Screening for inhibitors of mutacin synthesis in Streptococcus mutans using fluorescent reporter strains.

Background: Within the polymicrobial dental plaque biofilm, bacteria kill competitors by excreting mixtures of bacteriocins, resulting in improved fitness and survival. Inhibiting their bacteriocin synthesis might therefore be a useful strategy to eliminate specific pathogens. We used Streptococcus mutans, a highly acidogenic inhabitant of dental plaque, as a model and searched for natural products that reduced mutacin synthesis.

The natural product carolacton inhibits folate-dependent C1 metabolism by targeting FolD/MTHFD.

The natural product carolacton is a macrolide keto-carboxylic acid produced by the myxobacterium Sorangium cellulosum, and was originally described as an antibacterial compound. Here we show that carolacton targets FolD, a key enzyme from the folate-dependent C1 metabolism. We characterize the interaction between bacterial FolD and carolacton biophysically, structurally and biochemically.

The carolactam strategy is ineffective: synthesis and biological evaluation of carolactam.

Carolacton, a secondary metabolite isolated from the extracts of Sorangium cellulosum, causes membrane damage and cell death in biofilms of the caries- and endocarditis-associated bacterium Streptococcus mutans and Streptococcus pneumoniae. It is known that macrolactam derivatives can show improved pharmacological properties compared to the corresponding macrolactons (lactam strategy). Therefore, we here report the total synthesis and biological activity of the lactam derivative of carolacton ("carolactam").

The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of .

The myxobacterial secondary metabolite carolacton inhibits growth of and kills biofilm cells of the caries- and endocarditis-associated pathogen at nanomolar concentrations. Here, we studied the response to carolacton of an strain that lacked the outer membrane protein TolC. Whole-genome sequencing of the laboratory strain TolC revealed the integration of an insertion element, IS, at the locus and a close phylogenetic relationship to the ancient K-12.

The biofilm inhibitor Carolacton inhibits planktonic growth of virulent pneumococci via a conserved target.

New antibacterial compounds, preferentially exploiting novel cellular targets, are urgently needed to fight the increasing resistance of pathogens against conventional antibiotics. Here we demonstrate that Carolacton, a myxobacterial secondary metabolite previously shown to damage Streptococcus mutans biofilms, inhibits planktonic growth of Streptococcus pneumoniae TIGR4 and multidrug-resistant clinical isolates of serotype 19A at nanomolar concentrations. A Carolacton diastereomer is inactive in both streptococci, indicating a highly specific interaction with a conserved cellular target.

Carolacton Treatment Causes Delocalization of the Cell Division Proteins PknB and DivIVa in Streptococcus mutans in vivo.

The small inhibitory molecule Carolacton has been shown to cause chain formation and bulging in Streptococci, suggesting a defect in cell division, but it is not known how cell division is impaired on a molecular level. Fluorescent fusion proteins have successfully been applied to visualize protein localization and dynamics in vivo and have revolutionized our understanding of cell wall growth, cell division, chromosome replication and segregation. However, in Streptococci the required vectors are largely lacking.

Discovery of antiviral molecules for dengue: In silico search and biological evaluation.

Background: Dengue disease is a global disease that has no effective treatment. The dengue virus (DENV) NS2B/NS3 protease complex is a target for designing specific antivirals due to its importance in viral replication and its high degree of conservation.

Methods: NS2B/NS3 protease complex structural information was employed to find small molecules that are capable of inhibiting the activity of the enzyme complex.

The Alternative Sigma Factor SigX Controls Bacteriocin Synthesis and Competence, the Two Quorum Sensing Regulated Traits in Streptococcus mutans.

Two small quorum sensing (QS) peptides regulate competence in S. mutans in a cell density dependent manner: XIP (sigX inducing peptide) and CSP (competence stimulating peptide). Depending on the environmental conditions isogenic S.

Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation.

Background: The complex microbiome of the gut has an enormous impact on human health. Analysis of the transcriptional activity of microorganisms through mRNA sequencing (metatranscriptomics) opens a completely new window into their activity in vivo, but it is highly challenging due to numerous technical and bioinformatical obstacles. Here we present an optimized pipeline for extraction of high quality mRNA from stool samples.

Construction and verification of the transcriptional regulatory response network of Streptococcus mutans upon treatment with the biofilm inhibitor carolacton.

Background: Carolacton is a newly identified secondary metabolite causing altered cell morphology and death of Streptococcus mutans biofilm cells. To unravel key regulators mediating these effects, the transcriptional regulatory response network of S. mutans biofilms upon carolacton treatment was constructed and analyzed.

Cross-feeding and interkingdom communication in dual-species biofilms of Streptococcus mutans and Candida albicans.

Polymicrobial biofilms are of large medical importance, but relatively little is known about the role of interspecies interactions for their physiology and virulence. Here, we studied two human pathogens co-occuring in the oral cavity, the opportunistic fungus Candida albicans and the caries-promoting bacterium Streptococcus mutans. Dual-species biofilms reached higher biomass and cell numbers than mono-species biofilms, and the production of extracellular polymeric substances (EPSs) by S.

Genetic variability of mutans streptococci revealed by wide whole-genome sequencing.

Background: Mutans streptococci are a group of bacteria significantly contributing to tooth decay. Their genetic variability is however still not well understood.

Results: Genomes of 6 clinical S.

The biofilm inhibitor carolacton disturbs membrane integrity and cell division of Streptococcus mutans through the serine/threonine protein kinase PknB.

Carolacton, a secondary metabolite isolated from the myxobacterium Sorangium cellulosum, disturbs Streptococcus mutans biofilm viability at nanomolar concentrations. Here we show that carolacton causes leakage of cytoplasmic content (DNA and proteins) in growing cells at low pH and provide quantitative data on the membrane damage. Furthermore, we demonstrate that the biofilm-specific activity of carolacton is due to the strong acidification occurring during biofilm growth.

Subpopulation-specific transcriptome analysis of competence-stimulating-peptide-induced Streptococcus mutans.

Competence-stimulating-peptide (CSP)-mediated competence development in Streptococcus mutans is a transient and biphasic process, since only a subpopulation induces the expression of ComX in the presence of CSP, and the activation of the DNA uptake machinery in this fraction shuts down ~3 to 4 h postinduction. Here, we combine for the first time, to our knowledge, the bacterial flow-cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and noncompetent fraction of CSP-treated S. mutans cells.

Damage of Streptococcus mutans biofilms by carolacton, a secondary metabolite from the myxobacterium Sorangium cellulosum.

Background: Streptococcus mutans is a major pathogen in human dental caries. One of its important virulence properties is the ability to form biofilms (dental plaque) on tooth surfaces. Eradication of such biofilms is extremely difficult.

Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein.

Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment.

Optimization of a microarray sandwich-ELISA against hINF-gamma on a modified nitrocellulose membrane.

The highly specific and highly sensitive ELISA (enzyme linked immunosorbent assay) technique is the most commonly used method for immunological diagnostics in general. In combination with protein microarrays and their ability to allow performing thousands of experiments in parallel, a promising tool for global analytical approaches with reduced consumption of time, analytes, and reagents is given. In this study a protein microarray-based sandwich-ELISA for human interferon-gamma (hINF-gamma) is established.