Author:

Keywords:

biofilm, sRNAs, Csr, interference

Abstract:

Natural bacterial populations predominantly grow in biofilms. This is a structured community of cells, which are attached to a surface and/or to each other and are embedded in a self-produced polymeric matrix. Bacteria in a biofilm have an increased tolerance against antibiotics, disinfectants and components of the immune system. This makes it difficult to remove detrimental biofilms, for example on medical implants, surfaces in the food-processing industry or in industrial pipelines. Therefore, there is a need for compounds that prevent biofilm formation or have antibacterial activity against biofilm-associated bacterial infections or contaminations. Because of the increasing knowledge about the different processes that take place and the cellular components that are necessary when a biofilm is developing, target-based anti-biofilm molecules can be designed. In this way, studies already identified anti-biofilm molecules that specifically prevent attachment or destabilize the matrix. However, as biofilm formation is an energy-consuming and complex process that requires tight regulation, we speculate that interfering with the activity of important biofilm regulators, using the knowledge on the mode of action and the binding requirements of these molecules, might prevent or delay the formation of a mature biofilm as well. More specifically, the sRNA regulators of biofilm formation are of interest. sRNAs are small regulatory RNA molecules that regulate gene expression by affecting mRNA stability, translation efficiency or protein activity. Because these regulators are RNA molecules, it should be possible to interfere with their activity with complementary RNA sequences or RNA decoys. This hypothesis will be validated using the CsrA-based regulatory network. This regulatory network is involved in the regulation of biofilm formation and involves CsrA as the central global regulatory protein, of which the activity is regulated by the sRNAs CsrB, CsrC and McsA. Different complementary or decoy sequences that might interfere with CsrB or CsrA activity will be designed, expressed from a plasmid and tested for their biofilm inhibitory capacity after which the most promising biofilm inhibitory sequence will be made as a synthetic PNA-based analog. As this synthetic sequence can be added to the growth medium, it can be tested for its potential as a true biofilm inhibitor. Additionally, an alternative mechanism to control the stability of the sRNA MicA will be studied. This natural regulatory mechanism might be an inspiration for the development of other RNA sequences, besides complementary RNA sequences and decoys, that affect the activity of biofilm regulating sRNAs, broadening the future applicability of nucleic acid-based anti-biofilm molecules.