim 1. Identify the molecular basis underlying anaerobic acidified NO2- sensitivity in mucoid mucA mutant PA. Although our discovery in 2006 describes an "Achilles' Heel" of mucoid, mucA mutant bacteria, we still do not know the mechanism of killing of these organisms by A-NO2-. Specifically, the role of MucA, NO3-/NO2- transport, anaerobic regulatory machinery and NO-sensitive sulfhydryl/Fe-containing proteins is very much underappreciated. The molecular basis will be determined by (i) micoarray studies of mucA and wild-type strains grown under aerobic and anaerobic conditions; (ii) determination of the cellular MucA levels that allow nitrite sensitivity, (iii) determining the rates/levels of NO2- and NO3- transport in mucA and WT and mucA double (anaerobic regulatory hierarchy genes) and; (iv) elucidate the status of critical cellular proteins known to be targets of nitrosylation. Aim 2. Determine the effects of NO2- on viability of wild-type versus mucoid, DmucA mutants in complex, highly organized communities known as biofilms using 3 different established model systems. The biofilm mode of growth is that which has been determined to exist and actually thrive within the thick CF airway mucus. We will use 3 complimentary yet contrasting approaches that include (i) a static biofilm system, representing the stagnant mucus of COPD airways, (ii) a flow-through system that represents a contrasting biofilm mode of growth, and finally (iii) growth is static biofilms in airway surface liquid derived from human primary cells. Aim 3. Determine the effects of NO2- on viability of wild-type versus DmucA and double mucA anaerobic regulatory mutants in an established murine chronic lung infection model. Proof-of-principle animal studies are required to show the relative efficacy of the aforementioned treatments on not only mucA mutant organisms, but also mucA mutants with selected second site mutations in genes encoding proteins that are S-nitrosylated upon exposure to A-NO2-. PUBLIC HEALTH RELEVANCE: Chronic obstructive pulmonary disease (COPD) is a debilitating lung disorder encompassing chronic bronchitis and/or emphysema, and characterized by nonreversible airflow limitation. In 2005, COPD was the 4th most common discharge diagnosis at VA hospitals and care was estimated at $3 billion in direct healthcare costs in 1999. At the Cincinnati VAMC, the prevalence of COPD exceeds 40% and, in 2008, it accounted for over $20M in expenditures. Exacerbations are a major contributing factor to COPD morbidity/mortality. Episodes of breathlessness mediated by Pseudomonas aeruginosa (PA) infection often precipitate hospitalizations and is associated with increased COPD patient mortality. PA causes two distinct patterns of carriage: (1) short-term colonization with exacerbation followed by clearance and (2) long-term persistence which is associated with mucoid strains. Improved treatment options, in this case the use of sodium nitrite for the management of COPD exacerbations precipitated by PA infection are needed to reduce mortality and morbidity.

描述（由申请人提供）： 近40％的辛辛那提VA患者患有慢性阻塞性肺疾病（COPD），这些疾病通常受到机会性细菌的气道感染，其中最普遍的是铜绿假单胞菌（PA）。在长期感染的COPD气道中发现PA在高滴度下，许多菌株是粘液菌，这是由于粘性外多糖称为藻酸盐的过量而导致的。 PA的粘液转化的主要机制是通过突变。这些突变主要发生（> 84-92％），编码抗sigma因子。没有粘液，Sigma因子ALGT（U）指导参与藻酸盐生物合成的基因的转录，从而导致粘膜。 2006年，我们在《临床研究杂志》上发表了一篇论文，表明在厌氧暴露于酸化亚硝酸盐（A-NO2-）期间，粘液粘膜突变细菌被杀死。然而，在粘液背景中灭活ALGT（U）并不能减轻对敏化亚硝酸盐的敏感性，表明观察到的影响是粘液特异性的。在反式恢复的A-NO2抗性和随后的实验中，一氧化氮（NO）在细胞死亡中起作用。重要的是，当将A-NO2-应用于人气道上皮时，未观察到不良反应。总而言之，我们发现了一种新型的无毒剂，该剂有可能实现从COPD患者气道中根除粘液pa的转化目标。提出并设计了三个具体目标，以确定（i）粘液突变细菌中A-NO2-灵敏度的基本机制，（ii）粘膜和厌氧呼吸级联反应在生物膜上对A-NO2--敏感性的作用，并且（iii）测试粘膜和双重厌氧调节剂突变体在慢性肺部感染的尝试和真实小鼠模型中对A-NO2的敏感性更为敏感的假设。目的1。确定粘液粘膜突变体PA中的厌氧酸化NO2-敏感性的分子基础。尽管我们在2006年的发现描述了粘液，粘液突变细菌的“跟腱”，但我们仍然不知道A-NO2-杀死这些生物的机制。具体而言，粘膜，NO3-/NO2-转运，厌氧调节机制和无敏感性硫酰/Fe的蛋白质的作用非常低估。分子基础将由（i）在有氧和厌氧条件下生长的粘膜和野生型菌株的膜片研究确定； （ii）确定允许亚硝酸盐敏感性的细胞粘膜水平，（iii）确定粘膜和WT和粘液中的NO2和NO3转运的速率/水平以及粘膜双重（厌氧调节层次基因）和； （iv）阐明临界细胞蛋白的状态已知是硝基化靶标。 AIM2。使用3种不同的模型系统，在复杂的高度有组织的社区中，在复杂的高度组织社区中野生型与粘液，DMUCA突变体的生存能力，DMUCA突变体的影响。生物膜的生长模式是已确定存在并实际上在厚CF气道粘液中壮成长的模式。我们将使用3种免费但对比的方法，其中包括（i）静态生物膜系统，代表COPD气道停滞的粘液，（ii）一种流动系统，代表了对比鲜明的生物膜生长模式，最后（iii）生长是气道表面液体中的静态生物膜来自人类原代细胞。 AIM 3。确定NO2-在既定的鼠慢性肺部感染模型中野生型与DMUCA和双粘膜厌氧调节突变体的影响。原本动物研究需要显示上述处理不仅对粘液突变生物的相对疗效，而且还可以证明在编码蛋白质的基因中，粘膜突变体在编码S-硝基基于A-NO2-- NO2-- NO2--硝基基因的基因中具有选择的粘膜突变体。 。 公共卫生相关性： 慢性阻塞性肺疾病（COPD）是一种使人衰弱的肺部疾病，其中包括慢性支气管炎和/或肺气肿，其特征在于非可逆的气流限制。在2005年，COPD是VA医院的第四大最常见出院诊断，1999年，CARE估计直接医疗费用为30亿美元。在辛辛那提VAMC，COPD的患病率超过40％，2008年，它的估计超过208美元。支出。加重是导致COPD发病率/死亡率的主要因素。铜绿假单胞菌（PA）感染介导的呼吸困难发作通常会导致住院，并与COPD患者死亡率升高有关。 PA引起了两种不同的托架模式：（1）短期定植，随后发生了加重，然后清除和（2）与粘液菌株相关的长期持久性。改进的治疗选择，在这种情况下，需要使用亚硝酸钠来治疗PA感染引起的COPD加剧，以降低死亡率和发病率。