Identification of novel anti-virulence compounds against Pathogenic Burkholderia

项目摘要 该项目的长期目标是开发治疗革兰氏阴性菌的新疗法。 伯克霍尔德氏菌属中的病原体，包括洋葱伯克霍尔德氏菌复合体（BCC）， 类鼻疽伯克霍尔德菌这些病原体会导致严重的慢性肺部感染， 与医疗相关的感染。治疗由这些病原体引起的感染通常是困难的， 常见的广谱抗生素耐药性。新疗法的发展， 治疗这些病原体是迫切需要的。我们发表的工作分析了分离的基因组多样性的 BCC种B。dolosa和B.从CF患者中收集的多噬菌体发现， 基因似乎处于强正选择之下。FixL在根瘤菌属和柄杆菌属中被描述为 一种双组分系统的感觉性组氨酸激酶，其检测氧张力并磷酸化 转录因子FixJ在低氧条件下的表达。我们以前的工作已经发现BCC FixLJ系统 是致病所必需的，并且该途径的调节具有深远的毒力影响。有趣的是， 携带与患者临床衰退期相关的进化的FixL变体的构建体更多， 与携带祖先FixL变体的同基因构建体相比，在多种感染模型中具有毒性。这些 毒性更强的FixL序列比携带祖先FixL的毒性更弱的构建体具有更低的FixLJ活性 这些变体证明增加的FixLJ活性对毒力是决定性的。我们假设我们可以 通过激活FixLJ途径降低伯克霍尔德氏菌毒力。通过针对毒力，我们预测， 细菌将不再能够感染宿主或在宿主内持续存在。在本项目的第一个目标中，我们将确定 使用伯克霍尔德氏菌固定途径报告菌株的高通量筛选来活化FixLJ的化合物。 在目标2中，我们将评估目标1中确定的有希望的先导化合物在体外的有效性， 使用体外巨噬细胞测定降低多种伯克霍尔德氏菌属物种的毒力。我们还将评估 铅化合物的毒性使用细胞培养。我们计划鉴定出3-4种具有EC 50的先导化合物， 低微摩尔（1-10）范围和毒性至少比EC 50高5倍。这些先导化合物将 进一步发展为用于治疗伯克霍尔德氏菌感染的疗法。

Project Abstract The long-term goal of this project is to develop novel therapeutics for the treatment of the Gram-negative pathogens in the Burkholderia genus which includes the Burkholderia cepacia complex (BCC) and Burkholderia pseudomallei. These pathogens cause serious, chronic lung infections and are also major causes of healthcare-associated infections. Treatment of infections caused by these pathogens is often difficult due to the broad-spectrum antibiotic resistance that is commonly seen. The development of novel therapeutics to treat these pathogens is critically needed. Our published work analyzing the genomic diversity of isolates of the BCC species B. dolosa and B. multivorans collected from people with CF has led to the discovery that the fixL gene appears to be under strong positive selection. FixL has been described in Rhizobium and Caulobacter as a sensory histidine kinase of a two-component system that detects oxygen tension and phosphorylates the transcription factor FixJ under low oxygen conditions. Our previous work has found that the BCC FixLJ system is required for pathogenesis and modulation of this pathway has profound effects of virulence. Interestingly, constructs carrying evolved FixL variants associated with periods of clinical decline in patients were more virulent in multiple infection models compared to isogenic constructs carrying ancestral FixL variants. These more virulent FixL sequences had lower FixLJ activity than less virulent constructs carrying ancestral FixL variants demonstrating that increased FixLJ activity is determinantal to virulence. We hypothesize that we can reduce Burkholderia virulence by activating the FixLJ pathway. By targeting virulence, we predict that the bacteria would no longer be able to infect or persist within the host. In the first aim of this project we will identify compounds that activate FixLJ using a high-throughput screen with a Burkholderia fix pathway reporter strain. In Aim 2 we will evaluate promising lead compounds identified in Aim 1 for their in vitro effectiveness at reducing virulence of multiple Burkholderia species using an in vitro macrophage assay. We also will evaluate the toxicity of lead compounds using cell culture. We plan to identify 3-4 lead compounds with an EC50 in the low micromolar (1-10) range and a toxicity at least 5-fold higher than the EC50. These lead compounds will be further developed into therapies for use to treat Burkholderia infections.