Department of Microbiology and Immunology, College of Medicine, Tainan, 70101, Taiwan.
Institute of Bioinformatics and Biosignal Transduction, National Cheng Kung University, Tainan, 70101, Taiwan.
J Biomed Sci. 2017 Aug 11;24(1):54. doi: 10.1186/s12929-017-0361-9.
An attenuated mutant (designated NY303) of Vibrio vulnificus, which causes serious wound infection and septicemia in humans, was isolated fortuitously from a clinical strain YJ016. This mutant was defective in cytotoxicity, migration on soft agar and virulence in the mouse. The purpose of this study was to map the mutation in this attenuated mutant and further explore how the gene thus identified is involved in virulence.
The whole genome sequence of mutant NY303 determined by next-generation sequencing was compared with that of strain YJ016 to map the mutations. By isolating and characterizing the specific gene-knockout mutants, the gene associated with the phenotype of mutant NY303 was identified. This gene encodes a global regulator, Lrp. A mutant, YH01, deficient in Lrp was isolated and examined in vitro, in vivo and ex vivo to find the affected virulence mechanisms. The target genes of Lrp were further identified by comparing the transcriptomes, which were determined by RNA-seq, of strain YJ016 and mutant YH01. The promoters bound by Lrp were identified by genome footprinting-sequencing, and those related with virulence were further examined by electrophoretic mobility shift assay.
A mutation in lrp was shown to be associated with the reduced cytotoxicity, chemotaxis and virulence of mutant NY303. Mutant YH01 exhibited a phenotype resembling that of mutant NY303, and was defective in colonization in the mouse and growth in mouse serum, but not the antiphagocytosis ability. 596 and 95 genes were down- and up-regulated, respectively, in mutant YH01. Many of the genes involved in secretion of the MARTX cytotoxin, chemotaxis and iron-acquisition were down-regulated in mutant YH01. The lrp gene, which was shown to be negatively autoregulated, and 7 down-regulated virulence-associated genes were bound by Lrp in their promoters. A 14-bp consensus sequence, mkCrTTkwAyTsTG, putatively recognized by Lrp was identified in the promoters of these genes.
Lrp is a global regulator involved in regulation of cytotoxicity, chemotaxis and iron-acquisition in V. vulnificus. Down-regulation of many of the genes associated with these properties may be responsible, at least partly, for loss of virulence in mutant NY303.
一种减毒突变体（命名为 NY303）的创伤弧菌，它会导致人类严重的伤口感染和败血症，是从临床菌株 YJ016 中偶然分离出来的。这种突变体在细胞毒性、软琼脂上的迁移和在小鼠中的毒力方面存在缺陷。本研究的目的是绘制这个减毒突变体的突变图谱，并进一步探讨鉴定出的基因如何参与毒力。
通过下一代测序确定的突变体 NY303 的全基因组序列与菌株 YJ016 的序列进行比较，以绘制突变图谱。通过分离和表征特定的基因敲除突变体，鉴定与 NY303 表型相关的基因。该基因编码一个全局调控因子 Lrp。分离并研究了 Lrp 缺陷型突变体 YH01，以发现受影响的毒力机制。通过比较 RNA-seq 确定的菌株 YJ016 和突变体 YH01 的转录组，进一步鉴定 Lrp 的靶基因。通过基因组足迹测序确定 Lrp 结合的启动子，并通过电泳迁移率变动分析进一步研究与毒力相关的启动子。
表明 lrp 突变与突变体 NY303 的细胞毒性、趋化性和毒力降低有关。突变体 YH01 表现出类似于 NY303 的表型，在小鼠中定植和在小鼠血清中生长缺陷，但不具有抗吞噬能力。突变体 YH01 中分别有 596 个和 95 个基因下调和上调。许多参与 MARTX 细胞毒素分泌、趋化性和铁摄取的基因在突变体 YH01 中下调。lrp 基因被证明是负自调控的，并且 7 个下调的毒力相关基因在其启动子中被 Lrp 结合。在这些基因的启动子中，鉴定出一个推定由 Lrp 识别的 14 个碱基对的保守序列 mkCrTTkwAyTsTG。
Lrp 是一种全局调控因子，参与调控创伤弧菌的细胞毒性、趋化性和铁摄取。许多与这些特性相关的基因下调可能至少部分导致突变体 NY303 毒力丧失。
