Chen Li-Hong, Köseoğlu Volkan K, Güvener Zehra T, Myers-Morales Tanya, Reed Joseph M, D'Orazio Sarah E F, Miller Kurt W, Gomelsky Mark
Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, United States of America.
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky, United States of America.
PLoS Pathog. 2014 Aug 7;10(8):e1004301. doi: 10.1371/journal.ppat.1004301. eCollection 2014 Aug.
We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence.
我们对食源性病原体单核细胞增生李斯特菌中双鸟苷环化二磷酸（c-di-GMP）信号通路的关键成分和主要靶点进行了表征，鉴定出一种新的c-di-GMP诱导型胞外多糖，它负责抑制运动性、促进细胞聚集，并增强对消毒剂和干燥的耐受性，同时首次深入了解了c-di-GMP信号在李斯特菌毒力中的作用。对c-di-GMP信号通路进行全基因组遗传和生化分析发现，单核细胞增生李斯特菌有三种具有双鸟苷环化酶活性的GGDEF结构域蛋白，即DgcA（Lmo1911）、DgcB（Lmo1912）和DgcC（Lmo2174），以及三种具有c-di-GMP磷酸二酯酶活性的EAL结构域蛋白，即PdeB（Lmo0131）、PdeC（Lmo1914）和PdeD（Lmo0111）。删除所有磷酸二酯酶基因（ΔpdeB/C/D）或表达一种异源双鸟苷环化酶会刺激一种此前未知的胞外多糖的产生。这种胞外多糖的合成归因于pssA-E（lmo0527 - 0531）基因簇。该基因簇的最后一个基因编码第四个李斯特菌GGDEF结构域蛋白PssE，它作为一种I型位点c-di-GMP受体，对胞外多糖的合成至关重要。这种c-di-GMP诱导型胞外多糖在基本培养基中会导致细胞聚集，并损害细菌在半固体琼脂中的迁移，但它不会促进在非生物表面形成生物膜。这种胞外多糖还极大地增强了细菌对常用消毒剂以及干燥的耐受性，这可能有助于单核细胞增生李斯特菌在受污染食品和食品加工设施中存活。这种胞外多糖和另一个尚未明确的c-di-GMP依赖性靶点，在体外显著降低了李斯特菌对肠上皮细胞的侵袭性，并降低了经口感染小鼠肝脏和胆囊中的病原体载量，这表明升高的c-di-GMP水平在李斯特菌毒力中总体上起负面作用。
