Photorhabdus luminescens genes induced upon insect infection.
Photorhabdus luminescens genes induced upon insect infection.
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DOI：
10.1186/1471-2164-9-229
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发表时间：
2008-05-19
期刊：
影响因子：
4.4
通讯作者：
Heermann R
中科院分区：
文献类型：
--
作者：
Münch A;Stingl L;Jung K;Heermann R
发光小杆菌（Photorhabdus luminescens）是一种革兰氏阴性发光肠杆菌，是土壤线虫的共生菌，属于细菌异小杆菌（Heterorhabditis bacteriophora）。发光假单胞菌同时对昆虫具有高致病性。这种细菌表现出复杂的生命周期，包括一个共生阶段，其特征在于上部线虫肠道的定殖，和一个致病阶段，其特征在于从线虫释放到昆虫幼虫的血腔中，导致由细菌毒素引起的昆虫快速死亡。发光假单胞菌似乎感觉和适应新的宿主环境后，改变主机，这有利于生产的因素，涉及在主机内的生存，主机杀死，和开发。应用差异荧光诱导（DFI）方法来鉴定昆虫宿主大蜡螟（Galleria mellonella）感染后细菌中上调的基因。为此目的，构建了利用mCherry荧光团作为报道基因的发光假单胞菌启动子-陷阱文库，并筛选了约13，000个克隆在G.大蜡螟幼虫匀浆。由于发光假单胞菌具有多种可能感测化学分子（如激素）的调节剂，因此在体外进行上调基因或操纵子的筛选，排除物理化学信号（如氧气、温度或渗透压摩尔浓度）作为变量。获得表现出至少2.5倍诱导荧光的克隆（18），并认为其是对昆虫匀浆的特异性应答者。结合生物信息学方法，在这些DNA片段中鉴定出与发光假单胞菌基因组内的29种不同启动子相似的序列基序。通过克隆报告基因上游的每个预测的启动子，在体外验证了27个启动子的诱导，在活的G.大蜡螟幼虫在经验证的启动子中，一些已知调节毒素基因的表达，包括tccC 1（编码杀虫毒素复合物），以及其他编码推定毒素的启动子。在感染后观察到大量代谢基因或操纵子被诱导;其中包括eutABC、hutUH和agaerobic VCD，它们分别编码参与乙醇胺、组氨酸和塔格糖降解的蛋白质。这些结果反映了代谢的重排和昆虫其他代谢产物的利用。此外，观察到控制编码与抗生素产生和/或抗性相关的酶的基因表达的启动子的活性增强。抗生素的产生和耐药性可能会影响与其他细菌的竞争，因此可能对成功感染很重要。最后，几个未知功能的基因被确定，可能代表新的致病因子。我们表明，DFI屏幕是有用的识别基因或操纵子诱导的化学刺激，如稀释的昆虫匀浆。与已知启动子相似的基序的生物信息学比较是鉴定调控基因或操纵子的有力工具。我们的结论是，这些基因或操纵子的调控信号诱导发光拟青霉昆虫感染后，可能代表了各种各样的化合物，使昆虫宿主。我们的研究结果提供了对细菌病原体中发生的宿主复杂反应的深入了解，特别是反映了代谢变化和与毒力相关的其他特定变化的潜力。
Photorhabdus luminescens is a Gram-negative luminescent enterobacterium and a symbiote to soil nematodes belonging to the species Heterorhabditis bacteriophora. P.luminescens is simultaneously highly pathogenic to insects. This bacterium exhibits a complex life cycle, including one symbiotic stage characterized by colonization of the upper nematode gut, and a pathogenic stage, characterized by release from the nematode into the hemocoel of insect larvae, resulting in rapid insect death caused by bacterial toxins. P. luminescens appears to sense and adapt to the novel host environment upon changing hosts, which facilitates the production of factors involved in survival within the host, host-killing, and -exploitation. A differential fluorescence induction (DFI) approach was applied to identify genes that are up-regulated in the bacterium after infection of the insect host Galleria mellonella. For this purpose, a P. luminescens promoter-trap library utilizing the mCherry fluorophore as a reporter was constructed, and approximately 13,000 clones were screened for fluorescence induction in the presence of a G. mellonella larvae homogenate. Since P. luminescens has a variety of regulators that potentially sense chemical molecules, like hormones, the screen for up-regulated genes or operons was performed in vitro, excluding physicochemical signals like oxygen, temperature or osmolarity as variables. Clones (18) were obtained exhibiting at least 2.5-fold induced fluorescence and regarded as specific responders to insect homogenate. In combination with a bioinformatics approach, sequence motifs were identified in these DNA-fragments that are similar to 29 different promoters within the P. luminescens genome. By cloning each of the predicted promoters upstream of the reporter gene, induction was verified for 27 promoters in vitro, and for 24 promoters in viable G. mellonella larvae. Among the validated promoters are some known to regulate the expression of toxin genes, including tccC1 (encoding an insecticidal toxin complex), and others encoding putative toxins. A comparably high number of metabolic genes or operons were observed to be induced upon infection; among these were eutABC, hutUH, and agaZSVCD, which encode proteins involved in ethanolamine, histidine and tagatose degradation, respectively. The results reflect rearrangements in metabolism and the use of other metabolites available from the insect. Furthermore, enhanced activity of promoters controlling the expression of genes encoding enzymes linked to antibiotic production and/or resistance was observed. Antibiotic production and resistance may influence competition with other bacteria, and thus might be important for a successful infection. Lastly, several genes of unknown function were identified that may represent novel pathogenicity factors. We show that a DFI screen is useful for identifying genes or operons induced by chemical stimuli, such as diluted insect homogenate. A bioinformatics comparison of motifs similar to known promoters is a powerful tool for identifying regulated genes or operons. We conclude that signals for the regulation of those genes or operons induced in P. luminescens upon insect infection may represent a wide variety of compounds that make up the insect host. Our results provide insight into the complex response to the host that occurs in a bacterial pathogen, particularly reflecting the potential for metabolic shifts and other specific changes associated with virulence.
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影响因子：
2.9
作者：
Eleftherianos, Ioannis;Millichap, Peter J.;Reynolds, Stuart E.
通讯作者：
Reynolds, Stuart E.
DOI：
10.1073/pnas.102068099
发表时间：
2002-08-06
影响因子：
11.1
作者：
Daborn, PJ;Waterfield, N;Ffrench-Constant, RH
通讯作者：
Ffrench-Constant, RH
影响因子：
3.4
作者：
Dowling, AJ;Daborn, PJ;ffrench-Constant, RH
通讯作者：
ffrench-Constant, RH
影响因子：
5.4
作者：
Abee, T;Wouters, JA
通讯作者：
Wouters, JA
影响因子：
3.6
作者：
Blanc, V;Gil, P;Thibaut, D
通讯作者：
Thibaut, D