Masud Samrah, Xie Jiajun, Grijmans Bart J M, van der Kooij Sander, Zhang Rui, Prajsnar Tomasz K, Meijer Annemarie H
Institute of Biology Leiden, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Department of Evolutionary Immunology, Faculty of Biology, Jagiellonian University, Gronostajowa 9, 30-382 Krakow, Poland.
Autophagy Rep. 2023 Aug 24;2(1):2242715. doi: 10.1080/27694127.2023.2242715. eCollection 2023.
DRAM1 is an infection inducible autophagy modulator, previously shown to promote autophagic and lysosomal defense responses against the intracellular pathogen . However, its possible role in other anti-bacterial autophagic mechanisms remains unknown. Recently, LC3-associated phagocytosis (LAP) has emerged as autophagy-related mechanism that targets bacteria directly in phagosomes. Our previous work established LAP as the main autophagic mechanism by which macrophages restrict growth of Typhimurium in a systemically infected zebrafish host. We therefore employed this infection model to investigate the possible role of Dram1 in LAP. Morpholino knockdown or CRISPR/Cas9-mediated mutation of Dram1 led to reduced host survival and increased bacterial burden during . Typhimurium infection. In contrast, overexpression of by mRNA injection curtailed replication and reduced mortality of the infected host. During the early response to infection, GFP-Lc3- associations were reduced in knockdown or mutant embryos, and increased by overexpression. Since LAP is known to require the activity of the phagosomal NADPH oxidase, we used a biosensor strain to detect bacterial exposure to reactive oxygen species (ROS) and found that the ROS response was largely abolished with deficiency of , while it was increased with overexpression. Corroborating these results in a mammalian model, the LC3 and ROS responses to were similarly reduced or increased by knockdown or overexpression of , respectively, in murine RAW264.7 macrophages. Together, these results demonstrate the host protective role of Dram1/DRAM1 during . Typhimurium infection and suggest a functional link between Dram1/DRAM1 and the induction of LAP. ATG8: Autophagy related protein 8; ATG16: Autophagy related protein 16; CFU: colony-forming unit; DRAM1: DNA damage regulated autophagy modulator gene 1; dpf: days post fertilization; GFP: green fluorescent protein; hpi: hours post infection; LAP: LC3 associated phagocytosis; LC3, microtubule-associated protein 1 light chain 3; NADPH: Nicotinamide dinucleotide phosphate; p53: Tumor suppressor protein 53: ROS; reactive oxygen species; . Typhimurium: enterica serovar Typhimurium; TIPTP: 2(tetrahydroindazolyl) phenoxy-N-(thiadiazolyl)propenamide 2; UVRAG: UV radiation resistance associated protein.
DRAM1是一种感染诱导型自噬调节因子，先前已证明它能促进针对细胞内病原体的自噬和溶酶体防御反应。然而，其在其他抗菌自噬机制中的可能作用仍不清楚。最近，LC3相关吞噬作用（LAP）已成为一种自噬相关机制，可直接在吞噬体中靶向细菌。我们之前的工作确定LAP是巨噬细胞在系统性感染的斑马鱼宿主中限制鼠伤寒沙门氏菌生长的主要自噬机制。因此，我们采用这种感染模型来研究Dram1在LAP中的可能作用。Dram1的吗啉代敲低或CRISPR/Cas9介导的突变导致宿主存活率降低，且在鼠伤寒沙门氏菌感染期间细菌载量增加。相反，通过mRNA注射过表达可减少鼠伤寒沙门氏菌的复制并降低感染宿主的死亡率。在感染的早期反应中，在敲低或突变胚胎中GFP-Lc3的结合减少，而过表达则增加。由于已知LAP需要吞噬体NADPH氧化酶的活性，我们使用一种鼠伤寒沙门氏菌生物传感器菌株来检测细菌对活性氧（ROS）的暴露情况，发现随着Dram1缺乏，ROS反应基本消失，而过表达时ROS反应增加。在哺乳动物模型中证实了这些结果，在鼠RAW264.7巨噬细胞中，通过敲低或过表达Dram1，对鼠伤寒沙门氏菌的LC3和ROS反应分别同样降低或增加。总之，这些结果证明了Dram1/DRAM1在鼠伤寒沙门氏菌感染期间的宿主保护作用，并表明Dram1/DRAM1与LAP诱导之间存在功能联系。ATG8：自噬相关蛋白8；ATG16：自噬相关蛋白16；CFU：集落形成单位；DRAM1：DNA损伤调节自噬调节因子基因1；dpf：受精后天数；GFP：绿色荧光蛋白；hpi：感染后小时数；LAP：LC3相关吞噬作用；LC3：微管相关蛋白1轻链3；NADPH：烟酰胺腺嘌呤二核苷酸磷酸；p53：肿瘤抑制蛋白53；ROS：活性氧；鼠伤寒沙门氏菌：肠炎血清型鼠伤寒沙门氏菌；TIPTP：2（四氢吲唑基）苯氧基-N-（噻二唑基）丙烯酰胺2；UVRAG：紫外线抗性相关蛋白。
