Search bioRxiv⌕ Search

Biology subjects

Zimmermann, M.

Publications and source records attributed to Zimmermann, M..

At least 19 recordsLinked to original sources

Primary and higher-order thalamic nuclei make distinct contributions to cortical reorganization in congenital sensory loss

Early sensory loss transforms the functional organization of affected sensory systems, with regions that normally support vision or audition participating in noncanonical perceptual and cognitive functions. Such reorganization requires coordination across distributed networks. Higher-order thalamic nuclei are positioned to support these interactions through their widespread reciprocal cortical connectivity, yet studies of sensory-loss plasticity have focused primarily on cortex and first-order sensory pathways. Here, we combined structural and functional MRI in early blind and early deaf adults to test how reorganization after sensory loss is expressed across thalamocortical networks. In blindness, structural differences were concentrated along the primary input pathway, with reduced lateral geniculate nucleus volume that covaried with areal organization of primary visual cortex. Functional reorganization was more broadly distributed, with visual cortex showing greater similarity with higher-order (association) thalamic nuclei linked to cognitive control, paralleling a shift toward cognitive-control networks across cortex. This pattern was amplified during a nonvisual perceptual judgment task. Across visual cortex, cross-network functional differences were strongest in regions showing the largest structural effects. A related shift toward control-associated higher-order thalamus was observed in deafness, indicating that higher-order thalamic involvement generalizes across sensory modalities. Together, these results indicate that thalamocortical architecture shapes both the developmental consequences of early sensory loss and the capacity for distributed functional reorganization.

neuroscience↗

Bacterial metabolism of synthetic steroids across ecosystems reveals diverse biotransformation products, reactions, and enzymes

Biotransformation of synthetic steroid drugs by gut and environmental bacteria affects their activity within the host, persistence, and environmental fate of these widely used pharmaceuticals. However, the spectrum of bacterial transformation of synthetic steroids, the enzymes involved, and the role of cooperative microbial metabolism in these processes remain poorly understood. We systematically mapped the biotransformation of 22 steroids, comprising 20 synthetic and 2 natural compounds and spanning clinically used estrogens, progestogens, corticosteroids, and prodrugs, across 12 bacterial species (8 intestinal and 4 environmental), yielding 264 steroid-bacteria combinations. We identified 97 unique biotransformation products and found that the tested bacteria catalyze diverse reactions including ester hydrolysis, oxidation-reduction chemistry, and steroid side-chain cleavage. Notably, the environmental bacterium Sphingobium herbicidovorans catalyzed desmolase-like steroid side-chain cleavage under aerobic conditions, a transformation previously reported solely for anaerobic bacteria. Combining sequence homology searches, a genetic gain-of-function screen, and expression proteomics we identified six steroid-transforming enzymes in S. herbicidovorans. We further demonstrate that distinct bacterial species cooperatively metabolize synthetic steroids through cross-feeding, enabling sequential activation and metabolism of corticosteroids across microbial communities. Together, our findings uncover previously unrecognized bacterial enzymes and community-level interactions involved in synthetic steroid metabolism. By directly linking steroid metabolites, enzymes, and microbial community interactions, this study provides molecular-level mechanistic insights into microbial steroid biotransformation. Such insights are essential for ultimately predicting metabolic interactions within and across microbial communities, as well as their interactions with the host and the environment.

microbiology↗

13C flux ratio analysis with FRAPPPE reveals differences in metabolic fluxes between gut Bacteroidota and Escherichia coli

Gut bacteria shape the metabolism of their host and play an important role in human health. However, systems biology approaches to study their intracellular metabolic fluxes are largely underdeveloped. We present an experimental and computational workflow to quantify metabolic flux ratios in gut bacteria using 13C-labeled nutrient supplementation and a newly developed machine learning-based Flux Ratio Prediction Python PackagE (FRAPPPE). We apply FRAPPPE to investigate central carbon metabolism in two prevalent gut Bacteroidota, Bacteroides uniformis and Phocaeicola vulgatus, in comparison to Escherichia coli. FRAPPPE revealed altered tricarboxylic acid cycle bifurcation in Bacteroidota compared to E. coli under anaerobic conditions. Further, we used FRAPPPE to investigate co-metabolism of nucleosides and carbohydrates by B. uniformis and P. vulgatus. We found distinct species-specific patterns in how nucleosides affected growth and were utilized depending on the co-fed compound. We quantified co-metabolism and showed that the tested nucleosides were mainly contributing to anabolic metabolism closely related to the specific co-fed nucleoside. Together, these findings provide insights into central and nucleoside metabolism of two gut Bacteroidota, and showcase FRAPPPE as a generalizable workflow to investigate metabolic fluxes in gut bacteria.

