¿STUDY OF THE BEHAVIOR OF LACTOCOCCUS LACTIS AND LISTERIA MONOCYTOGENES IN COMPETITION FOR FOOD SAFETY IN DAIRY PRODUCTS¿

Microbial competition is a mechanism that occurs when two or more microbial species compete for ecological niches to support their survival and growth. Different factors can contribute to the outcome of microbial competition, such as molecules exchanged between the competing organisms for the regulation of cell densities and the initial spatial configuration of the microbe–microbe interaction. Specifically, production of compounds that kill or limit the growth of competing strains or species can promote niche monopolization [2]. The released compounds include secondary metabolite antibiotics, bacterial peptides or low-molecular-mass organic compounds. The same happens in food, and it could be possible to explore this 'molecular' communication to improve food safety. In that sense, it is very important to develop tools in the control of bacterial species for a better food safety. The present work focused on the study of microbial competition between Listeria monocytogenes (LM) and Lactococcus lactis (LAC) monitored through proteomics, peptidomics and metabolomics approach. We study the secretome of these two microorganisms (Listeria monocytogenes and Lactococcus lactis) alone and in co-culture. In particular, we studied by proteomic analysis the evaluation of proteins secreted by bacteria through one/two-dimensional electrophoresis coupled to mass spectrometry (MALDI TOF). Furthermore, in order to characterize each secretome, label free Shotgun analysis was conducted using nano UPLC-MS system. Furthermore, the secretome of these microorganisms has been studied through first an untargeted proteomics analysis in vitro, followed by validation directly in a system resuming cheese. The objective of the last part of the project has been the monitoring of bacterial competition between through a combination of microbial Imaging mass spectrometry and LC-MS/MS, in order to investigate the metabolic profile of each bacteria in the interacting microbial colonies. In according with obtained preliminary data (one-dimensional and two-dimensional electrophoresis), new data highlighted, during competition, the higher production by Listeria monocytogenes of moonlighting protein Enolase (C1KY94) and Glucose 6 Phosphate isomerase (Q71X61), of Septation ring formation regulator EzrA (B8DHE7), involved into cell replication in regulatory mechanisms of cell energetics or metabolism and the lower secretion Endopeptidase P60 (P21171), protein associated with the cell surface and involved in the process of invasion. In parallel, L. lactis produced higher amounts of Page 6 of 114 Secreted 45 kDa protein and switched from lantibiotic Nisin A production to Nisin Z production. In competition with LM, LAC strain investigated produce higher amounts of Secreted 45 kDa protein with peptidoglycan lytic activity and the selective secretion of Nisin Z probably to improve lantibiotic solubility in less acidic environment. Lastly, IMS analysis revealed several interesting compounds during interaction of microbial colonies. At least six compounds are uniquely expressed during the interaction between LM and LAC. Among these, we focused our attention on three compounds: Cyclo-(Leu-leu), Cyclo-(Phe-Tyr), Cyclo-(L-Phe-L-4-Hyp). These compounds are cyclic peptides, isolated by Lactobacilli, with a biological activity]. In particular, they play an important role in bacterial cell to cell communication. Probably, these peptides have a role in inducing of the transcription of gene coding for Nisine. These results could be useful to setup new molecular strategies in the control of bacterial species for a better food safety.