O 03
Increasing the cell surface expression of the adenovirus protein E3/49K to optimize its immunomodulatory function for potential therapeutic applications

Presenting author:

Aichane.Khadija@mh-hannover.de

Author(s):
Khadija Aichane, Mark Windheim

The early transcription unit 3 (E3) of human adenoviruses encodes immunomodulatory proteins, such as E3/49K. This protein is proteolytically processed at the cell surface by matrix metalloproteases. Its N-terminal ectodomain sec49K is released and interferes with leukocyte activation through binding to receptor-like protein tyrosine phosphatase CD45. Thus, E3/49K-mediated immunomodulation may provide a therapeutic strategy to prevent graft rejection after transplantation. Indeed, the expression of E3/49K by porcine cells was shown to reduce human anti-pig immune responses in vitro. It remains elusive whether secreted sec49K or membrane-anchored E3/49K or both are critical for the immunomodulatory function of E3/49K. We sought to generate an E3/49K mutant that is not proteolytically processed and could be more effective in reducing cell-mediated immune responses due to increased cell surface expression. We analyzed proteolytic cleavage sites, generated deletion mutants and investigated their effect on E3/49K cleavage with recombinant ADAM10 in vitro and in A549 and HEK293T cells. By replacing 35 juxtamembrane amino acid residues with a glycine-serine linker, proteolytic processing was reduced by ~96% in HEK293T cells. This mutation combined with a deletion of the C-terminal tail increased cell surface expression twofold. In sum, an E3/49K mutant with a strongly reduced proteolytic processing and an increased cell surface expression was generated.

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O 24
Just a side product? Tracking of 6S RNA activity in biofilms and microbiomes using product RNAs

Presenting author:

Philipps-Universität Marburg, SYNMIKRO, Karl-von-Frisch-Str. 14, 35032 Marburg [DE], alanati@staff.uni-marburg.de

Author(s):
Bayan Al-Anati, Michael Vockenhuber, Marcus Lechner

Bacterial 6S RNA that tunes the metabolism of cells by a regulation of transcription via binding to the active site of RNA polymerase holoenzymes.Typically encoded by a single gene. Bacillus subtilis encodes two paralogs. Deletion mutants of the undomesticated B. subtilis wild-type strain NCIB 3610 showed increased biofilm formation on solid media, the ability to form surface-attached biofilms in liquid culture and retarded swarming activity and earlier spore formation. 6S RNA also serves as a template for short transcripts, so called product RNAs, with a length of usually 7-15 nt

We hypothesize that product RNAs fulfill a cellular function that has yet to be identified. Notably, a number of mRNA products of genes that are affected by a frame shift in the laboratory strains 168 and PY79 of B. subtilis compared to the wild-type strain show reverse complementary regions to product RNAs. We aim to bioinformatically track this observation throughout the class of Bacilli and identity potential patterns. The presence of product RNAs can be used as an indicator of 6S RNA activity. Given its short length, an identification via blotting or sequencing proved ineffective. A synthetic riboswitch-based reporter system will be constructed to deferentially visualize 6S RNA activity in biofilms and microbiomes. In parallel, single-cell sequencing will enable us to track the differentiation of sub populations within bacterial communities comparing wild-type strain with 6S RNA deletion mutants.

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I 01
PUL-dependent cross-feeding mechanisms between the gut commensal Bacteroides thetaiotaomicron and the enteric pathogen Salmonella Typhimurium

Presenting author:

Helmholtz Institute for RNA-based Infection Research (HIRI), Host-Pathogen-Microbiota Interactions, Josef-Schneider-Straße 2/D15, 97080 Würzburg [DE], Lena.Amend@helmholtz-hiri.de

Author(s):
Lena Amend, Meina Neumann-Schaal, Alexander J. Westermann

The gut microbiota provides colonization resistance against pathogenic invaders. However, under conditions when this microbial ecosystem is perturbed, gut commensals can provide nutrients to enteric pathogens. For example, metabolites released during breakdown of complex glycans by Bacteroides—the dominant genus in the human intestine—are hypothesized to serve as nutrients for pathogens, facilitating their establishment in the gut. Consequently, targeting polysaccharide degradation in Bacteroides constitutes a promising therapeutic approach to intervene with pathogen invasion.

Here, we dissect cross-feeding mechanisms between Bacteroides thetaiotaomicron (B. theta) and the model enteropathogen Salmonella Typhimurium (S. Tm). We examined the in vitro growth of B. theta and gene expression of polysaccharide utilization loci (PULs) during cultivation in defined medium supplemented with diverse dietary- or host-derived glycans. In contrast to B. theta, S. Tm was unable to thrive on most of the glycans when provided as sole carbon source. Strikingly however, co-culture and spent media assays revealed glycan pre-degradation by B. theta to support S. Tm outgrowth. Using transposon-insertion mutants of B. theta, we identified PUL genes responsible for cross-feeding. In the future, we will pinpoint PUL-derived metabolites that constitute substrates for S. Tm. Together, our data suggest a metabolic interplay, in which B. theta can facilitate glycan utilization by S. Tm.

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M 06
Unraveling the role of microbiome-immune dysregulation in early onset chronic kidney disease

Presenting author:

Charité, ECRC, Lindenberger Weg 80, 13125 Berlin [DE], harithaa.anandakumar@charite.de

Author(s):
Harithaa Anandakumar, Johannes Holle, Julia Schlender, Valentin Vecera, Nicola Wilck, Hendrik Bartolomaeus, Anne Dueck

Chronic kidney disease (CKD) causes systemic issues including inflammation, cardiovascular dysfunction, and bone disease. A systems biology approach using omics techniques can provide insights into CKD pathophysiology to inform prevention and therapy. Gut microbiome dysbiosis is described in CKD, but comorbidities in adults limit disease-specific conclusions. Studying pediatric CKD could reveal mechanisms specific to CKD due to less comorbidity. We assessed cardiovascular phenotype, nutrition, fecal metagenomics, and immunome profiling in children with CKD (n=17), on hemodialysis (HD, n=11), and healthy controls (HC, n=10). CKD children exhibited cardiovascular dysregulation. Immune profiling showed broad alterations, confirming previous mucosa-associated invariant T cell and regulatory T cell changes. An inflammation-targeted proteomic panel revealed 55 dysregulated proteins out of 92. Microbiome analysis indicated diversity, compositional, and functional changes. strain-level differences in F. prausnitzii were noted. Transplanting microbiota from HD and HC into germ-free mice promoted immune aging signs and endothelial dysfunction. Despite lack of adult cardiovascular risk factors, pediatric CKD caused measurable cardiovascular changes. Our analysis demonstrated a strong inflammatory immune cell profile. Fecal transplantation provided preliminary evidence for the microbiota's causal role in CKD inflammation and cardiovascular risk through the microbiota-immune axis.

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M 17
Elucidating the influence of the human microbiome on the metabolism of pesticides: a case study on tebuconazole

Presenting author:

Bundesinstitut fuer Risikobewertung, 6.NG 1 Nachwuchsgruppe Hautmikrobiom , Max-Dohrn-Straße 8-10, 10589 Berlin [DE], miriam.arlt@bfr.bund.de

Author(s):
Miriam Arlt, Tessa Höper, Tewes Tralau, Lisa Lemoine

The human microbiome comprises a huge metabolic capacity which by far exceeds the human metagenome as well as the biochemical pathways therein. However, microbiome-mediated metabolization of xenobiotics is currently not taken into account during risk assessment although previous studies reported on the possibility of toxicity modulation.

It is well known that pesticides affect the function and composition of the gut microbiota. Besides the intestinal microbiota, also the skin microbiota is able to metabolize xenobiotics and thus influence toxification. In a comparative study, the metabolism of the triazole fungicide tebuconazole by the human gut and skin microbiota will be investigated. To date 51 bacterial transformation products of tebuconazole have been reported in e.g. soil and water, nonetheless metabolites of the human microbiome are not described, yet.

For this case study, gut microbes obtained from human faeces donations as well as an in situ commensal 3D skin model will be exposed to the fungicide. Based on high-resolution mass spectrometry, databases and prediction tools, a suspect screening will be performed to identify bacterial metabolites. Subsequently, in vitro assays with HaCaT and Caco-2-cells will be performed to compare the toxicity of the parent compound with its metabolites. Consequently, the results are intended to investigate the pesticide’s influence on and its potential toxicity modulation by the human microbiome.

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R 03
Maillard reaction products in the gut: unravelling the metabolism of Nε-carboxymethyllysine (CML) in Escherichia coli

Presenting author:

Ludwig-Maximilians-Universität, Mikrobiologie, Großhaderner Str. 2-4, 82152 Planegg-Martinsried [DE], Erica.Aveta@lmu.de

Author(s):
Erica Flora Aveta, Judith Mehler, Kim Ina Behringer, Nicola Gericke, Marlene Walczak, Patroklos Vougioukas, Michael Hellwig, Jürgen Lassak

Amino acids undergo numerous of enzymatic or non-enzymatic post-translational modifications. N_ε-Carboxymethyllysine (CML) results from the condensation of a reducing sugar with the free amino group of lysine, in the so called Maillard reaction or glycation (Maillard et al., 1912). This non-enzymatic event is responsible for the flavour and colour of thermally processed foods. Up to 11.3 mg of CML per kg of protein are ingested daily by humans, but the limited absorption in the gastrointestinal tract suggests a major role in its degradation by colonic bacteria (Delgato-Andrade et al., 2012; Lassak et al., 2023). When exposing E. coli to CML the predominant degradation product is carboxymethylcadaverine (Hellwig et al., 2019). However, no enzyme in the CML metabolism is known to date. We have now unveiled an unexpected high promiscuity of the ornithine decarboxylase SpeC toward many lysine derivatives including CML. The enzyme is the first in an underground metabolism, enabling E. coli to utilize CML as sole nitrogen source. We further discovered that proton consumption in the CML decarboxylation reaction helps to counteract the mild acid pH in the colon. Investigating the molecular “players” of CML metabolism in E. coli will help understand how glycated amino acids influence the ability of gut bacteria to thrive and compete, and gain insight about the effect those compounds and their degradation products have on our health.

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O 14
PHL7 - Breaking the Plastic Wave from Within the Compost Metagenome

Presenting author:

Leipzig University, Institute of Biochemistry, Brüderstraße 34, 04103 Leipzig [DE], yasmin.aydin@uni-leipzig.de

Author(s):
Yasmin Aydin, Ronny Frank, Christian Sonnendecker

Ecosystems worldwide are on the verge of collapse due to anthropogenic activities. Symptomatically, 350 million tons of plastic waste are generated yearly, with a significant portion mismanaged, resulting in unpredictable environmental damage. Despite efforts to return plastic waste to the material cycle, state-of-the-art segregation methods are too imprecise, rendering over 90% of used plastic unsuitable for high-level recycling.

One solution to this challenge is utilizing high-performance enzymes for chemical recycling. Here, we introduce PHL7, a polyethylene terephthalate (PET) hydrolase derived from a local compost metagenome. Engineered variants of PHL7 exhibit significantly increased activity and thermostability. To diversify our portfolio of high-performing enzymes, we developed a bioelectronic measurement tool capable of automated monitoring of the catalytic activities of numerous candidates. The impedance-based readout is universal for all substrate types and proves to be a robust method for the continuous assessment of plastic film degradation. Furthermore, we present a proof-of-concept batch reactor system for scaling up enzymatic plastic degradation from laboratory scale to a demo plant.

 

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M 09
Common silent and agonistic flagellins display temporally distinct binding profiles in interactions with Toll-Like Receptor 5

Presenting author:

Max Planck Institute for Biology Tübingen, Department of Microbiome Science, Max-Planck-Ring, 5., 72076 Tübingen [DE], michael.bell@tuebingen.mpg.de

Author(s):
Michael Bell, Sara Clasen, Andrea Borbon, Miriam Haag, Ruth Ley

Toll-like receptor 5 (TLR5) is a Pattern Recognition Receptor which targets and responds to conserved sites on bacterial flagellin, the protein subunit of bacterial flagella, which confers motility. TLR5-flagellin binding is widely used as a proxy for immune activity. However, we recently identified a class of flagellins termed “silent”. They contain the TLR5 epitope facilitating binding at the canonical nD1-domain binding site, but elicit a weak immune response.

 

To better characterise the relationship between TLR5-epitope binding and signalling, we determined the relative binding and activity profiles of 116 flagellins, with the TLR5 epitope, against a truncated human TLR5 construct, containing only TLR5-epitope binding site. This revealed a continuum in binding strengths with only a weak correlation to the immune response, decoupling binding and activity. We then explored the binding kinetics of the silent R. hominis FlaB and agonist S. typhimurium FliC flagellins to the truncated TLR5. This revealed temporal differences in their binding profiles, and diverging roles during binding for the ‘PIM’ residues, considered essential for TLR5 binding. Structural analysis was performed to uncover how differences in known binding residues influences their contrasting binding profiles.

 

Our findings reveal the complexity of TLR5-flagellin interactions, and indicate how silent innate immune evasion may be achieved. This could lead to new avenues for developing TLR5-targetted treatments.

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O 06
Single-cell analyses to dissect morphological heterogeneity in the gut commensal Bacteroides thetaiotaomicron

Presenting author:

Department of Molecular Infection Biology II, Institute of Molecular Infection Biology (IMIB), University of Würzburg, Germany, , Josef-Schneider Strasse 2, 97080 Wurzburg [DE], elise.bornet@yahoo.fr

Author(s):
Elise Bornet, Gianluca Prezza, Laura Cecchino, Jochen Behrends, Kerwyn Casey Huang, Emmanuel Saliba, Alexander Westermann

Genetically identical bacteria within the same micro-environment may not behave alike. This is exemplified by the abundant gut microbiota member Bacteroides thetaiotaomicron, whose name derives from the three morphotypes (dubbed ‘Theta’, ‘Iota’ and ‘Omicron’) that these bacteria can adopt. However, the molecular basis of this morphological heterogeneity and its functional consequences are unknown. Here, we map the transcriptome of Bacteroidesmorphotypes using sensitive ‘mini-bulk’ RNA-seq. We observed that across four biological replicates, the different morphotypes upregulate specific sets of marker genes, encoding proteins involved in primary metabolism and membrane-associated processes. A subset of morphotype-specific markers are validated using single-molecule fluorescence in-situ hybridization and by single-bacterium RNA-seq. Lastly, reanalysis of existing high-throughput imaging data derived from an ordered transposon insertion library of B.thetaiotaomicron in combination with confocal microscopy of clean deletion mutants, reveals the influence of individual genes on bacterial cell shape. Together, our work lays the ground to dissect the extent and impact of cell-to-cell variability in this dominant bacterial species of the human microbiota.

