[show abstract][hide abstract] ABSTRACT: A screening of a small-molecule library was conducted, in search of Salmonella biofilm inhibitors active in a broad temperature range, both in prevention and in eradication of biofilms. Moreover, the inhibitors were selected not to influence the planktonic growth of Salmonella to diminish the selective pressure and to prevent or slow down resistance development. Out of the 20,014 compounds screened at 16 and 37 °C, 140 hits were identified. After characterization of the most promising hits at a broader set of temperatures (16, 25, 30 and 37 °C), we identified 7-methoxy-4-[4-(3-phenyl-2-propen-1-yl)-1-piperazinyl]-5H-pyrimido[5,4-b]indole as an interesting preventive anti-biofilm compound. A first structure-activity relationship of this compound was delineated, revealing 8-fluoro-4-[4-(3-phenyl-2-propen-1-yl)-1-piperazinyl]-5H-pyrimido[5,4-b]indole as a promising analogue in the prevention of Salmonella biofilms.
[show abstract][hide abstract] ABSTRACT: The ability of Salmonella to form complex surface-associated communities, called biofilms, contributes to its resistance and persistence in both host and non-host environments and is especially important in food processing environments. In this review, the different types of abiotic (plastic, glass, cement, rubber, and stainless steel) and biotic surfaces (plant surfaces, epithelial cells, and gallstones) on which Salmonella biofilms have been described are discussed, as well as a number of commonly used laboratory setups to study Salmonella biofilm formation (rdar morphotype, pellicle formation, and biofilms on polystyrene pegs). Furthermore, the structural components important during Salmonella biofilm formation are described (curli and other fimbriae, BapA, flagella, cellulose, colanic acid, anionic O-antigen capsule and fatty acids), with special attention to the structural variations of biofilms grown on different surfaces and under different conditions. Indeed, biofilm formation is strongly influenced by different environmental signals, via a complex regulatory network. An extensive overview is given on the current understanding of this genetic network and the interactions between its different components (CsgD, RpoS, Crl, OmpR, IHF, H-NS, CpxR, MlrA, c-di-GMP, BarA/SirA, Csr, PhoPQ, RstA, Rcs, metabolic processes and quorum sensing). To further illustrate that biofilm formation is a mechanism of Salmonella to adapt to different environments, the resistance of Salmonella biofilms against different stress factors including desiccation stress, disinfectants (e.g. hypochlorite, glutaraldehyde, cationic tensides and triclosan) and antibiotics (e.g. ciprofloxacin) is described. Finally, a number of Salmonella biofilm inhibitors, identified through bottom-up- and top-down-approaches, are discussed, such as surfactin, glucose, halogenated furanones, 4(5)-aryl 2-aminoimidazoles, furocoumarins and salicylates. Also the potential of combination therapy (e.g. combinations of triclosan and quaternary ammonium salts or halogenated furanones and antibiotics/disinfectants) and nano- and micro-emulsions to inhibit Salmonella biofilm formation is discussed. Insight into the pathogen's complex biofilm process will eventually lead to further unraveling of its intricacies and more efficient strategies to combat Salmonella biofilms.
[show abstract][hide abstract] ABSTRACT: Lactobacillus rhamnosus GG (LGG) produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75) and Msp2 (LGG_00031 or p40), which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG) by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG.
PLoS ONE 01/2012; 7(2):e31588. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although the occurrence, biosynthesis and possible functions of glycoproteins are increasingly documented for pathogens, glycoproteins are not yet widely described in probiotic bacteria. Nevertheless, knowledge of protein glycosylation holds important potential for better understanding specific glycan-mediated interactions of probiotics and for glycoengineering in food-grade microbes.
Here, we provide evidence that the major secreted protein Msp1/p75 of the probiotic Lactobacillus rhamnosus GG is glycosylated. Msp1 was shown to stain positive with periodic-acid Schiff staining, to be susceptible to chemical deglycosylation, and to bind with the mannose-specific Concanavalin A (ConA) lectin. Recombinant expression in Escherichia coli resulted in a significant reduction in molecular mass, loss of ConA reactivity and increased sensitivity towards pronase E and proteinase K. Mass spectrometry showed that Msp1 is O-glycosylated and identified a glycopeptide TVETPSSA (amino acids 101-108) bearing hexoses presumably linked to the serine residues. Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA. The role of the glycan substitutions in known functions of Msp1 was also investigated. Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1. In addition, the glycan chain appeared not to be required for the activation of Akt signaling in intestinal epithelial cells by Msp1. On the other hand, examination of different cell extracts showed that Msp1 is a glycosylated protein in the supernatant, but not in the cell wall and cytosol fraction, suggesting a link between glycosylation and secretion of this protein.
