• New Deoxyenhygrolides from Provide Insights into Butenolide Core Biosynthesis.

      Hug, Joachim J; Kjaerulff, Louise; Garcia, Ronald; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2022-01-14)
      Marine myxobacteria present a virtually unexploited reservoir for the discovery of natural products with diverse biological functions and novel chemical scaffolds. We report here the isolation and structure elucidation of eight new deoxyenhygrolides (1-8) from the marine myxobacterium Plesiocystis pacifica DSM 14875T. The herein described deoxyenhygrolides C-J (1-8) feature a butenolide core with an ethyl residue at C-3 of the γ-lactone in contrast to the previously described derivatives, deoxyenhygrolides A and B, which feature an isobutyl residue at this position. The butenolide core is 2,4-substituted with a benzyl (1, 2 and 7), benzoyl (3 and 4) or benzyl alcohol (5, 6 and 8) moiety in the 2-position and a benzylidene (1-6) or benzylic hemiketal (7 and 8) in the 4-position. The description of these new deoxyenhygrolide derivatives, alongside genomic in silico investigation regarding putative biosynthetic genes, provides some new puzzle pieces on how this natural product class might be formed by marine myxobacteria.
    • Novel 2,4-disubstituted quinazoline analogs as antibacterial agents with improved cytotoxicity profile: Modification of the benzenoid part.

      Megahed, Sarah H; Rasheed, Sari; Herrmann, Jennifer; El-Hossary, Ebaa M; El-Shabrawy, Yahia I; Abadi, Ashraf H; Engel, Matthias; Müller, Rolf; Abdel-Halim, Mohammad; Hamed, Mostafa M; et al. (Elsevier Ltd., 2022-01-07)
      Bacterial resistance to currently used antibiotics demands the development of novel antibacterial agents with good safety margins and sufficient efficacy against multi-drug resistant isolates. We have previously described the synthesis of N-butyl-2-(butylthio)quinazolin-4-amine (I) as an optimized hit with broad-spectrum antibacterial activity and low cytotoxicity. In addition, we have identified a potential growing vector for this series of compounds. Herein, we describe further hit optimization which includes systematic diversifications of both the benzenoid part and the substituents at position 6 and 7 of compound I. Growing of the molecule beside the core modifications yielded several compounds with remarkable anti(myco)bacterial activity against a panel of pathogenic bacteria, including drug-resistant strains. Compound 12 showed a 2-4 fold improvement in activity than I against S. aureus Newman, S. pneumoniae DSM-20566 and E. faecalis DSM-20478. The compounds also showed a good safety profile towards human HepG2 cells.
    • Single-cell transcriptional profiling of splenic fibroblasts reveals subset-specific innate immune signatures in homeostasis and during viral infection.

      Pezoldt, Joern; Wiechers, Carolin; Erhard, Florian; Rand, Ulfert; Bulat, Tanja; Beckstette, Michael; Brendolan, Andrea; Huehn, Jochen; Kalinke, Ulrich; Mueller, Mathias; et al. (NPG, 2021-12-02)
      Our understanding of the composition and functions of splenic stromal cells remains incomplete. Here, based on analysis of over 20,000 single cell transcriptomes of splenic fibroblasts, we characterized the phenotypic and functional heterogeneity of these cells in healthy state and during virus infection. We describe eleven transcriptionally distinct fibroblastic cell clusters, reassuring known subsets and revealing yet unascertained heterogeneity amongst fibroblasts occupying diverse splenic niches. We further identify striking differences in innate immune signatures of distinct stromal compartments in vivo. Compared to other fibroblasts and to endothelial cells, Ly6C+ fibroblasts of the red pulp were selectively endowed with enhanced interferon-stimulated gene expression in homeostasis, upon systemic interferon stimulation and during virus infection in vivo. Collectively, we provide an updated map of fibroblastic cell diversity in the spleen that suggests a specialized innate immune function for splenic red pulp fibroblasts.
    • IgG seroprevalence of COVID-19 among people living with HIV or at high risk of HIV in south-west Germany: A seroprevalence study.

