Regulation of Ps. aeruginosa Virulence Factors by AlgR

诗篇的规定。

• 批准号: 7340222
• 负责人: Michael John Schurr
• 金额: $ 12.09万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7340222
• 关键词: DNA footprinting; Pseudomonas aeruginosa; alginates; apoptosis; bacteria infection mechanism; binding sites; biofilm; cellular respiration; gel mobility shift assay; gene induction /repression; genetic promoter element; genetic regulation; microarray technology; nitrogen metabolism; pathologic process; phosphorylation; posttranslational modifications; protein binding; protein sequence; protein structure function; proteoglycan; respiratory epithelium; site directed mutagenesis; transcription factor; virulence

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is ubiquitous, opportunistic pathogen that primarily infects immune-compromised individuals, including AIDS and transplant patients, severe burn patients, and those with cystic fibrosis (CF). In the context of CF, P. aeruginosa establishes a chronic condition whose morbidity and mortality results from lung damage. Due to the uncanny antibiotic resistance, CF patients infected with Pseudomonas often have chronic infections with limited therapeutic options. Therefore, for improved efficacy in treatment, a basic understanding of the pathogenic mechanisms utilized by this organism needs to be examined as possible therapeutic targets. While a majority of previous studies have focused on the initial stages of colonization and infection, we propose a new approach in searching for treatments. Mounting evidence indicates that microaerophilic metabolism and a biofilm mode of growth may be involved in P. aeruginosa pathogenesis; however, explanations for a mechanism have yet to be discussed. One virulence factor produced by P. aeruginosa under these growth conditions is HCN. Micromolar amounts of HCN inhibit the respiratory electron transport chain and several metalloenzymes (e.g., catalase, peroxidase, superoxide dismutase) of eukaryotic cells. We have discovered that AlgR, a regulator of the virulence factor, alginate, also activates HCN production in mucoid P. aeruginosa. Using the Pseudomonas Affymetrix GeneChip and S1 nuclease protection assays, we demonstrate that AlgR is controlling hcnA, encoding hydrogen cyanide synthase. Moreover, direct measurement of HCN production revealed that mucoid P. aeruginosa produce up to 2.5 mM of HCN in 4 h. Our preliminary data indicate two new roles for AlgR: i) AlgR controls HCN production and ii) AlgR is able to switch from a repressor in nonmcoid P. aeruginosa to an activator in mucoid bacteria on the hcnA promoter. Additionally, we demonstrate that AlgZ/FimS is playing a role in this process. The hypothesis to be tested is: AlgR activates HCN production in mucoid P. aeruginosa. We will test this hypothesis with four specific aims: i) we will determine the requirements for AlgR protein-DNA interaction within the hcnA promoter; ii) we will determine if phosphorylation is required for AlgR activation of hcnA expression in mucoid P. aeruginosa; iii) we will determine the amount of HCN production and hcnA expression within biofilms, and; iv) we will determine the effect of HCN production on lung epithelial and human neutrophil cells in vitro. Thus, at the end of our proposed studies, we hope to elucidate new possible therapeutic target as well as gaining a better understanding of Pseudomonas biology and pathogenesis.

描述（由申请人提供）：铜绿假单胞菌是一种普遍存在的机会性病原体，主要感染免疫功能低下的个体，包括艾滋病和移植患者、严重烧伤患者以及囊性纤维化 (CF) 患者。在 CF 的背景下，铜绿假单胞菌会形成一种慢性疾病，其发病率和死亡率是由肺损伤引起的。由于不可思议的抗生素耐药性，感染假单胞菌的 CF 患者通常患有慢性感染，治疗选择有限。因此，为了提高治疗效果，需要对该生物体利用的致病机制进行基本了解，将其作为可能的治疗靶点。虽然之前的大多数研究都集中在定植和感染的初始阶段，但我们提出了一种寻找治疗方法的新方法。越来越多的证据表明，微需氧代谢和生物膜生长模式可能与铜绿假单胞菌的发病机制有关。然而，对机制的解释仍有待讨论。铜绿假单胞菌在这些生长条件下产生的一种毒力因子是 HCN。微摩尔量的 HCN 会抑制真核细胞的呼吸电子传递链和几种金属酶（例如过氧化氢酶、过氧化物酶、超氧化物歧化酶）。我们发现 AlgR（毒力因子藻酸盐的调节剂）也能激活粘液铜绿假单胞菌中 HCN 的产生。使用 Pseudomonas Affymetrix GeneChip 和 S1 核酸酶保护测定，我们证明 AlgR 正在控制编码氰化氢合酶的 hcnA。此外，直接测量 HCN 产量表明，粘液铜绿假单胞菌在 4 小时内产生高达 2.5 mM 的 HCN。我们的初步数据表明 AlgR 的两个新作用：i) AlgR 控制 HCN 的产生，ii) AlgR 能够从非类粘液铜绿假单胞菌中的阻遏蛋白转变为粘液细菌中 hcnA 启动子上的激活蛋白。此外，我们证明 AlgZ/FimS 在此过程中发挥着作用。待检验的假设是：AlgR 激活粘液铜绿假单胞菌中 HCN 的产生。我们将通过四个具体目标来检验这一假设： i) 我们将确定 hcnA 启动子内 AlgR 蛋白-DNA 相互作用的要求； ii) 我们将确定粘液铜绿假单胞菌中 hcnA 表达的 AlgR 激活是否需要磷酸化； iii) 我们将确定生物膜内 HCN 产生量和 hcnA 表达量，以及； iv) 我们将在体外确定 HCN 产生对肺上皮细胞和人中性粒细胞的影响。因此，在我们提出的研究结束时，我们希望阐明新的可能的治疗靶点，并更好地了解假单胞菌生物学和发病机制。