In this study we have provided the first evidence of protein O-glycosylation in the probiotic L rhamnosus GG. The major secreted protein Msp1 is glycosylated with ConA reactive sugars at the serine residues at 106 and 107. Glycosylation is not required for the peptidoglycan hydrolase activity of Msp1 nor for Akt activation capacity in epithelial cells, but appears to be important for its stability and protection against proteases.
[show abstract][hide abstract] ABSTRACT: Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a spaCBA pilus knockout mutant in comparison with the wild type and other adhesin mutants. The SpaCBA pilus of L. rhamnosus GG showed to be key for efficient adherence to the Caco-2 intestinal epithelial cell (IEC) line and biofilm formation. Moreover, the spaCBA mutant induces an elevated level of interleukin-8 (IL-8) mRNA in Caco-2 cells compared to the wild type, possibly involving an interaction of lipoteichoic acid with Toll-like receptor 2. In contrast, an L. rhamnosus GG mutant without exopolysaccharides but with an increased exposure of pili leads to the reduced expression of IL-8. Using Transwells to partition bacteria from Caco-2 cells, IL-8 induction is blocked completely regardless of whether wild-type or mutant L. rhamnosus GG cells are used. Taken together, our data suggest that L. rhamnosus GG SpaCBA pili, while promoting strong adhesive interactions with IECs, have a functional role in balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid.
Applied and environmental microbiology 01/2012; 78(1):185-93. · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cell surface mucins configure the cell surface by presenting extended protein backbones that are heavily O-glycosylated. The glycopeptide structures establish physicochemical properties at the cell surface that enable and block the formation of biologically important molecular complexes. Some mucins, such as MUC1, associate with receptor tyrosine kinases and other cell surface receptors, and engage in signal transduction in order to communicate information regarding conditions at the cell surface to the nucleus. In that context, the MUC1 cytoplasmic tail (MUC1CT) receives phosphorylation signals from receptor tyrosine kinases and serine/threonine kinases, which enables its association with different signaling complexes that conduct these signals to the nucleus and perhaps other subcellular organelles. We have detected the MUC1CT at promoters of over 500 genes, in association with several different transcription factors, and have shown that promoter occupancy can vary under different growth factor conditions. However, the full biochemical nature of the nuclear forms of MUC1 and its function at these promoter regions remain undefined. I will present evidence that nuclear forms of the MUC1CT include extracellular and cytoplasmic tail domains. In addition, I will discuss evidence for a hypothesis that the MUC1CT possesses a novel catalytic function that enables remodeling of the transcription factor occupancy of promoters, and thereby engages in regulation of gene expression.
[show abstract][hide abstract] ABSTRACT: In inflammatory bowel diseases (IBD), it is known that besides genetic and environmental factors (e.g. diet, drugs, stress), the microbiota play an important role in the pathogenesis. Patients with IBD have an altered microbiota (dysbiosis) and therefore, probiotics, defined as 'live micro-organisms that when administered in adequate amounts can confer a health benefit on the host', have been suggested as nutritional supplements to restore these imbalances. The best response on probiotics among the different types of IBD appears to be in the case of ulcerative colitis. Although probiotics show promise in IBD in both clinical and animal studies, further mechanistic studies are necessary to optimize the use of probiotics as supporting therapy in IBD. Murine models of experimental colitis have been used for decades to study this pathology, and these models have been proven useful to search for new therapeutic approaches. The purpose of this review is to summarize probiotic-host interaction studies in murine models of experimental colitis and to evaluate how these models can further help in understanding these complex interactions. Unraveling the molecular mechanisms behind the beneficial effects will assist in better and possibly more efficient probiotic formulations.
