| Listeria monocytogenes(Lm)is a foodborne pathogen,widely existing in nature.It can survive at low temperature and is easy to contaminate cold refrigerated food.It can cause listeriosis such as abortion,meningitis and septicemia in humans and animals through the intestinal,placental and blood-brain barriers of humans and animals,and has a high fatality rate of 20～30%.As the specific virulence factor of Lm,LLO is encoded by the hly gene,which destroy the phagosome membrane after Lm invades host cells and help Lm escape.As a highly specific and residue free antimicrobial agents,phages can effectively inhibit Lm contamination,However,the ratio of inhibition are difference among the Lm isolate,and the interaction between phages and Lm is still unclear.In addition,Lm has good environmental adaptability,and the biofilm formed by Lm is closely related to Lm contamination.In this study,we constructed the hly gene defectivestrain Lm NJ05-Δhly based on the wide-type NJ05,which isolated from food,by homologous recombination,and identified the biological characteristics of growth,motility,adhesion and invasion.The mechanism of virulence factor LLO affecting biofilm formation and phage sensitivity was analyzed.The results can be summarized as follows:The defective strain Lm NJ05-Δhly was successfully constructed by homologous recombination technique and its growth characteristics were analyzed.The growth performance of the defective strain Lm NJ05-Δhly was higher than the wild strain Lm NJ05 in the stable period.After hly gene deletion,the motility of Lm NJ05 increased and hemolytic disappeared.The adhesion rate and invasion rate of Lm NJ05-Δhly on RAW264.7 cells were decreased by 84.93%and 43.88%,respectively.Compared with Lm NJ05,Lm NJ05-Δhly didn’t affect mitochondrial membrane potential of RAW264.7,and the accumulation of mitochondrial ROS,suggested that hly deletion reduced mitochondrial damage caused by Lm.In order to explore the effect of LLO on the Lm biofilm,the formation ability of biofilm was determined by crystal violet staining.The results showed that the formation ability of biofilm decreased significantly after hly gene deletion,and the OD590nm decreased from 2.40 to 0.70.The number of Lm in biofilm was analyzed,and the colony form unitof Lm NJ05-Δhly in biofilm decreased by 1log CFU/mL.When 0.5%glucose was added,the biofllm formation capacity of Lm NJ05 was enhanced,and the biofilm formation capacity increased at beginning and decreased with the increase of glucose concentration.However,Lm NJ05-Δhly biofilm formation was negatively correlated with glucose concentration,and the maximum biofilm formation occurred at 8%glucose.The effect of Lm NJ05 interaction induced by hly gene was investigated by analyzing the efficiency of plaque formation,lysis activity,adsorption and one-step growth curve of phage vB-LmoM-NJ05.After the interaction between hly defective strain and phage vB-LmoM-NJ05,the plaque efficiency decreased by about 100 times.In vitro lysis analysis showed that phage vB-LmoM-NJ05 decreased the lysis effect of deletion strain Lm NJ05-Δhly.The adsorption characteristics showed that the adsorption capacity of Lm NJ05-Δhly(15 min)is delayed and weaker than that of Lm NJ05.The one-step growth curve showed that Lm NJ05-Δhly had no obvious outbreak period,and the average outbreak amount was about 0.122 PFU/bacteria,much lower than Lm NJ05(78.29 PFU/bacteria).When treated at 108 PFU/mL,the biofilm of Lm NJ05-Δhly was completely suppressed and cleared.At the same time,the phage vB-LmoM-NJ05 could alleviate the damage caused by Lm NJ05 to RAW264.7 macrophages:inhibit the decrease of mitochondrial membrane potential and ROS accumulation,and reduce the adhesion and invasion ability of Lm to cells.The wild-type strain Lm NJ05 and the defective strain Lm NJ05-Δhly were analyzed by Transcriptome.The differential expression of genes in Lm NJ05 and defective strain were compared.The results showed that 2218 differentially expressed genes were found after deletion of hly gene,including 1896 genes down-regulated and 322 genes up-regulated.GO functional enrichment and KEGG pathway enrichment were performed for 2218 differentially expressed genes.The results showed that GO enrichment was mainly manifested in DNA repair,cellular response to stress,cellular protein metabolic process and hydrolase activity.KEGG enrichment pathways mainly focus on peptidoglycan biosynthesis,purine metabolism,O-antigen nucleotide sugar biosynthesis and D-amino acid metabolism.After the deletion of hly gene,genes related to Lm phage sensitivity,pathogenicity,biofilm formation and motility were significantly down-regulated.These results indicated that LLO could significantly regulate Lm pathogenicity,phage sensitivity and biofilm formation.In conclusion,as a crucial virulence factor of Lm,LLO played an important role in regulating the pathogenicity,biofilm formation and phage sensitivity of Lm.Our study would provide an important target for the pathogenicity and control of Lm,and lay a theoretical basis for the future application of phage in food bio-control. |