microbiology↗

SYBR Gold staining of single stranded nucleic acids is strongly influenced by the presence of guanosines

SYBR Gold is one of the most strongly fluorescent members of the SYBR family of asymmetrical cyanine dyes commonly used for staining double and single-stranded DNA and RNA. The dye fluorescence increases dramatically upon nucleic acid binding. The primary mode of binding to double-stranded DNA is through intercalation between the base-paired deoxyribonucleotides, while the precise mode of binding to single-stranded DNA and to RNA is less well understood. Here we show that the fluorescence activity of SYBR Gold is strongly influenced by the presence of guanines in single-stranded nucleic acids. We expect this insight to aid in the evaluation of SYBR Gold-based measurements of nucleic acid abundance and spark further studies into the binding behaviour of the SYBR family dyes.

molecular biology↗

Affinity-tag-based microfluidic protein isolation enables high-resolution Cryo-EM from minimal starting material

Cryo-EM is central to high-resolution structure determination, but conventional sample preparation consumes substantial amounts of protein and can destabilize sensitive complexes. Microfluidic approaches can miniaturize and accelerate these workflows while retaining the particle numbers required for single-particle analysis. Here, we present a generalized microfluidic isolation strategy that captures proteins directly from cell lysates or in vitro translation reactions using genetically encoded tags. The platform supports both affinitybased (ALFA-nanobody) and covalent (SpyTag3/SpyCatcher3) capture. Specificity derives from two sequential, orthogonal steps: (i) tag-mediated capture and concentration of the target protein on beads, and (ii) photoelution from the bead surface. By confining the sample to nanoliter volumes and minimizing transport, the approach suppresses non-specific carryover. Importantly, the isolation workflow is coupled to grid preparation, enabling preparation of three cryo-EM or negative-stain grids from a 25 nL eluate. Using this workflow, we isolated E. coli ferritin A, {beta}-galactosidase, and P. aeruginosa VgrG1 from cell lysate, obtaining reconstructions at 1.9-2.6 [A] resolution with B-factors comparable to optimized conventional workflows. We further isolated ferritin A from an in vitro translation reaction, yielding a 2.04 [A] reconstruction. A standardized 50 {micro}L starting volume enabled more than 16 independent microfluidic isolations. Tag-based microfluidic isolation thus provides a broadly applicable route to cryo-EM sample preparation, reducing sample volumes more than 1000-fold, reducing preparation times more than 3-fold for lysate-based workflows and up to 10-fold when combined with cell-free expression, and enabling high-throughput structural screening directly from in vitro translation reactions. Significance StatementCryo-electron microscopy (cryo-EM) enables protein structure determination at atomic resolution, but conventional sample preparation requires large amounts of purified protein and laborious workflows that can damage fragile complexes. We present a microfluidic platform that captures tagged proteins directly from minute amounts of crude lysates or cell-free synthesis reactions and deposits them onto cryo-EM grids in a single integrated workflow. Using this approach, we determine three protein structures at high resolution. By reducing sample volumes by roughly three orders of magnitude, the platform expands cryo-EM to proteins inaccessible to conventional workflows and provides a foundation for automated, high-throughput structural analysis.

molecular biology↗

Dynamic transcriptional remodeling in alcohol use disorder reveals immune dysregulation and adaptive shifts in coagulation during therapy

