Targeting Pseudomonas biofilm proteases to control Pseudomonas-pathogen interactions

Pseudomonas are among the most widespread bacteria in the food industry, where they persist in the form of biofilms on surfaces and cause food contamination from there. These biofilms create new niches for other bacteria that are present in this environment but would not themselves survive stressful influences. The potential of Pseudomonas for food spoilage is determined by hydrolytic enzymes, of which proteases, characterized by great structural diversity and specificity, are responsible for spoilage of refrigerated, protein-rich foods. Despite the well-studied AprX protease causing spoilage of milk and dairy products, proteases involved in spoilage of meat remain unexplored at a fundamental level. Their role in the biofilm of Pseudomonas bacteria in interaction with pathogens has also not been investigated. The proposed project adds to the lack of basic knowledge on proteases of Pseudomonas isolated from meat. The aim of the project is to clarify the role of extracellular proteases in the Pseudomonas biofilm to improve the survival of pathogenic bacteria in meat. The project will determine the metabolic activity of Pseudomonas with emphasis on protease activity at the genome and phenotype level. This will also enable us to identify and characterize new proteases of Pseudomonas and fill the gap in understanding their role in meat spoilage. We will further determine the association between the protease activity of Pseudomonas and their biofilm formation, which will be determined based on cell viability, biofilm biomass, and metabolic activity. In vitro research will determine the type of interaction between the selected Pseudomonas strain and pathogenic bacteria (gram-negative Campylobacter jejuni, Salmonella enterica and Escherichia coli; gram-positive Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus). We will determine the role of simultaneous and pre-cultivation of Pseudomonas biofilm for effects on pathogens and the role of Pseudomonas protease activity for providing nutrients to pathogens. With the selected combination of Pseudomonas-pathogen, where there will be a positive interaction, we will determine the basic mechanism of Pseudomonas protease activity by transcriptome and proteome analysis, which will reveal the response of Pseudomonas in interaction with the pathogen. The integration of the obtained data will enable us to understand the mechanisms underlying the process of biofilm formation of Pseudomonas, which is modulated by extracellular proteases. Thus, we will highlight the fundamental mechanisms of action of Pseudomonas biofilm proteases not only for spoilage but also for human health risk due to interactions with pathogenic bacteria. The project is designed as a continuation of research conducted during the applicant’s doctoral studies (Pseudomonas bacteria causing spoilage) and the core research group’s research work (microbial interactions). The postdoctoral project applicant has the necessary knowledge and skills to carry out the project. With established work in the core research group and collaborative research network with domestic and international researchers, the project applicant will have access to all necessary equipment and support to successfully complete the project. The results obtained will be original by combining data at different levels - genomic, transcriptomic, proteomic and phenotypic. The results of the project will highlight new insights, making the results original and publishable in high impact scientific journals. The objectives of the project are aimed at acquiring new fundamental knowledge, which is crucial for finding new targets for the control of Pseudomonas.