[show abstract][hide abstract] ABSTRACT: In spite of the wealth of clinical evidence supporting the health benefits of Lactobacillus rhamnosus GG in humans, there is still a lack of understanding of the molecular mechanisms behind its probiosis. Current knowledge suggests that the health-promoting effects of this probiotic strain might be partly dependent on its persistence in the intestine and adhesion to mucosal surfaces. Moreover, L. rhamnosus GG contains mucus-binding pili that might also explain the occupation of its ecological niche as a comparatively less stringent allochthonous intestine-dwelling bacterium. To uncover additional surface proteins involved in mucosal adhesion, we investigated the adherence properties of the only predicted protein (LGG_02337) in L. rhamnosus GG that exhibits homology with a known mucus-binding domain. We cloned a recombinant form of the gene for this putative mucus adhesin and established that the purified protein readily adheres to human intestinal mucus. We also showed that this mucus adhesin is visibly distributed throughout the cell surface and participates in the adhesive interaction between L. rhamnosus GG and mucus, although less prominently than the mucus-binding pili in this strain. Based on primary structural comparisons, we concluded that the current annotation of the LGG_02337 protein likely does not accurately reflect its predicted properties, and we propose that this mucus-specific adhesin be called the mucus-binding factor (MBF). Finally, we interpret our results to mean that L. rhamnosus GG MBF, as an active mucus-specific surface adhesin with a presumed ancillary involvement in pilus-mediated mucosal adhesion, plays a part in the adherent mechanisms during intestinal colonization by this probiotic.
Applied and environmental microbiology 07/2011; 77(13):4465-72. · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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, cyclo-pentyl 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.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Medicinal Chemistry 01/2011; 54(2):472-84. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bacterial biofilm formation is an important cause of environmental persistence of food-borne pathogens, such as Salmonella Typhimurium. As the ensemble of bacterial cells within a biofilm represents different physiological states, even for monospecies biofilms, gene expression patterns in these multicellular assemblages show a high degree of heterogeneity. This heterogeneity might mask differential gene expression that occurs only in subpopulations of the entire biofilm population when using methods that average expression output. In an attempt to address this problem and to refine expression analysis in biofilm studies, we used the Differential Fluorescence Induction (DFI) technique to gain more insight in S. Typhimurium biofilm gene expression. Using this single cell approach, we were able to identify 26 genetic loci showing biofilm specific increased expression. For a selected number of identified genes, we confirmed the DFI results by the construction of defined promoter fusions, measurement of relative gene expression levels and construction of mutants. Overall, we have shown for the first time that the DFI technique can be used in biofilm research. The fact that this analysis revealed genes that have not been linked with Salmonella biofilm formation in previous studies using different approaches illustrates that no single technique, in casu biofilm formation, is able to identify all genes related to a given phenotype.
Journal of microbiological methods 01/2011; 84(3):467-78. · 2.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: Aims: To investigate the spatial organization of endogenous and exogenously applied Lactobacillus communities at specific locations in the adult gastrointestinal tract of different hosts. Methods and Results: Samples of the human, murine and avian gastrointestinal tract of subjects that received or not received a Lactobacillus probiotic were analysed by fluorescence in situ hybridization (FISH) with rRNA-targeted probes. High levels of endogenous lactobacilli were observed on the nonsecretory, stratified squamous epithelia present in the forestomach of mice and crop of chickens, respectively. These epithelial associations showed characteristics of bacterial biofilms, i.e. bacteria attached to a surface and embedded in a matrix of extracellular polymeric substances. In other regions of the analysed intestines, lactobacilli seemed to occur mainly as dispersed bacterial cells or as microcolonies. Exogenous administration of a Lactobacillus probiotic did increase the levels of loosely adherent Lactobacillus cells detected. However, the probiotic strains were unable to establish themselves inside the gastrointestinal biofilms. Conclusions: Gastrointestinal biofilms of lactobacilli occur only in specific niches in certain hosts, such as the murine forestomach and avian crop. Significance and Impact of the Study: Biofilm formation by lactobacilli in specific parts of animal gastrointestinal tracts was documented for the first time by FISH.
