DISTILLER: a data integration framework to reveal condition dependency of complex regulons in Escherichia coli

DISTILLER, a data integration framework for the inference of transcriptional module networks, is presented and used to investigate the condition dependency and modularity in Escherichia coli networks

Rational design of small molecules that modulate the transcriptional function of the response regulator PhoP

The response regulator PhoP, which is part of the PhoP/PhoQ two-component system, regulates the expression of multiple genes involved in controlling virulence in Salmonella enterica serovar Typhimurium and other species of Gram-negative bacteria. Modulating the phosphorylation-mediated dimerization in the receiver domain may interfere with the transcriptional function of PhoP. In this study, we analyzed the therapeutic potential of the PhoP receiver domain by exploring it as a potential target for drug design. The structural information was then applied to identify the first hit compounds from commercial chemical libraries by combining pharmacophore modelling and docking methods with a GFP (Green Fluorescent Protein)-based promoter-fusion bioassay. In total, one hundred and forty compounds were selected, purchased, and tested for biological activity. Several novel scaffolds showed acceptable potency to modulate the transcriptional function of PhoP, either by enhancing or inhibiting the expression of PhoP-dependent genes. These compounds may be used as the starting point for developing modulators that target the protein-protein interface of the PhoP protein as an alternative strategy against antibiotic resistance.status: publishe

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

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Impedimetric fingerprinting and structural analysis of isogenic E. coli biofilms using multielectrode arrays

© 2018 Elsevier B.V. Microbial biofilm contamination is an ubiquitous and persistent problem in industry and clinics. The structure of the biofilm, its extracellular matrix and its formation process are very complex. At present, there are only limited options to investigate biofilms outside the lab, as most in situ techniques lack sensitivity and resolution. Impedance-based sensors provide a fast, label-free and sensitive manner to characterize biofilms, although mainly large electrodes have been used so far. Here, we used 60 μm-sized electrode arrays (MEAs) to characterize the structure of biofilms formed by wild type (WT) Escherichia coli TG1 and the isogenic ΔcsgD, ΔcsgB and ΔbcsA mutants. At 24 h of growth, the interfacial resistance at 2 Hz increased by 3.4% and 0.3% for the curli producing strains (WT and ΔbcsA), yet it decreased by 5.7% and 4% for the curli non-producing strains (ΔcsgD and ΔcsgB). The imaginary impedance at 2 Hz decreased for all the strains by 7.2%, 6.9%, 5.1% and 2.5% (WT, ΔbcsA, ΔcsgB and ΔcsgD, respectively). Interestingly, the spatial variation of impedance within each biofilm, resulting from physiological and structural heterogeneity, was significantly different for each biofilm and most pronounced in the WT. Depending on the strain, the biofilm attachment phase lasted between 6 and 10 h, and was characterized by an increase in the interfacial resistance of up to 6% for the WT, 5.5% for ΔcsgD, 3.5% for ΔcsgB and 5% for ΔbcsA, as opposed to the decrease in medium resistance observed during the maturation phase. Overall, impedance-based MEA assays proved effective to differentiate between biofilms with varying structure, detect spatial diversity and explain biofilm life-cycle in terms of attachment and maturation.status: 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

Structure-activity relationship of brominated 3-alkyl-5-methylene-2(5H)-furanones and alkylmaleic anhydrides as inhibitors of Salmonella biofilm formation and quorum sensing regulated bioluminescence in Vibrio harveyi

A library of 25 1'-unsubstituted and 1'-bromo or 1'-acetoxy 3-alkyl-5-methylene-2(5H)-furanones and two 3-alkylmaleic anhydrides was synthesized using existing and new methods. This library was tested for the antagonistic effect against the biofilm formation by Salmonella Typhimurium and the quorum sensing regulated bioluminescence of Vibrio harveyi. The length of the 3-alkyl chain and the bromination pattern of the ring structure were found to have a major effect on the biological activity of the 1'-unsubstituted furanones. Remarkably, the introduction of a bromine atom on the 1' position of the 3-alkyl chain did drastically enhance the activity of the furanones in both biological test systems. The introduction of an acetoxy function in this position did in general not improve the activity. Finally, the potential of the (bromo)alkylmaleic anhydrides as a new and chemically easily accessible class of biofilm and quorum sensing inhibitors was demonstrated.status: publishe

Structure-Activity Relationship of 4(5)-Aryl-2-amino-1H-imidazoles, N1-Substituted 2-Aminoimidazoles and Imidazo[1,2-a]pyrimidinium Salts as Inhibitors of Biofilm Formation by Salmonella Typhimurium and Pseudomonas aeruginosa

