Zhou Ze-jun, Zhang Lu, Sun Li
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
Vet Microbiol. 2015 Jan 30;175(1):145-9. doi: 10.1016/j.vetmic.2014.11.001. Epub 2014 Nov 11.
Pseudomonas fluorescens, a Gram-negative bacterium, is an aquaculture pathogen with a broad host range. In a previous study, we had demonstrated that knockout of the fur gene of a pathogenic P. fluorescens strain, TSS, resulted in profound virulence attenuation. In this work, we studied the properties of the fur knockout mutant, TFM, in comparison with the wild type strain TSS. We found that compared to TSS, TFM (i) was impaired in siderophore production and extracellular enzyme activities, (ii) exhibited altered global polarity, (iii) was dramatically reduced in the ability to resist oxidative stress, (iv) showed higher tolerance to manganese, and (v) exhibited significantly reduced cytotoxicity. When incubated with cultured host cells, TFM displayed a cellular binding index much lower than that of TSS. Neither TFM nor TSS was able to survive and replicate in host cells. Following inoculation into Japanese flounder (Paralichthys olivaceus), TSS upregulated the expression of a wide range of genes involved in innate immunity, notably IL-1β and two CC chemokines. In contrast, TFM caused significant inductions of only a few genes and to much lower magnitudes than TSS. Given the strong inductions of IL-1β and the two chemokines by TSS, the effect of these three genes on P. fluorescens invasion was examined. The results showed that overexpression of these genes in flounder significantly inhibited TSS dissemination into and colonization of host tissues. Taken together, these results indicate that Fur is required for multiple processes associated with virulence, and that proinflammatory cytokines and chemokines likely play important roles in the clearance of P. fluorescens infection.
荧光假单胞菌是一种革兰氏阴性菌，是一种宿主范围广泛的水产养殖病原菌。在先前的一项研究中，我们已经证明，致病性荧光假单胞菌菌株TSS的fur基因敲除导致其毒力大幅减弱。在这项工作中，我们研究了fur基因敲除突变体TFM与野生型菌株TSS相比的特性。我们发现，与TSS相比，TFM：（i）在铁载体产生和胞外酶活性方面受损；（ii）表现出整体极性改变；（iii）抵抗氧化应激的能力显著降低；（iv）对锰的耐受性更高；（v）细胞毒性显著降低。当与培养的宿主细胞一起孵育时，TFM的细胞结合指数远低于TSS。TFM和TSS都不能在宿主细胞中存活和复制。接种到日本牙鲆（Paralichthys olivaceus）后，TSS上调了一系列参与先天免疫的基因的表达，特别是IL-1β和两种CC趋化因子。相比之下，TFM仅显著诱导了少数基因，且诱导程度远低于TSS。鉴于TSS对IL-1β和这两种趋化因子的强烈诱导，研究了这三个基因对荧光假单胞菌入侵的影响。结果表明，这些基因在牙鲆中的过表达显著抑制了TSS向宿主组织的扩散和定植。综上所述，这些结果表明Fur是与毒力相关的多个过程所必需的，并且促炎细胞因子和趋化因子可能在清除荧光假单胞菌感染中发挥重要作用。