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M 22
Maturation of the infant microbiome in time and space

Presenting author:

MDC Berlin, ECRC, Robert-Rössle-Straße 10, 13125 Berlin [DE], robert.buecking@mdc-berlin.de

Author(s):
Robert Bücking, Víctor Hugo Jarquin Diaz, Georgina Sket, Claudia Buß, Dorothee Viemann, Sofia Forslund

The neonatal gut after birth is a vast empty habitat that provides a good model to investigate microbial colonization dynamics. While the number of published studies describing infant gut colonization is steadily rising, comparisons across cohorts, geographic locations and lifestyles are rare. The increasing number of available microbiome datasets enables meta-analyses to detect generalizable microbial dynamics across cohorts.
We aim to define a generalizable model of infant gut colonization that will provide insight into host-microbiome interactions and microbial ecological succession in a sterile environment. We analyzed published longitudinal 16S sequencing data of children up to two years of age from different cohorts, countries, and lifestyles. Our preliminary results based on 15,565 samples from 19 studies revealed a decline in cohort-specific effects with the inclusion of additional datasets. We identified key taxa predictive of host age across studies. We showed that lifestyle-specific models are more accurate than a global model.
The assembled dataset allows for a comprehensive analysis of variables influencing infant gut colonization. Our findings emphasize the importance of diverse datasets for deriving generalizable models for microbial dynamics. Next, our models will incorporate functional information from shotgun sequencing data. In the future, we aim to use these models to detect deviations from a healthy maturation.

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N 04
The ability of Staphylococcus aureus to proliferate within the nasal ecosystem is strain- and community-specific

Presenting author:

Interfaculty Institute of Microbiology and Infection Medicine Tübingen (IMIT), Infection Biology, Auf der Morgenstelle 28, 72076 Tübingen [DE], laura.camus@uni-tuebingen.de

Author(s):
Laura Camus, Jessica Franz, Jeffrey John Power, Marcelo Navarro Díaz, Anna Lange, David Gerlach, Simon Heilbronner

Asymptomatic nasal colonization of Staphylococcus aureus increases the risk of infection and concerns about 30% of the population. The contribution of the endogenous microbiota to this pattern of carriage is poorly understood. We hypothesize that, depending on their composition and origin, nasal bacterial communities can promote or inhibit the proliferation of S. aureus within nares.

Three nasal communities of different composition were reconstituted by isolating nasal bacterial species from healthy volunteers. Using pairwise co-culture assays, we observed that the interaction networks of two communities were dominated by negative interactions. When assembled as synthetic consortia, these two communities inhibited the proliferation of four different S. aureus strains. However, the third community presented an interaction network mostly cooperative and specifically promoted the survival of the S. aureus nasal strain that was co-isolated from the same nose, suggesting the existence of co-adaptation mechanisms. In vitro metabolomics assays and in vivo nasal colonization experiments in gnotobiotic mice will reveal the impact of these interactions on S. aureus metabolism and ability to thrive in the nose.

Altogether, our results indicate that nasal communities significantly affect the proliferation of S. aureus in a strain-specific manner. This could explain why certain communities are invaded by this pathogen, and inform strategies for preventing nasal colonization by S. aureus.

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M 21
The dynamics of the structure and the function of the human respiratory microbiome

Presenting author:

Heidelberg University Hospital, Infectious diseases, Division of Medical microbiology and Hygeine , Im Neuenheimer Feld 324, Room 104, 69120 Heidelberg [DE], bachar.cheaib@med.uni-heidelberg.de

Author(s):
Bachar Cheaib, Olaf Sommerburg, Selina Mayer, Alexander Dalpke, Sébastien Boutin

Understanding the fundamental process of the respiratory tract microbiota ontogenesis can help us to understand the function and the development of the lung in the context of health and disease, especially in patients with Cystic Fibrosis. We performed a microbiome study including a cohort of 210 samples of nasal and throat swabs from 73 patients (infants and children) with CF. The total MRI scores increase with age for 30 % of the patients. The analyses of alpha-diversity indexes are significantly higher in the throat compared to the nasal microbiome. Fitting a linear mixed model with the patient as a random effect, we observed an increase over age of the ratio’s Richness/evenness in the throat versus a decrease in the nasal niches. The beta diversity revealed substantial variations in the microbiome community structure between the nose and the throat, suggesting higher stability in the throat microbiome. The bacterial composition analysis indicates a significant differential abundance between the nose and the throat. A differential abundance analysis was also performed on the taxa composition between consecutive age ranges in the throat and the nose. The results suggest stabilisation over age in both sites but higher stability in the throat niches. The network analysis of the throat microbiome across sequential ages compared to the nose’s microbiome showed a significantly higher persistence of positive interactions between commensals despite the prevalence of pathogens.

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R 04
The Role of the Gut Microbiota in Determining Pyrrolizidine Alkaloid Toxicity

Presenting author:

UNIVERSITY OF CAMBRIDGE, MRC Toxicology Unit, Tennis Court Road, CB2 1QR Cambridge [GB], tfd26@cam.ac.uk

Author(s):
Tara Davis

Pyrrolizidine alkaloids (PAs) are produced by various plant species worldwide and have been identified as frequent contaminants of several common food items, such as teas, herbs and honey. The toxicity of PAs is highly dependent on the balance between their free necine base form and their N-oxidised form (PANOs), as higher levels of reduced PAs leads to the formation of pyrrole adducts with DNA and cellular proteins in the liver, potentially causing serious clinical complications. Evidence suggests that the metabolic conversion of PANOs to their corresponding PAs by the gut microbiota is an important pathway for increasing their toxic potential. However, the small species overlap in the gut microbiota between humans and animal models means that there is uncertainty around the accuracy of toxicity predictions made using rodents. Through the screening of 87 gut bacterial strains prevalent in human and mouse microbiota for the ability to reduce PANOs, we have identified several species responsible for catalysing this reaction. This, combined with insight into the mechanism of microbial PANO biotransformation, will be used to simulate the impact that gut microbiota variability between humans and rodent models has on PA-induced toxicity.

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I 05
Drug-induced alterations in community composition drive the loss of colonization resistance against Salmonella Typhimurium

Presenting author:

Universitätsklinikum Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, Otfried-Müller-Str. 37, 72076 Tübingen [DE], jacobo.delacuesta@med.uni-tuebingen.de

Author(s):
Juan Jacobo de la Cuesta Zuluaga, Grießhammer Anne, Taiyeb Zahir, Cordula Gekeler, Lisa Maier

The gut microbiome can be altered by non-antibiotic drugs, although implications for the host are not well understood. Here, we evaluated the effect of non-antibiotics on the microbiome’s ability to resist colonization by Salmonella Typhimurium (S.Tm). We assessed the growth of S.Tm on a synthetic community comprising 20 species from the human gut after a challenge with >50 human-targeted drugs; 35% of the drugs resulted in an increased abundance of S.Tm. Based on pathogen growth, we classified drugs as S.Tm-favoring, S.Tm-restricting or having no effect. Using 16S rRNA gene sequencing, we identified species significantly different in communities treated with S.Tm-favoring drugs compared to controls. S.Tm-favoring communities were enriched in Collinsella aerofaciens, Enterocloster bolteae, Dorea formicigenerans, and Agathobacter rectalis, while Bacteroidales taxa and Streptococcus parasanguinis were depleted. Drug treatment altered the predicted functional capacity of the microbiome, independent of its ability to resist pathogens. S.Tm-favoring communities had an increased abundance of pathways of allantoin metabolism, glycolysis and gluconeogenesis, while lower abundances of pathways of histidine metabolism, and purine and pyrimidine biosynthesis. Our results might help the development of strategies to minimize microbiome-mediated side effects of human-targeted drugs.

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M 18
A novel information theory framework to study microbial co-abundance networks in ageing

Presenting author:

Leibniz Institute on Aging - Fritz Lipmann Institute (FLI), , Beutenbergstraße 11, 07745 Jena [DE], melike.donertas@leibniz-fli.de

Author(s):
Handan Melike Donertas

The host-associated microbiome plays a pivotal role in maintaining health and homeostasis. Age-related changes in the microbiome affect host physiology and disease vulnerability. We leverage network biology to explore age-related changes in microbial community interactions, key species, and community interaction topology. Our novel framework integrates network biology with information theory, surpassing traditional methods by: 1) merging network structure with individual microbial abundances to analyze interactions per sample; 2) addressing the limitations of beta diversity variations with age, enhancing power to detect key species in late ages; 3) enabling longitudinal analysis of individual-specific networks across ages. We reanalysed public cross-sectional and longitudinal datasets, identifying species critical for community dynamics and aging prediction. Currently, our analysis focuses on positive interactions between microbial taxa, with plans to include negative interactions and multi-omic integration. Our method innovates network biology approaches for microbiome research, promising new diagnostic and therapeutic avenues.

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M 08
Effect of Probiotic Supplementation on the Intestinal Microbiome of Preterm Infants

Presenting author:

TH Nürnberg Georg Simon Ohm, Fakultät Angewandte Chemie, Prinzregentenufer 47, 90489 Nürnberg [DE], ronald.ebbert@th-nuernberg.de

Author(s):
Sabrina Hofmann, Krisztina Somogyi, Adrian Rosenberg-Granzner, Niels Rochow, Katja Knab, Christoph Fusch, Ronald Ebbert

Probiotic supplements in preterm infants can reduce mortality, late-onset-sepsis and necrotizing enterocolitis. In the current study, 30 preterm infants received the probiotic ProPrems containing Bifidobacterium longum subsp. infantis, Streptococcus thermophilus and Bifidobacterium animalis subsp. lactis. Longitudinal stool samples were collected, 28 samples before, 118 during and 69 after supplementation. 44 samples from 14 preterm infants without supplementation served as control. Bacteria were identified by Nanopore sequencing of the complete 16S gene. Sequence analysis was performed by a workflow based on QIIME 2.

PCoA based on Bray-Curtis distances separates samples “During” supplementation (1), “After” supplementation (2) and the overlapping groups “Before” and “Control” (3). Bifidobacterium longum has the strongest influence on beta-diversity. PCoA results are supported by PERMANOVA analysis, where similarity between “Before” and “Control” is stronger than for all other comparisons.
ANCOM analysis shows significantly higher abundances of the ProPrems species during supplementation than “Before”. Unexpectedly, both S. thermophilus and B. animalis are strongly reduced in “After” samples compared to “During”. Of the probiotic species, only B. longum increases significantly in abundance from “Before” to “After”.
In summary, all three probiotic strains are found during supplementation, but apparently only B. longum subsp. infantis successfully colonizes the preterm gut.

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R 02
An essential small RNA for gut colonization of the prevalent human commensal Segatella copri

Presenting author:

Helmholt Center for Infections Research - HZI, Microbial Immune Regulation, Inhoffenstraße 7, 38124 Braunschweig [DE], youssef.elmouali@helmholtz-hzi.de

Author(s):
Youssef El Mouali, Caroline Tawk, Kun Huang, Lena Amend, Till Robin Lesker, Falk Ponath, Jörg Vogel, Till Strowig

Segatella copri (former Prevotella copri) is a prevalent and abundant member of the human gut microbiota that has been associated with both health and disease traits. Despite the increasing interest in the biology of S. copri, a significant gap exists in understanding the fundamental gene expression regulation and physiological responses crucial for effective gut colonization. Here, by mapping the primary transcriptome of S. copri and examining gene expression in human-derived samples we uncover an essential regulator of gut colonization in S. copri, the small RNA here named SrcF. Identified as the most abundant sRNA in the human gut, SrcF deletion rendered S. copri unable to colonize the gut in a mouse model. SrcF regulates genes involved in nutrient acquisition, and its expression is controlled by metabolizing specific dietary complex carbohydrates, particularly fructans. The HTCS regulator of the inulin polysaccharide utilization locus senses fructose, leading to SrcF downregulation. Human microbiome composition and fructan breakdown by cohabitating commensals impact SrcF expression, suggesting that complex carbohydrate breakdown not only contributes as an energy source but also mediates inter-species signaling among commensals. This study establishes the foundation of S. copri RNA biology, highlighting non-coding sRNAs as key regulators for gut colonization and potential targets for targeted microbiome intervention.

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M 01
Genetic Fitness of Gut Bacteroidales during Host Colitis

Presenting author:

University of Chicago, Microbiology, 900 E 57th St, 60637 Chicago [US], nelmekki@uchicago.edu

Author(s):
Nazik M. Elmekki, Leonor Garcia-Bayona, Michael J. Coyne, Laurie E. Comstock

The human gut microbiome is a complex community including diverse microbes that affect host health. The most predominant bacterial order in the human gut is the Bacteroidales, which includes highly prevalent species from the genera Bacteroides, Phocaeicola, Parabacteroides, and Prevotella among others. Phocaeicola vulgatus is one of the most ubiquitous and abundant members of the healthy human gut, but far less studied than other Bacteroidales such as B. thetaiotaomicron and B. fragilis. In this study, we are investigating how P. vulgatus responds to inflammation in the mammalian gut using a high-throughput genetic screening method, Barcoded Transposon-Insertion Sequencing (BarSeq)¹. BarSeq allows for the calculation of fitness scores for all genes in the bacterial genome under specific conditions and determination of which genes are conditionally essential. We have performed this genetic screen in mono-associated gnotobiotic C57BL/6 mice to identify genes important for survival during host colitis, using the well-established Dextran Sodium Sulfate (DSS) model. Candidate genes with the highest conditional fitness scores have been identified using the RTISAn pipeline². We are confirming the importance of these genes during colitis using genetic approaches to understand how they help these bacteria survive in an inflamed gut.

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M 08
Differentiating evolution via point mutation or structural change on a per-species basis in complex microbiomes

Presenting author:

Max Planck Institute for Biology Tübingen, Microbiome Science, Max-Planck-Ring, 72076 Tuebingen [DE], hagay.enav@tuebingen.mpg.de

Author(s):
Hagay Enav, Inbal Paz, Ruth E. Ley

Microbial species diversify into separate strains through mutation, recombination, and gene loss/acquisition. Elucidating mechanisms driving the genomic diversity of species residing within complex microbiomes remains biased towards mutation, because current strain tracking methods are focused on SNPs and are relatively insensitive to structural changes in genomes. To complement these methods, we developed a tool that compares strains using synteny - the conservation of the order of sequence blocks in homologous genomic regions in pairs of metagenomic assemblies. As a standalone tool, our tool allows improved microbial strain tracking within and between hosts. The combined use of our tool and SNP-based tools in metagenome analysis allows the identification of species undergoing high rates of structural change with low rates of mutation, or conversely, high rates of structural change with low mutation rates, providing a novel window into different modes of evolution on a per-species basis in complex microbiomes.

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I 08
How gut bacteria modulate brain-resident immune cells

Presenting author:

Institute of Neuropathology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany, , Breisacher Straße 64, 79106 Freiburg [DE], daniel.erny@uniklinik-freiburg.de

Author(s):
Daniel Erny

 

Microglia represent the main tissue resident macrophages of the central nervous system (CNS). As innate immune cells microglia are first responders to pathological conditions. Microglia are also crucial for proper brain development and physiological CNS function during adulthood. They maintain tissue homeostasis e.g. by removing cellular debris and remodeling synapses.

In my studies, we have uncovered that gut bacteria critically shape microglial maturation, function and metabolic state in homeostasis and disease by examining various gnotobiotic mouse models (Erny et al., Nature Neuroscience 2015; Erny et al., Cell Metabolism 2021). Further, we determined that gut bacteria critically modulate the pathogenesis In the 5xFAD mouse model for Alzheimer’s disease (AD) via microglia and their capacity to degrade toxic amyloid beta (Aβ) deposits (Mezö et al., Acta Neuropathologica Communications 2020). Mechanistically, we identified the gut-derived acetate as critical gut bacteria-derived mediator for microglia properties and their role in the gut-brain axis (Erny et al., Cell Metabolism 2021).

