Zhou Ze-jun, Sun Bo-guang, Sun Li
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
Vet Microbiol. 2015 Jun 12;177(3-4):332-40. doi: 10.1016/j.vetmic.2015.03.030. Epub 2015 Apr 13.
Edwardsiella tarda is a severe bacterial pathogen to a wide arrange of farmed fish. One salient virulent feature of E. tarda is a remarkable ability to survive in host serum. In this study, in order to identify E. tarda proteins involved in serum resistance, we conducted proteomic analysis to examine the extracellular protein profiles of TX01, a pathogenic E. tarda isolate, in response to serum treatment. Five differentially expressed proteins were identified, one of which was a putative zinc protease (named Sip1). Western blot confirmed extracellular production of Sip1 by E. tarda. Sequence analysis revealed that Sip1 possesses a conserved zinc metalloprotease motif and shares low homology with the putative zinc proteases/aureolysin of several bacterial species. Purified recombinant Sip1 (rSip1) exhibited zinc-dependent proteolytic activity that reached maximum at 40°C and pH 8. Compared to the wild type, the sip1 knockout mutant, TXΔsip1, was dramatically reduced in the ability to cause mortality in the host (Japanese flounder) and to survive in host serum. These lost virulence capacities of TXΔsip1 were restored by complementation with the sip1 gene. Further study showed that rSip1 enhanced the serum resistance of TX01 and TXΔsip1, whereas antibody blocking of the Sip1 produced naturally by TX01 impaired serum resistance. Vaccination study showed that rSip1 as a subunit vaccine was able to induce effective protection in flounder against E. tarda challenge. Taken together, these results indicate that Sip1 is a novel zinc metalloprotease that is essential to serum resistance and host infection.
迟缓爱德华氏菌是多种养殖鱼类的严重细菌病原体。迟缓爱德华氏菌的一个显著毒力特征是在宿主血清中具有很强的存活能力。在本研究中，为了鉴定与血清抗性相关的迟缓爱德华氏菌蛋白，我们进行了蛋白质组学分析，以检测致病性迟缓爱德华氏菌分离株TX01在血清处理后的细胞外蛋白谱。鉴定出了5种差异表达蛋白，其中一种是假定的锌蛋白酶（命名为Sip1）。蛋白质免疫印迹证实迟缓爱德华氏菌可在细胞外产生Sip1。序列分析表明，Sip1具有保守的锌金属蛋白酶基序，与几种细菌的假定锌蛋白酶/金葡菌溶素的同源性较低。纯化的重组Sip1（rSip1）表现出锌依赖性蛋白水解活性，在40°C和pH 8时达到最大值。与野生型相比，sip1基因敲除突变体TXΔsip1在宿主（牙鲆）中导致死亡的能力以及在宿主血清中存活的能力显著降低。通过sip1基因互补恢复了TXΔsip1这些丧失的毒力能力。进一步研究表明，rSip1增强了TX01和TXΔsip1的血清抗性，而对TX01天然产生的Sip1进行抗体阻断则损害了血清抗性。疫苗接种研究表明，rSip1作为亚单位疫苗能够在牙鲆中诱导对迟缓爱德华氏菌攻击的有效保护。综上所述，这些结果表明Sip1是一种新型锌金属蛋白酶，对血清抗性和宿主感染至关重要。
