FabR regulates Salmonella biofilm formation via its direct target FabB

Background: Biofilm formation is an important survival strategy of Salmonella in all environments. By mutant screening, we showed a knock-out mutant of fabR, encoding a repressor of unsaturated fatty acid biosynthesis (UFA), to have impaired biofilm formation. In order to unravel how this regulator impinges on Salmonella biofilm formation, we aimed at elucidating the S. Typhimurium FabR regulon. Hereto, we applied a combinatorial high-throughput approach, combining ChIP-chip with transcriptomics. Results: All the previously identified E. coli FabR transcriptional target genes (fabA, fabB and yqfA) were shown to be direct S. Typhimurium FabR targets as well. As we found a fabB overexpressing strain to partly mimic the biofilm defect of the fabR mutant, the effect of FabR on biofilms can be attributed at least partly to FabB, which plays a key role in UFA biosynthesis. Additionally, ChIP-chip identified a number of novel direct FabR targets (the intergenic regions between hpaR/hpaG and ddg/ydfZ) and yet putative direct targets (i.a. genes involved in tRNA metabolism, ribosome synthesis and translation). Next to UFA biosynthesis, a number of these direct targets and other indirect targets identified by transcriptomics (e.g. ribosomal genes, ompA, ompC, ompX, osmB, osmC, sseI), could possibly contribute to the effect of FabR on biofilm formation. Conclusion: Overall, our results point at the importance of FabR and UFA biosynthesis in Salmonella biofilm formation and their role as potential targets for biofilm inhibitory strategies

Gene expression variability in clonal populations: causes and consequences

During the last decade it has been shown that among cell variation in gene expression plays an important role within clonal populations. Here, we provide an overview of the different mechanisms contributing to gene expression variability in clonal populations. These are ranging from inherent variations in the biochemical process of gene expression itself, such as intrinsic noise, extrinsic noise and bistability to individual responses to variations in the local micro-environment, a phenomenon called phenotypic plasticity. Also genotypic variations caused by clonal evolution and phase variation can contribute to gene expression variability. Consequently, gene expression studies need to take these fluctuations in expression into account. However, frequently used techniques for expression quantification, such as microarrays, RNA sequencing, quantitative PCR and gene reporter fusions classically determine the population average of gene expression. Here, we discuss how these techniques can be adapted towards single cell analysis by integration with single cell isolation, RNA amplification and microscopy. Alternatively more qualitative selection-based techniques, such as mutant screenings, in vivo expression technology (IVET) and recombination-based IVET (RIVET) can be applied for detection of genes expressed only within a subpopulation. Finally, differential fluorescence induction (DFI), a protocol specially designed for single cell expression is discussed.peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=imby20status: publishe

A GFP promoter fusion library for the study of Salmonella biofilm formation and the mode of action of biofilm inhibitors

status: publishe

New broad applicable small molecule inhibitors of Salmonella biofilmformation: identification, characterization and determination of their mode of action

A major difficulty in the prevention and treatment of Salmonella infections is the fact that Salmonella is able to form biofilms on various biotic and abiotic surfaces. Within these biofilms, Salmonella is protected against i.a.. antibiotics and the host immune system. As such, biofilm formation is an important survival strategy of Salmonella, both in- and outside the host. Therefore, the prevention and/or eradication of these biofilms could be an effective way to limit the spread, and prevent infections of Salmonella. To identify new Salmonella biofilm inhibitors we conducted a high-throughput screening 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 therefore have potential to be used both in- and outside the host. Additionally we aim to identify compounds that specifically inhibit the biofilm formation, but do not kill the bacteria in order to prevent or slow down the development of resistance to these compounds. We identified 144 (0.72 %) possible biofilm inhibitors. Subsequently, the dose-response relationship of these ‘hits’ was determined, as well as the effect of the compounds on the planktonic growth of Salmonella, using a ‘bioscreen’ (Labsystems). The compounds with maximum biofilm inhibitory activity and minimal effect on planktonic growth were studied further, both with respect to prevention and eradication of biofilms from Salmonella Typhimurium and Pseudomonas aeruginosa, at different temperatures (16 °C, 25 °C, 30 °C and 37 °C). Using these results we identified 11 compound classes, from which analogues were purchased (± 20 analogues/family resulting in a library of 227 analogues). Using these analogues an early “structure-activity relationship” was delineated, to select and optimize the most potent compounds. Of the most interesting compounds the ‘mode of action’ will be determined using amongst others reporter gene fusions. Therefore we created a library of 81 GFP promoter fusions of important Salmonella biofilm genes, selected out of literature and in-house tests (Steenackers and Hermans et al., in press). This way we can quickly (semi-high throughput) identify the effect of the compounds on specific biofilm-related processes. One of the advantages of using reporter-fusions is that it is cheap, fast and easy to use and thus can be conducted for several compounds. This will yield valuable knowledge about the ‘mode of action’ of our compounds, and will also be used to select a compound class for more extensive studies using microarray analyses and mutant libraries. Reference: Steenackers, H.P.L.#, Hermans, K.#, Vanderleyden, J., De Keersmaecker, S.C.J. 2011. Salmonella Biofilms: an Overview on Occurrence, Structure, Regulation and Eradication. Food Res Int. In press, doi:10.1016/j.foodres.2011.01.038. # Equal contribution.Posterstatus: publishe