A library of 112 4(5)-aryl-2-amino-1H-imidazoles, 4,5-diphenyl-2-amino-1H-imidazoles, and N1-substituted 4(5)-phenyl-2-aminoimidazoles was synthesized and tested for the antagonistic effect against biofilm formation by Salmonella Typhimurium and Pseudomonas aeruginosa. The substitution pattern of the 4(5)phenyl group and the nature of the N1-substituent were found to have a major effect on the biofilm inhibitory activity. The most active compounds of this series were shown to inhibit the biofilm formation at low micromolar concentrations. Furthermore, the influence of 6 imidazo[1,2-a]pyrimidines and 18 imidazo[1,2-a]pyrimidinium salts on the biofilm formation was tested. These compounds are the chemical precursors of the 2-aminoimidazoles in our synthesis pathway. A good correlation was found between the activity of the imidazo[1,2-a]pyrimidinium salts and their corresponding 2-aminoimidazoles, supporting the hypothesis that the imidazo[1,2-a]pyrimidinium salts are possibly cleaved by cellular nucleophiles to form the active 2-aminoimidazoles. However, the imidazo[1,2-a]pyrimidines did not show any biofilm inhibitory activity, indicating that these molecules are not susceptible to in situ degradation to 2-aminoimidazoles. Finally, we demonstrated the lack of biofilm inhibitory activity of an array of 37 2N-substituted 2-aminopyrimidines, which are the chemical precursors of the imidazo[1,2-a]pyrimidinium salts in our synthesis pathway.status: publishe

Structure-activity relationship of 2-hydroxy-2-aryl-2,3-dihydro-imidazo[1,2-a]pyrimidinium salts and 2N-substituted 4(5)-aryl-2-amino-1H-imidazoles as inhibitors of biofilm formation by Salmonella Typhimurium and Pseudomonas aeruginosa

A library of 80 1-substituted 2-hydroxy-2-aryl-2,3-dihydro-imidazo[1,2-a]pyrimidinium salts and 54 2N-substituted 4(5)-aryl-2-amino-1H-imidazoles was synthesized and tested for the antagonistic effect against biofilm formation by Salmonella Typhimurium and Pseudomonas aeruginosa. The nature of the substituent at the 1-position of the salts was found to have a major effect on their biofilm inhibitory activity. Salts with an intermediate length n-alkyl or cyclo-alkyl chain (C7-C10) substituted at the 1-position in general prevented the biofilm formation of both species at low micromolar concentrations, while salts with a shorter n-alkyl or cyclo-alkyl chain (C1-C5) or longer n-alkyl chain (C11-C14) were much less potent. Salts with a long cyclo-alkyl chain however were found to be strong biofilm inhibitors. Furthermore, we demonstrated the biofilm inhibitory potential of salts with certain aromatic substituents at the 1-position, such as piperonyl or 3-methoxyphenetyl. The activity of the 2-aminomidazoles was found to be dependent on the nature of the 2N-substituent. Compounds with a n-butyl, iso-butyl, n-pentyl, cyclopentyl or n-hexyl chain at the 2N-position have an improved activity as compared to their unsubstituted counterparts, whereas compounds with shorter 2N-alkyl chains do have a reduced activity and compounds with longer 2N-alkyl chains do have an effect that is dependent on the nature of the substitution pattern of the 4(5)-phenyl ring. Finally, we demonstrated that introduction of a 3-methoxyphenethyl or piperonyl group at the 2N-position of the imidazoles could also result in an enhanced biofilm inhibition. (C) 2011 Elsevier Ltd. All rights reserved.status: publishe

Microwave-assisted one-pot synthesis and anti-biofilm activity of 2-amino-1H-imidazole/triazole conjugates

A microwave-assisted protocol was developed for the construction of 2-amino-1H-imidazole/triazole conjugates starting from the previously described 2-hydroxy-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium salts. The process involves a one-pot hydrazinolysis/Dimroth-rearrangement of these salts followed by a ligand-free copper nanoparticle-catalyzed azide-alkyne Huisgen cycloaddition. The 2-amino-1H-imidazole/triazole conjugates showed moderate to high preventive activity against biofilms of S. Typhimurium, E. coli, P. aeruginosa and S. aureus. The most active compounds had BIC50 values between 1.3 and 8 μM. A remarkable finding was that introduction of the triazole moiety into the side chain of 2-aminoimidazoles with a long (C8-C13) 2N-alkyl chain did drastically improve their activity. Conclusively, the 2-amino-1H-imidazole/triazole scaffold provides a lead structure for further design and development of novel biofilm inhibitors.crosscheck: This document is CrossCheck deposited related_data: Supplementary Information copyright_licence: The Royal Society of Chemistry has an exclusive publication licence for this journal copyright_licence: The accepted version of this article will be made freely available after a 12 month embargo period history: Received 15 November 2013; Accepted 18 March 2014; Accepted Manuscript published 18 March 2014; Advance Article published 24 April 2014; Version of Record published 14 May 2014status: publishe