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

基本信息

项目摘要

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-.
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),这种疾病经常受到机会细菌的呼吸道感染,其中最常见的是铜绿假单胞菌(PA)。在慢性感染的COPD呼吸道中发现PA的效价很高,许多菌株是粘液状的,这是由于一种名为海藻酸盐的粘性胞外多糖的过度生产造成的。PA粘液转化的主要机制是通过突变。这些突变主要(>84-92%)发生在粘蛋白A内,编码一种抗西格玛因子。在没有粘蛋白A的情况下,西格玛因子ALGT(U)指导参与藻酸盐生物合成的基因的转录,导致粘液性。2006年,我们在临床调查杂志上发表了一篇论文,证明了粘液A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,在粘液A背景中灭活ALGT(U)并不能减轻对酸性亚硝酸盐的敏感性,这强烈表明观察到的影响是MUA特异性的。反式提供粘液A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-应用于人的呼吸道上皮细胞时,没有观察到不良反应。综上所述,我们发现了一种新的、无毒的制剂,可以潜在地实现从COPD患者的呼吸道中根除粘液PA的翻译目标。我们提出并设计了三个特定的目标来确定(I)粘液A突变细菌对A-NO2-敏感的机制(S),(Ii)粘液A和厌氧呼吸级联成员在A-NO2-敏感生物膜中的作用,以及(Iii)在一个久经考验的慢性肺部感染小鼠模型中,测试粘液A和双厌氧调节基因突变体将对A-NO2-更加敏感的假设。目的1.鉴定粘液性粘液A突变株对厌氧酸化NO2敏感性的分子基础。虽然我们在2006年的发现描述了一种粘液状、粘液A突变细菌的“阿喀琉斯跟腱”,但我们仍然不知道A-NO2-杀死这些微生物的机制。具体地说,粘液A、NO3-/NO2-转运、厌氧调节机制和不敏感的巯基/含铁蛋白的作用被严重低估。分子基础将通过(I)对在好氧和厌氧条件下生长的粘液A和野生型菌株的微阵列研究;(Ii)允许亚硝酸盐敏感的细胞粘液A水平的测定;(Iii)测定粘液A和WT以及粘液A双重(厌氧调节层级基因)中NO2-和NO3-运输的速率/水平;以及(Iv)阐明已知为亚硝酸化靶标的关键细胞蛋白的状态。目的2.使用3种不同的已建立的模型系统,确定NO2-对被称为生物膜的复杂、高度组织的群落中野生型和粘液型DmucA突变体的活性的影响。生物膜的生长模式是已经确定存在并实际上在厚厚的CF呼吸道粘液中生长的生物膜。我们将使用3种互补但不同的方法,包括(I)静态生物膜系统,代表COPD呼吸道的停滞粘液,(Ii)流动系统,代表对比生物膜的生长模式,以及(Iii)生长是来自人类原代细胞的呼吸道表面液体中的静态生物膜。目的3.在已建立的小鼠慢性肺部感染模型中,确定NO2-对野生型和双黏液A厌氧调节突变体活性的影响。需要进行原理验证的动物研究,以证明上述处理的相对有效性,不仅是对MUTA突变生物,而且对编码蛋白质的基因具有选定第二位点突变的MUA突变,这些蛋白质在暴露于A-NO2-时被S亚硝化。

项目成果

期刊论文数量(1)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)

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DANIEL J. HASSETT其他文献

DANIEL J. HASSETT的其他文献

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{{ truncateString('DANIEL J. HASSETT', 18)}}的其他基金

An innovative treatment for Pneumocystis pneumonia
肺孢子菌肺炎的创新治疗方法
  • 批准号:
    8873445
  • 财政年份:
    2015
  • 资助金额:
    --
  • 项目类别:
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
  • 财政年份:
    2010
  • 资助金额:
    --
  • 项目类别:
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
  • 财政年份:
    2010
  • 资助金额:
    --
  • 项目类别:
Role of OxyR in P. aeruginosa Biofilm Resistance to H202
OxyR 在铜绿假单胞菌生物膜 H2O2 抗性中的作用
  • 批准号:
    7271227
  • 财政年份:
    2004
  • 资助金额:
    --
  • 项目类别:
Role of OxyR in P. aeruginosa Biofilm Resistance to H202
OxyR 在铜绿假单胞菌生物膜 H2O2 抗性中的作用
  • 批准号:
    6831086
  • 财政年份:
    2004
  • 资助金额:
    --
  • 项目类别:
Role of OxyR in P. aeruginosa Biofilm Resistance to H202
OxyR 在铜绿假单胞菌生物膜 H2O2 抗性中的作用
  • 批准号:
    6931186
  • 财政年份:
    2004
  • 资助金额:
    --
  • 项目类别:
Role of OxyR in P. aeruginosa Biofilm Resistance to H202
OxyR 在铜绿假单胞菌生物膜 H2O2 抗性中的作用
  • 批准号:
    7111811
  • 财政年份:
    2004
  • 资助金额:
    --
  • 项目类别:
Proteogenome of Anaerobic P. aeruginosa in CF Mucus
CF 粘液中厌氧铜绿假单胞菌的蛋白质组
  • 批准号:
    6609992
  • 财政年份:
    2003
  • 资助金额:
    --
  • 项目类别:
B.pseudomallei bioterrorism and quorum sensing
B.pseudomallei 生物恐怖主义和群体感应
  • 批准号:
    6659914
  • 财政年份:
    2002
  • 资助金额:
    --
  • 项目类别:
B.pseudomallei bioterrorism and quorum sensing
B.pseudomallei 生物恐怖主义和群体感应
  • 批准号:
    6556955
  • 财政年份:
    2002
  • 资助金额:
    --
  • 项目类别:

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