      Kaddu-Mulindwa, Dominic; Keuser, Lukas; Lesan, Vadim; Rissland, Jürgen; Smola, Sigrun; Werdecker, Victoria; Stilgenbauer, Stephan; Christofyllakis, Konstantinos; Thurner, Lorenz; Bewarder, Moritz; et al. (John Wiley & Sons LTD, 2021-11-22)
      Objectives: Seroprevalence studies of SARS-CoV-2 have shown that there is a high number of undiagnosed missing cases. Seroprevalence of SARS-CoV-2 in people living with HIV (PLWH) is lacking. Therefore, we conducted a prospective cross-sectional study to estimate the seroprevalence of SARS-CoV-2 among PLWH without known diagnosis of COVID-19 in the south-west of Germany. Methods: Serological testing for SARS-CoV-2 immunoglobulin G (IgG) antibodies based on two assays was performed in PLWH who visited the outpatient HIV centre of two hospitals from April to June 2020. Additionally, patients had to answer questionnaires about possible COVID-19-related symptoms and predefined risk factors. Moreover, we tested 50 non-HIV-infected patients receiving post- or pre-exposure (PEP/PrEP) HIV prophylaxis. Results: In all, 594 (488 male, 106 female) PLWH (median age 51 years) and 50 PEP/PrEP-users were included in the study. The estimated seroprevalence of the PLWH cohort was 1.85% (11/594), with 11 positive tested cases in the cohort. Among all patients, only five had COVID-19-related symptoms. One PCR-positive patient did not show any antibody response in repeatedly carried out tests. None of the patients was hospitalized due to COVID-19. Three PrEP users were tested positive. Three patients had been previously diagnosed with SARS-COV-2 infection before inclusion. The used questionnaire did not help to detect SARS-CoV-2 positive patients.
    • Biotechnological production optimization of argyrins - a potent immunomodulatory natural product class.

      Pogorevc, Domen; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (John Wiley & Sons LTD, 2021-11-01)
      Argyrins represent a family of cyclic octapeptides exhibiting promising immunomodulatory activity via inhibiting mitochondrial protein synthesis, which leads to reduced IL-17 production by the T-helper 17 cells. Argyrins are formed by a non-ribosomal peptide synthetase (NRPS), originating from the myxobacterial producer strains Archangium gephyra Ar8082 and Cystobacter sp. SBCb004. In this work, a previously established heterologous production platform was employed to provide evidence of direct D-configured amino acid incorporation by the argyrin assembly line. An adenylation domain of the argyrin NRPS was characterized and shown to have a high preference for D-configured amino acids. Eight novel argyrin derivatives were generated via biosynthetic engineering of the heterologous production system. The system was also optimized to enable formation of methylated argyrin C and D derivatives with improved immunosuppressive activity compared with their unmethylated counterparts. Furthermore, the optimization of cultivation conditions allowed exclusive production of one major derivative at a time, drastically improving the purification process. Importantly, engineering of transcription and translation initiation resulted in a substantially improved production titre reaching 350-400 mg l-1 . The optimized system presented herein thus provides a versatile platform for production of this promising class of immunosuppressants at a scale that should provide sufficient supply for upcoming pre-clinical development.
    • Distinct Patterns of Blood Cytokines Beyond a Cytokine Storm Predict Mortality in COVID-19.

