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): 辛辛那提近40%的VA患者患有慢性阻塞性肺疾病(COPD),其经常遭受由机会性细菌引起的气道感染,其中最普遍的是铜绿假单胞菌(PA)。PA在慢性感染的COPD气道中以高滴度存在,并且许多菌株是粘液样的,这是由称为藻酸盐的粘性胞外多糖的过度产生引起的。PA的粘液样转化的主要机制是通过突变。这些突变主要(>84-92%)发生在编码抗σ因子的mucA内。在没有MucA的情况下,σ因子AlgT(U)指导参与藻酸盐生物合成的基因的转录,从而导致粘液性。2006年,我们在《临床研究杂志》上发表了一篇论文,证明了粘液样mucA突变细菌在厌氧暴露于酸化亚硝酸盐(A- NO2-)期间被杀死。然而,在mucA背景下algT(U)的失活并没有减轻对酸化亚硝酸盐的敏感性,这强烈表明观察到的影响是MucA特异性的。反式提供mucA恢复了A-NO2-抗性,随后的实验证实一氧化氮(NO)在细胞死亡中起作用。重要的是,当A-NO2-应用于人气道上皮细胞时,未观察到不良反应。总之,我们发现了一种新型无毒药物,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-敏感性的潜在机制,(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,和(iii)测试mucA和双厌氧调节子突变体在试验和-慢性肺部感染的真实小鼠模型。目标1.确定粘液mucA突变体PA中厌氧酸化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“阿喀琉斯之踵”,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-运输,厌氧调节机制和NO敏感性巯基/含铁蛋白的作用是非常低估。分子基础将通过以下方式确定:(i)在需氧和厌氧条件下生长的mucA和野生型菌株的微阵列研究;(ii)确定允许亚硝酸盐敏感性的细胞MucA水平;(iii)确定mucA和WT以及mucA双链中NO2-和NO3-转运的速率/水平(厌氧调节层次基因)和;(iv)阐明已知为亚硝基化的目标的关键细胞蛋白质的状态。目标2.使用3种不同的已建立的模型系统,确定NO2-对野生型与粘液型、DmucA突变体在称为生物膜的复杂、高度组织化的群落中的活力的影响。生物膜生长模式是已经确定存在并实际上在厚CF气道粘液中生长的生物膜。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,以及最后(iii)生长是源自人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。需要原理证明动物研究来显示上述处理不仅对mucA突变生物体,而且对在编码暴露于A-NO2-时S-亚硝基化的蛋白质的基因中具有选定的第二位点突变的mucA突变体的相对功效。

项目成果

期刊论文数量(1)
专著数量(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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