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: 7931027
• 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/7931027
• 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; 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病 (COPD)，这种疾病经常受到机会性细菌的气道感染，其中最常见的是铜绿假单胞菌 (PA)。在慢性感染的慢性阻塞性肺病气道中，PA 的滴度很高，并且许多菌株呈粘液状，这是由于一种称为藻酸盐的粘性胞外多糖的过量产生而产生的。 PA 粘液转化的主要机制是通过突变。这些突变主要发生在编码抗 Sigma 因子的 mucA 内（>84-92%）。如果没有 MucA，sigma 因子 AlgT(U) 会指导参与藻酸盐生物合成的基因转录，从而产生粘液性。 2006 年，我们在《临床研究杂志》上发表了一篇论文，证明粘液性 mucA 突变细菌在厌氧暴露于酸化亚硝酸盐 (A-NO2-) 时会被杀死。然而，mucA 背景中 algT(U) 的失活并没有减轻对酸化亚硝酸盐的敏感性，强烈表明观察到的影响是 MucA 特异性的。反式提供 mucA 可恢复 A-NO2 抗性，随后的实验证实一氧化氮 (NO) 在细胞死亡中发挥作用。重要的是，当 A-NO2- 用于人类气道上皮细胞时，没有观察到任何副作用。总之，我们发现了一种新型无毒药物，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) mucA 突变细菌中 A-NO2- 敏感性的机制，(ii) MucA 和厌氧呼吸级联成员在生物膜对 A-NO2- 敏感性中的作用，以及 (iii) 检验 mucA 和双厌氧调节突变体对 A-NO2- 更加敏感的假设 A-NO2- 在经过验证的慢性肺部感染小鼠模型中。目标 1. 确定粘液 mucA 突变体 PA 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样 mucA 突变细菌的“致命弱点”，但我们仍然不知道 A-NO2- 杀死这些生物体的机制。具体而言，MucA、NO3-/NO2-转运、厌氧调节机制和 NO 敏感的巯基/含铁蛋白的作用被严重低估。分子基础将通过 (i) 对在有氧和厌氧条件下生长的 mucA 和野生型菌株进行微阵列研究来确定； (ii) 测定允许亚硝酸盐敏感性的细胞 MucA 水平，(iii) 测定 mucA 和 WT 以及 mucA double（厌氧调节等级基因）中 NO2- 和 NO3- 转运的速率/水平； (iv) 阐明已知为亚硝基化靶点的关键细胞蛋白的状态。目标 2. 使用 3 种不同的已建立模型系统，确定 NO2- 对野生型与粘液样 DmucA 突变体在复杂、高度组织的群落（称为生物膜）中活力的影响。生物膜的生长模式已被确定为在厚厚的 CF 气道粘液中存在并实际生长的模式。我们将使用 3 种互补但对比的方法，包括 (i) 静态生物膜系统，代表 COPD 气道的停滞粘液，(ii) 流通系统，代表对比的生物膜生长模式，最后 (iii) 生长是源自人类原代细胞的气道表面液体中的静态生物膜。目标 3. 在已建立的小鼠慢性肺部感染模型中确定 NO2- 对野生型与 DmucA 和双 mucA 厌氧调节突变体活力的影响。需要原理验证动物研究来证明上述治疗方法不仅对 mucA 突变生物体，而且对编码在暴露于 A-NO2- 后发生 S-亚硝基化的蛋白质的基因中具有选定第二位点突变的 mucA 突变体具有相对功效。 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，包括慢性支气管炎和/或肺气肿，其特征是不可逆的气流受限。 2005 年，COPD 是 VA 医院第四大常见的出院诊断，1999 年的直接医疗费用估计为 30 亿美元。在辛辛那提 VAMC，COPD 的患病率超过 40%，2008 年，其支出超过 2000 万美元。病情加重是 COPD 发病/死亡的一个主要因素。由铜绿假单胞菌 (PA) 感染介导的呼吸困难发作常常会导致住院，并与慢性阻塞性肺病 (COPD) 患者死亡率增加相关。 PA 引起两种不同的携带模式：(1) 短期定植，恶化后清除；(2) 与粘液菌株相关的长期持续存在。需要改进治疗方案，在这种情况下，需要使用亚硝酸钠来治疗 PA 感染引起的 COPD 恶化，以降低死亡率和发病率。