【作者】 潘海建；

【导师】 莫照兰；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2012， 硕士

【摘要】 Cpx（conjugative plasmid expression）是存在于革兰氏阴性菌细胞膜上的双组分调节系统，能够感应外界环境变化，参与细菌细胞膜压力调节，维持细胞膜蛋白稳定性。迟缓爱德华氏菌Cpx的功能未知，本研究开展了如下工作：（1）对表型的影响。利用同源克隆获得LSE40菌株全长2,713bp的cpx基因簇，该Cpx含有编码组氨酸蛋白激酶CpxA、反应调节蛋白CpxR和抑制因子CpxP的基因。利用框内基因缺失突变技术，构建了cpx基因簇缺失突变株Δcpx和cpxR基因缺失株ΔcpxR。在正常培养条件下，两个突变株的生长、运动力、对抗生素的敏感性、自凝集、三型分泌系统（T3SS）输送器蛋白（EseB，EseC,EseD）分泌与野生型相比没有显著变化（P>0.05），cpx的缺失使细菌的菌膜形成能力减弱（P <0.05），而ΔcpxR的菌膜形成没有显著变化（P>0.05）；在缺铁和3.5%NaCl的培养条件下，两个突变株的生长无显著差异（P>0.05）；在1%H₂O₂和0.05%SDS培养条件下，两个突变株的生长显著下降（P <0.05）。上述结果表明Cpx参与了迟缓爱德华氏菌对氧化剂和表面活性剂等压力的响应。（2）对毒力的影响。检测了突变株对蓝曼龙（Trichogaster trichopterus）的毒力，浸泡感染结果显示Δcpx和ΔcpxR对蓝曼龙的毒力分别下降1.5倍和3.0倍，肌肉注射感染结果显示两突变株的毒力分别下降7.33倍和4.58倍，两株突变株在鱼体内的生存能力显著下降（P <0.05）。上述结果表明Cpx系统参与了对迟缓爱德华氏菌毒力的调控。（3）对基因转录水平的影响。qRT-PCR结果显示，用1mM H₂O₂处理迟缓爱德华氏菌LSE40后，cpxR基因的转录水平显著升高（P <0.05），表明H₂O₂处理可激活迟缓爱德华氏菌LSE40cpxR的表达。cpx和cpxR缺失后，一些涉及抗氧化、蛋白正确折叠、细胞分离等过程的蛋白基因的转录水平与野生型细菌有显著性差异，在cpxR互补株中的表达量又回复到野生型水平，表明CpxR参与对这些基因的调控。更多还原

【Abstract】 The Cpx（conjugative plasmid expression）two-component regulatory system existson the membrane of a variety of gram-negative bacteria. It senses the change ofnatural environment or the systemic infection niches of hosts, and is involved in theresponse to envelope stress and the maintenance of envelope proteins. Function of theCpx in E. tarda is not clarified. Here we studied its role:（1） The impact on bacterial phenotypes. We obtained the2,713bp cpx gene clusterof Edwardsiella tarda LSE40using a homologous cloning strategy. The Cpx iscomposed of a histidine kinase activity protein CpxA, a cognate cytoplasmic responseregulator CpxR and a periplasmic inhibitor CpxP. To investigate the function of Cpx,the cpxR gene and the cpx cluster in-frame deletion mutants were constructed fromLSE40. Neither ΔcpxR or Δcpx mutants showed significant differences with thewild-type strain in the celluar growth, mobility, sensitivities to antibiotics,auto-aggregation and secretion of T3SS component protein EseB, EseC, EseD（P>0.05） in TSA or TSB medium. The Δcpx mutant showed aberrant biofilmformation （P <0.05）, while the ΔcpxR mutant exihibited significant difference（P>0.05）. Mutants didn’t showed significant differences in3.5%NaCl density TSBand Fe2+limited medium （P>0.05）. While and both mutants exhibited increasedsensitivities under1%（w/v） H₂O₂and0.05%（w/v） SDS stresses （P <0.05）. Thesedata indicated that E. tarda Cpx participated in sensing environmental oxidant andsurfactant signal.（2） The impact on bacterial virulence. Bacteria were administrated to blue guoramifish Trichogaster trichopterus by immersing infection and intramuscular injection. Inthe injection route, the Δcpx and ΔcpxR mutants decreased4.58-fold and7.33-fold in virulence compared with the wild-type strain. In the immersion route, the Δcpx andΔcpxR mutants showed1.5-fold and3.0-fold decrease. Both mutants exhibiteddecreased bacterial proliferation ability in host’s liver tissue as well, which indicatedthat Cpx pathway was involved in the virulence of E. tarda.（3） The impact on genes transcription. Expression of LSE40cpxR was dected byreal-time quantitative RT-PCR （qRT-PCR）. The transcriptional level was significantlyaraised in strain under1mM H₂O₂treatment compared with untreated strain, whichindicated H₂O₂could probably activate the expression of cpxR. We further studied theimpact on the expression of the putative CpxR-regulated genes, the functions ofwhich range from antioxidation, protein folding to cell division processes.Transcriptional levels were observed significantly different in deletion mutants andreturned to wild type levels in complementary strain, which indicated the regulationof CpxR to these genes.更多还原