An efficient synthesis of 1,6-anhydro-N-acetylmuramic acid from N-acetylglucosamine

A novel synthesis of 1,6-anhydro-N-acetylmuramic acid is described, which proceeds in only five steps from the cheap starting material N-acetylglucosamine. This efficient synthesis should enable future studies into the importance of 1,6-anhydromuramic acid in bacterial cell wall recycling processes

Pathoblockers or antivirulence drugs as a new option for the treatment of bacterial infections

The rapid development of antimicrobial resistance is threatening mankind to such an extent that the World Health Organization expects more deaths from infections than from cancer in 2050 if current trends continue. To avoid this scenario, new classes of antiinfectives must urgently be developed. Antibiotics with new modes of action are needed, but other concepts are also currently being pursued. Targeting bacterial virulence as a means of blocking pathogenicity is a promising new strategy for disarming pathogens. Furthermore, it is believed that this new approach is less susceptible towards resistance development. In this review, recent examples of anti-infective compounds acting on several types of bacterial targets, e.g., adhesins, toxins and bacterial communication, are described

N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism

Tannerella forsythia is an anaerobic, Gram-negative oral pathogen that thrives in multispecies gingival biofilms associated with periodontitis. The bacterium is auxotrophic for the commonly essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) and, thus, strictly depends on an exogenous supply of MurNAc for growth and maintenance of cell morphology. A MurNAc transporter (Tf_MurT; Tanf_08375) and an ortholog of the Escherichia coli etherase MurQ (Tf_MurQ; Tanf_08385) converting MurNAc-6-phosphate to GlcNAc-6-phosphate were recently described for T. forsythia. In between the respective genes on the T. forsythia genome, a putative kinase gene is located. In this study, the putative kinase (Tf_MurK; Tanf_08380) was produced as a recombinant protein and biochemically characterized. Kinetic studies revealed Tf_MurK to be a 6-kinase with stringent substrate specificity for MurNAc exhibiting a 6 × 104 - fold higher catalytic efficiency (kcat/Km) for MurNAc than for N-acetylglucosamine (GlcNAc) with kcat values of 10.5 s−1 and 0.1 s−1 and Km values of 200 µM and 116 mM, respectively. The enzyme kinetic data suggest that Tf_MurK is subject to substrate inhibition (Ki[S] = 4.2 mM). To assess the role of Tf_MurK in the cell wall metabolism of T. forsythia, a kinase deletion mutant (1Tf_murK::erm) was constructed. This mutant accumulated MurNAc intracellularly in the exponential phase, indicating the capability to take up MurNAc, but inability to catabolize MurNAc. In the stationary phase, the MurNAc level was reduced in the mutant, while the level of the peptidoglycan precursor UDP-MurNAc-pentapeptide was highly elevated. Further, according to scanning electron microscopy evidence, the 1Tf_murK::erm mutant was more tolerant toward low MurNAc concentration in the medium (below 0.5 µg/ml) before transition from healthy, rod-shaped to fusiform cells occurred, while the parent strain required > 1 µg/ml MurNAc for optimal growth. These data reveal that T. forsythia readily catabolizes exogenous MurNAc but simultaneously channels a proportion of the sugar into peptidoglycan biosynthesis. Deletion of Tf_murK blocks MurNAc catabolism and allows the direction of MurNAc solely to peptidoglycan biosynthesis, resulting in a growth advantage in MurNAc-depleted medium. This work increases our understanding of the T. forsythia cell wall metabolism and may pave new routes for lead finding in the treatment of periodontitis

Chemical synthesis of tripeptide thioesters for the biotechnological incorporation into the myxobacterial secondary metabolite argyrin via mutasynthesis

The argyrins are secondary metabolites from myxobacteria with antibiotic activity against Pseudomonas aeruginosa. Studying their structure–activity relationship is hampered by the complexity of the chemical total synthesis. Mutasynthesis is a promising approach where simpler and fully synthetic intermediates of the natural product’s biosynthesis can be biotechnologically incorporated. Here, we report the synthesis of a series of tripeptide thioesters as mutasynthons containing the native sequence with a dehydroalanine (Dha) Michael acceptor attached to a sarcosine (Sar) and derivatives. Chemical synthesis of the native sequence ᴅ-Ala-DhaSar thioester required revision of the sequential peptide synthesis into a convergent strategy where the thioester with sarcosine was formed before coupling to the Dha-containing dipeptide

Ground-based photometry of space-based transit detections: Photometric follow-up of the CoRoT mission

