Novel Modular Megasynthases in Myxobacteria: The Stigmatellin- and Myxochelin- Biosynthesis in Stigmatella aurantiaca

Neben den Actinomyceten zeichnen sich vor allem die Myxobakterien als Quelle für neuartige biologisch aktive Naturstoffe aus. Stigmatella aurantiaca produziert neben den Myxalamiden, den Myxothiazolen, den Aurachinen, die elektronentransportinhibitorisch wirken, das Stigmatellin A und die Myxocheline A und B. Bei den letzteren handelt es sich um sogenannte Eisenchelatoren vom Katecholat-Typ, welche unter Myxobakterien weit verbreitet sind. Mit der Charakterisierung der Myxochelin-Biosynthese in S. aurantiaca Sg a15 ist es erstmalig gelungen den vollständigen Stoffwechsel-Weg eines myxobakteriellen Metaboliten in vitro nachzustellen. Insgesamt 6 Enzyme darunter eine nicht ribosomale Peptidsynthetase (MxcG) mit ungewöhlichem Aufbau sind an der Biosynthese der Myxocheline beteiligt. Auf diese Weise konnte ein bislang noch nicht charakterisierter Terminationsmechanismus in der Biosynthese von nicht ribosomal synthetisierten Peptiden dargestellt werden, bei dem das enzymatisch gebundenen Thioesterintermediat durch eine einzelne katalytische Domäne über eine zweistufige Reduktion zum Alkohol überführt wird. Schließlich resultierten aus der DNA- Sequenzanalyse des Myxochelin Biosyntheseoperons aus Stigmatella aurantiaca Sg a15 Einblicke in die EisenTransportmechanismen von Myxobakterien. Bei dem aromatischen Polyketid Stigmatellin A handelt es sich um einen Elektronentransportinhibitor. Nachdem das gesamte Stigmatellin-Biosynthese-Gencluster aus S. aurantiaca Sg a15 kloniert wurde, ergaben die DNASequenzanalysen neun für modulare Typ I Polyketidsynthase codierende Gene. Durch Fütterungsexperimente wurde bestätigt, dass eines der Module während des Stigmatellin Biosynthese-Prozesses ständig iterativ verwendet wird, da zehn Acetat bzw. Propionat-Einheiten in das StigmatellinGrundgerüst inkorporiert werden. Die iterative Verwendung eines Modules verlangt eine ungewöhnliche Rückacylierung des an der Acetyl Carrier Protein-Domäne lokalisierten Thioester-Intermediates auf die ß-Ketoacyl Synthase des gleichen Proteins. Die Stigmatellin Biosynthese verläuft damit nicht co-linear zum Proteinaufbau, was dem klassischen Modell der Wirkungsweise einer bakteriellen Typ I Polyketidsynthase widerspricht. Durch Geninaktivierungen mittels gerichteter Insertion konnten neue aktive Stigmatellin-Derivate generiert werden, deren Struktur mit Hilfe von HNMR- Untersuchungen aufgeklärt wurden.Beside actinomycetes myxobacteria is a major source for novel bioactive compounds. The electron transport inhibitors myxalamides, myxothiazoles and aurachines are produced by different strains of Stigmatella aurantiaca as stigmatellin A and the myxochelins A and B are. The myxochelins are catecholic acid containing iron chelating compounds produced by myxobacteria. In this work the task was undertaken to heterologously express the myxochelin biosynthetic machinery in Escherichia coli. By reconstituting the myxochelin biosynthesis in vitro a novel type of reductive release mechanism during non ribosomal peptide synthesis was characterized: A single enzymatic domain is responsible for two reductive rounds in which the enzyme bound thioester-intermediate is converted to the free alcohol. Additionally and for the first time the analysis of the myxochelin biosynthetic operon revealed novel insights in the iron transport mechanisms of myxobacteria. The aromatic polyketide stigmatellin A reveals a highly potent electron transport inhibitor. After cloning and analysing the stigmatellin biosynthetic operon nine type I modular polyketide synthase genes were identified. Feeding experiments showed that one of the modules has to be used constantly iterative to enable the incorporation of ten acetate and propionate units into the stigmatellin backbone. For the first time a bacterial type one polyketide synthase was described, where the biosynthesis of a polyketide was not collinear to the protein structure. This was the first contradiction to the standard model of type I polyketide synthesis. By using gene inactivations novel bioactive stigmatellin derivatives were generated. Their structures were confirmed by HNMR analysis

Treatment with the IL-17 blocking antibody secukinumab (AIN457) does not interfere with the efficacy of Influenza and Meningococcal vaccination in healthy subjects. Results of an open-label, parallel group, randomized, single-center study

