Huiyuan Jiang1, Minhui Cao, Xuesong Cao, Hongwei Gu, Ke Zeng. 1. State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China. janghuiyuannju@163.com
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 delta 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 delta sraB (sraB deletion), SE2472 delta 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 delta sraB was only about 61%-70% of that of SE2472; while SE2472 delta sraB-comp improve the survival rate of SE2472 delta sraB by 10%-33% . In the transferrin bactericidal experiment, the survival rate of SE2472 delta sraB was 38% of that of SE2472 at 8 h incubation, and 23% at 24 h incubation. SE2472 delta sraB-comp played an important role in improving the survival rate rescued the defect by 14% than SE2472 delta 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 delta sraB was 41% of that of SE2472 at 8 h incubation, and 27% at 24 h incubation, compared with SE2472 delta sraB, the expression of sraB of SE2472 delta 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.
OBJECTIVE:Salmonella species are important food-borne pathogens of human and animal. S. enterica serovar Enteritidis is the only serovar that routinely causes humaninfection 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 delta 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 delta sraB (sraB deletion), SE2472 delta 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 delta sraB was only about 61%-70% of that of SE2472; while SE2472 delta sraB-comp improve the survival rate of SE2472 delta sraB by 10%-33% . In the transferrin bactericidal experiment, the survival rate of SE2472 delta sraB was 38% of that of SE2472 at 8 h incubation, and 23% at 24 h incubation. SE2472 delta sraB-comp played an important role in improving the survival rate rescued the defect by 14% than SE2472 delta 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 delta sraB was 41% of that of SE2472 at 8 h incubation, and 27% at 24 h incubation, compared with SE2472 delta sraB, the expression of sraB of SE2472 delta 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.