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M 24
Bifidobacteria compensate for genotype in lactose tolerance

Presenting author:

Max Planck Institute for Biology Tübingen, Microbiome Science, Max-Planck-Ring 5, 72076 Tübingen [DE], liam.fitzstevens@tuebingen.mpg.de

Author(s):
Liam Fitzstevens, Xiaoying Liu, Albane Ruaud, Victor T. Schmidt, Mirabeau Mbong Ngwese, Nicholas D. Youngblut, Julia Rauch, Taichi Suzuki, Nguyen Thu Ha, Aleksandr Arzamasov, Dmitry Rodionov, Niklas Pfister, Nikolaj Thams, Bayode R. Adegbite, Jeannot F. Zinsou, Meral Esen, Thirumalaisamy P. Velavan, Ayola A. Adegnika, Le Huu Song, Peter Kremsner, Andrei Ostermann, Alexander V. Tyakht, Ruth Ley

The lactase-persistence (LP) genotype allows digestion of the milk sugar lactose in adults and confers lactose tolerance. Genetically lactase non-persistent (LNP) individuals can also be lactose tolerant, but responsible microbiota remain elusive. Here, we assessed lactose tolerance as H₂-production in breath after lactose dose, LP/LNP genotype, and gut microbiome metagenomic diversity in 480 adults from Gabon (100% LNP), Vietnam (99% LNP), and Germany (23% LNP). In all three populations, ~ 13% of LNP were lactose tolerant though microbiomes differed. In-vitro lactose addition to stool showed low H₂ production stemmed either from minimal breakdown of lactose, or breakdown producing metabolites of the Bifid shunt pathway - lactate and acetate - and the growth of Bifidobacterium. Our results indicate that Bifidobacterium can confer lactose tolerance across populations, including where the LP genotype is rare, and may have facilitated functional take-over by the human genome when dairying first began 12,000 years ago.

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I 07
Mouse strain-dependent effect of host microbiota on microglia during homeostasis and disease

Presenting author:

Uniklinik Freiburg, Institut für Neuropathologie, Breisacher Str. 64, 79106 Freiburg [DE], vidmante.fuchs@uniklinik-freiburg.de

Author(s):
Daniel Erny, Vidmante Fuchs, Marco Prinz

Being resident innate immune cells, microglia fulfil tissue homeostasis and immune surveillance functions in the central nervous system. It has been shown that the gut microbiota is able to modulate microglia, including their cell density, morphology, transcription and immunological functions. Gut microbiota derived SCFA, especially acetate, were determined as the key factors driving microglial properties. Here we observed microglia and the gut microbiota relations in a mouse strain-dependent context, which to this day is largely unknown, despite mice being widely used as an animal model in biomedical research. We characterized microglial phenotype across mouse strains and revealed distinct microglial features in C57BL/6, BALB/c and FVB mice. Additionally, we determined the microbiota differences across the mouse strains that differentially modulate the microglia phenotype. Microglia’s phenotype and transcriptional changes were further observed in germ free (GF) condition as well as viral challenge model across the mouse strains. Furthermore, the C57BL/6 microbiota composition transplantation to GF BALB/c and FVB animals was able to partially overcome the host genetics and modulate microglia properties. These findings indicate that mouse strains harbour strain-specific compositions of the gut microbiota which in turn is able to differentially affect the microglia cells, driving functions of microglia during health and disease.

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O 15
Development of an Engineered Living Materials System in a Microfluidic Bioelectrochemical Reactor

Presenting author:

Karlsruhe Insitute of Technology (KIT), Institute of Biological Interfaces 1, Hermann-von-Hemholtz-Platz-1, 76344 Karlsruhe [DE], Philipp.Gaspers@kit.edu

Author(s):
Philipp Gaspers, Christof Niemeyer, Kersten Rabe

Engineered Living Materials (ELM) connect synthetic biology with artificial materials to create living systems that have the capability for replication, self-repair and regulation towards external stimuli. Due to their ability to self-optimise, these systems have great potential for applications like bioelectrochemical systems (BES) in which bacteria like Shewanella oneidensis (S. oneidensis) are used for wastewater treatment or the clean production of valuable metabolites while producing electrical energy.

However, current BES do not yet produce enough electricity to operate in an economically viable way. One solution could be the generation of self-optimizing conductive biofilms by the use of DNA composite materials that contain silica nanoparticles and carbon nanotubes for enhanced electron transfer to the systems electrodes. These materials have been developed by our group recently and are well suited for the cultivation of S. oneidensis

Our research focuses on the development of an ELM system in which S. oneidensis is able to produce or degrade these materials by itself within a microfluidic BES to adjust the composition of the biofilm for optimal process performance. To this end, we have generated S. oneidensis strains that present the necessary DNA enzymes on their surface. Preliminary results show that these strains can generate DNA hydrogels and are still able to produce electricity, thus serving as a robust platform for further development.

 

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O 21
Precision editing of non-model bacteria in native microbiomes using mobile CRISPR-associated transposases

Presenting author:

Columbia University Irving Medical Center, Systems Biology, 3960 Broadway, 10032 New York City [US], drg2165@cumc.columbia.edu

Author(s):
Diego Gelsinger, Carlotta Ronda, Tyler Perdue, Samuel H. Sternberg, Harris H. Wang

Deep sequencing shed light on the vast microbial diversity in nature, changing understanding of the crucial roles that microbiomes play in environments. Knowledge of gene functions comes from manipulating the DNA of species in isolation from their natural communities, yet fewer than 1% of microbes are culturable and even fewer are genetically tractable. Moreover, in nature, microbes live in open, dynamic, complex habitats that are difficult to recapitulate in a laboratory. To address these shortcomings, we developed a novel methodology that enables in vivo precision microbiome engineering by leveraging highly mobile vectors to effectively shuttle payloads between diverse microbes, with programmable transposases to catalyze targeted DNA integration, a technology we term MAGICAST. We optimized delivery and activity of CRISPR-associated transposases in 4 non-model Bacteroides species isolated from humans, towards development of a suite of highly mobile gene editing systems. We next introduced novel payloads with high specificity (>90% on-target), using computationally designed metagenomic spacers, and temporal persistence in Bacteroides species within the gut microbiome of live mice. We used this to enable genetic tagging of target species in vivo, to facilitate their isolation from the environment and investigation into bile acid metabolism. This work defines a new paradigm for genetic studies of gut microbiome and establishes a pipeline for genetic engineering of bacteria from nature into the laboratory. Future work will focus on expanding this technology into other microbiomes (e.g., clinical, extremophilic) to enable discovery of novel functions from undomesticated bacteria with implications in biotechnological, therapeutic, and environmental fields

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O 16
Translation at the Phage Nucleus in ΦKZ

Presenting author:

Würzburg University, IMIB, HIRI, AG Vogel, Josef-Schneider-Str. 2, 97080 Würzburg [DE], milan.gerovac@uni-wuerzburg.de

Author(s):
Milan Gerovac, Kotaro Chihara, Laura Wicke, Bettina Böttcher, Rob Lavigne, Jörg Vogel

Bacteriophages must seize control of the host gene expression machinery to promote their own protein synthesis. Since the bacterial hosts are armed with numerous anti-phage defence systems, it is essential that mechanisms of host takeover act immediately upon infection. Although individual proteins that modulate components of the bacterial gene expression apparatus have been described in several different phages, systematic approaches which capture the phage’s arsenal for immediate targeting of host transcription and translation processes have been lacking. In particular, there are no known phage factors that associate directly with host ribosomes to modulate protein synthesis. Here, we take an integrative high-throughput approach to uncover numerous new proteins encoded by the jumbo phage ΦKZ that target the gene expression machinery of the Gram-negative human pathogen Pseudomonas aeruginosa immediately upon infection. By integrating biochemical and structural analyses, we identify a conserved phage factor that associates with the large ribosomal subunit by binding the 5S ribosomal RNA. This highly abundant factor is amongst the earliest ΦKZ proteins expressed after infection and stays bound to ribosomes during the entire translation cycle. Our study provides a general strategy to decipher molecular components of phage-mediated host take-over. Gerovac et al. 2023 bioRxiv

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M 19
Gut-derived metabolite phenylacetylglutamine induces cardiac remodeling in chronic kidney disease

Presenting author:

Uniklinik RWTH Aachen, Department of Cardiology, Angiology and Critical Care (Medical Clinic 1), Pauwelsstr. 30, 52074 Aachen [DE], mgesper@ukaachen.de

Author(s):
Maren Gesper, Henriette de Loor, Björn Meijers, Chao-Chung Kuo, Julia Franzen, Nikolaus Marx, Ben A. Kappel

Cardiovascular disease is the main cause of death in patients with chronic kidney disease (CKD). Within the paradigm of gut-heart-kidney cross talk, the pivotal role of gut microbiota and their metabolites has emerged as a focal point in understanding the intricate communication between organs. The gut-derived serum metabolite phenylacetylglutamine (PAG) has formerly been associated with mortality and cardiovascular disease in patients with CKD. In this study, we examined the influence of PAG on cardiac remodeling in a mouse model of CKD and unraveled molecular pathways that underlie the effect of PAG on the heart in vitro. After long-term treatment of PAG in C57Bl/6J mice with 5/6-nephrectomy, left ventricular volume measured by echocardiography was significantly enlarged after 7 weeks. While left ventricular ejection fraction remained unaltered, heart-to-body weight ratio as well as diastolic dysfunction significantly increased. Based on histological and gene expression analysis, enhanced left ventricular fibrosis was observed. To understand underlying molecular mechanisms, we performed 3’mRNA sequencing in HL-1 cardiomyocytes. One of the significantly upregulated genes was Ccn2, a paracrine factor to induce fibrosis in the heart. We further confirmed this in heart tissue of C57Bl/6J mice treated with PAG. Therefore, PAG induced cardiac remodeling characterized by increased fibrosis and diastolic dysfunction and could be a potential mediator in the gut-heart-kidney axis.

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M 14
Impact of non-antibiotic treatment on antimicrobial resistance of human gut bacteria

Presenting author:

Contributed equally; European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany, , Meyerhofstraße 1, 69117 Heidelberg [DE], samir.giri@embl.de

Author(s):
Camille Goemans, Samir Giri, Nazgul Sakenova, Athanasios Typas

Mounting evidence indicates that medication is one of the central extrinsic factors affecting the gut microbiota composition, including antibiotics and non-antibiotic drugs that can directly impact the growth of various abundant and prevalent gut bacterial species. However, the extent to which exposure to non-antibiotic drugs accelerates antibiotic resistance or sensitivity remains unclear. Here, we first investigated whether gut bacteria can evolve resistance to non-antibiotics by experimentally evolving eight abundant and prevalent gut bacterial species in the presence of increasing concentrations of 12 non-antibiotics. The majority of the species developed several-fold resistance to the non-antibiotics. We then examined whether this resistance impacts the evolved lineages’ MIC in 12 major classes of antibiotics. This analysis revealed cases where resistance to non-antibiotics increases antibiotic resistance (cross-resistance), but also, surprisingly, many cases in which antibiotic sensitivity was increased (collateral sensitivity). In particular, we found bacterial populations that, by evolving resistance to one of the tested non-antibiotics, became broadly collateral sensitive to many antibiotics. This suggests that a microbiota exposed to such a drug would be prone to increased dysbiosis after antibiotic treatment. Altogether, these findings provide insight into drug-microbiota interactions and offer ways to mitigate the deleterious effect of drugs on the gut microbiota.

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O 20
Recognition of an intestinal helminth by the C-type lectin receptor Mincle

Presenting author:

University of Veterinary Medicine Hanover, Germany, Institute of Immunology, Bünteweg 2, Building 261 , 30559 Hannover [DE], Vinayaga.Srinivasan.Gnanapragassam@tiho-hannover.de

Author(s):
Vinayaga Srinivasan Gnanapragassam, Lara Linnemann, Jennifer Antwi-Ekwuruke, Minka Breloer, Bernd Lepenies

Strongyloides ratti is a pathogenic helminth that infects rat species. Transition from the larval stage to the adult stage occurs in the host intestine. S.ratti needs to resist innate immune recognition and the host immune response, in order to persist in the host. We hypothesize that during the early phase of infection, S.ratti interacts with myeloid C-type lectin receptors (CLRs), expressed by macrophages and dendritic cells to modulate the host immune response.

The Macrophage inhibitory C-type lectin receptor (MINCLE) is a myeloid CLR, that acts as a pattern recognition receptor (PRR) involved in sensing pathogen associated molecular patterns (PAMPs), thereby modulating the initated immune response.

S.ratti infection with C57BL/6 mice exhibit highest infection on the day 6, with reduced worm burden in the small intestine of the Mincle deficient mice in comparision to wild type littermates. This finding suggests that Mincle may be exploited by parasitic helminths for immune evasion. Since the interaction of S.ratti-derived ligands with Mincle has not yet been examined, we prepared infective stage larvae lysate and analyzed for Mincle binding and activation by ELISA, using Mincle-Fc fusion protein and by Mincle reporter cell assay, respectively. The putative Mincle ligand was affinity purified from the lysate and identified by mass spectrometry. Currently, the immune modulatory properties of the identified S.ratti-derived protein ligand of Mincle are under investigation.

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O 25
A parallel pathway for inositol sphingolipid synthesis in gut Bacteroidota

Presenting author:

Max Planck Institute for Biology Tübingen, Microbiome Science, Max-Planck-Ring 5, 72076 Tübingen [DE], sheaver@tuebingen.mpg.de

Author(s):
Stacey Heaver, D.L. Vu, A.E. Bryson, Ruth Ley

Inositol lipid production is phylogenetically restricted among bacteria but prevalent in host-associated Bacteroidota. The inositol lipid metabolic pathway in the human gut symbiont Bacteroides thetaiotaomicron (BT) is similar to the mycobacterial pathway for phosphatidylinositol synthesis and proceeds through a phosphatidylinositol-phosphate (PIP-DAG) intermediate. However, some Bacteroidota spp. lacking homology to the BT-like pathway for inositol lipid synthesis nevertheless produce inositol sphingolipids through a pathway we predicted to lack the PIP-DAG intermediate, instead generating an array of soluble inositol intermediates leading to inositol sphingolipid synthesis. Here, we characterize this alternative inositol lipid gene cluster via heterologous expression in BT and gene knockout in Phocaeicola dorei. We define the activity of key enzymes in the gene cluster in vitro and describe inositol lipid structural diversity in a panel of 29 Bacteroidota species. As inositol lipids are potent bioactive signaling molecules in humans, the inositol lipid contribution from gut-associated Bacteroidota, via one of two metabolic pathways, offers future potential mechanisms for host-microbe interactions.

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O 18
NFDI4Microbiota supporting microbiome research providing data access, services, training and workflows

Presenting author:

Helmholtz Center for Infections Research - HZI, Bioinformatik der Infektionsforschung, Inhoffenstraße 7, 38124 Braunschweig [DE], cordula.hege@helmholtz-hzi.de

Author(s):
Cordula Hege, Barbara Götz, Mattea Müller, Konrad Förstner, Alice McHardy

NFDI4Microbiota aims to support the microbiome research by providing access to data, analysis services, data/metadata standards and training. It belongs to the National Research data Infrastructure (NFDI), which aims to develop a comprehensive research data management. NFDI4Microbiota intends to facilitate the digital transformation in the microbiological community (bacteriology, virology, mycology and parasitology).

