非编码小RNA（sraB）调控肠炎型沙门菌的抗蛋清抑菌作用

sRNA (sraB) regulate the resistant ability of Salmonella enterica serovar Enteritidis to egg albumen

摘要:

摘要: 【目的】肠炎型沙门菌是一种食源性人畜共患病病原菌，可在禽蛋中存活并传播。sRNA为新近发现的基因表达调控分子，本实验以sRNA（sraB）为对象，探索sRNA与肠炎型沙门菌在禽蛋中存活的相关性，及研究其在细菌抵抗蛋清抑菌作用中的调控功能。【方法】参考已报道的沙门菌全基因组及sraB序列，设计并扩增sraB突变用基因片段，运用Red重组系统（red recombination system）对肠炎沙门菌野生株（SE2472）sraB基因进行定点敲除，构建出sraB敲除株（SE2472△sraB）。分析比较sraB敲除对沙门菌在蛋清中存活的影响；另构建表达sraB的回复表达质粒pHDB3-sraB，将其转入sraB敲除株构建回复株SE2472△sraB-comp，以回复表达sraB，分析sraB表达对沙门菌敲除株的回复作用；并分别以野生株、敲除株及回复株研究sraB在抵抗蛋清中几种抑菌因子（如溶菌酶和卵转铁蛋白）作用中可能的调控作用。【结果】敲除株在蛋清中存活率为野生型的61%－70%，回复株相比野生型的存活率比敲除株提高10%－33%；在转铁蛋白抑菌实验中，孵育8 h和24 h，敲除株的存活率分别为野生型存活率的 38%和23%，孵育8 h回复株相比野生型的存活率比敲除株提高15%，但孵育24 h回复株的存活率未见提高；在溶菌酶抑菌实验中，孵育8 h和24 h后，敲除株存活率分别为野生型的41%和27%，回复株相比野生型的存活率分别比敲除株提高35%和23%。【结论】通过比较sraB敲除与否，研究肠炎型沙门菌在禽蛋中的存活及对抑菌因子的抵抗作用，结果表明sraB在肠炎沙门菌抵抗蛋清抑菌作用中起着重要调控功能。

Abstract:

Abstract: [Objective] Salmonella species are important food-borne pathogens of human and animal. S. enterica serovar Enteritidis is the only serovar that routinely causes human infection through intact egg, the molecular basis of its ability to survive in egg is poorly understood. The importance of post-transcriptional regulation by small non-coding RNAs ( sRNA ) has recently been recognized. The sRNAs play diverse physiological roles in stress responses, regulation of metabolism, control of bacterial envelope composition and bacterial virulence. In this study, we studied regulatory function of salmonella sRNA sraB associated with survival ability of S. enterica serovar Enteritidis in egg albumen. [Methods] To study the contribution of sraB to the survival ability of S. Enteritidis in egg albumen, we constructed sraB deletion strain (SE2472△sraB) with wild type S. Enteritidis SE2472, using red recombination system. For complementation of sraB, complete fragment sraB was amplified from SE2472 and inserted into plasmid pHDB3 to overexpress sraB. We carried out the egg albumen bactericidal experiment with strains of SE2472, SE2472△sraB(sraB deletion), SE2472△sraB-comp(sraB complement) and control. To explore the regulatory role of sraB, we assayed the bactericidal activity of the two important antimicrobial components of egg albumen: lysozyme and transferrin. [Results] In the egg albumen bactericidal experiment, the survival rate of SE2472△sraB was only about 61%－70% of that of SE2472; while SE2472△sraB-comp improve the survival rate of SE2472△sraB by 10%－33% . In the tranferrin bactericidal experiment, the survival rate of SE2472△sraB was 38% of that of SE2472 at 8 h incubation, and 23% at 24 h incubation. SE2472△sraB-comp played an important role in improving the survival rate rescued the defect by 14% than SE2472△sraB at 8 h of incubation, but failed to rescue the defect at 24 h incubation. In the Lysozyme experiment, the survival rate of SE2472△sraB was 41% of that of SE2472 at 8 h incubation, and 27% at 24 h incubation, compared with SE2472△sraB, the expression of sraB of SE2472△sraB-comp has improved the survival rate by 35% after 8 h of incubation and 23% after 24 h of incubation. [Conclusion] Based on our results, we conclude that small RNA (sraB) plays important role during the survival of S. Enteritidis in egg albumen, and may contribute regulatory role in response to antimicrobial components of egg albumen such as lysozyme and transferrin.