      Herr, Christian; Mang, Sebastian; Mozafari, Bahareh; Guenther, Katharina; Speer, Thimoteus; Seibert, Martina; Srikakulam, Sanjay Kumar; Beisswenger, Christoph; Ritzmann, Felix; Keller, Andreas; et al. (Dove Press, 2021-09-15)
      Background: COVID-19 comprises several severity stages ranging from oligosymptomatic disease to multi-organ failure and fatal outcomes. The mechanisms why COVID-19 is a mild disease in some patients and progresses to a severe multi-organ and often fatal disease with respiratory failure are not known. Biomarkers that predict the course of disease are urgently needed. The aim of this study was to evaluate a large spectrum of established laboratory measurements. Patients and methods: Patients from the prospective PULMPOHOM and CORSAAR studies were recruited and comprised 35 patients with COVID-19, 23 with conventional pneumonia, and 28 control patients undergoing elective non-pulmonary surgery. Venous blood was used to measure the serum concentrations of 79 proteins by Luminex multiplex immunoassay technology. Distribution of biomarkers between groups and association with disease severity and outcomes were analyzed. Results: The biomarker profiles between the three groups differed significantly with elevation of specific proteins specific for the respective conditions. Several biomarkers correlated significantly with disease severity and death. Uniform manifold approximation and projection (UMAP) analysis revealed a significant separation of the three disease groups and separated between survivors and deceased patients. Different models were developed to predict mortality based on the baseline measurements of several protein markers. A score combining IL-1ra, IL-8, IL-10, MCP-1, SCF and CA-9 was associated with significantly higher mortality (AUC 0.929). Discussion: Several newly identified blood markers were significantly increased in patients with severe COVID-19 (AAT, EN-RAGE, myoglobin, SAP, TIMP-1, vWF, decorin) or in patients that died (IL-1ra, IL-8, IL-10, MCP-1, SCF, CA-9). The use of established assay technologies allows for rapid translation into clinical practice.
    • Towards the sustainable discovery and development of new antibiotics.

      Miethke, Marcus; Pieroni, Marco; Weber, Tilmann; Brönstrup, Mark; Hammann, Peter; Halby, Ludovic; Arimondo, Paola B; Glaser, Philippe; Aigle, Bertrand; Bode, Helge B; et al. (Springer Nature, 2021-08-19)
      An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.
    • Rational construction of genome-reduced Burkholderiales chassis facilitates efficient heterologous production of natural products from proteobacteria.

      Liu, Jiaqi; Zhou, Haibo; Yang, Zhiyu; Wang, Xue; Chen, Hanna; Zhong, Lin; Zheng, Wentao; Niu, Weijing; Wang, Sen; Ren, Xiangmei; et al. (Nature publishing group, 2021-07-23)
      Heterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM 7029 exhibits potential for heterologous BGC expression, but its cells undergo early autolysis, hindering further applications. Herein, we rationally construct DC and DT series genome-reduced S. brevitalea mutants by sequential deletions of endogenous BGCs and the nonessential genomic regions, respectively. The DC5 to DC7 mutants affect growth, while the DT series mutants show improved growth characteristics with alleviated cell autolysis. The yield improvements of six proteobacterial natural products and successful identification of chitinimides from Chitinimonas koreensis via heterologous expression in DT mutants demonstrate their superiority to wild-type DSM 7029 and two commonly used Gram-negative chassis Escherichia coli and Pseudomonas putida. Our study expands the panel of Gram-negative chassis and facilitates the discovery of natural products by heterologous expression.
    • Zebrafish: An Attractive Model to Study Staphylococcus aureus Infection and Its Use as a Drug Discovery Tool.

      Rasheed, Sari; Fries, Franziska; Müller, Rolf; Herrmann, Jennifer; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MDPI, 2021-06-21)
      Non-mammalian in vivo disease models are particularly popular in early drug discovery. Zebrafish (Danio rerio) is an attractive vertebrate model, the success of which is driven by several advantages, such as the optical transparency of larvae, the small and completely sequenced genome, the small size of embryos and larvae enabling high-throughput screening, and low costs. In this review, we highlight zebrafish models of Staphyloccoccus aureus infection, which are used in drug discovery and for studying disease pathogenesis and virulence. Further, these infection models are discussed in the context of other relevant zebrafish models for pharmacological and toxicological studies as part of early drug profiling. In addition, we examine key differences to commonly applied models of S.aureus infection based on invertebrate organisms, and we compare their frequency of use in academic research covering the period of January 2011 to January 2021.
    • Insights into evolution and coexistence of the colibactin- and yersiniabactin secondary metabolite determinants in enterobacterial populations.

