Regulation of Ps. aeruginosa Virulence Factors by AlgR

诗篇的规定。

• 批准号: 7171785
• 负责人: Michael John Schurr
• 金额: $ 25.55万
• 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/7171785
• 关键词: Acquired Immunodeficiency Syndrome; Affinity; Alginates; Anabolism; Antibiotic Resistance; Apoptosis; Aspartate; Aspergillus Nuclease S1; Bacteremia; Bacteria; Binding; Binding Sites; Biological Assay; Biology; Burn injury; Cells; Chronic; Commit; Condition; Cystic Fibrosis; DNA-Protein Interaction; DNase-I Footprinting; Data; Dermatitis; Electron Transport; Enzymes; Epithelial; Epithelial Cells; Eukaryotic Cell; Gene Expression; Genes; Genetic Transcription; Goals; Growth; Human; Hydrogen Cyanide; Immune; In Vitro; Individual; Infection; LacZ Genes; Lung; Malignant Neoplasms; Measurement; Measures; Mediating; Metabolism; Microbial Biofilms; Modeling; Morbidity - disease rate; Necrosis; Nuclease Protection Assays; Organism; Pathogenesis; Patients; Peroxidase; Peroxidases; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Pilum; Play; Poisoning; Process; Process Measure; Production; Pseudomonas; Pseudomonas Infections; Pseudomonas aeruginosa; Regulation; Regulon; Relative (related person); Respiratory System; Respiratory tract structure; Role; Septicemia; Site-Directed Mutagenesis; Soft Tissue Infections; Staging; Superoxide Dismutase; Testing; Therapeutic; Time; Tissue Transplantation; Transcription Coactivator; Transplant Recipients; Urinary tract; Virulence; Virulence Factors; catalase; cell killing; cell motility; cystic fibrosis patients; gel mobility shift assay; hydrogen cyanide synthase; improved; metalloenzyme; mortality; mucoid; mutant; neutrophil; nitrogen metabolism; novel strategies; nuclease; pathogen; promoter; respiratory; rhamnolipid; therapeutic target

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(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患者，包括艾滋病和移植患者、严重烧伤患者和囊性纤维症患者。在慢性肺病方面，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。由于奇怪的抗生素耐药性，感染假单胞菌的CF患者经常患有慢性感染，治疗选择有限。因此，为了提高治疗效果，需要对这种微生物利用的致病机制进行基本了解，作为可能的治疗靶点。虽然以前的大多数研究都集中在定植和感染的初始阶段，但我们提出了一种新的方法来寻找治疗方法。越来越多的证据表明，微需氧代谢和生物膜生长模式可能参与了铜绿假单胞菌的致病过程；然而，对其机制的解释尚不清楚。铜绿假单胞菌在这些生长条件下产生的一个毒力因子是HCN。微摩尔量的HCN抑制真核细胞的呼吸电子传递链和几种金属酶(如过氧化氢酶、过氧化物酶、超氧化物歧化酶)。我们已经发现，作为毒力因子海藻酸盐的调节剂，ALGR也能激活粘液型铜绿假单胞菌中HCN的产生。利用假单胞菌Affymetrix基因芯片和S1核酸酶保护实验，我们证明了ALGR控制着编码氰化氢合成酶的hcnA。此外，对HCN产生的直接测量表明，粘液型铜绿假单胞菌在4小时内产生多达2.5 mM的HCN。我们的初步数据表明，ALGR有两个新的作用：i)ALGR控制HCN的产生；II)ALGR能够从非mcoid铜绿假单胞菌的抑制者转换为hcnA启动子上的粘液性细菌的激活剂。此外，我们还证明了ALGZ/FIMS在这一过程中发挥了作用。需要检验的假设是：ALGR激活粘液型铜绿假单胞菌中HCN的产生。我们将通过四个具体目标来验证这一假说：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ALGR激活粘液性铜绿假单胞菌hCNA表达是否需要磷酸化；iii)我们将确定HCN产量和hCNA在生物膜中的表达；以及IV)我们将确定HCN产量对体外肺上皮细胞和人中性粒细胞的影响。因此，在我们拟议的研究结束时，我们希望阐明新的可能的治疗靶点，并对假单胞菌的生物学和发病机制有更好的了解。