The microbiome research network will be boosted through training and community-building activities, and by creating a cloud-based system that will make the storage, integration and analysis of microbial data, especially omics data, consistent, reproducible, and accessible. Thereby, NFDI4Microbiota will promote the FAIR (Findable, Accessible, Interoperable and Re-usable) principles and Open Science.

The NFDI4Microbiota consortium develops and provides computational infrastructure and analytical workflows required to store, access, process, and interpret various data types. Here, NFDI4Microbiota works on designing and implementing software and standardized workflows.

NFDI4Microbiota offers trainings, spanning from metagenomics, over courses about programming in R, to research data management. To interact with young scientists, the consortium launched an ambassador program. Technical solutions are developed, tested and refined in use cases from different fields of microbiology. All relevant information and specific services are available via the web portal.

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N 01
Nasal commensals reduce Staphylococcus aureus proliferation by restricting siderophore availa-bility

Presenting author:

LMU - Ludwig Maximilian University of Munich, Microbiology, Großhadener Str. 2-4, 82152 Martinsried [DE], simon.heilbronner@lmu.de

Author(s):
Simon Heilbronner, Yanfeng Zhao, Alina Bitzer, Jeffrey Power, Darya Belikova, Benjamin Torres-Salazar, Lea Adolf, David Gerlach, Bernhard Krismer

 

The human microbiome is critically associated with human health and disease. Staphylococcus aureus can reside within the nasal microbiota which increases the risk of infections. Epidemiological studies of the nasal microbiome have revealed positive and negative correlations between non-pathogenic nasal commensals and S. aureus, but the underlying molecular mechanisms are unclear. The nasal cavity is iron-limited and bacteria are known to produce iron-scavenging siderophores to proliferate in such environments. Siderophores are public goods that can be consumed by all members of a community. Accordingly, siderophores are known to mediate bacterial interaction. We show that siderophore acquisition is crucial for S. aureus nasal colonization in vivo. We screened 94 nasal bacterial strains for their capacity to produce siderophores as well as to consume the siderophores produced by S. aureus. We found 80% of the strains to engaged in siderophore-mediated interactions with S. aureus. Non-pathogenic corynebacterial species are prominent consumers of S. aureus siderophores and reduced S. aureus growth in an iron-dependent fashion. Our data show a wide network of siderophore-mediated interactions between the species of the human nasal microbiome which impacts pathogen proliferation. This opens avenues for designing nasal probiotics to displace S. aureus from the nasal cavity of humans.

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M 07
Quantifying the bidirectional interactions between anti-cancer drugs and the human gut microbiome

Presenting author:

EMBL Heidelberg, Genome Biology, Meyerhofstraße 1, 69117 Heidelberg [DE], zachariah.henseler@embl.de

Author(s):
Zachariah Henseler

The human gut harbors trillions of commensal microbes that influence health and wellbeing in various ways. One manner in which the gut microbiome influences human health is through the chemical alteration or degradation of human-targeted drugs, processes which can lead to changes in drug activity and availability. A group of human-targeted drugs of particular interest due to their mechanistic diversity are anti-cancer drugs (ACDs). Some ACDs can act as antibiotics, altering the composition and function of the gut microbiome. Conversely, certain gut bacteria have been shown to chemically alter ACDs, modulating their toxicity to both microbial and cancer cells.

This work aims to systematically quantify these bi-directional interactions between gut microbes and ACDs. Using a culture-based method, we tested the effects of 94 different ACDs on the gut microbe using 16S rRNA gene sequencing. In addition, we examined changes in the microbial metabolome using untargeted LC-MS. We found that both the microbiome and its associated metabolome shift in a drug-specific manner upon ACD treatment. By further examining the minimal inhibitory concentration of these ACDs on select bacterial isolates, we identified regions of drug protein targets that may confer drug resistance. Finally, we plan to further examine how these drug-induced changes in the microbiome may affect host physiology by measuring supernatant-induced changes in macrophage polarization.

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N 02
Supporting microbiome research by latest metaproteomics software development

Presenting author:

Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., , , 44139 Dortmund [DE], Robert.Heyer@ISAS.de

Author(s):
Robert Heyer, Emanuel Lange, Maximilian Wolf, Daniel Kautzner, Kay Schallert

 

The LC-MS/MS-based analysis of microbiomes called metaproteomics, plays a crucial role in understanding the impact of gut microbiomes on diseases, optimizing biotechnological applications, and exploring environmental processes. Ongoing advancements in experimental workflows and mass spectrometry have significantly enhanced the resolution of metaproteomics analysis, providing a more comprehensive view of microbial phenotypes through increased protein identifications. However, automated analysis of metaproteomics data faces challenges due to data volume and microbiome complexity.

The MetaProteomeAnalyzer, introduced in 2015, addresses these challenges by enabling protein identification, homologous protein grouping, and taxonomic/functional evaluation. A recent cloud-based version enhances scalability and accessibility through a user-friendly web interface. Furthermore, we developed a novel tool for metaproteomics metadata handling.

The MPA_Pathway_Tool complements these efforts by facilitating metabolic phenotype reconstruction from metaproteomics data. It enables mapping of protein identifications to molecular networks, modification or creation of new networks, and enhancing metabolic models using experimental data, potentially increasing precision by a factor of five for Escherichia coli.

In conclusion, our tools expedite the evaluation of (meta-) proteomics data, advancing the use of LC-MS/MS-based analyses in microbiome research.

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M 20
High-throughput anaerobic cultivation facilitates host-specific culture collections and the creation of synthetic communities for targeted functional study of microbiomes

Presenting author:

University Hospital RWTH Aachen, Aachen, Germany, Institute of Medical Microbiology, Pauwelsstraße, 52074 Aachen [DE], thitch@ukaachen.de

Author(s):
Thomas Hitch, Johanna Bosch, Johannes Masson, Afrizal Afrizal, David Wylensek, Charlie Pauvert, Thomas Clavel

Host-associated microbiota consist of hundreds of species, each containing thousands of proteins, making them functionally complex. Similar to our previous work on public collections of isolates from the pig and mouse gut, we have established a comprehensive Human Intestinal Bacterial Collection (HiBC), containing 395 publicly available bacterial isolates, including full taxonomic description of >40 novel species. Genomic study of Faecalibacterium strains within HiBC suggests the separation of F. prausnitzii into four species, each varying in butyrate production. Application of single-cell dispensing approaches to cultivation has enhanced the throughput, leading to the isolation of 11 novel genera and 10 novel species. This technology has now been integrated into an automated workflow, termed iSOMiC, which enhances our ability to generate personalised collections of bacterial isolates.

To utilise these isolates, we have developed a tool for the creation of synthetic communities (SynComs) that are functionally representative of the ecosystem of interest. Firstly, the functional repertoire of each isolate is compared to metagenomes, individually, and then in combinations. The most functionally representative isolate combinations is then metabolically modelled to predict interactions and community stability. Using this method, we have developed condition-specific SynComs (healthy Vs diseased) and SynComs representative of an entire ecosystem (the human gut).

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O 08
Spatiotemperal Probing of Microbial Communities from Winogradsky Columns as a Model Ecosystem

Presenting author:

ahmad.itani@kit.edu

Author(s):
Ahmad Itani, Laura Meisch, Marta Velaz Martin, Phillip Lemke, Tim Scharnweber, Julia Weisser, Saskia Gimmel, Anna Edinger, Islam Khattab, Anne-Kristin Kaster, Kersten S. Rabe, Christof M. Niemeyer

To better understand the cultivation conditions of rare taxa, it is essential to study their development in their natural environments and communities. This is possible through introducing a biomimetic 3D culture platoform, such as the macroporous elastomeric silicone foam (MESIF), that can be colonized by bacteria when interfaced with their natural environments.[1]

As an alternative, Winogradsky columns, prepared by cultivating a sample of sediment in a closed column, can be used as model ecosystems to cultivate and study rare taxa.[2] For this purpose, spatiotemporal sampling is essential.

In this study, we develop a system for the continuous spatiotemporal sampling of Winogradsky columns as a foundation to study the growth conditions of rare taxa. MESIF pieces are suspended via metallic frames at different heights inside various columns prepared and cultivated under different environmental conditions. The frames are then extracted after a specific incubation time for subsequent DNA extraction and sequencing. Preliminary results suggest that MESIF pieces attract communities that reflect the growth conditions in the respective environments inside the columns.

References

[1] Zoheir et al., “Macroporous Silicone Chips for Hunting Microbial Dark Matter”, ACS Appl. Mater. Interfaces 2022, 14, 44, 49592-49603.
[2] Esteban DJ et al., “Temporal and Spatial Distribution of the Microbial Community of Winogradsky Columns”, PLoS ONE 2015, 10, 8, e0134588.

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N 07
Wild-ARGI Map: Molecular-based profiling of the gastrointestinal resistome in wild mice

Presenting author:

Max-Delbrück-Centrum für Molekulare Medizin in der Helmholtz-Gemeinschaft, , Robert-Rössle-Straße 10 , 13125 Berlin [DE], VictorHugo.JarquinDiaz@mdc-berlin.de

Author(s):
Robin Julian Edelmann, Hendrik Bartolomaeus, Uli Klümper, Ulrike Löber, Susana Carolina Martins Ferreira, Aimara Planillo, Johan Bengtsson-Palme, Stephanie Kramer-Schadt, Emanuel Hetlinger, Sofia Kirke Forslund, Víctor Hugo Jarquín-Díaz

Antimicrobial resistance represents a global challenge, primarily driven by antibiotic misuse, leading to the spread of bacteria carrying antimicrobial resistance genes (ARGs) in various environments. However, the dynamics and maintenance of ARGs within host-derived microbiomes remain to be fully understood, especially in hosts with close interactions with the environment and humans. Here, we conducted a molecular profiling of the collection of all ARGs (resistome) along the gastrointestinal tract of house mice from their natural environment. Utilizing metagenomic sequencing of intestinal content, we assessed the richness and composition of the microbiome and resistome. Microbiome differences accounted for approximately 60% of resistome composition, with mouse individual and gastrointestinal segments as the primary predictors. We additionally employed qPCR to trace and quantify indicator ARGs associated with transmission risk and anthropogenic impact. Notably, we identified a low abundance but high prevalence of the vancomycin ARG vanA along the gastrointestinal tract. Our findings highlight the impact of the microbial composition of intestinal niches on ARG content. The results suggest a crucial role of interspecies bacterial interactions in maintaining pre-existing resistance independent of direct antibiotic exposure. Microbiomes of mice from their natural environment are a reservoir for resistant bacteria linked to human exposure and an experimentally traceable model.

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M 04
Lipid metabolism as critical link between gut microbiota and colorectal cancer

(withdrawn)

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I 02
Spatial Host-Microbiome Interactions in the Murine Intestine: Dynamics between Altered Microbiota and Host Immunity

Presenting author:

Heidelberg University, Computational Biomedicine, Institute for Computational Biomedicine Im Neuenheimer Feld 130.3, 69120 Heidelberg [DE], ece.kartal@uni-heidelberg.de

Author(s):
Ece Kartal

Deciphering the intricate spatial dynamics of host-microbiome interactions in the murine intestine is crucial for understanding the complex interplay between microbial communities and host physiology. In this study, we investigate the impact of altered microbiota on host immunity and gene expression profiles within the intestinal microenvironment. Using germ-free mice colonized with diverse microbiota and specific pathogen-free mice subjected to antibiotic and antifungal treatments, we employ spatial transcriptomics (visium) to analyze gene expression patterns across the entire intestine. Our preliminary results reveal distinct transcriptional signatures associated with different microbiota compositions, with differential activation of signaling pathways observed. Further analysis involves deconvoluting spatial niches and assessing their association with specific microbiota compositions. By integrating prior knowledge resources, we also estimate transcription factors and pathway activities, uncovering condition- and location-specific alterations. Utilizing advanced methodologies, we investigate cell-cell communication within the intestinal microenvironment, revealing local ligand-receptor relationships and spatial patterns of intercellular communication. Overall, our study provides insights into the spatial host-microbiome interactions in the murine intestine, contributing to a deeper understanding of the complex dynamics governing gut homeostasis and immunity.

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O 12
Hypochlorous acid (HOCl)-derived lipid N-chloramines in host-microbe interactions

Presenting author:

Ruhr Universität Bochum Fakultät für Medizin, Microbial Biochemistry, Universitätsstraße 150, 44801 Bochum [DE], lisa.knoke@rub.de

Author(s):
Lisa Roxanne Knoke, Sara Ursula Abad Herrera, Sascha Heinrich, Natalie Lupilov, Julia E. Bandow, Thomas Günther-Pomorski, Lars I. Leichert

 

Neutrophils are the first line of defence against pathogens in humans. Highly specialized in phagocytosis, they kill engulfed pathogens with a cocktail of oxidants, including the toxic hypochlorous acid (HOCl). In vitro studies have shown that HOCl results in the chlorination of some membrane lipids’ head group amino residues. However, reliable methods for in vivo detection of lipid N-chloramines are lacking and hence their physiological role is still under investigation.

Here, we used the dansyl derivative dansyl sulfinic acid (DANSO₂H) to investigate N-chlorination of membrane lipids in living bacteria and mammalian cells exposed to HOCl.

We showed that exposure of living mammalian or bacterial cells to HOCl results in formation of lipid N-chloramines, and cellular proteins. In vitro studies of model membranes in large unilamellar vesicles (LUVs) single out phosphatidylethanolamine (PE), a major phospholipid in E. coli, also present in the mammalian cytoplasmic membrane, as a main target of HOCl. These resulting N-chloramines were reversed by the cellular antioxidant glutathione (GSH), indicating a role of GSH in counteracting N-chlorination. HOCl-treated LUVs induce probe oxidation in the redox sensitive roGFP2 protein, indicating oxidative activity towards biomolecules. Taken together, our experiments suggest that lipid N-chloramines have a biologically relevant oxidative activity, and potentially accelerate the host immune response similar to protein N-chloramines.

 

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M 23
Microbial context-dependence in the evolution of antibiotic resistant Klebsiella pneumoniae

Presenting author:

EMBL Heidelberg, Genome Biology, Meyerhofstr. 1, 69117 Heidelberg [DE], knopp@embl.de

Author(s):
Michael Knopp, Sarela Santamarina, Denise Selegato, Vitor Cabral, Lina Michel, Nicolai Karcher, Joshua Wong, Michael Zimmermann, Karina Xavier, Nassos Typas

Antibiotic resistance (AR) can impose a fitness cost to the bacterium. Identifying conditions that enhance this cost offers a way to counter-select AR populations. Typically, fitness is assessed in monocultures under artificial conditions, which are far from the complex natural habitats of enteric pathogens. To determine the influence of the microbiome composition on the fitness of AR bacteria, we established a highly sensitive method to determine fitness in complex communities using flow cytometry. We identified a microbiome-specific selection of a carbapenem-resistant K. pneumoniae strain mediated by the acquisition of a secondary mutation in the transcriptional repressor cscR causing upregulation of a carbohydrate-catabolic operon. The selective advantage was largely due to a specific focal E. coli strain, it is contact independent, not-transferable to other microbiomes, the effect size can be modulated by further competitors and it is driven by differential carbohydrate preferences. We are currently applying our findings to an in vivo mouse model to show that nutrient availability shaped by microbiome specific factors can play an important role in competitiveness of AR subpopulations. Our results highlight the microbiome as an overlooked factor when characterizing the fitness of AR-mutants, which could potentially be exploited to minimize the reservoir of AR pathogens in the human gastrointestinal tract.