      Wami, Haleluya; Wallenstein, Alexander; Sauer, Daniel; Stoll, Monika; von Bünau, Rudolf; Oswald, Eric; Müller, Rolf; Dobrindt, Ulrich; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Microbiology Society, 2021-06-15)
      The bacterial genotoxin colibactin interferes with the eukaryotic cell cycle by causing dsDNA breaks. It has been linked to bacterially induced colorectal cancer in humans. Colibactin is encoded by a 54 kb genomic region in Enterobacteriaceae. The colibactin genes commonly co-occur with the yersiniabactin biosynthetic determinant. Investigating the prevalence and sequence diversity of the colibactin determinant and its linkage to the yersiniabactin operon in prokaryotic genomes, we discovered mainly species-specific lineages of the colibactin determinant and classified three main structural settings of the colibactin-yersiniabactin genomic region in Enterobacteriaceae. The colibactin gene cluster has a similar but not identical evolutionary track to that of the yersiniabactin operon. Both determinants could have been acquired on several occasions and/or exchanged independently between enterobacteria by horizontal gene transfer. Integrative and conjugative elements play(ed) a central role in the evolution and structural diversity of the colibactin-yersiniabactin genomic region. Addition of an activating and regulating module (clbAR) to the biosynthesis and transport module (clbB-S) represents the most recent step in the evolution of the colibactin determinant. In a first attempt to correlate colibactin expression with individual lineages of colibactin determinants and different bacterial genetic backgrounds, we compared colibactin expression of selected enterobacterial isolates in vitro. Colibactin production in the tested Klebsiella species and Citrobacter koseri strains was more homogeneous and generally higher than that in most of the Escherichia coli isolates studied. Our results improve the understanding of the diversity of colibactin determinants and its expression level, and may contribute to risk assessment of colibactin-producing enterobacteria.
    • Structure and biosynthesis of sorangipyranone - a new γ-dihydropyrone from the myxobacterial strain MSr12020.

      Okoth, Dorothy A; Hug, Joachim J; Mándi, Attila; Kurtán, Tibor; Garcia, Ronald; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Oxford Academic, 2021-05-18)
      Sorangipyranone was isolated as a novel natural product featuring a unique 2,3-dihydro-γ-4H-pyrone scaffold from cultures of the myxobacterial strain MSr12020. We report here the full structure elucidation of sorangipyranone by spectroscopic techniques including 2D NMR and high-resolution mass spectrometry together with the analysis of the biosynthetic pathway. Determination of the absolute configuration was performed by time-dependent density functional theory-electronic circular dichroism calculations and determination of the applicability of the Snatzke's helicity rule, to correlate the high-wavelength n→π* electronic circular dichroism (ECD) transition and the absolute configuration of the 2,3-dihydro-4H-γ-pyrone, was done by the analysis of low-energy conformers and the Kohn-Sham orbitals. Sorangipyranone outlines a new class of a γ-dihydropyrone-containing natural product comprised of malonyl-CoA-derived building blocks and features a unique polyketide scaffold. In silico analysis of the genome sequence of the myxobacterial strain MSr12020 complemented with feeding experiments employing stable isotope-labeled precursors allowed the identification and annotation of a candidate biosynthetic gene cluster that encodes a modular polyketide synthase assembly line. A model for the biosynthetic pathway leading to the formation of the γ-dihydropyrone scaffold is presented in this study.
    • miRMaster 2.0: multi-species non-coding RNA sequencing analyses at scale.

