Title: Determination of the Mode of Action of New Broad Applicable Small Molecule Inhibitors of Salmonella Biofilm Formation
Authors: Robijns, Stijn ×
Roberfroid, Stefanie
De Pauw, B.
Uceda Santamaría, E.
Marchand, Arnaud
Chaltin, Patrick
De Keersmaecker, Bart
Vanderleyden, Jos
Steenackers, Hans #
Issue Date: Nov-2012
Conference: Gutday 2012 edition:14 location:Leuven, Belgium date:9 November 2012
Abstract: Salmonella, the second most important food pathogen worldwide, is able to form biofilms on various biotic and abiotic surfaces, both in- and outside a host. The prevention and/or eradication of these biofilms can be an effective way to limit the spread, and infections of Salmonella.

Therefore we screened a library of > 20,000 small molecules in search of Salmonella biofilm inhibitors, which are active at a temperature ranging from 16 °C to 37 °C, and hence have potential to be used both in- and outside the host. Additionally we aim to identify compounds that specifically inhibit biofilm formation, but do not influence the (planktonic) growth, in order to prevent or slow down the development of resistance to these compounds. Compounds with maximum biofilm inhibitory activity and minimal effect on planktonic growth were studied further, both with respect to prevention and eradication of Salmonella Typhimurium biofilms at different temperatures (16 °C, 25 °C, 30 °C and 37 °C). After delineation of an early “structure-activity relationship” 3 chemically diverse compound families were selected (Robijns et al., 2012; FEMS Immunol Med Microbiol 65: 390-394).

Of these compound families, the ‘mode of action’ is currently being determined. Therefore we constructed a library of so far 81 different GFP promoter fusions of important Salmonella biofilm genes, based on literature and in-house data. These include genes involved in biofilm regulation, matrix production, quorum sensing, metabolic genes and genes involved in motility and c-di-GMP synthesis and degradation. By measuring differences of fluorescence in time, we can quickly identify the effect of the compounds on specific biofilm-related processes. This is a quick, inexpensive and easy to use assay, and thus can be conducted for several compounds in different conditions (e.g. different temperatures, static or shaking conditions,...). In addition this system is very versatile since also analysis with e.g. fluorescence-microscopy or FACS (Fluorescence-activated cell sorting) can be conducted.

Results with one of the compounds tested indicate that i.a. the expression of csgD, which encodes for the master regulator of Salmonella (and E. coli) biofilm formation, is inhibited. This inhibition can be followed downstream the regulatory pathway of CsgD, as also directly regulated genes like adrA and csgB show a similar inhibition. Currently we are further investigating this mode of action to determine which factors inhibit the csgD expression and which regulators are involved. The observed effects will be confirmed via several other test systems like qRT-PCR, mutant analysis and/or phenotypical tests.

In the end, this will yield new, broadly applicable, (Salmonella) biofilm inhibitors, of which the full ‘Mode of Action’ is known. This can be used for further optimalisation of the compounds.
Description: Poster
Publication status: published
KU Leuven publication type: IMa
Appears in Collections:Centre of Microbial and Plant Genetics
Intellectual Property
Studiegebied Gezondheidszorg VIVES-Zuid
× corresponding author
# (joint) last author

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