Inter-kingdom communication in the marine environment: bacterial modulation of growth, sexual reproduction and biofilm formation of marine microalgae

Mikroalgen, und insbesondere Kieselalgen, spielen eine grundlegende ökologische Rolle und haben ein breites Feld von industriellen und technologischen Anwendungen. In den letzten Jahren hat sich die Forschung in der chemischen Ökologie und Biotechnologie auf molekulare Mechanismen, die die Wechselwirkung zwischen Algen und Bakterien regulieren, fokussiert. Diese Dissertation untersucht die bakterielle Beeinflussung der sexuellen Fortpflanzung und des Wachstumes der pennaten Modelldiatomee Seminavis robusta. Durch die Kombination von Metabolomics und Transcriptomics-Ansatz, sowie physiologische Bioassays und gezielte Analysen, konnten wir neuartige Wechselbeziehung zwischen Algen und Bakterien entdecken. Die mit S. robusta Paarungstyp (MT-) assoziierten Bakterien haben verschiedene Einflüsse auf die sexuelle Fortpflanzung von Diatomeen. Bakterien haben ein besonderer Einfluss auf dem Aminosäuremetabolismus des S. robustas und die lösen eine Oxidative Stressreaktion in Diatomeen aus. Weiterhin, die beeinflussen das Wachstum und das Metabolismus von einer anderen Mikroalge, Nannochloropsis sp., aber die induzieren keine Biofilmproduktion in Bioreaktoren. Diese Erkenntnisse sind wichtig, um die herausragende Bedeutung der Algen-Bakterien-Kommunikation unter der Perspektive der Algenkultivierung und -produktion zu verstehen. Diese könnte den Einsatz von mikrobiologischen Gemeinschaften mit erhöhter Produktivität in Bioreaktoren fördern. Um durch Wasser übertragene Signalmoleküle besser zu erfinden, entwickelten wir schließlich eine neue, zerstörungsfreie Methode für die Extraktion von primär- und Sekundärmetaboliten von Algenoberflächen. Die Ergebnisse dieser Doktorarbeit zeigen neue Erkenntnisse in dem relativ jungen Gebiet der Mikroalgen-Bakterien-Wechselwirkungen, indem verschiedene Ansätze (-omische Techniken, physiologische Bioassays, innovative analytische Verfahren) in einen breiten interdisziplinären Ansatz angewendet und miteinander verknüpft wurden

Disruption-free Solid Phase Extraction of Surface Metabolites from Macroalgae

This is an Accepted Manuscript of a book chapter published by CRC Press in Protocols for Macroalgae Research on 28.04.2018, available online: https://www.crcpress.com/Protocols-for-Macroalgae-Research/Charrier-Wichard-Reddy/p/book/9781498796422 The surface chemistry of aquatic organisms is decisive for their biotic interactions. Metabolites in the spatially limited laminar boundary layer mediate processes, such as fouling, allelopathy and chemical defense against herbivores. However, very few methods are available for the investigation of such surface metabolites. Here we give a detailed protocol in which surfaces are extracted by means of C18 solid phase material, elution of the solid phase extraction material with solvent and analysis via liquid chromatography / mass spectrometry (LC/MS) and/or gas chromatography / mass spectrometry (GC/MS). The protocol introduced here is based on a previous publication (Cirri et al. 2016) where validation is described. The method is robust, picks up metabolites of a broad polarity range and is easy to handle. It was developed for the macroalgae Fucus vesiculosus, Caulerpa taxifolia and Gracilaria vermiculophylla, but can be easily transferred to other algae and to other aquatic organisms in general

Associated bacteria affect sexual reproduction by altering gene expression and metabolic processes in a biofilm inhabiting diatom