      Fehlmann, Tobias; Kern, Fabian; Laham, Omar; Backes, Christina; Solomon, Jeffrey; Hirsch, Pascal; Volz, Carsten; Müller, Rolf; Keller, Andreas; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Oxford University Press, 2021-04-19)
      Analyzing all features of small non-coding RNA sequencing data can be demanding and challenging. To facilitate this process, we developed miRMaster. After the analysis of over 125 000 human samples and 1.5 trillion human small RNA reads over 4 years, we present miRMaster 2 with a wide range of updates and new features. We extended our reference data sets so that miRMaster 2 now supports the analysis of eight species (e.g. human, mouse, chicken, dog, cow) and 10 non-coding RNA classes (e.g. microRNAs, piRNAs, tRNAs, rRNAs, circRNAs). We also incorporated new downstream analysis modules such as batch effect analysis or sample embeddings using UMAP, and updated annotation data bases included by default (miRBase, Ensembl, GtRNAdb). To accommodate the increasing popularity of single cell small-RNA sequencing data, we incorporated a module for unique molecular identifier (UMI) processing. Further, the output tables and graphics have been improved based on user feedback and new output formats that emerged in the community are now supported (e.g. miRGFF3). Finally, we integrated differential expression analysis with the miRNA enrichment analysis tool miEAA. miRMaster is freely available at https://www.ccb.uni-saarland.de/mirmaster2.
    • Search for the Active Ingredients from a 2-Aminothiazole DMSO Stock Solution with Antimalarial Activity.

      Ropponen, Henni-Karoliina; Bader, Chantal D; Diamanti, Eleonora; Illarionov, Boris; Rottmann, Matthias; Fischer, Markus; Witschel, Matthias; Müller, Rolf; Hirsch, Anna K H; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Wiley-VCH, 2021-04-12)
      Chemical decomposition of DMSO stock solutions is a common incident that can mislead biological screening campaigns. Here, we share our case study of 2-aminothiazole 1, originating from an antimalarial class that undergoes chemical decomposition in DMSO at room temperature. As previously measured biological activities observed against Plasmodium falciparum NF54 and for the target enzyme PfIspE were not reproducible for a fresh batch, we tackled the challenge to understand where the activity originated from. Solvent- and temperature-dependent studies using HRMS and NMR spectroscopy to monitor the decomposition led to the isolation and in vitro evaluation of several fractions against PfIspE. After four days of decomposition, we successfully isolated the oxygenated and dimerised compounds using SFC purification and correlated the observed activities to them. Due to the unstable nature of the two isolates, it is likely that they undergo further decomposition contributing to the overall instability of the compound.
    • Synergizing the potential of bacterial genomics and metabolomics to find novel antibiotics.

      Panter, Fabian; Bader, Chantal D; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Royal Society of Chemistry, 2021-03-29)
      Antibiotic development based on natural products has faced a long lasting decline since the 1970s, while both the speed and the extent of antimicrobial resistance (AMR) development have been severely underestimated. The discovery of antimicrobial natural products of bacterial and fungal origin featuring new chemistry and previously unknown mode of actions is increasingly challenged by rediscovery issues. Natural products that are abundantly produced by the corresponding wild type organisms often featuring strong UV signals have been extensively characterized, especially the ones produced by extensively screened microbial genera such as streptomycetes. Purely synthetic chemistry approaches aiming to replace the declining supply from natural products as starting materials to develop novel antibiotics largely failed to provide significant numbers of antibiotic drug leads. To cope with this fundamental issue, microbial natural products science is being transformed from a 'grind-and-find' study to an integrated approach based on bacterial genomics and metabolomics. Novel technologies in instrumental analytics are increasingly employed to lower detection limits and expand the space of detectable substance classes, while broadening the scope of accessible and potentially bioactive natural products. Furthermore, the almost exponential increase in publicly available bacterial genome data has shown that the biosynthetic potential of the investigated strains by far exceeds the amount of detected metabolites. This can be judged by the discrepancy between the number of biosynthetic gene clusters (BGC) encoded in the genome of each microbial strain and the number of secondary metabolites actually detected, even when considering the increased sensitivity provided by novel analytical instrumentation. In silico annotation tools for biosynthetic gene cluster classification and analysis allow fast prioritization in BGC-to-compound workflows, which is highly important to be able to process the enormous underlying data volumes. BGC prioritization is currently accompanied by novel molecular biology-based approaches to access the so-called orphan BGCs not yet correlated with a secondary metabolite. Integration of metabolomics, in silico genomics and molecular biology approaches into the mainstream of natural product research will critically influence future success and impact the natural product field in pharmaceutical, nutritional and agrochemical applications and especially in anti-infective research.
    • In vivo and in vitro reconstitution of unique key steps in cystobactamid antibiotic biosynthesis.