Objectives: to evaluate the efficacy of influenza and meningococcal vaccinations in healthy subjects exposed to the anti-IL-1 mAb secukinumab. Methods: open-label, parallel groups, randomized single-center study in 50 healthy subjects. Subjects received a single secukinumab (AIN457) 150 mg s.c. dose or no treatment, followed by influenza and meningococcal vaccinations two weeks later. Primary efficacy variables: responses to vaccinations as ≥4-fold increase in Ab titer in ≥2/3 serotypes (influenza) and ≥4-fold increase (meningococcus), both at 4 weeks post-vaccination. Immunogenicity was assesed as hemagglutination inhibition (HI, influenza) and Serum Bactericidal Assay (SBA, Neisseria meningitidis). Results: all 50 subjects, randomized to secukinumab (n=25) or control (n=25) completed the study. Antibody responses to vaccinations measured at four weeks post-vaccination were comparable in both groups, with responses following influenza vaccine of 20/25(80%) for both groups and following MenC vaccine of 19/25(76%) for secukinumab and 18/25(72%) for control. The differences between groups were 0% (90% CI -19%,19%) and 4% (90% CI -16%,24%) for influenza and MenC vaccines respectively. Antibody responses were also comparable in the two groups at different time-points assessed. Headache was the most frequently reported adverse event. No deaths or serious adverse events were reported during the study. Conclusions: Blockade of IL-17 by secukinumab (AIN457) does not appear to interfere with the efficacy of influenza and meningococcal vaccinations, as assessed by the achievement of antibody protective levels. A protective (≥4 fold) immune response to both vaccinations at 4 weeks was achieved in 80 and 76% of subjects exposed to secukinumab and control, respectively

Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads.

Type III polyketide synthases (PKS) were regarded as typical for plant secondary metabolism before they were found in microorganisms recently. Due to microbial genome sequencing efforts, more and more type III PKS are found, most of which of unknown function. In this manuscript, we report a comprehensive analysis of the phylogeny of bacterial type III PKS and report the expression of a type III PKS from the myxobacterium Sorangium cellulosum in pseudomonads. There is no precedent of a secondary metabolite that might be biosynthetically correlated to a type III PKS from any myxobacterium. Additionally, an inactivation mutant of the S. cellulosum gene shows no physiological difference compared to the wild-type strain which is why these type III PKS are assumed to be "silent" under the laboratory conditions administered. One type III PKS (SoceCHS1) was expressed in different Pseudomonas sp. after the heterologous expression in Escherichia coli failed. Cultures of recombinant Pseudomonas sp. harbouring SoceCHS1 turned red upon incubation and the diffusible pigment formed was identified as 2,5,7-trihydroxy-1,4-naphthoquinone, the autooxidation product of 1,3,6,8-tetrahydroxynaphthalene. The successful heterologous production of a secondary metabolite using a gene not expressed under administered laboratory conditions provides evidence for the usefulness of our approach to activate such secondary metabolite genes for the production of novel metabolites

Evaluation of strain coverage of the multicomponent meningococcal serogroup B vaccine (4CMenB) administered in infants according to different immunisation schedules

The 4-component vaccine 4CMenB, developed against invasive disease caused by meningococcal serogroup B, is approved for use in infants in several countries worldwide. 4CMenB is mostly used as 3 + 1 schedule, except for the UK, where a 2 + 1 schedule is used, and where the vaccine showed an effectiveness of 82.9%. Here we compared the coverage of two 4CMenB vaccination schedules (3 + 1 [2.5, 3.5, 5, 11 months] versus 2 + 1 [3.5, 5, 11 months of age]) against 40 serogroup B strains, representative of epidemiologically-relevant isolates circulating in England and Wales in 2007–2008, using sera from a previous phase 3b clinical trial. The strains were tested using hSBA on pooled sera of infants, collected at one month post-primary and booster vaccination. 4CMenB coverage was defined as the percentage of strains with positive killing (hSBA titres ≥ 4 after immunisation and negative baseline hSBA titres < 2). Coverage of 4CMenB was 40.0% (95% confidence interval [CI]: 24.9–56.7) and 87.5% (95%CI: 73.2–95.8) at one month post-primary and booster vaccination, respectively, regardless of immunisation schedule. Using a more conservative threshold (post-immunisation hSBA titres ≥ 8; baseline ≤ 2), at one month post-booster dose, strain coverages were 80% (3 + 1) and 70% (2 + 1). We used a linear regression model to assess correlation between post-immunisation hSBA data for each strain in the two groups; Pearson’s correlation coefficients were 0.93 and 0.99 at one month post-primary and booster vaccination. Overall, there is no evidence for a difference in strain coverage when 4CMenB is administered according to a 3 + 1 or 2 + 1 infant vaccination schedule