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R 05
Microbial glycan coating may be modulated by disease-associated factors

Presenting author:

DRFZ, Schwiete laboratory for microbiota and inflammation, Charitéplatz 1, 10117 Berlin [DE], jannike.krause@drfz.de

Author(s):
Jannike Krause, Hyun-Dong Chang

The intestinal microbiota is essential for human health. The cell surfaces of these microbes are densely coated with a glycans, which contribute to host-microbe interactions via specific glycan-binding molecules, i.e. lectins. Recent data from our group show that the microbiota of diseased individuals can be discriminated from the microbiota of healthy individuals by their lectin-signature resembling their glycan coating. However, it is not clear which factors modulate glycan coating. To identify potential modulating factors, we utilized a fecal E. coli isolate. We investigated the effect of individual carbohydrates, metabolic activity, temperature and “inflammatory” fecal supernatant in vitro. Potential changes in glycan coating were analyzed with lectin staining and microbiota flow cytometry.

The metabolic activity most prominently affected lectin-binding, resembling glycan decoration. More specifically, bacteria from non-metabolizing cultures showed higher lectin-binding than from metabolizing cultures, while individual carbohydrates were not obviously driving the lectin-signatures. Temperature induced minor changes in glycan coating and inflammatory fecal supernatant reduced lectin-binding in E. coli. Our data demonstrate that microbial glycan coating may be modulated by disease-associated factors.

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O 22
Regulation of sialic acid synthesis pathway enzymes after supplementation with N-acetylmannosamine (ManNAc) and N-Acetylneuraminic (Neu5Ac) in C2C12 and HEK cell culture models.

Presenting author:

Jonas.krause.heist@gmail.com

Author(s):
Jonas Krause, Kaya Bork, Astrid Gesper

 

GNE myopathy (GNEM) is a rare condition characterized by muscle dystrophy, which usually arises in early adulthood and progresses slowly, until most affected patients become wheelchair dependent. It arises from mutations in the gene responsible for producing a crucial precursor involved in sialic acid production, known as UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). With a prevalence ranging from one to nine cases per million individuals, GNEM is considered an ultra-rare autosomal recessive disorder for which no effective treatment currently exists. Various efforts have been made to alleviate GNEM symptoms, including oral supplementation with precursors of sialic acid such as N-acetylmannosamine (ManNAc), aimed at restoring proper sialylation of muscle cell membranes and thereby enhancing muscle strength. However, most studies have not shown significant improvement in patients treated with these approaches. We examined the expression of the key sialic pathway Enzymes GNE, N-acetylneuraminate synthase (NANS), N-acylneuraminate-9-phosphatase (NANP) and N-acylneuraminate cytidylyltransferase (CMAS), as well as pathway adjacent Enzymes such as N-acetylneuraminate pyruvate lyase (NPL) and N-acetylglucosamine kinase (NAGK). For this we used C2C12 as well as HEK cell culture models, in which GNE was knocked out via CRISPR based gene editing.

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O 13
Degradation of Advanced glycation endproducts by the soil microbiota: Pseudomonas bavariensis as the first Nε-carboxy(m)ethyllysine degrading bacterium

Presenting author:

LMU - Ludwig-Maximilians-Universität München, Faculty of Biology, Großhaderner Straße 2, 82152 Planegg-Martinsried [DE], juergen.lassak@lmu.de

Author(s):
Jürgen Lassak

Thermal food processing leads to the formation of advanced glycation end products (AGE) such as N_ε-carboxymethyllysine (CML). Accordingly, these non-canonical amino acids are an important part of the human diet. However, CML is only partially decomposed by human gut bacteria and up to 30% are excreted via feces. We asked whether and how the soil microbiota can utilize CML. We isolated a bacterium that can grow on CML as well as its higher homologue Nε-carboxyethyllysine (CEL) as sole source of carbon. Bioinformatic analyses upon whole genome sequencing revealed a subspecies of Pseudomonas asiatica, which we named ‘bavariensis’. We performed a metabolite screening of P. asiatica subsp. bavariensis str. JM1 grown either on CML or CEL and identified N-carboxymethylaminopentanoic acid (CM-APA), and N-carboxyethylaminopentanoic acid (CE-APA), respectively. We further detected α-aminoadipate as intermediate in the metabolism of CML. These reaction products suggest two routes of degradation: While CEL seems to be predominantly processed from the α-C-atom, decomposition of CML can also be initiated with cleavage of the carboxymethyl group and under the release of acetate. Thus, our study provides novel insights into the metabolism of two important AGEs and how these are processed by environmental bacteria.

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O 23
Synthesis of Multivalent Cell-Penetrating Peptide Conjugates as Potential Tools for Drug Delivery

Presenting author:

Leipzig University, Institute of Biochemistry, Brüderstraße 34, 04103 Leipzig [DE], nicolas.lindholm@uni-leipzig.de

Author(s):
Nicolas Lindholm, Moritz List, Maik Tretbar, Annette G. Beck-Sickinger

Cell-penetrating peptides (CPP) display an interesting class of biomolecules for various biomedical applications, including the potential use as drug-delivery systems or to generate antimicrobial compounds. With the aim to generate a cell type-selective shuttle for cancer therapeutics, we established a multistep synthesis process to obtain a trimeric derivative of the cell-penetrating peptide Tat (Transactivator or transcription) named tri-cTatB, which was shown to deliver various cargoes into eucaryotic cells in previous studies. The construction of the peptide monomer, a cyclic variant of Tat (cTatB), was straightforward possible using solid-phase-peptide synthesis (SPPS). Here, an azido-lysine moiety was introduced N-terminally to enable subsequent conjugation reactions via homogeneous copper-catalyzed azide-alkye cycloaddition reactions (CuAAC), and the peptide was subsequently cyclized on-resin via head-to-sidechain lactamization. A tetra-alkyne functionalized small-molecule linker was then synthesized via propargylation of pentaerythrit, on which the cTatB peptides as well as a fluorophore were afterwards conjugated in-solution in two sequential CuAAC reactions. After the CuAAC reactions were initially hampered due to sterical and electrostatic restraints of the reactands, optimization of the synthesis procedure yielded the final product tri-cTatB in sufficient amounts for further application in cell-based internalization assays.

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D 05
Effects of trace element modulation on the intestine of mice with different age

Presenting author:

Friedrich Schiller University Jena, Institute of Nutritional Sciences, Nutritional Physiology, Dornburger Str. 24, Jena 07743 [DE], kristina.lossow@uni-jena.de

Author(s):
Kristina Lossow, Tanja Schwerdtle, Anna Patricia Kipp

The intestine is not only relevant for absorption of macronutrients, but also for that of micronutrients. Depending on geographical location, supply of trace elements is more or less satisfactory. Particularly in case of iron, zinc and selenium, a supply fulfilling the recommendations is often not ensured. In addition, ageing leads to a modulation of trace element concentrations in serum and liver.

To investigate effects of modulating trace element supply of mice on integrity and function of the gut and on the composition of the gut microbiota, 3- and 40-week-old C57BL/6Jrj mice were fed low or sufficient amounts of the trace elements copper, iron, selenium and zinc for a period of 6 months.

While the trace element concentrations of faecal samples were primarily determined by trace element intake, a diet-related reduction in intestinal trace element concentrations was only be observed for iron and copper when comparing suboptimal to adequate supply. Age further led to pronounced changes in bacterial composition up to phylum level, whereas low trace element supply only led to minor changes in the microbiota.

This suggests that the intestine, which is the first to be confronted with a changed nutrient supply and reacts in the short term with corresponding adaptations, is modulated in the long term with regard to intestinal parameters and the composition of the microbiota, however, primarily by age and less by the trace element supply.

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N 12
Inferring Horizontal Gene Transfer and Gene Fusion/Fission in Microbial Communities from Orthology Data

Presenting author:

Philipps-Universität Marburg, SYNMIKRO, Karl-von-Frisch-Str. 14, 35032 Marburg [DE], masoodf@staff.uni-marburg.de

Author(s):
Fareha Masood, Paul Klemm, Marcus Lechner

The microbiome, a complex ecosystem of microorganisms residing within and around us, profoundly influences environmental dynamics and human health. In host-associated microbiomes with high bacterial densities horizontal gene transfer is assumed to be ubiquitous. This non-sexual movement of genetic information between genomes is an important evolutionary force, facilitating bacterial diversity and also key for the rapid dissemination of antibiotic resistance. In parallel, gene fusion/fission is a major contributor to evolution of multi-domain bacterial proteins.

In this contribution, we aim to facilitate orthology data predicted by orthology inference tools such as Proteinortho, SonicParanoid or OrthoFinder to depict horizontal gene transfer. We employ statistical tests that identify noticeable deviations in the similarity of any two genes from an orthologous group relative to their peers within the same set of species. This method also enables us to identify the most likely source of the transfer. Gene fusion/fission will be predicted using a combinatorial approach that works on gaps within predicted orthologous groups. It evaluates whether these can be explained by fusion/fission events and includes these in the orthology prediction.

Both algorithms in conjunction enable to track complex evolutionary events within microbiomes. Our work will provide means to elucidate the mechanisms driving genetic diversity and adaptation in microbial communities.

 

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O 04
Surface-Patterned DNA Origami Rulers for the Study of the Nanoscale Distance-Dependency of the Epidermal Growth Factor Receptor Activation

Presenting author:

Karsruhe Institute of Technology (KIT), Institut für biologische Grenzflächen 1, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen [DE], ivy.mayer@kit.edu

Author(s):
Ivy Mayer

 

Over the last two decades, DNA origami has evolved into a well-established, versatile method for the specific arrangement of molecules in the nanometre range.

Within the framework of MOSAIC (Multiscale Origami Structures as Interfaces for cells), we structure DNA-Origami on a surface, to form micro arrays for the analysis of biological processes. Here, we focus on the phenomenon of receptor clustering, were receptors form clusters and show cooperative behaviour that can lead to an amplification of the intensity of a signaling cascade after ligand binding. With the complete control over the number and spatial arrangement of ligands bound on DNA‑Origami, MOSAIC is a compelling way to influence the clustering process and study it in detail. Predominately, we examine the interaction between the Epidermal Growth Factor Receptor (EGFR) and its ligand, the epidermal growth factor (EGF) in various arrangements.

In our current work, we use MOSAIC to gain a better understanding of the influence of nanostructured EGF arrangements on the activation of EGFR while utilizing a simple and highly precise tool.

Additionally, we show that MOSAIC is a versatile method that can be transferred to different cell lines and platforms while still maintaining consistent results which solidifies its potential use as a broadly applicable tool to study the activation of early cell signaling cascades.

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N 06
Macroporous Silicone Chips for Decoding Microbial Dark Matter in Environmental Microbiomes

Presenting author:

Karlsruhe Institute of Technologie (KIT), IBG-1, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen [DE], laura.meisch@kit.edu

Author(s):
Laura Meisch, Marta Velaz Martín, Anne-Kristin Kaster, Kersten S. Rabe, Christof M. Niemeyer

Microorganisms appear in an immense variety on earth, colonizing a huge range of diverse habitats. The majority of these microorganisms is still unknown and are therefore called microbial dark matter (MDM). Since most of these microorganisms do not follow an isolated planktonic lifestyle, cultivation in the laboratory is difficult and their biotechnological potential unexploited. A novel cultivation method using a macroporous elastomeric silicone foam (MESIF) as a cultivation matrix addresses this problem. The MESIF is integrated in a chip design with a media reservoir that can be used for decoding MDM in environmental microbiomes. The model organism Escherichia coli expressing a fluorescent protein, was used to validate the rapid colonization of the low-cost and bioinert chip. Since the surface of the MESIF can be chemically modified via established chemistry, the influence of the surface properties e.g. hydrophobicity on the colonization could also be shown. Environmental microbiomes were enriched in the MESIF chip within a few days. Especially organisms belonging to the Candidate Phyla Radiation could be enriched in chips placed in a moving bed biofilter of a fish tank. From dry air from a chicken coop a variety of biotechnologically interesting Actinobacteriota could be detected in the chip. Sampling with the herbicide glyphosate as the sole carbon source enriched Desulfobacteria from chemical wastewater, which are known to include species metabolizing glyphosate.

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O 02
DNA-based Hydrogels as Functional Materials in Cell Culture and Their Use as Templates for Cell-Free Protein Synthesis

Presenting author:

Karlsruhe Institute of Technologie (KIT), Institute for Biological Interfaces (IBG-1), Hermann-von-Helmholtz-Platz 1, Gebäude 601, Spöcker Straße, 76344 Eggenstein-Leopoldshafen [DE], svenja.moench@kit.edu

Author(s):
Svenja Moench, Alessa Schipperges, Kersten Rabe, Christof M. Niemeyer

 

DNA-based hydrogels are a type of biomaterial with significant potential for various biotechnological applications. Recently, we have developed them into more complex DNA nanocomposite materials using DNA-functionalized nanoparticles as starting points for the polymerization of ultra-long single strands of DNA produced by rolling circle amplification.

These hydrogels possess unique properties of nucleic acids and exhibit excellent biocompatibility. Their nanocomposite material-specific characteristics make them suitable for use in cell culture. Flow cytometry and confocal microscopy have demonstrated that these nanocomposites are efficiently taken up by various eukaryotic cell lines (> 90%).

Materials with transcribable genetic information are produced by incorporating plasmids encoding fluorescent proteins. The use of plasmid-integrated nanocomposites does not result in protein expression in living cells. However, they serve as effective templates for cell-free protein synthesis (CFPS).

Our research focuses on the impact of various DNA-based hydrogels on the efficiency of CFPS. Specifically, we are examining RCA-based materials in both polymerized and non-polymerized states, as well as ligation-based and acrylamide-based hydrogels. The final yields can be easily compared by fluorescence spectroscopy when expressing green fluorescent protein (GFP). These results will aid in efforts to increase the efficiency of CFPS.

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D 02
Antisense oligomers repress the genotoxic activity of colibactin producing E. coli

Presenting author:

Institute for Molecular Infection Biology (IMIB), University of Würzburg, Germany, , Josef-Schneider-Str. 2, 97080 Würzburg [DE], sarah.nentwich@uni-wuerzburg.de

Author(s):
Sarah Nentwich, Linda Popella, Jörg Vogel

The genotoxin colibactin is a secondary metabolite produced by several Enterobacteriaceae that triggers interstrand crosslinking of DNA and thereby induces DNA damage in eukaryotic cells. The presence of colibactin-producing bacteria has been correlated to the promotion of colorectal cancer and urinary tract tumors. Further, a specific mutational signature is associated with the genotoxic activity of colibactin. Colibactin is encoded by a 54-kb gene cluster, referred to as pks (polyketide synthase) island, which consists of 19 clb genes (clbA-clbS). Here, we present a new strategy to repress the production of colibactin via targeted gene knockdown using antisense oligomers. Specifically, various peptide nucleic acid (PNAs) 10mers were designed to inhibit translation of clbA, clbP, clbQ, and clbR. The efficiency of PNAs to dampen colibactin production was evaluated at different levels. First, PNA-mediated decrease of target protein level was validated in vitro as well as in living bacterial cells. Second, we provide evidence that PNAs can decrease the in vitro crosslinking capacity of colibactin-producing bacteria. Third, cell culture infection experiments revealed a reduction of the colibactin-induced mammalian DNA damage response when bacteria were pretreated with specific PNAs. In summary, we identified three promising PNAs, two of them targeting clbA, involved in the activation of colibactin synthesis, and one targeting the key transcriptional activator ClbR.