      Groß, Sebastian; Schnell, Bastien; Haack, Patrick A; Auerbach, David; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Nature Research, 2021-03-16)
      Cystobactamids are myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity. They are formed by a non-ribosomal peptide synthetase (NRPS) and consist of tailored para-aminobenzoic acids, connected by a unique α-methoxy-L-isoasparagine or a β-methoxy-L-asparagine linker moiety. We describe the heterologous expression of the cystobactamid biosynthetic gene cluster (BGC) in Myxococcus xanthus. Targeted gene deletions produce several unnatural cystobactamids. Using in vitro experiments, we reconstitute the key biosynthetic steps of linker formation and shuttling via CysB to the NRPS. The biosynthetic logic involves a previously uncharacterized bifunctional domain found in the stand-alone NRPS module CysH, albicidin biosynthesis and numerous BGCs of unknown natural products. This domain performs either an aminomutase (AM) or an amide dehydratase (DH) type of reaction, depending on the activity of CysJ which hydroxylates CysH-bound L-asparagine. Furthermore, CysQ O-methylates hydroxyl-L-(iso)asparagine only in the presence of the AMDH domain. Taken together, these findings provide direct evidence for unique steps in cystobactamid biosynthesis.
    • Natural products in drug discovery: advances and opportunities.

      Atanasov, Atanas G; Zotchev, Sergey B; Dirsch, Verena M; Supuran, Claudiu T; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer Nature, 2021-01-28)
      Natural products and their structural analogues have historically made a major contribution to pharmacotherapy, especially for cancer and infectious diseases. Nevertheless, natural products also present challenges for drug discovery, such as technical barriers to screening, isolation, characterization and optimization, which contributed to a decline in their pursuit by the pharmaceutical industry from the 1990s onwards. In recent years, several technological and scientific developments - including improved analytical tools, genome mining and engineering strategies, and microbial culturing advances - are addressing such challenges and opening up new opportunities. Consequently, interest in natural products as drug leads is being revitalized, particularly for tackling antimicrobial resistance. Here, we summarize recent technological developments that are enabling natural product-based drug discovery, highlight selected applications and discuss key opportunities.
    • Expanding the Scope of Detectable Microbial Natural Products by Complementary Analytical Methods and Cultivation Systems.

      Bader, Chantal D; Haack, Patrick A; Panter, Fabian; Krug, Daniel; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Chemical Society (ACS), 2021-01-15)
      Outer membrane vesicles (OMVs) are universally produced by prokaryotes and play important roles in symbiotic and pathogenic interactions. They often contain DNA, but a mechanism for its incorporation is lacking. Here, we show that Dinoroseobacter shibae, a dinoflagellate symbiont, constitutively secretes OMVs containing DNA. Time-lapse microscopy captured instances of multiple OMV production at the septum during cell division. DNA from the vesicle lumen was up to 22-fold enriched for the region around the terminus of replication (ter). The peak of coverage was located at dif, a conserved 28-bp palindromic sequence required for binding of the site-specific tyrosine recombinases XerC/XerD. These enzymes are activated at the last stage of cell division immediately prior to septum formation when they are bound by the divisome protein FtsK. We suggest that overreplicated regions around the terminus have been repaired by the FtsK-dif-XerC/XerD molecular machinery. The vesicle proteome was clearly dominated by outer membrane and periplasmic proteins. Some of the most abundant vesicle membrane proteins were predicted to be required for direct interaction with peptidoglycan during cell division (LysM, Tol-Pal, Spol, lytic murein transglycosylase). OMVs were 15-fold enriched for the saturated fatty acid 16:00. We hypothesize that constitutive OMV secretion in D. shibae is coupled to cell division. The footprint of the FtsK-dif-XerC/XerD molecular machinery suggests a novel potentially highly conserved route for incorporation of DNA into OMVs. Clearing the division site from small DNA fragments might be an important function of vesicles produced during exponential growth under optimal conditions.IMPORTANCE Gram-negative bacteria continually form vesicles from their outer membrane (outer membrane vesicles [OMVs]) during normal growth. OMVs frequently contain DNA, and it is unclear how DNA can be shuffled from the cytoplasm to the OMVs. We studied OMV cargo in Dinoroseobacter shibae, a symbiont of dinoflagellates, using microscopy and a multi-omics approach. We found that vesicles formed during undisturbed exponential growth contain DNA which is enriched for genes around the replication terminus, specifically, the binding site for an enzyme complex that is activated at the last stage of cell division. We suggest that the enriched genes are the result of overreplication which is repaired by their excision and excretion via membrane vesicles to clear the divisome from waste DNA.
    • Kibdelosporangium persicum sp. nov., a new member of the Actinomycetes from a hot desert in Iran.