Diatoms are unicellular algae with a fundamental role in global biogeochemical cycles as major primary producers at the base of aquatic food webs. In recent years, chemical communication between diatoms and associated bacteria has emerged as a key factor in diatom ecology, spurred by conceptual and technological advancements to study the mechanisms underlying these interactions. Here, we use a combination of physiological, transcriptomic, and metabolomic approaches to study the influence of naturally coexisting bacteria, Maribacter sp. and Roseovarius sp., on the sexual reproduction of the biofilm inhabiting marine pennate diatom Seminavis robusta. While Maribacter sp. severely reduces the reproductive success of S. robusta cultures, Roseovarius sp. slightly enhances it. Contrary to our expectation, we demonstrate that the effect of the bacterial exudates is not caused by altered cell-cycle regulation prior to the switch to meiosis. Instead, Maribacter sp. exudates cause a reduced production of diproline, the sexual attraction pheromone of S. robusta. Transcriptomic analyses show that this is likely an indirect consequence of altered intracellular metabolic fluxes in the diatom, especially those related to amino acid biosynthesis, oxidative stress response, and biosynthesis of defense molecules. This study provides the first insights into the influence of bacteria on diatom sexual reproduction and adds a new dimension to the complexity of a still understudied phenomenon in natural diatom populations

Molecular characterization of hematopoietic stem cells after in vitro amplification on biomimetic 3D PDMS cell culture scaffolds

Hematopoietic stem cell (HSC) transplantation is successfully applied since the late 1950s. However, its efficacy can be impaired by insufficient numbers of donor HSCs. A promising strategy to overcome this hurdle is the use of an advanced ex vivo culture system that supports the proliferation and, at the same time, maintains the pluripotency of HSCs. Therefore, we have developed artificial 3D bone marrow-like scaffolds made of polydimethylsiloxane (PDMS) that model the natural HSC niche in vitro. These 3D PDMS scaffolds in combination with an optimized HSC culture medium allow the amplification of high numbers of undifferentiated HSCs. After 14 days in vitro cell culture, we performed transcriptome and proteome analysis. Ingenuity pathway analysis indicated that the 3D PDMS cell culture scaffolds altered PI3K/AKT/mTOR pathways and activated SREBP, HIF1α and FOXO signaling, leading to metabolic adaptations, as judged by ELISA, Western blot and metabolic flux analysis. These molecular signaling pathways can promote the expansion of HSCs and are involved in the maintenance of their pluripotency. Thus, we have shown that the 3D PDMS scaffolds activate key molecular signaling pathways to amplify the numbers of undifferentiated HSCs ex vivo effectively

Proteomic analysis of peripheral nerve myelin during murine aging

Aging of the peripheral nervous system (PNS) is associated with structural and functional changes that lead to a reduction in regenerative capacity and the development of age-related peripheral neuropathy. Myelin is central to maintaining physiological peripheral nerve function and differences in myelin maintenance, degradation, formation and clearance have been suggested to contribute to age-related PNS changes. Recent proteomic studies have elucidated the complex composition of the total myelin proteome in health and its changes in neuropathy models. However, changes in the myelin proteome of peripheral nerves during aging have not been investigated. Here we show that the proteomes of myelin fractions isolated from young and old nerves show only subtle changes. In particular, we found that the three most abundant peripheral myelin proteins (MPZ, MBP, and PRX) do not change in old myelin fractions. We also show a tendency for high-abundance myelin proteins other than these three to be downregulated, with only a small number of ribosome-related proteins significantly downregulated and extracellular matrix proteins such as collagens upregulated. In addition, we illustrate that the peripheral nerve myelin proteome reported in this study is suitable for assessing myelin degradation and renewal during peripheral nerve degeneration and regeneration. Our results suggest that the peripheral nerve myelin proteome is relatively stable and undergoes only subtle changes in composition during mouse aging. We proffer the resultant dataset as a resource and starting point for future studies aimed at investigating peripheral nerve myelin during aging. Said datasets are available in the PRIDE archive under the identifier PXD040719 (aging myelin proteome) and PXD041026 (sciatic nerve injury proteome)

Biofilm interactions-bacteria modulate sexual reproduction success of the diatom Seminavis robusta