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M 10
Fecal microbial load is a major determinant of gut microbiome variation and a confounder for disease associations

Presenting author:

European Molecular Biology Laboratory, Structural and Computational Biology, Friedrichstraße 13, 69117 Heidelberg [DE], suguru.nishijima@embl.de

Author(s):
Suguru Nishijima, Michael Kuhn, Evelina Stankevic, Oliver Aasmets, Helene Juel, Maja Thiele, Elin Org, Naoyoshi Nagata, Aleksander Krag, Torben Hansen, Peer Bork

Sequencing-based approaches capture only relative abundances of taxa and genes in microbiome studies. Recognizing the limitations of existing labor-intensive and costly methods to perform quantitative analysis, we aimed to develop a novel computational method that quantifies the microbial load (total microbial cells per gram) directly from the sequencing data. Leveraging paired gut metagenomes and cell count data from two independent large study populations, GALAXY (n = 1,894) and MetaCardis (n = 1,812), we constructed machine-learning models to predict the fecal microbial load based on the relative species abundances. When applied to the public dataset from 161 previous studies (n = 34,539), the predicted loads were significantly associated with numerous host factors, including 15 different diseases with significant changes in the microbial load. We found that the change in the microbial load was a major driver in shifting the microbiome composition in those patients, and more than half of the disease-associated species could be more strongly associated with the load than the disease status. Adjusting for the effect of the microbial load substantially reduced the statistical significance of the disease-associated species. Our large-scale analysis reveals that the fecal microbial load is a major determinant of gut microbiome variation and confounder in microbiome studies, highlighting its importance for understanding microbiome variation in health and disease.

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I 03
Eat or die - microbial context in the gut shapes toxin-dependent and independent protection against Salmonella

Presenting author:

Helmholtz Centre for Infection Research, Microbial Immune Regulation, Inhoffenstraße 7, 38124 Braunschweig [DE], lisa.osbelt-block@helmholtz-hzi.de

Author(s):
Lisa Osbelt, Éva d.H. Almasi, Marie Wende, Sabine Kienesberger, Ellen Zechner, Till Strowig

 

The Klebsiella oxytoca species complex (KoSC) is part of the human microbiome and is particularly prevalent during infancy and childhood. KoSC strains can produce two enterotoxic natural products, tilimycin and tilivalline, while also contributing to colonization resistance (CR) against pathogenic Enterobacteriaceae. The relationship between these seemingly contradictory roles has remained underexplored.

In this study, we show that K. oxytoca provides CR against Salmonella infections, a major health threat, especially in infants, via two distinct but interconnected mechanisms that are largely shaped by the microbial context. In situations with high sugar accessibility, such as disturbed microbiomes, antimicrobial activity against Salmonella strains depended on tilimycin production, while it was largely toxin-independent in mice with residual microbiota. This was linked to the relative levels of toxin-inducing carbohydrates in vivo.

These findings demonstrate that the environment-dependent production of antibacterial secondary metabolites is intimately linked to the availability of carbon sources and further show the potential relevance of these to interspecies interactions. This is a remarkable example of how metabolic flexible keystone species confer significant functional variability in the developing or disturbed human gut microbiome and might also be a relevant finding for future intervention strategies to prevent the negative effects of acute or chronic toxin production.

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N 05
Adenylyl-Cyclases of Sinorhizobium meliloti and cAMP-Signaling-Models

Presenting author:

Philipps-Universität Marburg, FB15 Chemie, Hans-Meerwein-Str. 4 , 35033 Marburg [DE], stefan.petschak@chemie.uni-marburg.de

Author(s):
Stefan Petschak, Laura Werel, Hagen Nußhär, Ankan Banerjee, Elizaveta Krol, Anke Becker, Lars-Oliver Essen

In many bacteria, cyclic 3',5'-AMP (cAMP) serves as crucial secondary nucleotide messenger and contributes to the regulation of metabolism, motility and host colonization. Predominantly, cAMP is synthesized by different classes of adenylyl cyclases (ACs). In the case of the plant symbiotic bacterium Sinorhizobium meliloti, the genome encodes not only 28 class III ACs, but also one putative class VI AC. This raises the question of how the rhizobial signal transduction of class VI ACs differs from the signaling of other AC classes.

Crystallization of the Clr-effector-DNA-complex together with cyclic 3',5'-GMP (cGMP) or cAMP shows that both cyclic nucleotides elicit a similar active conformation of the transcriptional regulator Clr and thus trigger expression of reporter genes in promoter activity assays. Furthermore, we crystallized the monomeric cyclase homology domain of CyaD1 and reconstructed the active dimer using a published X-ray structure of CyaC from Arthrospira platensis.

We were able to heterologously express various class VI rhizobial ACs and characterize their activity. In silico structural modeling and sequence analysis indicate a highly conserved structure among the investigated class VI ACs and CyaC. Therefore, further studies will clarify whether the enzymatic mechanism of rhizobial class VI ACs rather supports a signaling model of freely diffusing cAMP, indicated by crystallographic data for Clr, or a caged cAMP-Clr-signaling-complex, as published for CyaC.

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O 07
Probing transcriptional dynamics of gut epithelium colonization by the human commensal Bacteroides thetaiotaomicron

Presenting author:

Helmholtz Institute for RNA-based Infection Research, , Josef-Schneider-Straße 2/D15, 97080 Würzburg [DE], gianluca.prezza@helmholtz-hiri.de

Author(s):
Gianluca Prezza, Gohar Mädler, Thomas Guest, Alexander J. Westermann

 

Microbial communities colonize the gut of humans throughout their host’s entire life and have multiple consequences on its health. Clarifying host-microbiota interactions is therefore key to a complete understanding of human physiology. Cross-species transcriptomic technologies (Westermann and Vogel, Nat Rev Genet, 2021) lend themselves as tools to uncover the molecular basis of these interactions. Here, we apply dual RNA-seq to follow gene expression kinetics during B. thetaiotaomicron colonization of a human gut epithelium cell culture model and reveal Bacteroides gene sets differentially regulated between bacteria adherent to the host mucus and the luminal population. Among the identified differentially expressed genes, several encode small RNAs (sRNAs)—short noncoding transcripts commonly used across the bacterial kingdom to quickly respond to external and intrinsic cues by post-transcriptionally regulating target gene expression. Through a CRISPR-based reverse genetics screen (Prezza et al., PNAS, in press), we identify sRNAs that functionally contribute to the interaction with the human mucosa and begin to characterize one of them, BTnc60, which is predicted to regulate mucin-utilization genes. This ongoing work provides insights into the spatio-temporal aspects of gut colonization and underscores the significance of bacterial sRNAs in shaping mutualistic interactions within the human microbiota.

 

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O 10
Metabolic Profile Analysis of an Omnivorous and Vegetarian Diet using 1H-Nuclear Magnetic Resonance Spectroscopy on Human Faeces Samples

Presenting author:

luna.reifenrath@gmail.com

Author(s):
Luna Reifenrath

Clinical relevance of the gut microbiome and metabolic profile of faeces samples in relation to health and disease has been demonstrated recently. Linkage of the faeces metabolic profile to nutrition has not yet been well understood but is receiving growing interest. Therefore, the metabolic profiles of a short-term vegetarian diet and a long-term omnivorous diet are investigated by nuclear magnetic resonance (NMR) and distinguished using multivariate analysis. Faeces have a significantly complex matrix, and sample preparation techniques vary between different studies. Today no standardized protocol including at home sample collection has been established. Samples collected for this study were stored in ethanol for sample preservation. Drying in a vacuum centrifuge to remove the ethanol proved to be efficient while preserving short chain fatty acids. Partial Least Squares Discriminant Analysis (PLS-DA) revealed a separation of the samples obtained during vegetarian and omnivorous dietary periods. Interestingly, short chain fatty acids were associated with the signals that induced the separation of the two groups. Intraindividual differences in the concentration of various metabolites were seen between diet groups, but interindividual variability led to concentration variations in opposing directions. These are already promising findings considering the relatively short intervention period and no controlled macro- and micronutrient intake.

 

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M 02
Structural basis of Fusobacterium nucleatum adhesin Fap2 interaction with receptors on cancer and immune cells

Presenting author:

Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), , Robert Roessle Str. 10, 13125 Berlin [DE], roderer@fmp-berlin.de

Author(s):
Daniel Roderer, Felix Schöpf

The intestinal microbiome (IM) is decisive for the human host’s health. Numerous microbiota drive the progression of colorectal cancer (CRC), the third-most common cancer worldwide. The gram-negative Fusobacterium nucleatum (Fn) is overrepresented in the IM of CRC patients and has been correlated with the emergence, progression, and metastasis of tumors. A key pathogenic factor of Fn is the adhesin Fap2, a 400 kDa autotransporter protein that facilitates association to cancer and immune cells via two receptors, the glycan Gal-GalNAc and the T-cell protein TIGIT, respectively. The latter interaction leads to deactivation of immune cells. Mechanistic details of the Fap2/TIGIT interaction remain elusive due to the lack of high-resolution structural data. Here, we report a system to recombinantly express functional Fap2 on the Escherichia coli surface, which interacts with Gal-GalNAc on cancer cells and with purified TIGIT with submicromolar affinity. Cryo-EM structures of Fap2, alone and in complex with TIGIT, show that the 50 nm long rod-shaped Fap2 extracellular region binds to TIGIT on its membrane-distal tip via an extension of a β-helix domain. Moreover, by combining structure predictions, cryo-EM, docking and MD simulations, we identified a binding grove for Gal-GalNac on the tip of Fap2. Our data provide the first mechanistic insight into the role of Fap2 in Fn tumor colonization and lay the foundation for the inhibitor design to interfere with Fap2 receptor interaction.

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M 11
Airway microbial metagenomics from premature birth to end-stage lung disease

Presenting author:

Hannover Medical School (MHH), Department for Pediatric Pneumology, Allergology and Neonatology, Carl-Neuberg-Straße 1, 30625 Hannover [DE], rosenboom.ilona@mh-hannover.de

Author(s):
Ilona Rosenboom, Ajith Thavarasa, Hollian Richardson, Colin F. Davenport, Lutz Wiehlmann, Dorothee Viemann, James D. Chalmers, Burkhard Tümmler

Background: Progressive respiratory diseases are characterised by chronic infections. Shotgun metagenomics identifies bacteria, fungi and DNA viruses at higher resolution than amplicon sequencing commonly applied to study this habitat.
Objectives: The airway metagenome of preterm infants (n=24), healthy adults (n=88) and people with bronchiectasis (n=101) was examined to identify microbial community members that distinguish disease from lung health.
Methods: Shotgun metagenomics sequencing was performed on an Illumina NextSeq. The generated data sets were processed by our in-house pipeline Wochenende.
Results: During their stay at the neonatal intensive care unit, preterm neonates acquired individual non-maternal airway metagenome signatures from the hospital environment. After hospital discharge, airway metagenomes developed towards a common taxonomic structure, but did not achieve the stable bacterial community structures seen in healthy full-term infants. In case of the people with bronchiectasis, the individual metagenomes clustered by the absence or presence of H. influenzae and P. aeruginosa. Comparison with the sputum metagenome of healthy non-smokers revealed a gradient of depletion of commensal taxa in bronchiectasis, even in the absence of any respiratory pathogen.
Conclusion: As signs of microbial dysbiosis, commensal species become gradually suppressed in people with bronchiectasis and the bacterial metagenome of preterm infants is still immature by 15 months of age.

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M 16
A commensal 3D skin model to study the interplay of skin bacteria and melanoma progression in situ

Presenting author:

Bundesinstitut fuer Risikobewertung, Nachwuchsgruppe Hautmikrobiom, Max-Dohrn-Str. 8-10, 10589 Berlin [DE], aline.rosin@bfr.bund.de

Author(s):
Aline Rosin, Maya Kissner, Tewes Tralau, Tessa Hoeper, Lisa Lemoine

The human skin harbours millions of microorganisms, including bacteria, fungi and viruses. Together they are referred to as the skin microbiome. Especially bacteria are involved in critical cellular processes, like pathogen protection or immune modulation. However, a lack of balance among bacterial communities, termed dysbiosis, is implied to play a role in the development of some skin diseases including the skin cancer type melanoma. Nevertheless, systematic data on the underlying mechanisms between the microbiome and melanoma progression are still elusive.

To our knowledge, we are the first to establish a co-culture system capable to study host microbiome interactions during melanoma progression in situ. This system consists of the commercial 3D skin model MelanomaFT^TM colonized with skin bacteria obtained from a skin swab of a healthy volunteer.

The models showed a stable co-colonization, yet significant differences regarding the transcriptome profile were detected in colonized models relative to control on day twelve. In particular, pathways involved in melanoma progression, like RAF/MAP and PI3K kinase, showed an upregulation in colonized models. This correlates with the evaluated release of the cytokines VEGF and IL-8 as well as the melanoma marker MIA. MIA along with the secreted cytokines are known to be involved in tumorigenesis. Further, NGS analysis will be performed in order to prove dysbiosis and identify the bacteria species associated with the noticed effects.

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O 19
When clinical goes sweet – Early detection of hepatocellular cancer and other inflammation-related diseases using NMR glycosylation profiles from serum samples

Presenting author:

l.rudolph@uni-luebeck.de

Author(s):
Lorena Rudolph, Darko Castven, Lina Jegodzinski, Franziska Schmelter, Jarne Mölbitz, Renia Krellmann, Jens Marquardt, Alvaro Mallagaray, Ulrich L. Günther

Nuclear Magnetic Resonance (NMR) spectroscopy is an established technique for blood metabolomics. NMR spectra of blood provide vital data, revealing signals from metabolites, lipoproteins, and acute-phase glycoproteins. Particularly, glycosylation profiles hold significant diagnostic potential since they are modulated under inflammatory conditions. Traditionally, NMR-based biomarkers for such conditions were limited to two prominent signals from N-acetyl groups of glycoproteins. Our latest research represents a breakthrough as we decomposed these signals, offering detailed, quantitative insights into the glycosylation types and nature of the associated proteins. Building on this, we developed NMR experiments that can detect a wide range of characteristic, previously elusive glycoprotein signals, achieving this in under 10 minutes and without sample pre-processing. The clinical potential of this method is demonstrated for hepatocellular cancer samples. Our technique identifies alterations in glycosylation patterns, including linkage-specific levels of sialylation, galactosylation, and fucosylation in prevalent N-glycans, along with their branching complexity. Notably, we can detect Lewis antigen, a biomarker for early detection of several diseases, including cancer. The rapid, cost-effective nature of this method, coupled with its ability to analyze native plasma or serum samples, opens up new avenues in the application of glycosylation biomarkers for clinical diagnostics.