Letters in Applied Microbiology 01/2011; · 1.63 Impact Factor
[show abstract][hide abstract] ABSTRACT: Metabolic reconstructions (MRs) are common denominators in systems biology and represent biochemical, genetic, and genomic (BiGG) knowledge-bases for target organisms by capturing currently available information in a consistent, structured manner. Salmonella enterica subspecies I serovar Typhimurium is a human pathogen, causes various diseases and its increasing antibiotic resistance poses a public health problem.
Here, we describe a community-driven effort, in which more than 20 experts in S. Typhimurium biology and systems biology collaborated to reconcile and expand the S. Typhimurium BiGG knowledge-base. The consensus MR was obtained starting from two independently developed MRs for S. Typhimurium. Key results of this reconstruction jamboree include i) development and implementation of a community-based workflow for MR annotation and reconciliation; ii) incorporation of thermodynamic information; and iii) use of the consensus MR to identify potential multi-target drug therapy approaches.
Taken together, with the growing number of parallel MRs a structured, community-driven approach will be necessary to maximize quality while increasing adoption of MRs in experimental design and interpretation.
BMC Systems Biology 01/2011; 5:8. · 2.98 Impact Factor
[show abstract][hide abstract] ABSTRACT: LuxS is the synthase enzyme of the quorum sensing signal AI-2. In Salmonella Typhimurium, it was previously shown that a luxS deletion mutant is impaired in biofilm formation. However, this phenotype could not be complemented by extracellular addition of quorum sensing signal molecules.
Analysis of additional S. Typhimurium luxS mutants indicated that the LuxS enzyme itself is not a prerequisite for a wild type mature biofilm. However, in close proximity of the luxS coding sequence, a small RNA molecule, MicA, is encoded on the opposite DNA strand. Interference with the MicA expression level showed that a balanced MicA level is essential for mature Salmonella biofilm formation. Several MicA targets known to date have previously been reported to be implicated in biofilm formation in Salmonella or in other bacterial species. Additionally, we showed by RT-qPCR analysis that MicA levels are indeed altered in some luxS mutants, corresponding to their biofilm formation phenotype.
We show that the S. Typhimurium biofilm formation phenotype of a luxS mutant in which the complete coding region is deleted, is dependent on the sRNA molecule MicA, encoded in the luxS adjacent genomic region, rather than on LuxS itself. Future studies are required to fully elucidate the role of MicA in Salmonella biofilm formation.
[show abstract][hide abstract] ABSTRACT: While some probiotic strains might have adjuvant effects in the therapy for inflammatory bowel diseases (IBD), these effects remain controversial and cannot be generalized. In this study, a dltD mutant of the model probiotic Lactobacillus rhamnosus GG (LGG), having a drastic modification in its lipoteichoic acid (LTA) molecules, was analysed for its effects in an experimental colitis model. Dextran sulphate sodium (DSS) was used to induce either moderate to severe or mild chronic colitis in mice. Mice received either phosphate-buffered saline (PBS), LGG wild-type or the dltD mutant via the drinking water. Macroscopic parameters, histological abnormalities, cytokine and Toll-like receptor (TLR) expression were analysed to assess disease activity. LGG wild-type did not show efficacy in the different experimental colitis set-ups. This wild-type strain even seemed to exacerbate the severity of colitic parameters in the moderate to severe colitis model compared to untreated mice. In contrast, mice treated with the dltD mutant showed an improvement of some colitic parameters compared to LGG wild-type-treated mice in both experimental models. In addition, treatment with the dltD mutant correlated with a significant down-regulation of Toll-like receptor-2 expression and of downstream proinflammatory cytokine expression in the colitic mice. These results show that molecular cell surface characteristics of probiotics are crucial when probiotics are considered for use as supporting therapy in IBD.
[show abstract][hide abstract] ABSTRACT: Probiotic bacteria are administered as live micro-organisms to provide a health benefit to the host. Knowledge on adaptation factors that promote the survival and persistence of probiotics in the intestine is key to understand and improve their ecological and probiotic performance. Adaptation factors include adhesins, molecules conferring stress tolerance and nutritional versatility, antimicrobial products against competing microbes, and factors promoting resistance against the host immune system. Here, we present an overview of the current knowledge on adaptation factors of probiotic lactobacilli, with focus on the prototypical and widely documented probiotic strain Lactobacillus rhamnosus GG.