Determination of the mode of action of new broad applicable small molecule inhibitors of salmonella biofilm formation

Salmonella is able to form biofilms on many surfaces. Within these biofilms Salmonella is protected against many stress factors and therefore is an important survival strategy of Salmonella. Therefore, the prevention and eradication of biofilms can be an effective way to limit the spread and prevent infections of Salmonella. Initially 144 possible biofilm inhibitors were identified and after further characterization and early structure-activity relationship studies, 3 chemically distinct compound families were selected. Of those the ‘mode of action’ will be determined using a newly created library of 81 reporter biofilm-gene fusions. This way we can quickly identify the effect of the compounds on specific biofilm-related processes and determine their precise ‘mode of action’.Poster Winner P&G Innovation Award 2012status: accepte

Identification, characterization and determination of the mode of action of new broad applicable small molecule inhibitors of Salmonella biofilm formation

A major difficulty in the prevention and treatment of Salmonella infections is the fact that Salmonella is able to form biofilms on various biotic and abiotic surfaces. Within these biofilms, Salmonella is protected against i.a. UV-radiation, desiccation, antibiotics, disinfectants and the host immune system. As such, biofilm formation is an important survival strategy of Salmonella, both in- and outside the host. Therefore, the prevention and/or eradication of these biofilms could be an effective way to limit the spread, and prevent infections of Salmonella. To identify new Salmonella biofilm inhibitors a high-throughput screening of > 20,000 small molecules was conducted, in search of Salmonella biofilm inhibitors which are active at a temperature ranging from 16 °C to 37 °C, and therefore have potential to be used both in- and outside the host. Additionally we aim to identify compounds that specifically inhibit the biofilm formation, but do not kill the bacteria in order to prevent or slow down the development of resistance to these compounds. In total 144 (0.72 %) possible biofilm inhibitors were identified. Subsequently, the dose-response relationship of these ‘hits’ was determined, as well as the effect of the compounds on the planktonic growth of Salmonella, using a ‘bioscreen’ (Labsystems). The compounds with maximum biofilm inhibitory activity and minimal effect on planktonic growth were studied further, both with respect to prevention and eradication of biofilms from Salmonella Typhimurium and Pseudomonas aeruginosa, at different temperatures (16 °C, 25 °C, 30 °C and 37 °C). Using these results we identified 11 compound classes, from which analogues were purchased (± 20 analogues/family resulting in a library of 227 analogues). Using these analogues an early “structure-activity relationship” was delineated, to select and optimize the most potent compounds. This way 3 chemically diverse compound families were selected (Robijns et al., 2012). Of these compound families the ‘mode of action’ is currently being determined using amongst others reporter gene fusions. Therefore we created a library of 81 GFP promoter fusions of important Salmonella biofilm genes, selected out of literature and in-house knowledge (Steenackers et al., 2012). By following the difference in fluorescence, i.e. gene-expression, with and without compound present, we can quickly (semi-high throughput) identify the effect of the compounds on specific biofilm-related processes. One of the advantages of using reporter-fusions is that it is cheap, fast and easy to use and thus can be conducted for several compounds. This system is also very versatile and can be used in different set-ups like fluorescence-microscopy or FACS (Fluorescence-activated cell sorting)-analysis. This will yield valuable knowledge about the ‘mode of action’ of our compounds, and will also be used to select a compound class for more extensive studies using microarray analyses and mutant libraries. Preliminary results show that using this system it is indeed possible to follow the differences in gene expression throughout the biofilm regulatory pathways. Since both up- and down regulation of genes could be observed following described pathways. By analyzing the differences in expression both up and down stream of the biofilm genes, the direct target and full ‘Mode of Action’ of the Biofilm inhibitors can quickly be determined. 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 deliver us new, broadly applicable, (Salmonella) biofilm inhibitors, of which the full ‘Mode of Action’ is known, which will strengthen the IP position of the inhibitors and can be used for further optimalisation of the compounds. Robijns, S. C., De Pauw, B., Loosen, B., Marchand, A., Chaltin, P., De Keersmaecker, S. C., Vanderleyden, J. en Steenackers, H. P. (2012). Identification And Characterization Of 4-[4-(3-Phenyl-2-propen-1-yl)-1-Piperazinyl]-5H-Pyrimido[5,4-B]Indole Derivatives As Salmonella Biofilm Inhibitors. FEMS Immunol Med Microbiol, Steenackers, H., Hermans, K., Vanderleyden, J. en De Keersmaecker, S. C. J. (2012). Salmonella biofilms: An overview on occurrence, structure, regulation and eradication. Food Research International, 45:502-531Posterstatus: publishe