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O 17
Structural insights into the µ-opioid receptor, β-arrestin, GRK interaction

Presenting author:

Leipzig University, Institut of Biochemistry, Südliches Heimfeld 111, 04509 Delitzsch [DE], annalena.roehlig@icloud.com

Author(s):
Annalena Marie Röhlig, Irene Coin

Understanding the molecular dynamics of the µ-opioid receptor, a rhodopsin-like G protein-coupled receptor (GPCR), is not only crucial for unraveling the receptor's physiology but also for exploring its pharmacology, which facilitates the development of opioid alternatives with less side effects. G protein-coupled receptor kinases (GRKs) and β-arrestins are crucial regulators of the µ‑opioid receptor desensitization and internalization. In the lack of 3D structures of signaling complexes of the µ-opioid receptor, we use the genetic code expansion technique to examine the interaction between GRKs, β‑arrestin and the µ‑opioid receptor. We apply a photo-activatable genetically encoded unnatural amino acid (UAA) as a proximity probe, which enables us to explore the µ-opioid receptor-GRK or ‑β‑arrestin interface directly in mammalian cells with a single-residue resolution. Here, we reveal first structural insights into the interaction between the µ-opioid receptor, GRKs and β‑arrestins.

 

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R 01
The RNA-binding protein RbpB is a central regulator of polysaccharide utilization in gut Bacteroides

Presenting author:

Institute for Molecular Infection Biology (IMIB) , , Josef-Schneider-Straße 2, 97080 Würzburg [DE], ann-sophie.ruettiger@uni-wuerzburg.de

Author(s):
Ann-Sophie Rüttiger, Daniel Ryan, Luisella Spiga, Vanessa Lamm-Schmidt, Gianluca Prezza, Sarah Reichardt, Lars Barquist, Franziska Faber, Wenhan Zhu, Alexander J. Westermann

Paramount to human health, symbiotic bacteria in the gastrointestinal tract rely on the breakdown of complex polysaccharides to thrive in this sugar-deprived environment. Gut Bacteroides, as metabolic generalists, deploy dozens of polysaccharide utilization loci (PULs) to forage diverse dietary and host-derived glycans. The expression of the multi-protein PUL complexes is tightly regulated at the transcriptional level. However, how PULs are orchestrated at translational level in response to fluctuating substrate levels is unknown. Here, we identify the RNA-binding protein RbpB and a family of noncoding RNAs as key players in post-transcriptional PUL regulation. Ablation of RbpB in Bacteroides thetaiotaomicron compromises colonization in the mouse gut, dependent on the host diet. Current dogma holds that individual PULs are regulated by dedicated transcriptional regulators. We demonstrate that RbpB acts as a global RNA binder interacting with several hundred cellular transcripts. This includes a paralogous noncoding RNA family comprised of 14 members, the FopS (family of paralogous sRNAs) cluster. Through a series of in-vitro and in-vivo assays, we reveal that FopS sRNAs repress the translation of a SusC-like glycan transporter when substrates are limited —an effect antagonized by RbpB. Together, this study implicates RNA-coordinated metabolic control as an important, yet overlooked, factor contributing to the in-vivo fitness of Bacteroides in dynamic nutrient landscapes.

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I 06
18S rRNA sequencing to detect microbial eukaryotes in human samples from rural Madagascar

Presenting author:

Institut für Klinische Molekularbiologie, AG Franke - Genetics & Bioinformatics, Am Botanischen Garten 11, 24118 Kiel [DE], j.saalfrank@ikmb.uni-kiel.de

Author(s):
Johanna Saalfrank, Corinna Bang, Daniela Fusco, Andre Franke

Majority of world’s population is affected by intestinal helminths. Despite potential benefits, eukaryotic intestinal parasites are often neglected in host-microbiome studies. In Madagascar, the prevalence of schistosomiasis is 52% and thus, human stool samples from this area were used to specifically detect Schistosoma mansoni, one causative agent for this neglected disease to obtain further insights into host-parasite interactions.

We established a Metabarcoding Next Generation Sequencing method, targeting eukaryotic 18S rDNA and investigated 803 human stool samples from Malagasy communities. The workflow included DNA amplification with the 616*F/1132R primer pair, paired-end MiSeq® Illumina sequencing, concatenation of reads, annotations to PR2 database, and data analysis. In addition, the bacterial microbiota was investigated using 16S V3V4 rDNA sequencing.

Besides Schistosoma, additional intestinal helminths and various fungi were found. Significant differences in eukaryotic microbiota composition were observed, based on region of sample collection and participant’s gender. Similarly, bacterial microbiota composition showed significant differences related to gender and location, but surprisingly not concerning S. mansoniinfection. In addition, diagnosis of schistosomiasis in stool samples was comparable to results of specific PCR from human plasma. Thus, the established NGS-method could be used for research, but also for diagnostics on those samples on site in future.

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D 03
Quest for Novel Species-specific Carriers for Antisense Oligonucleotides into Bacteria

Presenting author:

University of Wuerzburg, Insitute for Molecular Infection Biology, Josef-Schneider-Straße 2/D-15, 97080 Würzburg [DE], paramita.sarkar@uni-wuerzburg.de

Author(s):
Paramita Sarkar, Linda Popella, Jörg Vogel

Programmable antisense oligomers (ASOs) bear great potential for species-specific killing of bacterial pathogens and modulation of gene functions in genetically intractable microbes. However, they are limited by a lack of efficient carriers for the translocation of antisense oligonucleotides (ASOs) across the bacterial membrane. Additionally, there is no strategy to directly report their uptake. To address this, we develop a reporter assay to screen ASO carriers’ penetration into the cell. The assay exploits antisense-based activation of a synthetic toehold switch with a cell-penetrating peptide-peptide nucleic acid conjugate (CPP-PNA) in Escherichia coli and Salmonella enterica. Screening different carriers, we observe up to 120-fold activation, depending on the peptide and bacterial strain. The screening platform represents a portable tool to enable the discovery of species-specific ASO carriers for targeted manipulation of complex communities of bacteria.

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N 10
A phenotypic landscape of key Bacteroides species

Presenting author:

EMBL Heidelberg, Genome Biology Unit, Friedrich-Ebert-Anlage 46, 69117 Heidelberg [DE], eva.schorr@embl.de

Author(s):
Eva-Magdalena Schorr, Carlos G. P. Voogdt, Athanasios Typas

Most of our knowledge on bacterial gene function stems from few model species that do not represent the large genetic diversity of the gut microbiota. Consequently, about half of the bacterial genes in the human intestinal microbiome remain poorly characterized. We set out to systematically discover functions of this so called “genetic dark matter” in two core species of the human gut microbiome, Bacteroides uniformis and Phocaeicola vulgatus.

We will use barcoded transposon mutant libraries and massively paralleled barcode sequencing to determine mutant fitness across hundreds of biological, chemical and physical conditions. This enables us to link genes to phenotypes. Genes with similar phenotypes across many conditions are likely functionally related, and together with other types of information (physical complexes, structural predictions, genomic context) will give insights into gene function and organization.

Bacteroides and Phocaeicola species are well known for their capacity to degrade dietary polysaccharides but the genes involved are not all discovered yet. From an initial screen with 20 different carbon sources, we found that mutants in BACUNI_01153 to BACUNI_01163 (PUL 17) cannot grow on Fructans and BACUNI_01201 to BACUNI_01214 (PUL 18) on Glycogen. With this data we can link 2 new PULs to their respective substrate in B. uniformis. We expect that our data on other conditions will help uncover gene functions in other pathways.

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M 03
Multi-parameter microbiota flow cytometry for disease-specific microbiota phenotyping and identification of disease-associated bacteria

Presenting author:

German Rheumatism Research Center (DRFZ) Berlin, A Leibniz Institute, Schwiete Laboratory for Microbiota and Inflammation, Charitéplatz 1, 10117 Berlin [DE], toni.sempert@drfz.de

Author(s):
Toni Sempert, Hyun-Dong Chang

Chronic-inflammatory diseases (CID) show alterations in mucosal immune response and composition of intestinal microbiota, known as dysbiosis. Alterations of the intestinal microbial community are primarily determined by 16S rRNA gene amplicon sequencing of the entire bacterial community present in a stool sample. We developed an approach to analyze cellular properties of human gut microbiota from stool samples on single cell-level by multi-parameter microbiota flow cytometry (mMFC). We acquire information about coating with endogenous host immunoglobulins by isotype-specific stainings to capture the mucosal immune response of the host. Further, the expression of specific sugar moieties on the bacterial surface by lectin staining potentially reflects adaption of the bacteria to altered micro-environmental conditions in the intestine. By using machine-learning, we identified disease-specific phenotypic signatures for various CIDs. The direct isolation of immunoglobulin-coated and lectin-stained bacterial populations by fluorescence-activated cell sorting followed by whole 16S rRNA gene sequencing revealed that the phenotypic signature as represented by immunoglobulin coating not only correlates with but also enhances the observed taxonomic dysbiosis. Thus, mMFC can not only be applied as a diagnostic tool for disease classification but can also help to understand host-microbiota crosstalk in disease by enabling the identification and isolation of disease-associated bacteria.

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M 13
Antibiotic-induced gut microbiome dysbiosis negatively modulate CNS serotonin levels and affects anxiety-like behavior

Presenting author:

ICCBS - University of Karachi , Molecular Medicine, Lab P-134, PCMD, ICCBS, University of Karachi, 75270 Karachi [PK], jazibshafiq@gmail.com

Author(s):
Jazib Shafiq, Ayaz Ahmed

The complex ecosystem of gut harbors diverse microbial communities that regulate host physiological functions through neural, immune, and metabolite interactions. Variations in gut microbiome composition contribute to behavioral disorders. Gut microbiome also has an impact on host anxiety through mechanisms not completely understood. In 1^st phase of our study, we developed gut dysbiosis mouse model using antibiotic cocktail to dissect the microbiome-mediated anxiety regulation mechanism, and in 2^nd phase, we analyzed the post-antibiotic recovery trajectory of gut microbiome and correlated it with anxiety-like behavior. Following anxiety evaluation, neurotransmitter, and targeted gene transcripts were assessed in mice hippocampus. Further, cecum was subjected to metagenomics. Our results revealed decreased hippocampal tryptophan (Trp), serotonin (5-HT), and 5-HIAA levels with anxiolytic behavior in dysbiosis mice. Hippocampal expression of the 5-HT transporter was also downregulated. Metagenomics analysis showed increased dominance of Klebsiella and Escherichia species in dysbiosis group that metabolize Trp and decreases its accessibility to the host. Further our analysis confirmed that reversal of anxiety phenotype and recovery in gut bacterial diversity and richness after 42 days of antibiotic cessation. Together, our data suggest that gut microbiome perturbations alter hippocampal 5-HT bioavailability and modulate anxiety-like behavior via Trp metabolism pathway.

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N 09
Genome-scale mapping of genetic regulators of uracil exchange in yeast

Presenting author:

Leibniz Institute on Aging - Fritz Lipman Institute, , Beutenbergstraße 11, 07745 Jena [DE], scherbakovaa625@gmail.com

Author(s):
Gerbren Spoelstra, Michael Muelleder, Stephan Kamrad, Anastasiia Shcherbakova, Clara Correia-Melo, Markus Ralser

Metabolism takes place both within and outside of cells. Metabolite exchange interactions between cells drive cell diversity and specialization, with the generation of specific exometabolomes that impact, for example, survival, lifespan, antioxidant response, and even drug tolerance. However, how metabolites exchange interactions are regulated at the genome level is still not clear.

In our study, we engineered a library of ~5000 Saccharomyces cerevisiae knockouts (YKO) to track and enhance metabolite exchange. For this, we generated self-establishing metabolically cooperating communities (SeMeCo) in each yeast KO of the library. These communities, auxotrophic for uracil, leucine, and histidine, were transformed with three plasmids compensating for the respective auxotrophies that, upon plasmid loss, rely on the exchange of these metabolites for survival. To identify the genetic regulators for uracil exchange, we performed DNA-barcode Next Generation Sequencing on the pooled SeMeCo-YKO cultured with or without uracil.

Mutant enrichment analysis revealed notable variation in mutant composition within the community depending on the presence/absence of uracil. Enriched and depleted mutants not only included KO genes for uracil metabolism, but also those associated with protein synthesis, and some unannotated genes. This study will enable the genome-scale mapping of uracil exchange regulators, with the potential to reveal new gene functions.

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M 15
Mapping microbes in the tumor microenvironment

Presenting author:

EMBL Heidelberg, SCB, Mark Twain Strasse, 69126 Heidelberg [DE], shraddha.shitut@embl.de

Author(s):
Shraddha Shitut, Nicolai Karcher, Ruby Ponnudurai, Matthias Kloor, Georg Zeller

The interaction between microbes and their host significantly impacts host survival, growth, and disease development, including cancer. Recent studies have revealed a significant correlation between specific bacterial taxa and tumors in various regions of the body, known as microbial signatures. The cause and consequence of these specific taxa on the tumor microenvironment or vice versa is unclear. To better understand how tumor-infiltrating bacteria interact with host cells and impact tumor progression, a sequential FISH (fluorescence in situ hybridization) approach is presented. This method allows for the identification and visualization of different bacterial taxa associated with colorectal cancer (CRC) tissue. By using custom oligonucleotide probes and fluorescence microscopy, multiple bacterial species can be spatially mapped on the tumor landscape. Comparing a tumor region to an adjacent normal region, clear differences in the density and relative proportions of the bacterial signal are observed. Although the overall diversity across the tissue remains similar, there is an enrichment of certain taxa in distinct physiological zones (lumen, mucosa, submucosa). This method, which capitalizes on metagenomic data for probe design, offers a deeper understanding of the spatial interactions between microbes and the host. Identifying the role of microbes in cancer progression will potentially aid in the development of targeted treatments and better diagnostic approaches.

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O 05
Monitoring of antimicrobial resistance carrying plasmids – adaptation to long-term antibiotic treatment

Presenting author:

University Hospital Münster, Institute of Hygiene, Robert-Koch-Straße 51, 48149 Münster [DE], annika.sobkowiak@ukmuenster.de

Author(s):
Annika Sobkowiak, Natalie Scherff, Franziska Schuler, Alexander Mellmann, Vera Schwierzeck, Vincent van Almsick

Antimicrobial resistance (AMR) is a global burden on human health. Little is known about the adaptation of multidrug resistant bacteria (MDRB) in vivo and the role of AMR plasmids in the clinical setting. To get insights, we analysed consecutive MDRB isolates and their AMR plasmids of one patient.

All MDRB were collected between 02/2022 and 05/2023 and AMR was tested. Whole genomes were sequenced with a PacBio^® Sequel IIe system and de novo assembled. For further analysis, Ridom SeqSphere+ (core genome MLST), DFAST (annotation) and MAUVE (alignment) software tools were used.

The patient received antibiotic therapy for recurrent infections of the liver and biliary system since 12/2021. Since 11/2022, the patient was treated with meropenem. Five multidrug resistant Klebsiella isolates were collected from this patient.
First, the predominant Klebsiella species identified was K. quasipneumoniae, from 03/2022 on K. pneumoniae was detected. Two different phenotypic K. pneumoniae isolates with an allelic distance of 46 were detected in 03/2023.
Despite the genetic diversity of the isolates, we identified similarities in the plasmids of the isolates, suggesting a well-adapted AMR plasmid.

This case shows the in vivo adaptation of different Klebsiella isolates under antibiotic selection pressure. We speculate that the plasmid has been transferred among the isolates; however, the role of the plasmid to this adaptation have to be clarified in future in vitro experiments.

