Schaefers Matthew M, Liao Tiffany L, Boisvert Nicole M, Roux Damien, Yoder-Himes Deborah, Priebe Gregory P
Division of Critical Care Medicine, Department of Anesthesiology, Perioperative and Pain Medicine, Boston Children's Hospital, Boston, Massachusetts, United States of America.
Department of Anaesthesia, Harvard Medical School, Boston, Massachusetts, United States of America.
PLoS Pathog. 2017 Jan 3;13(1):e1006116. doi: 10.1371/journal.ppat.1006116. eCollection 2017 Jan.
Burkholderia dolosa is a member of the Burkholderia cepacia complex (BCC), which is a group of bacteria that cause chronic lung infection in patients with cystic fibrosis (CF) and can be associated with outbreaks carrying high morbidity and mortality. While investigating the genomic diversity of B. dolosa strains collected from an outbreak among CF patients, we previously identified fixL as a gene showing signs of strong positive selection. This gene has homology to fixL of the rhizobial FixL/FixJ two-component system. The goals of this study were to determine the functions of FixLJ and their role in virulence in B. dolosa. We generated a fixLJ deletion mutant and complemented controls in B. dolosa strain AU0158. Using a fixK-lacZ reporter we found that FixLJ was activated in low oxygen in multiple BCC species. In a murine pneumonia model, the B. dolosa fixLJ deletion mutant was cleared faster from the lungs and spleen than wild-type B. dolosa strain AU0158 at 7 days post infection. Interestingly, the fixLJ deletion mutant made more biofilm, albeit with altered structure, but was less motile than strain AU0158. Using RNA-seq with in vitro grown bacteria, we found ~11% of the genome was differentially expressed in the fixLJ deletion mutant relative to strain AU0158. Multiple flagella-associated genes were down-regulated in the fixLJ deletion mutant, so we also evaluated virulence of a fliC deletion mutant, which lacks a flagellum. We saw no difference in the ability of the fliC deletion mutant to persist in the murine model relative to strain AU0158, suggesting factors other than flagella caused the phenotype of decreased persistence. We found the fixLJ deletion mutant to be less invasive in human lung epithelial and macrophage-like cells. In conclusion, B. dolosa fixLJ is a global regulator that controls biofilm formation, motility, intracellular invasion/persistence, and virulence.
多氏伯克霍尔德菌是洋葱伯克霍尔德菌复合体（BCC）的成员之一，该复合体是一组可导致囊性纤维化（CF）患者慢性肺部感染的细菌，且可能与高发病率和死亡率的疾病暴发相关。在调查从CF患者疾病暴发中分离出的多氏伯克霍尔德菌菌株的基因组多样性时，我们之前鉴定出fixL是一个显示出强烈正选择迹象的基因。该基因与根瘤菌FixL/FixJ双组分系统的FixL具有同源性。本研究的目的是确定FixLJ的功能及其在多氏伯克霍尔德菌毒力中的作用。我们构建了多氏伯克霍尔德菌菌株AU0158的fixLJ缺失突变体及其互补对照。使用fixK - lacZ报告基因，我们发现FixLJ在多种BCC物种的低氧环境中被激活。在小鼠肺炎模型中，感染后7天，多氏伯克霍尔德菌fixLJ缺失突变体从肺和脾脏中的清除速度比野生型多氏伯克霍尔德菌菌株AU0158更快。有趣的是，fixLJ缺失突变体形成了更多的生物膜，尽管结构有所改变，但比菌株AU0158的运动性更低。通过对体外培养细菌进行RNA测序，我们发现相对于菌株AU0158，fixLJ缺失突变体中有约11%的基因组存在差异表达。多个与鞭毛相关的基因在fixLJ缺失突变体中表达下调，因此我们还评估了缺乏鞭毛的fliC缺失突变体的毒力。我们发现fliC缺失突变体在小鼠模型中相对于菌株AU0158的持续存在能力没有差异，这表明除鞭毛外的其他因素导致了持续存在能力下降的表型。我们发现fixLJ缺失突变体在人肺上皮细胞和巨噬细胞样细胞中的侵袭性较低。总之，多氏伯克霍尔德菌fixLJ是一种全局调节因子，可控制生物膜形成、运动性、细胞内侵袭/持续存在以及毒力。