      Safaei, Nasim; Nouioui, Imen; Mast, Yvonne; Zaburannyi, Nestor; Rohde, Manfred; Schumann, Peter; Müller, Rolf; Wink, Joachim; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.;HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Microbiology Society, 2021-01-11)
      Isolate 4NS15T was isolated from a neglected arid habitat in Kerman, Iran. The strain showed 16S rRNA gene sequence similarity values of 98.9 % to the type strains of Kibdelosporangium aridum subsp. aridum, Kibdelosporangium phytohabitans and Kibdelosporangium philippinense and 98.6 % to the type strain K. aridum subsp. largum, respectively. Genome-based phylogenetic analysis revealed that isolate 4NS15T is closely related to Kibdelosporangium aridum subsp. aridum DSM 43828T. The digital DNA-DNA hybridization value between the genome sequences of 4NS15T and strain DSM 43828T is 29.8 %. Strain 4NS15T produces long chains of spores without a sporangium-like structure which can be distinguished from other Kibdelosporangium species. Isolate 4NS15T has a genome size of 10.35 Mbp with a G+C content of 68.1 mol%. Whole-cell hydrolysates of isolate 4NS15T are rich in meso-diaminopimelic acid and cell-wall sugars such as arabinose, galactose, glucose and ribose. Major fatty acids (>10 %) are C16 : 0, iso-C16 : 0 and iso-C15 : 0. The phospholipid profile contains diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylhydroxyethanolamine, aminolipid and glycoaminolipid. The predominant menaquinone is MK-9(H4). Based on its phenotypic and genotypic characteristics, isolate 4NS15T (NCCB 100701=CIP 111705=DSM 110728) merits recognition as representing a novel species of the genus Kibdelosporangium, for which the name Kibdelosporangium persicum sp. nov. is proposed.
    • Heterologous expression of the atypical tetracycline chelocardin reveals the full set of genes required for its biosynthesis.

