Ali Muhammad, Sun Yu, Xie Li, Yu Huafu, Bashir Anum, Li Lin
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural UniversityWuhan, China; Biotechnology Program, Department of Environmental Sciences, COMSATS Institute of Information TechnologyAbbottabad, Pakistan.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University Wuhan, China.
Front Microbiol. 2016 May 30;7:805. doi: 10.3389/fmicb.2016.00805. eCollection 2016.
Different species of the Pseudomonas genus have been reported for their pathogenic potential against animal cells. However, the pathogenicity of Pseudomonas syringae against Caenorhabditis elegans has never been reported. In this study, the interaction of P. syringae MB03 with C. elegans was studied. Different bioassays such as killing assay, lawn leaving assay, food preference assay, L4 growth assay and newly developed "secretion assay" were performed to evaluate the pathogenic potential of P. syringae on different growth media. The results of the killing assay showed that P. syringae MB03 was able to kill C. elegans under specific conditions, as the interaction between the host and the pathogen varied from non-pathogenic (assay on NGM medium) to pathogenic (assay on PG medium). The lawn leaving assay and the food preference assay illustrated that C. elegans identified P. syringae MB03 as a pathogen when assays were performed on PG medium. Green fluorescent protein was used as the reporter protein to study gut colonization by P. syringae MB03. Our results suggested that MB03 has the ability to colonize the gut of C. elegans. Furthermore, to probe the role of selected virulence determinants, qRT-PCR was used. The genes for pyoverdine, phoQ/phoP, phoR/phoB, and flagella were up regulated during the interaction of P. syringae MB03 and C. elegans on PG medium. Other than these, the genes for some proteases, such as pepP, clpA, and clpS, were also up regulated. On the other hand, kdpD and kdpB were down regulated more than threefold in the NGM - C. elegans interaction model. The deletion of the kdpD and kdpE genes altered the pathogenicity of the bacterial strain against C. elegans. Overall, our results suggested that the killing of C. elegans by P. syringae requires a prolonged interaction between the host and pathogen in an agar-based assay. Moreover, it seemed that some toxic metabolites were secreted by the bacterial strain that were sensed by C. elegans. Previously, it was believed that P. syringae could not damage animal cells. However, this study provides evidence of the pathogenic behavior of P. syringae against C. elegans.
据报道，假单胞菌属的不同物种对动物细胞具有致病潜力。然而，丁香假单胞菌对秀丽隐杆线虫的致病性从未有过报道。在本研究中，对丁香假单胞菌MB03与秀丽隐杆线虫的相互作用进行了研究。进行了不同的生物测定，如杀伤测定、离菌苔测定、食物偏好测定、L4生长测定以及新开发的“分泌测定”，以评估丁香假单胞菌在不同生长培养基上的致病潜力。杀伤测定结果表明，丁香假单胞菌MB03在特定条件下能够杀死秀丽隐杆线虫，因为宿主与病原体之间的相互作用从非致病性（在NGM培养基上的测定）到致病性（在PG培养基上的测定）有所不同。离菌苔测定和食物偏好测定表明，当在PG培养基上进行测定时，秀丽隐杆线虫将丁香假单胞菌MB03识别为病原体。绿色荧光蛋白被用作报告蛋白来研究丁香假单胞菌MB03在肠道中的定殖情况。我们的结果表明，MB03具有定殖于秀丽隐杆线虫肠道的能力。此外，为了探究选定毒力决定因素的作用，使用了定量逆转录聚合酶链反应（qRT-PCR）。在PG培养基上丁香假单胞菌MB03与秀丽隐杆线虫相互作用期间，铁载体、phoQ/phoP、phoR/phoB和鞭毛的基因上调。除此之外，一些蛋白酶的基因，如pepP、clpA和clpS也上调。另一方面，在NGM-秀丽隐杆线虫相互作用模型中，kdpD和kdpB下调了三倍以上。kdpD和kdpE基因的缺失改变了该细菌菌株对秀丽隐杆线虫的致病性。总体而言，我们的结果表明，在基于琼脂的测定中，丁香假单胞菌对秀丽隐杆线虫的杀伤需要宿主与病原体之间长时间的相互作用。此外，似乎该细菌菌株分泌了一些被秀丽隐杆线虫感知到的有毒代谢产物。以前，人们认为丁香假单胞菌不会损害动物细胞。然而，本研究提供了丁香假单胞菌对秀丽隐杆线虫致病行为的证据。