Marine biofilms are complex multi-species communities where chemical signaling regulates a substantial share of interactions. The involved natural products represent targets for competition strategies by signal interference. Diatoms, that often dominate biofilms, rely on a complex pheromone system during sexual reproduction, involving synchronizing and attracting metabolites. The present study addresses the effect of bacteria on sexual reproduction of the model pennate diatom Seminavis robusta. We observe that sexual reproduction is most efficient under axenic conditions. Bacteria isolated from field collected biofilms modulate sexual reproduction in the algae. A species-specific effect on the diatom mating efficiency could be observed, with Maribacter sp. and Marinobacter sp. significantly reducing the sexual reproduction rate. Spent medium from these bacteria has the same effect, demonstrating that chemically mediated cross kingdom interactions take place. In contrast, proportion of auxospores. We further observed a lower concentration of the diatom attraction pheromone diproline in the presence of bacteria compared to axenic conditions. In agreement, the Seminavis-associated bacterial community as well as isolated bacterial strains degraded the pheromone over time. Our results highlight that the pheromone system of diatoms is subject to interference strategies of the associated bacterial community by modulation of the signal landscape

A solid phase extraction based non-disruptive sampling technique to investigate the surface chemistry of macroalgae

The surface chemistry of aquatic organisms determines their biotic interactions. Metabolites in the spatially limited laminar boundary layer mediate processes, such as antifouling, allelopathy and chemical defense against herbivores. However, very few methods are available for the investigation of such surface metabolites. An approach is described in which surfaces are extracted by means of C18 solid phase material. By powdering wet algal surfaces with this material, organic compounds are adsorbed and can be easily recovered for subsequent liquid chromatography/mass spectrometry (LC/MS) and gas chromatography/mass spectrometry (GC/MS) investigations. The method is robust, picks up metabolites of a broad polarity range and is easy to handle. It is more universal compared to established solvent dipping protocols and it does not cause damage to the test organisms. A protocol is introduced for the macroalgae Fucus vesiculosus, Caulerpa taxifolia and Gracilaria vermiculophylla, but it can be easily transferred to other aquatic organisms

Multi-omics analysis identifies RFX7 targets involved in tumor suppression and neuronal processes

Recurrently mutated in lymphoid neoplasms, the transcription factor RFX7 is emerging as a tumor suppressor. Previous reports suggested that RFX7 may also have a role in neurological and metabolic disorders. We recently reported that RFX7 responds to p53 signaling and cellular stress. Furthermore, we found RFX7 target genes to be dysregulated in numerous cancer types also beyond the hematological system. However, our understanding of RFX7's target gene network and its role in health and disease remains limited. Here, we generated RFX7 knock-out cells and employed a multi-omics approach integrating transcriptome, cistrome, and proteome data to obtain a more comprehensive picture of RFX7 targets. We identify novel target genes linked to RFX7's tumor suppressor function and underscoring its potential role in neurological disorders. Importantly, our data reveal RFX7 as a mechanistic link that enables the activation of these genes in response to p53 signaling

Extensive remodeling of the extracellular matrix during aging contributes to age-dependent impairments of muscle stem cell functionality

During aging, the regenerative capacity of skeletal muscle decreases due to intrinsic changes in muscle stem cells (MuSCs) and alterations in their niche. Here, we use quantitative mass spectrometry to characterize intrinsic changes in the MuSC proteome and remodeling of the MuSC niche during aging. We generate a network connecting age-affected ligands located in the niche and cell surface receptors on MuSCs. Thereby, we reveal signaling by integrins, Lrp1, Egfr, and Cd44 as the major cell communication axes perturbed through aging. We investigate the effect of Smoc2, a secreted protein that accumulates with aging, primarily originating from fibro-adipogenic progenitors. Increased levels of Smoc2 contribute to the aberrant Integrin beta-1 (Itgb1)/mitogen-activated protein kinase (MAPK) signaling observed during aging, thereby causing impaired MuSC functionality and muscle regeneration. By connecting changes in the proteome of MuSCs to alterations of their niche, our work will enable a better understanding of how MuSCs are affected during aging