Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, Iowa, United States of America.
PLoS Pathog. 2010 Aug 26;6(8):e1001075. doi: 10.1371/journal.ppat.1001075.
Pseudomonas aeruginosa is a nearly ubiquitous human pathogen, and infections can be lethal to patients with impaired respiratory and immune systems. Prior studies have established that strong loss-of-function mutations in the egl-9 gene protect the nematode C. elegans from P. aeruginosa PAO1 fast killing. EGL-9 inhibits the HIF-1 transcription factor via two pathways. First, EGL-9 is the enzyme that targets HIF-1 for oxygen-dependent degradation via the VHL-1 E3 ligase. Second, EGL-9 inhibits HIF-1-mediated gene expression through a VHL-1-independent mechanism. Here, we show that a loss-of-function mutation in hif-1 suppresses P. aeruginosa PAO1 resistance in egl-9 mutants. Importantly, we find stabilization of HIF-1 protein is not sufficient to protect C. elegans from P. aeruginosa PAO1 fast killing. However, mutations that inhibit both EGL-9 pathways result in higher levels of HIF-1 activity and confer resistance to the pathogen. Using forward genetic screens, we identify additional mutations that confer resistance to P. aeruginosa. In genetic backgrounds that stabilize C. elegans HIF-1 protein, loss-of-function mutations in swan-1 increase the expression of hypoxia response genes and protect C. elegans from P. aeruginosa fast killing. SWAN-1 is an evolutionarily conserved WD-repeat protein belonging to the AN11 family. Yeast two-hybrid and co-immunoprecipitation assays show that EGL-9 forms a complex with SWAN-1. Additionally, we present genetic evidence that the DYRK kinase MBK-1 acts downstream of SWAN-1 to promote HIF-1-mediated transcription and to increase resistance to P. aeruginosa. These data support a model in which SWAN-1, MBK-1 and EGL-9 regulate HIF-1 transcriptional activity and modulate resistance to P. aeruginosa PAO1 fast killing.
铜绿假单胞菌是一种几乎无处不在的人类病原体，感染会对呼吸和免疫系统受损的患者造成致命威胁。先前的研究已经证实，egl-9 基因的强功能丧失突变可保护秀丽隐杆线虫免受铜绿假单胞菌 PAO1 的快速杀伤。EGL-9 通过两种途径抑制 HIF-1 转录因子。首先，EGL-9 是通过 VHL-1 E3 连接酶靶向 HIF-1 进行氧依赖性降解的酶。其次，EGL-9 通过 VHL-1 独立机制抑制 HIF-1 介导的基因表达。在这里，我们表明 hif-1 的功能丧失突变可抑制 egl-9 突变体中铜绿假单胞菌 PAO1 的抗性。重要的是，我们发现 HIF-1 蛋白的稳定不足以保护秀丽隐杆线虫免受铜绿假单胞菌 PAO1 的快速杀伤。然而，抑制 EGL-9 两条途径的突变会导致更高水平的 HIF-1 活性，并赋予对病原体的抗性。通过正向遗传筛选，我们鉴定出其他赋予铜绿假单胞菌抗性的突变。在稳定秀丽隐杆线虫 HIF-1 蛋白的遗传背景下，swan-1 的功能丧失突变会增加低氧反应基因的表达，并保护秀丽隐杆线虫免受铜绿假单胞菌 PAO1 的快速杀伤。SWAN-1 是一种进化上保守的 WD-重复蛋白，属于 AN11 家族。酵母双杂交和共免疫沉淀实验表明，EGL-9 与 SWAN-1 形成复合物。此外，我们提供了遗传证据表明 DYRK 激酶 MBK-1 作为 SWAN-1 的下游作用，以促进 HIF-1 介导的转录并增加对铜绿假单胞菌 PAO1 的抗性。这些数据支持了这样一种模型，即 SWAN-1、MBK-1 和 EGL-9 调节 HIF-1 转录活性，并调节对铜绿假单胞菌 PAO1 快速杀伤的抗性。