The motivation, techniques and performance of the ground-based photometric follow-up of transit detections by the CoRoT space mission are presented. Its principal raison d'\^{e}tre arises from the much higher spatial resolution of common ground-based telescopes in comparison to CoRoT's cameras. This allows the identification of many transit candidates as arising from eclipsing binaries that are contaminating CoRoT's lightcurves, even in low-amplitude transit events that cannot be detected with ground-based obervations. For the ground observations, 'on'-'off' photometry is now largely employed, in which only a short timeseries during a transit and a section outside a transit is observed and compared photometrically. CoRoT planet candidates' transits are being observed by a dedicated team with access to telescopes with sizes ranging from 0.2 to 2 m. As an example, the process that led to the rejection of contaminating eclipsing binaries near the host star of the Super-Earth planet CoRoT-7b is shown. Experiences and techniques from this work may also be useful for other transit-detection experiments, when the discovery instrument obtains data with a relatively low angular resolution.Comment: Accepted for the A&A special issue on CoRo

A remote secondary binding pocket promotes heteromultivalent targeting of DC-SIGN

Dendritic cells (DC) are antigen-presenting cells coordinating the interplay of the innate and the adaptive immune response. The endocytic C-type lectin receptors DC-SIGN and Langerin display expression profiles restricted to distinct DC subtypes and have emerged as prime targets for next-generation immunotherapies and anti-infectives. Using heteromultivalent liposomes copresenting mannosides bearing aromatic aglycones with natural glycan ligands, we serendipitously discovered striking cooperativity effects for DC-SIGN+ but not for Langerin+ cell lines. Mechanistic investigations combining NMR spectroscopy with molecular docking and molecular dynamics simulations led to the identification of a secondary binding pocket for the glycomimetics. This pocket, located remotely of DC-SIGN’s carbohydrate bindings site, can be leveraged by heteromultivalent avidity enhancement. We further present preliminary evidence that the aglycone allosterically activates glycan recognition and thereby contributes to DC-SIGN-specific cell targeting. Our findings have important implications for both translational and basic glycoscience, showcasing heteromultivalent targeting of DCs to improve specificity and supporting potential allosteric regulation of DC-SIGN and CLRs in general

The Binary Protein Interactome of Treponema pallidum – The Syphilis Spirochete

Protein interaction networks shed light on the global organization of proteomes but can also place individual proteins into a functional context. If we know the function of bacterial proteins we will be able to understand how these species have adapted to diverse environments including many extreme habitats. Here we present the protein interaction network for the syphilis spirochete Treponema pallidum which encodes 1,039 proteins, 726 (or 70%) of which interact via 3,649 interactions as revealed by systematic yeast two-hybrid screens. A high-confidence subset of 991 interactions links 576 proteins. To derive further biological insights from our data, we constructed an integrated network of proteins involved in DNA metabolism. Combining our data with additional evidences, we provide improved annotations for at least 18 proteins (including TP0004, TP0050, and TP0183 which are suggested to be involved in DNA metabolism). We estimate that this “minimal” bacterium contains on the order of 3,000 protein interactions. Profiles of functional interconnections indicate that bacterial proteins interact more promiscuously than eukaryotic proteins, reflecting the non-compartmentalized structure of the bacterial cell. Using our high-confidence interactions, we also predict 417,329 homologous interactions (“interologs”) for 372 completely sequenced genomes and provide evidence that at least one third of them can be experimentally confirmed

Integrating protein-protein interactions and text mining for protein function prediction

<p>Abstract</p> <p>Background</p> <p>Functional annotation of proteins remains a challenging task. Currently the scientific literature serves as the main source for yet uncurated functional annotations, but curation work is slow and expensive. Automatic techniques that support this work are still lacking reliability. We developed a method to identify conserved protein interaction graphs and to predict missing protein functions from orthologs in these graphs. To enhance the precision of the results, we furthermore implemented a procedure that validates all predictions based on findings reported in the literature.</p> <p>Results</p> <p>Using this procedure, more than 80% of the GO annotations for proteins with highly conserved orthologs that are available in UniProtKb/Swiss-Prot could be verified automatically. For a subset of proteins we predicted new GO annotations that were not available in UniProtKb/Swiss-Prot. All predictions were correct (100% precision) according to the verifications from a trained curator.</p> <p>Conclusion</p> <p>Our method of integrating CCSs and literature mining is thus a highly reliable approach to predict GO annotations for weakly characterized proteins with orthologs.</p