The Effect Of Carbon Source And PrfA On The Biofilm Formation Of Listeria Monocytogenes

The foodborne pathogen Listeria monocytogenes that becomes a major concern for the food safety has the ability to develop biofilm in food-processing environment. PrfA is one of the key transcription activator, which regulates most listerial virulence gene expression. In this thesis, in order to investgate the effect of different carbon sources on the PrfA activity and biofilm formation in L. monocytogenes, the abilities of different strains (EGDe, EGDeApr/A, EGDeΔprfA+pPrfA, EGDe+pPrfA, EGDeAprfA+pPrfA*and EGDe+pPrfA*) to form biofilm in the media of brain heart infusion (BHI), the minimal essential medium (MEM) with glucose, cellobiose or glycerol are compared. Moreover, to gain better insight into the conserved protein expression profile in biofilms formed by L. monocytogenes in rich BHI media and poor MEM, the total proteins from biofilm-and planktonic-grown cells from MEM with50mmol/L glucose and BHI were compared using2-dimensional polyacrlamide gel electrophoresis followed by MALDI-TOF/TOF analysis.The experimental results show that:1) L. monocytogenes can form stronger biofilm in poor MEM than in rich BHI;2) Loss of prfA gene reduced the biofilm forming ability of L. monocytogenes in media with different carbon sources, which shows that PrfA can promote the formation of L. monocytogenes biofilm. However, the role of PrfA on L. monocytogenes biofilm might be affected by the types of carbon sources, the amount of PrfA protein and its activate state;3) The biofilm-forming ability of EGDsAprfA can be resumed in rich BHI medium but not not in poor MEM medium by high expression of PrfA protein in L. monocytogenes. Moreover, the overexpression of PrfA*could not resume the ability of biofilm formation of EGDeΔprfA strain, but even greatly reduce the biofilm formation of these strains;4) Using2-dimensional polyacrylamide gel electrophoresis followed by MALDI-TOF/TOF analysis, forty seven proteins were successfully identified to be either up (19proteins) or down (28proteins) regulated in the biofilm states. Most (30proteins) of them were assigned to the metabolism functional category in COG (Cluster of Orthologous Groups of proteins). Among them, up-regulated proteins were mainly associated with the pentose phosphate pathway and glycolysis, whereas a key enzyme CitC involved in tricarboxylic acid cycle was down-regulated in biofilms compared to the planktonic states.The above results indicate the importance of carbon catabolite control for L. monocytogenes biofilm formation in response to nutrient availability. But we should do the further experiments to elucidate the molecular basis of the carbon metabolism and PrfA activity and the biofilm formation in Listeria monocytogenes.