Regulation of Ps. aeruginosa Virulence Factors by AlgR

诗篇的规定。

基本信息

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
项目类别：
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

项目摘要

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）：铜绿假单胞菌是普遍存在的机会性病原体，主要感染包括艾滋病和移植患者在内的免疫功能低下的个体，严重的烧伤患者以及患有囊性纤维化的患者（CF）。在CF的背景下，铜绿假单胞菌建立了一种慢性疾病，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，为了提高治疗的疗效，需要检查对该生物使用的致病机制的基本理解，以作为可能的治疗靶标。尽管以前的大多数研究都集中在殖民和感染的初始阶段，但我们提出了一种新方法来寻找治疗方法。越来越多的证据表明，铜绿假单胞菌发病机理可能涉及微量潜水的代谢和生物生长模式。但是，关于一种机制的解释尚未讨论。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抑制真核细胞的几种呼吸电子传输链和几种金属酶（例如，过氧化氢酶，过氧化物酶，过氧化物酶，超氧化物歧化酶）。我们已经发现，毒力因子的调节剂Algr也激活了粘液藻中的HCN产生。使用假单胞菌的假单胞菌Affymetrix Genechip和S1核酸酶保护测定法，我们证明了Algr正在控制HCNA，编码氰化氢合酶。此外，直接测量HCN的产生表明，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了ALGR的两个新角色：i）ALGR对照HCN的产生，II）ALGR能够从非木质P.铜绿假单胞菌中的阻遏物转换为HCNA启动子上粘液菌细菌中的激活剂。此外，我们证明了Algz/FIMS在此过程中发挥了作用。要检验的假设是：ALGR激活粘液藻中的HCN产生。我们将以四个特定的目的检验这一假设：i）我们将确定HCNA启动子内Algr蛋白-DNA相互作用的要求； ii）我们将确定在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的HCN产生和HCNA表达的量，并且； iv）我们将在体外确定HCN产生对肺上皮和人类嗜中性粒细胞的影响。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标，并更好地了解假单胞菌生物学和发病机理。