Effect Of LuxS Gene On Environmental Tolerance And Pathogenicity From Food-borne Staphylococcus Aureus

Staphylococcus aureus is a major pathogen for bacterial-caused food poisoning,which caused food poisoning has become an imporant factor for the problems associated with food safety.Staphylococcal enterotoxin A(SEA)is considered to be the most critical enterotoxin involved in food poisoning,and about 75% of food poisoning was caused by SEA.ST6,ST5 and ST1 are common strains in food contamination and food poisoning strains.Meanwhile,Staphylococcus aureus is also one of the main pathogens to produce biofilm,which will adhere to the surface of vessels and difficult to remove.It makes the possibility of the long-term colonization and transimission of germs and increases the risk of Staphylococcus aureus contamination in food.Recntly,Quorum sensing(QS),a type of bacterial cell-to-cell communication,has been a hot topic to study.Thereinto,more and more studies have revealed that AI-2 quorum sensing system exists in various bacteria and regulate a dicerse array of physiological and pathological acticities in bacteria.As the AI-2 synthase gene,the luxS gene plays very imporant roles in many bacteria in terms of biofilm formation and pooduction of virulence factors.For the reasons above,the aim of this study is to investigate the effects of luxS gene on biofilm formation and virulence from food-borne Staphylococcus aureus.In this study,We selected food-borne Staphylococcus aureus strains 124 and 265 isolated from the previous experiment in our own laboratory.These two strains can produce high yield production of SEA and belong to type of ST6,spa-t701 and type of ST7,spa-t091,respectively.In the follow-up experiment,we constructed luxS knockout mutants as well as the corresponding complementary strain through homologous recombination.And then we compared the differences in envirommental stress reponse and pathogencity among the wild-type strain,luxS mutant strain and complemented strain.Finally,our study confirmed the effects of luxS in food-borne Staphylococcus aureus and provided a theoretical basis for the prevention and control of food poisoning caused by Staphylococcus aureus.The key discoveries were showen as follows:(1)The homologous recombination vector pBT2-LuxS was transformed into Staphylococcus aureus strains 124 and 265 by electroporation.Subsequently,the lux S mutant was seleted by homologous recombination at 42 ? and confirmed by PCR.The successfully constru-ced luxS mutants were named ?124 and ?265,respectively.In addition,the corresponding complementary strains were successfully constructed using similar methods and named r124 and r265,respectively.(2)The luxS gene had no effevt on the growth of Staphylococcus aureus.There was no sharp defference in the growth curve among the wild-type strain,luxS mutant strain and complemented strain.However,we found that resistance to heat stress was highly strengthened and the acid tolerance was slightly increased after the luxS gene was knocked.But the osmotic tolerance did not change.(3)Biofilm formation was assessed using a 96-well microtiter plate.The results showed that the biofilm formation ability of strains was obviously increased after the luxS gene was knocked.It indicated that luxS gene inhibited biofilm formation.Furhermore,quantitative real-time PCR showed that expression of icaA and sarA genes in correlation to bilfilm synthesis were up-regulated.It suggested that lux S may regulate the transcription of icaA and sarA to affect biofilm formation.While the level of cidA transcription almost unchanged,suggeseting that cidA may have nothing to do with the process of biofilm formation by luxS.(4)There was no distinct change on the phemomenon of haemolysis observed on blood agar plates after the luxS gene was knocked.Furhermore,quantitative real-time PCR showed that expression of hla?hld and hlg in correlation to haemolysis synthesis were no variation.It indicated that luxS was not involved in the regulation of hemolysin.(5)The Staphylococcus aureus strains 124 and 265 possessed enterotoxin sea and see gene by our previously experimental identification.Furhermore,quantitative real-time PCR showed that expression of sea and see were no variation.It indicated that luxS was not involved in the regulation of SEA and SEE.