New broad applicable small molecule inhibitors of salmonella biofilm formation: Identification, characterization and determination of their mode of action

A major difficulty in the prevention and treatment of Salmonella infections is the fact that Salmonella is able to form biofilms on various biotic and abiotic surfaces. Within these biofilms, Salmonella is protected against i.a.. antibiotics and the host immune system. As such, biofilm formation is an important survival strategy of Salmonella, both in- and outside the host. Therefore, the prevention and/or eradication of these biofilms could be an effective way to limit the spread of Salmonella. To identify new Salmonella biofilm inhibitors we conducted a high-throughput screening 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 therefore have potential to be used both in- and outside the host. Additionally we aim to identify compounds that inhibit biofilm formation, but do not kill the bacteria in order to prevent or slow down the development of resistance to these compounds. We identified 144 (0.72 %) possible biofilm inhibitors. Subsequently, the dose-response relationship of these ‘hits’ was determined, as well as the effect of the compounds on the planktonic growth of Salmonella, using a ‘bioscreen’ (Labsystems). The compounds with maximum biofilm inhibitory activity and minimal effect on planktonic growth were studied further, both with respect to prevention and destruction of biofilms from Salmonella Typhimurium and Pseudomonas aeruginosa, at different temperatures (16 °C, 25 °C, 30 °C and 37 °C). Using these results we identified 11 compound classes, from which analogues were purchased (± 20 analogues/family resulting in a library of 227 analogues). Using these analogues an early “structure-activity relationship” was determined, to select and optimize the most potent compounds. Of the most interesting compounds the ‘mode of action’ will be determined using, a.o. reporter gene fusions. Therefore we created a library of 78 GFP promoter fusions of important Salmonella biofilm genes, selected out of literature and in-house tests (Steenackers and Hermans et al., in press). This way we can quickly (semi-high throughput) identify the effect of the compounds on specific biofilm-related processes. One of the advantages of using reporter-fusions is that it is cheap, fast and easy to use and thus can be conducted for several compounds. This will yield valuable knowledge about the ‘mode of action’ of our compounds, and will also be used to select a compound class for more extensive studies using microarray analyses and mutant libraries. Steenackers, H.P.L.#, Hermans, K.#, Vanderleyden, J., De Keersmaecker, S.C.J. 2011. Salmonella Biofilms: an Overview on Occurrence, Structure, Regulation and Eradication. Food Res Int. In press, doi:10.1016/j.foodres.2011.01.038. # Equal contribution.Posterstatus: publishe

A GFP promoter fusion library for the mode of action study of biofilm inhibitors and the identification of anti-biofilm targets

To study the gene expression and regulation in Salmonella biofilms, we constructed a library of 81 different GFP-promoter fusions of important S. Typhimurium 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. This library is currently being used to study the mode of action of biofilm inhibitors and to identify anti-biofilm targets. For the mode of action studies, the effect of Salmonella biofilm inhibitors on the GFP expression is measured in time. As such we can quickly identify the effect of the compounds on specific biofilm-related processes. This is a fast, inexpensive and easy to use assay, and thus can be conducted for several compounds in different conditions. Results with one of our biofilm inhibitors indicate that this compound inhibits the expression of csgD, which encodes for the master regulator of Salmonella biofilms. This inhibition can be followed downstream the regulatory pathway of CsgD, as also directly regulated genes like adrA and csgB show a similar inhibition. Since in situ biofilms can exist of more than one microbial species, it is essential for the future identification of new inhibitors to identify anti-biofilm targets which are of importance both in monospecies and multispecies biofilms. Therefore we compared by FACS analysis the expression of the promoter fusions in monospecies Salmonella biofilms and multispecies biofilms containing Salmonella. Some genes showed a high and similar expression in monospecies and multispecies biofilms (without being expressed in free-living cultures) and therefore form good targets for new anti-biofilm strategies. On the other hand we also found unique expression patterns of Salmonella in multispecies versus monospecies biofilms, indicating that the increased complexity and higher heterogeneity in multispecies biofilms influences gene expression of Salmonella.Presentationstatus: publishe