Research On The Mechanisms Of Biofilm Formation Of Listeria Monocytogenes At Transcription Levels

In china, there is an old saying "Hunger breeds discontentment", which shows that food is the basic material prerequisites for the people’s right of existence. There is still a word "A closed mouth catches no flies". The quality of the food insecurity will affect the people’s health. Food safety concerns the mankind’s lives, survival and continuance, which is an important topic of human development. The foodborne disease caused by microbes is one of the most serious problems that influence the food safety. Many outbreaks of infectious diseases have been found to be associated with biofilm. It is well documented that biofilm has become a problem in food industries as it renders its inhabitants resistant to antimicrobial agents and cleaning.The foodborne pathogen Listeria monocytogenes has the ability to develop biofilm, therefore, the molecular mechanism of this bacterial biofilm formation is worth investigation in deeply. To adte, investigations on biofilm formatiom have mainly been focused the following aspects:Firstly, establishment of the standard methods to culture and analyze the biofilm formed by strains under different culture conditions including nutrition, temperature and the properties of materials; Secondly, comparison of biofilm-forming ability and morphological differences by different strains from diverse sources, such as ready-to-eat food products, freshwater products or clinical and so on as well as serotype or pathogenic lineage; Thirdly, construction of gene mutagenesis libraries involved in biofilm formation; Finally, studies of the roles of the major transcriptional activator PrfA and SigB in biofilm formation.It has been shown that PrfA promote Lm biofilm formation, but the molecular mechanism remains unknown. Previous data indicated that PrfA activity and nutrient conditions had a significant impact on the biofilm formation. According to comparative proteomic, we found that CcpA might play a role in L. monocytogenes biofilm foemation. In order to explore the relationship between PrfA and CcpA, we compared the relative expression of different strains(EGDe, EGDe△PrfA, EGDe△PrfApPrfA and EGDeAPrfApPrfA*) by qRT-PCR at transcriptional level. The results suggest that PrfA may negatively regulate CcpA.Futhermore, the whole genome microarray was used to identify differentially expressed genes associated with the putative interaction between biofilm formation and PrfA in L. monocytogenes in this study. Our results indicated that nutritional conditions had a significant influence on biofilm formation. The global intensity of the biofilm cells grown in BHI from six independent repeats was much lower, indicating that the physiological state and gene expression of L. monocytogenes EGDe within the mature biofilm (after48h of incubation) formed in the complex nutrient-rich BHI medium went through quiescent stage and most gene expressions were arrested. Therefore, comparison of differential gene expression between biofilm and planktonic cells could be performed solely with the nutrient-poor minimal essential medium (MEM) growth condition. Over21.95%(627out of2857) of the EGDe genes grown in MEM containing glucose were altered in their expression in biofilm compared to the planktonic phase. These genes were classified into different functional categories which cover most of the biochemical functions encountered in bacterial cells, indicating that L. monocytogenes biofilm formation is probably controlled by a complex regulation network involved in variable genes required for the different biological pathways. Further comparison of gene expression profiles of biofilms between L. monocytogenes EGDe and its PrfA deletion mutant revealed185genes associated with PrfA and biofilm formation. Except for10genes, transcription levels of175genes were completely opposite between△prA and wild type during the biofilm formation, i.e, up-regulated genes in△prfA were down-regulated in the wild-type strain, and vice versa, indicating that function loss of PrfA dramatically altered gene expression patterns in L. monocytogenes biofilm and resulted in reduced ability of the biofilm formation.To confirm results of the microarray analysis, transcription levels of eight genes and19genes with differential expression in L. monocytogenes EGDe biofilm and planktonic cells as well as in its PrfA deletion mutant were quantified using quantitative real time polymerase chain reaction (qRT-PCR) and reverse transcription PCR(RT-PCR), respectively. The results from qRT-PCR and RT-PCR were consistent with the microarray data.The genes identified in our whole genome microarray analysise that are associated with listerial biofilm formation and PrfA will be studied further. An in-depth understanding of pathogenetic mechanism of L. monocytogenes will provide the theoretical base for prevention and treatment of bacterial infections, and the establishment of cost-effective food production and preservation methods, as well as improvement of food hygiene and safety.