      Lukežič, Tadeja; Pikl, Špela; Zaburannyi, Nestor; Remškar, Maja; Petković, Hrvoje; Müller, Rolf; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (BMC (part of Springer), 2020-12-19)
      Background: Chelocardin (CHD) exhibits a broad-spectrum antibiotic activity and showed promising results in a small phase II clinical study conducted on patients with urinary tract infections. Importantly, CHD was shown to be active also against tetracycline-resistant Gram-negative pathogens, which is gaining even more importance in today’s antibiotic crisis. We have demonstrated that modifications of CHD through genetic engineering of its producer, the actinomycete Amycolatopsis sulphurea, are not only possible but yielded even more potent antibiotics than CHD itself, like 2-carboxamido-2-deacetyl-chelocardin (CD-CHD), which is currently in preclinical evaluation. A. sulphurea is difficult to genetically manipulate and therefore manipulation of the chd biosynthetic gene cluster in a genetically amenable heterologous host would be of high importance for further drug-discovery efforts. Results: We report heterologous expression of the CHD biosynthetic gene cluster in the model organism Streptomyces albus del14 strain. Unexpectedly, we found that the originally defined CHD gene cluster fails to provide all genes required for CHD formation, including an additional cyclase and two regulatory genes. Overexpression of the putative pathway-specific streptomyces antibiotic regulatory protein chdB in A. sulphurea resulted in an increase of both, CHD and CD-CHD production. Applying a metabolic-engineering approach, it was also possible to generate the potent CHD analogue, CD-CHD in S. albus. Finally, an additional yield increase was achieved in S. albus del14 by in-trans overexpression of the chdR exporter gene, which provides resistance to CHD and CDCHD. Conclusions: We identified previously unknown genes in the CHD cluster, which were shown to be essential for chelocardin biosynthesis by expression of the full biosynthetic gene cluster in S. albus as heterologous host. When comparing to oxytetracycline biosynthesis, we observed that the CHD gene cluster contains additional enzymes not found in gene clusters encoding the biosynthesis of typical tetracyclines (such as oxytetracycline). This finding probably explains the different chemistries and modes of action, which make CHD/CD-CHD valuable lead structures for clinical candidates. Even though the CHD genes are derived from a rare actinomycete A. sulphurea, the yield of CHD in the heterologous host was very good. The corrected nucleotide sequence of the CHD gene cluster now contains allgene products required for the production of CHD in a genetically amenable heterologous host, thus opening new possibilities towards production of novel and potent tetracycline analogues with a new mode of action.
    • Corallopyronin A for short-course anti-wolbachial, macrofilaricidal treatment of filarial infections.

      Schiefer, Andrea; Hübner, Marc P; Krome, Anna; Lämmer, Christine; Ehrens, Alexandra; Aden, Tilman; Koschel, Marianne; Neufeld, Helene; Chaverra-Muñoz, Lillibeth; Jansen, Rolf; et al. (PLOS, 2020-12-07)
      Current efforts to eliminate the neglected tropical diseases onchocerciasis and lymphatic filariasis, caused by the filarial nematodes Onchocerca volvulus and Wuchereria bancrofti or Brugia spp., respectively, are hampered by lack of a short-course macrofilaricidal-adult-worm killing-treatment. Anti-wolbachial antibiotics, e.g. doxycycline, target the essential Wolbachia endosymbionts of filariae and are a safe prototype adult-worm-sterilizing and macrofilaricidal regimen, in contrast to standard treatments with ivermectin or diethylcarbamazine, which mainly target the microfilariae. However, treatment regimens of 4-5 weeks necessary for doxycycline and contraindications limit its use. Therefore, we tested the preclinical anti-Wolbachia drug candidate Corallopyronin A (CorA) for in vivo efficacy during initial and chronic filarial infections in the Litomosoides sigmodontis rodent model. CorA treatment for 14 days beginning immediately after infection cleared >90% of Wolbachia endosymbionts from filariae and prevented development into adult worms. CorA treatment of patently infected microfilaremic gerbils for 14 days with 30 mg/kg twice a day (BID) achieved a sustained reduction of >99% of Wolbachia endosymbionts from adult filariae and microfilariae, followed by complete inhibition of filarial embryogenesis resulting in clearance of microfilariae. Combined treatment of CorA and albendazole, a drug currently co-administered during mass drug administrations and previously shown to enhance efficacy of anti-Wolbachia drugs, achieved microfilarial clearance after 7 days of treatment at a lower BID dose of 10 mg/kg CorA, a Human Equivalent Dose of 1.4 mg/kg. Importantly, this combination led to a significant reduction in the adult worm burden, which has not yet been published with other anti-Wolbachia candidates tested in this model. In summary, CorA is a preclinical candidate for filariasis, which significantly reduces treatment times required to achieve sustained Wolbachia depletion, clearance of microfilariae, and inhibition of embryogenesis. In combination with albendazole, CorA is robustly macrofilaricidal after 7 days of treatment and fulfills the Target Product Profile for a macrofilaricidal drug.