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O 09
A microfluidic chip for adaptation and selection of whole-cells

Presenting author:

Karlsruhe Insitute of Technology (KIT), Institute for Biological Interfaces (IBG-1) , Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen [DE], camilla.stolle@kit.edu

Author(s):
Camilla Stolle, Christof M. Niemeyer, Kersten S. Rabe

Adaptive laboratory evolution (ALE) is a key tool in the study of evolutionary processes and biotechnology. Microfluidics have revolutionized microbial ALE as it can mimic naturally occurring microenvironments promoting the growth of biofilms alongside planktonic cells.

This poster introduces a novel microfluidic chip which is used for the adaptation of microbial cells to stressors such as antibiotics by cultivating them in consecutive microcompartments within a customizable stressor concentration gradient.^[1] The chip was successfully used to adapt Escherichia coli to different antibiotics and importantly lead to the discovery of previously unknown mutations that confer resistance to nalidixic acid. The controlled change in the antibiotic concentration revealed the chip’s capacity to differentiate between persistence and resistance. Recently, the organism scope of the chip was extended to extremophiles and Thermus thermophilus was successfully adapted to kanamycin using the chip system.

The chip presented in this poster enhances the occurrence of mutations, resulting in the generation of stress-resistant strains. This kind of miniaturized chip-based ALE offers insights into the mechanism of antibiotic resistance and can be applied to adapt virtually any microorganism.

 

References

[1] Zoheir, A. E., Späth, G. P., Niemeyer, C. M., Rabe, K. S., Microfluidic Evolution-On-A-Chip Reveals New Mutations that Cause Antibiotic Resistance. Small 2021, 17, 2007166.

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O 01
Access to thermostable enzymes and their application in flow biocatalysis

Presenting author:

KIT - Karlsruher Institut für Technologie, IBG-1, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen [DE], marius.stoeckle@kit.edu

Author(s):
Marius Stöckle, Martin Peng, Christof Niemeyer, Kersten Rabe

 

The immobilization of biocatalysts in a continuous fluidic setup is one way to achieve compartmentalization and thus precise control over artificial reaction cascades for synthetic chemistry. We recently demonstrated the encapsulation of unmodified thermostable enzymes in a 3D printed, agarose-based hydrogel to enable multi-step sequential biotransformations.¹ To test the feasibility of the encapsulation strategy, we used a naturally thermostable alcohol dehydrogenase as well as a ketoisovalerate decarboxylase (KIVD) from a mesophile organism. KIVD was thermostabilized by different computational or evolutionary methods to increase the T₅₀ value by up to 9°C.^1,2

Thermostable enzymes also offer improved process stability when covalently attached onto beads in a packed-bed reactor and we selected a benzaldehyde lyase as an example, since only one enzyme had been biochemically characterized before, which was rather instable.³ To this end, we employed a computational prediction tool for the identification of a novel thermostable benzaldehyde lyase and employed the enzyme for the continuous production of α-hydroxy-ketones. A homology-model based approach was used to create enzyme variants with altered substrate scope, which also showed further increased thermal stability (up to 22°C increase in T₅₀).

1) M. Maier et al. (2018) Angew. Chem., Int. Ed., 57, 5539.

2) M. Peng et al. (2019) Biol. Chem., 400, 1519.

3) M. Peng et al. (2022) ChemBioChem, 23, e202100468.

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D 01
Revealing the processes that shape the ecosystem of Bacteroidales-dominated gut communities

Presenting author:

Helmholtz Center for Infection Research, Microbial Immune Regulation, Inhoffenstraße 7, 38124 Braunschweig [DE], caroline.taouk@helmholtz-hzi.de

Author(s):
Caroline Tawk, Youssef El Mouali, Johanna Rapp, Hannes Link, Athanasios Typas, Till Strowig

The gut microbiota of healthy individuals is commonly dominated by the Bacteroidota phylum. Within this phylum, members of the Bacteroidaceae and Prevotellaceae family tend to exclusively predominate the microbiomes of industrialized and agrarian populations, respectively. These predominance patterns are mainly attributed to distinct diets and are associated with ambiguous health outcomes. Understanding how such compositional patterns are shaped in the gut of human populations is critical for elucidating microbiome health biomarkers. We designed a defined community of human gut commensal isolates to explore niche interactions between members of Bacteroidaceae and Prevotellaceae. We assessed the effect of ~100 dietary components on the interaction between members of these two families within the robust and reproducible ecological network of the defined community. We found that a single strain Segatella copri (former Prevotella copri) can displace Bacteroidaceae strains in the presence of specific dietary polysaccharides in a process that requires the presence of other key commensals. These results suggest that a combination of nutrient competition and specific microbe-microbe relationships determine major microbiota signatures. These results bring us closer to answering the long-standing question of what drives the major human microbiota signatures involving Bacteroidota members.

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I 04
Decoding Persistent Salmonella Infections: High Throughput Exploration of Molecular Decision Points

Presenting author:

Julius-Maximilians-Universität Würzburg, Microbiology, Hechinger 10, 72072 Tübingen [DE], alexandra.tietze@uni-wuerzburg.de

Author(s):
Alexandra Tietze, David Gerlach, Ana Rita Brochado

Bacterial pathogens exhibit resistance to antibiotic treatments through either antimicrobial resistance mechanisms or the adoption of bet-hedging strategies, where a slow-growing subpopulation becomes tolerant to antibiotics (persisters). Such treatment-resistant pathogens not only contribute to the spread of antibiotic resistance but are also linked to chronic and recurring infections.

While extensive research is conducted to decipher the mechanisms of antibiotic resistance, it is largely unknow how the existence of persister subpopulations is triggered and sustained during infections, hindering the development of efficient treatment strategies.

To address this research gap, we have designed a high-throughput approach to uncover key molecular decisions for sustaining or aborting a persistent state during Salmonella infections within macrophages.

Our approach involves the simultaneous targeting of the bacterium and the macrophage using a combination of antibiotics and immunomodulatory drugs to identify conditions that modify Salmonella's intracellular survival.

Subsequent in-depth exploration of the most promising conditions will unveil the genetic landscape of bacterial and host pathways facilitating intracellular pathogen survival and or exit from the persistence state. In the long term, we aim to investigate how pathogen- or host cell-specific our findings are, which will hint towards the need for broad-spectrum or targeted therapies.

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M 05
Discretizing bacterial abundances for data integration

Presenting author:

Goethe University Frankfurt am Main, , Robert-Mayer-Straße 11-15, 60325 Frankfurt [DE], Tsenova@med.uni-frankfurt.de

Author(s):
Kristiyana Tsenova, Jörg Ackermann, Maria Vehreschild, Ina Koch

The intestinal microbiota is the community of microorganisms in the gut and has immunological and gut-protective functions. Over the past decade, numerous studies have indicated a link between the gut microbiota and various diseases. Based on microbiome data, machine learning techniques have been applied for association and clinical use for disease diagnosis and prognosis. However, these studies have used small datasets and different sequencing and processing methods, making cross-study comparisons difficult. A suitable transformation of the microbiome data could enable the integration and comparability of different data sets. Therefore, we propose the discretization of the microbiome data.

Our approach involves discretizing microbial data into three categories: normal, low abundance, and increased abundance. Several discretization methods were employed, including using a healthy population from the data base GMrepo as a reference and considering the absence and non-absence of bacteria. To assess the diversity of intestinal microbiota, we applied the Lempel-Ziv complexity. We applied the discretization approaches to data sets from the American Gut Project and the Microbiome Learning Repo, considering liver disease and cirrhosis. We found significant differences between healthy subjects and patients with liver disease. Our work represents a first step towards discretization of microbial abundances, paving the way for improved data integration.

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N 03
Merging two worlds: Synergy between a photobioreactor and a chicken coop from the microbiome point of view

Presenting author:

Karlsruhe Institute of Technology (KIT), , Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen [DE], marta.martin@kit.edu

Author(s):
Marta Velaz Martín, Laura Meisch, Till Glockow, Anne-Kristin Kaster, Kersten S. Rabe, Christof M. Niemeyer

The growing global demand for food, coupled with diminishing arable land and environmental concerns, requires the development of sustainable agricultural practices. While urban controlled environment agriculture is effective for plant agriculture, sustainable approaches in circular farming for animals are underdeveloped. Photosynthetic microorganisms (PMO’s), such as microalgae and Cyanobacteriota (Spirulina), in mixed populations can be harnessed in photobioreactors within conventional animal houses to utilize exhaust components for growth. We investigated the capacity of the PMOs to transform animal housing emissions into valuable biomass as well as potential community shifts in the mixed population. To this end, a suspended, innovative cone-shaped, helical tubular photobioreactor was introduced into a chicken coop and demonstrated its efficiency in purifying exhaust air while producing Spirulina. Metagenomics analysis revealed the impact of the chicken house exhaust on the algal culture and provided insights into the NH₃ metabolism. The taxonomic classification of the microbial community in the photobioreactor revealed a predominance of bacterial organisms before aeration, with a notable shift to a more diverse community, including eukaryotes, after exposure to exhaust air from the chicken coop. The emergence of Viridiplantae, and Stramenopiles, particularly Chlorella and diatoms, suggests a significant impact on the microbial composition influenced by the exhaust air.

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O 11
Novel tools for making pooled and ordered transposon mutant libraries in intestinal Bacteriodales species

Presenting author:

EMBL, Genome Biology unit, Structural and Computational Biology unit, Meyerhofstrasse 1, 69117 Heidelberg [DE], carlos.voogdt@embl.de

Author(s):
Carlos Voogdt, Nicolai Karcher, Katharina Müller, Georg Zeller, Michael Zimmermann, Nassos Typas

The Bacteriodales order includes numerous commensal species in the intestinal microbiota. Despite their influence on host physiology, a significant fraction of Bacteriodales genes remain uncharacterized, which hinders our understanding of their biology. To accelerate the discovery of Bacteriodales gene functions we developed new tools for performing genome-wide barcoded transposon mutagenesis in Bacteriodales species. We could efficiently mutagenize 24 strains of 14 species among the Bacteroides, Parabacteroides and Phocaeicola genera. We created dense, barcoded libraries in B. uniformis and P. vulgatus that are well-suited to link genotypes to phenotypes and assign functions to genes using fitness-related phenotypes. In order to also screen for fitness-unrelated phenotypes we created a high quality ordered collection of 6144 P. vulgatus transposon mutants, which target 3108 of the 4172 genes (74.4%) in this strain. We are now using the tools and libraries (pooled and ordered) in a wide setting of screens such as for drug sensitivity/metabolism, bacterial cross-feeding, secretion, microbial interactions, phage defense, host colonization and more. The use of these and future libraries will empower the systematic investigation and discovery of gene functions in Bacteriodales species.

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M 12
Bacterial aggregation in the disbalanced intestinal microbiota

Presenting author:

Friedrich-Schiller-Universität Jena, Institute of Microbiology, Theoretical Microbial Ecology Group, Winzerlaer Str. 2, 07745 Jena [DE], stefanie.wagner@uni-jena.de

Author(s):
Stefanie Wagner, Anne Busch, Michael Bauer, Rosalind Allen

Bacterial aggregation is widespread among environmental and pathogenic bacteria. This cellular clumping may protect the prokaryotes from external stress and has been suggested to be an important step in biofilm formation. The beneficial properties of multicellular aggregates may lead to a selective advantage for specific species, especially in highly competitive environments like the human intestine. However, the specific role of bacterial aggregation in this dysbiosis is as yet unknown.

To investigate the aggregation and biofilm formation ability of a disbalanced microbiota, we isolated Enterobacteriaceae from fecal samples of intensive care patients (from study MS-ICU) who were treated with the antibiotics meropenem, piperacillin/tazobactam, or with no antibiotics. As bile acids accumulate in patients’ blood during sepsis, we also assessed the influence of bile acids on cell size, shape and aggregation via cell counting and microscopy.

Our study demonstrates that several aggregate- and biofilm-forming Enterobacteriaceae are abundant among the isolated surviving strains, for all patient treatment groups. We suggest that aggregation and biofilm formation might play a universal role in bacterial survival in the gut. Further experiments will consider the host-pathogen interaction and the influence of the antibiotics. Integrating our results, we aim to understand the role of bacterial aggregation in the maintenance of a balanced human intestinal microbiome.

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D 04
Antibiotic Modulation of the Gut Microbiome Ameliorates Hypertensive Organ Damage

Presenting author:

Charite-Universitätsmedizin, ECRC - AG Wilck, Lindenberger Weg 80, 13125 Berlin [DE], moritz.wimmer@charite.de

Author(s):
Moritz Immanuel Wimmer, Hendrik Bartolomaeus, Valentin Vecera, Harithaa Anandakumar, Ulrike Löber, Dominik N. Müller, Sofia Kirke Forslund-Startceva, Nicola Wilck

To better understand the potential of microbiome-targeting therapeutic approaches for organ protection in hypertension, we used narrow-spectrum antibiotics without enteral absorption to deplete gram-negative or -positive bacteria in double transgenic rats (dTGR, transgenic for human renin and angiotensinogen).

The microbiome, clinical and immune phenotype of dTGRs treated with oral Vancomycin (Vanco), Polymyxin B (Poly) or Vehicle (Veh) for 3 weeks were analyzed by shotgun metagenomic sequencing, radiotelemetric blood pressure (BP) measurements, clinical chemistry and flow cytometry.

Independent of BP levels, hypertensive organ damage was ameliorated by Vanco treatment as assessed by albuminuria and cardiac hypertrophy indices. Vanco treatment resulted in a large increase of Lactobacillus murinus. This shift was accompanied by an increase in the functional potential of tryptophan synthesis, and metabolism towards immunomodulatory catabolites like indole lactic acid (ILA, via araT). We previously showed that ILA inhibits the differentiation of pathological Th17 cells through AhR modulation. Similarly, Vanco treatment reduced the elevated number of pathological Th17 in the kidney and intestine of dTGR.

Modulation of the intestinal microbiome by narrow-spectrum antibiotics ameliorates hypertensive organ damage. Our data underscores the importance of the gut microbiome in modulating hypertensive organ damage and helps to identify novel microbiome-directed therapeutic strategies.

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N 11
Genetic and environmental drivers of biofilm formation in Escherichia coli

Presenting author:

EMBL Heidelberg, Genome Biology, Konstanzer str 15, 69126 Heidelberg [DE], luo.yan.yong@embl.de

Author(s):
Luoyan Yong, Alexandra Koumoutsi, Nassos Typas

The formation of biofilms by bacterial communities is crucial for their ability to withstand physical and chemical stresses and responses to changes in the environment, such as temperature fluctuations. Although Escherichia coli K12 has been studied extensively for its biofilm formation, the significant genetic variability of the E. coli species offers numerous opportunities for developing novel adaptive traits, including in biofilm formation. In this study, we profiled colony biofilms of approximately 500 natural E. coli isolates under different growth temperatures and identified a small subset of strains that could form biofilms at body temperature. By using forward genetics on a saturated transposon library of a representative strain which exhibited preferential biofilm formation at 37°C, we identified genetic elements that control biofilm formation at this temperature in the uropathogenic E. coli IAI33. Specifically, we discovered that a transcriptional regulator, FabR, plays a significant role in biofilm formation at both 37°C and 25°C. These findings demonstrate the significance of investigating biofilm formation in different E. coli strains. Novel regulation and possible differences in biofilm structural elements can explain the great variability in biofilm structures and phenotypes we observed.

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