Impact of Alginate Overproduction on P. aeruginosa LPS O Antigen Expression

项目摘要/摘要。 铜绿假单胞菌是一种重要的条件致病菌，为革兰氏阴性杆菌。 囊性纤维化(CF)患者慢性危及生命的肺部感染的主要原因。铜绿假单胞菌 天然的抗生素耐药性和这种细菌的感染是出了名的难以治疗。约70%的 Cf患者在18岁之前感染铜绿假单胞菌，平均预期寿命不到40岁。一个 需要更好地了解这种细菌是如何进化而导致这些慢性呼吸道感染的。 当铜绿假单胞菌从环境过渡到引起疾病时，会发生许多表型变化 缩短生命的慢性CF型肺部感染。粘液表型分离株的出现，是由于 胞外多糖海藻酸盐的过量生产最为突出；粘液菌株的分离是一种 这些患者的临床衰退的预测指标。粘液样菌株还有一个不太明显的 多糖变化：这些菌株通常缺乏脂多糖O抗原，这是一种表型 被称为“LPS-Rough”。有趣的是，这种向粘液样、内毒素粗糙形式的转化在P. 铜绿假单胞菌分离自非慢性支气管炎患者。在罕见的粘液性菌株中确实表达了 LPSO抗原、不同长度的LPSO抗原的表达与非粘液型LPSO抗原表达的比较 菌株：从非粘液性菌株中分离的内毒素包括长O抗原和超长O抗原，而从非粘液性菌株中分离的内毒素 粘液菌株的很长O抗原的表达减少，但长O的表达相同 抗原，我们认为它代表了初始感染形式和慢性感染形式之间的过渡状态。 这项为期两年的探索/发展研究补助金的目标是确定粘液样物质和 O抗原(以及其他基因类型)的丢失在慢性定植过程中出现，以及细菌和 宿主对这些感染做出反应。在特定目标1中，我们将利用实验室菌株PAO1及其粘液 衍生物PDO300在一种新描述的慢性呼吸道定植的小鼠模型中，并将遵循 定植过程中出现和进化的菌株以及细菌的表型和基因型别 以及宿主对感染的反应。在具体目标2中，我们将采用分子方法来鉴定基因 控制粘液型内毒素平滑菌株中超长O抗原的调控机制及其评估 藻酸盐能抑制很长的O抗原。抑制因子wzz2(基因)的发现 其编码超长的O抗原链长度调节器)将为治疗提供相关靶点 干预。如果我们能抑制抑制wzz2，这可能会造成细菌不能 保持粘液表型，因此不会受到天然宿主免疫反应或 演变成一种长期殖民的形式。这些研究的长期目标将是确定细菌因素 可以靶向抑制慢性肺部感染和宿主因素，这些因素可以增加细菌的清除和 从而改善CF患者的生活。

PROJECT SUMMARY/ABSTRACT. Pseudomonas aeruginosa, a Gram-negative bacterium, is an important opportunistic pathogen and the leading cause of chronic life-threatening lung infections in cystic fibrosis (CF) patients. P. aeruginosa is naturally antibiotic resistant and infections with this bacterium are notoriously difficult to treat. About 70% of CF patients are infected with P. aeruginosa by age 18 and the mean life expectancy is less than 40 years. A better understanding of how this bacterium evolves to cause these chronic respiratory infections is needed. Numerous phenotypic changes take place as P. aeruginosa transitions from the environment to causing life-shortening chronic CF lung infections. The emergence of isolates with a mucoid phenotype, due to the overproduction of the exopolysaccharide alginate is the most conspicuous; the isolation of mucoid strains is a prognosticator of clinical decline in these patients. Mucoid strains also have an additional less obvious polysaccharide alteration: these strains are generally lack lipopolysaccharide (LPS) O antigen, a phenotype referred to as “LPS-rough”. Interestingly, this same conversion to a mucoid, LPS-rough form is prominent in P. aeruginosa isolates from patients with non-CF chronic bronchiectasis. In rare mucoid strains that do express LPS O antigen, different lengths of LPS O antigen are expressed compared to non-mucoid LPS-smooth strains: while LPS isolated from non-mucoid strains includes both long and very long O antigen, LPS isolated from mucoid strains shows decreased expression of very long O antigen but the same expression of long O antigen, which we suggest represents a transition state between the initial- and chronic-infecting forms. The goal of this 2-year Exploratory/Developmental Research Grant is to determine how mucoidy and loss of O antigen (as well as other genotypes) emerge during chronic colonization and how the bacteria and host respond to these infections. In Specific Aim 1, we will utilize the laboratory strain PAO1 and its mucoid derivative, PDO300, in a newly described murine model of chronic respiratory colonization and will follow the phenotypes and genotypes of the strains emerging and evolving during colonization as well as the bacterial and host response to infection. In Specific Aim 2, we will take a molecular approach to identify the genetic regulatory mechanisms controlling very long O antigen in a mucoid LPS-smooth strain and assess how alginate represses very long O antigen. The discovery of factors involved in the repression wzz2 (the gene which encodes the very long O antigen chain length regulator) will provide the relevant targets for therapeutic intervention. If we can inhibit the repression wzz2, this may set up a situation where the bacteria cannot maintain the mucoid phenotype and therefore would not be shielded from the innate host immune response or evolve into a chronic-colonizing form. The long-term goal of these studies will be to identify bacterial factors that can be targeted to inhibit chronic lung infection and host factors that can increase bacterial clearance and therefore improve the lives of CF patients.