The Role Of PrfA And SigB In The Biofilm Formation In Listeria Monocytogenes

The ability of the foodborne pathogen Listeria monocytogenes(LM), to develop biofilm in food-processing environment is a major concern for the food safety, because formation of biofilm facilitates bacteria to survive in the adverse environment and resist desiccation, UV light and treatment with antimicrobial and sanitizing agents. However, the molecular mechanism of biofilm formation in L. monocytogenes has not been fully understood.PrfA is a key transcriptional activator that positively regulates most of the known listerial virulence genes expression. SigB is a stress response factor in LM, which contribute it to live under the stress conditions. In order to explore the role of PrfA and SigB on Listeria biofilm development, we designed the followed experiments:1) we compared the ability of strains (EGD, EGDeΔprfA, EGDeΔsigB, EGDΔprfAΔsigB and LI) biofilm formation in BHI media.2) Then in order to research the factor PrfA, another two groups of the experiments followed. Group one we compared the abilities of biofilm formation in this study for LM wild type strains (EGD and EGDe) and their prfA deletion mutants (EGDΔprfA and EGDeΔprfA), nonpathogenic Listeria innocua, as well as the recombinant strains that can constitutively express PrfA in L.innocua (LI-pERL3-prfA*) and in EGDeΔprfA (EGDeAprfA-pERL3-prfA*). Group two we compared the wild type strain EGDe and its PrfA deletion mutant EGDeAprfA, as well as the recombinant strain that can constitutively express PrfA in EGDeΔprfA (EGDeΔprfA-pERL3-prfA*) in the medias BHI (Brain Heart Infusion), MM+Glu (Minimal Essential Medium added the PTS sugar glucose) and the MM+Gly (Minimal Essential Medium added the non-PTS sugar glycerol).3) In order to research the factor SigB, we compared the biofilm formation of strains EGD, EGDΔsigB and LI in the BHI and BHI added0.3%bile.Our results can be showed in four aspects.1) The wild types of LM had strong abilities to develop "a network of knitted chains" biofilm structures on polyvinyl chloride microtiter plates, while unstructured biofilm was observed in LI. Biofilm formation was reduced in LM mutants lacking PrfA and rescued in the strain with constitutive expression of PrfA. However, PrfA had no impact on LI biofilm formation.2) The wild type of LI had strong ability to develop"a network of knitted chains" biofilm structures on polyvinyl chloride microtiter plates, while biofilm formation was reduced in the mutant lacking PrfA no matter cultured in the BHI or MM media, suggesting that PrfA plays an important role in the LM biofilm formation. However, the ability of EGDeAprfA to form biofilm could be rescued by the highly expression of PrfA only in BHI, but not in MM supplemented with glucose (the PTS carbohydrate) or glycerol (the non-PTS carbohydrate), and the strain EGDeΔprfA-pERL3-prfA*had a lowest biofilm production among the strains tested when cultured in MM with glucose, indicating that different productions generated during the different carbohydrate catabolism might affect the activity of PrfA, which in turn has an impact on the ability of biofilm formation by strains cultured in different media;3) All strains had a stronger abilities to form biofilm in poor MM than in rich BHI, suggesting that the medium components affect bacterial biofilm development.4) The0.3%bile can promote the biofilm formation of LM, but not the sigB deletion mutant EGDeAsigB.Our results suggest that PrfA plays a significant role only in the LM biofilm formation but not in LI. However, this role may influenced by conditions of media, especially by the sugars in media. PrfA and SigB might indirectly regulate expression of certain genes involving in LM biofilm formation.