BackgroundChronic alcohol use disorder (AUD) is associated with profound dysregulation of immune function, neuroinflammation, and systemic stress responses, which contribute to both the maintenance of addiction and alcohol-related organ damage. While brain transcriptomic studies have established neuroimmune signaling and synaptic remodeling as central features of AUD, peripheral blood signatures during early withdrawal and recovery remain underexplored. Understanding the dynamic transcriptional changes in peripheral blood accompanying supervised withdrawal therapy is critical for identifying reversible molecular processes versus persistent trait-like alterations. MethodsRNA sequencing (RNA-seq) was performed on peripheral blood from individuals with alcohol use disorder (AUD, n = 100) and healthy controls (n = 74) at baseline and after three weeks of supervised withdrawal therapy. Differentially expressed genes (DEGs) were identified using linear mixed models assessing main effects of group, time, and their interaction. Functional enrichment and co-expression network analyses were performed to identify coordinated biological processes. ResultsAt baseline, more than 1,000 genes were differentially expressed between AUD and control participants, showing robust dysregulation of immune-related pathways. After three weeks of withdrawal, the number of DEGs decreased markedly to 141, indicating partial transcriptomic normalization. Nevertheless, immune dysregulation persisted despite treatment, particularly linked to B cell activation and cell-cell junctions. Interaction analyses (group x time) identified 16 genes whose expression dynamically changed with therapy, highlighting strong enrichment for fatty acid pathways. Co-expression network analysis revealed that baseline modules were enriched for genes associated with secretory granules and immune signaling, while therapy-related co-expression shifts involved coagulation and platelet activation processes. ConclusionsAUD is associated with widespread but partly reversible transcriptomic dysregulation in peripheral blood. These findings support a system-level view of AUD as a disorder of intertwined immune, metabolic, and coagulation biology and suggest that longitudinal blood transcriptomics may help identify both rapidly therapy-responsive and more stable molecular targets for relapse prevention.

molecular biology↗

Lithocholic acid modulates the growth of butyrate-producing bacteria and is decreased in the feces of stunted children

Bile acids modulate the intestinal microbiota and serve as key signaling molecules in host physiology. Bile acid dysregulation has been implicated in nutritional and inflammatory diseases; however, data on the pool of bile acids present in stunted children or children suffering of environmental enteric dysfunction (EED) is limited, particularly in the upper intestinal compartment where disease phenotypes are most relevant. In this study, we performed a targeted metabolomics approach on 75 bile acids and their derivatives, including gastric and duodenal aspirates and fecal samples from almost 1000 children from two Sub-Saharan cities. We found that levels of secondary bile acids, especially lithocholic acid, are significantly lower in the feces of stunted and EED children, while ursocholic acid and its derivatives are significantly higher. Levels of primary and sulfated bile acids are also increased in the feces of children with EED. Microbiota sequencing revealed that high lithocholic acid levels are positively associated with butyrate-producing bacteria, while negatively associated with oral taxa like Streptococcus and Veillonella. In vitro tests on a panel of reference strains showed that oral bacteria bioaccumulate and are inhibited by a variety of bile acids, while lithocholic and chenodeoxycholic acids modulate the growth of several butyrate-producing bacteria. This effect was even stronger with tauro- or glycol-conjugated bile acids. Exposing stool-derived in vitro communities from children in Afribiota to these bile acids confirmed their positive impact on butyrate producers and negative effect on overgrowing oral taxa. Our findings suggest that secondary bile acids, reduced in stunting and EED, modulate the growth of butyrate-producing bacteria while suppressing harmful oral taxa, highlighting their potential as tools to modulate microbiota composition.

microbiology↗

Domain-Specific Agonist Binding Affinities Explain Structural and Functional Regulation of TRPM2

TRPM2 is a Ca{superscript 2}-permeable cation channel activated by ADP-ribose (ADPR) and oxidative stress, yet the relative contributions of its two nucleotide-binding domains, MHR1/2 and NUDT9H, remain incompletely understood. Here, we quantitatively determine the affinities of the isolated human TRPM2 MHR1/2 and NUDT9H domains for ADPR, 2-deoxy-dADPR (dADPR), and 8-Br-cADPR using biophysical approaches. The MHR1/2 domain binds ADPR with high affinity (Kd {approx} 0.5 {micro}M), whereas the NUDT9H domain displays substantially lower affinity (Kd {approx} 192 {micro}M), revealing a difference of nearly three orders of magnitude. Mutational analysis demonstrates that alterations in MHR1/2 strongly affect ligand binding and channel activation, while mutations within NUDT9H that markedly reduce ligand affinity exert only modest effects on gating. In parallel, we quantify intracellular ADPR concentrations in resting and hydrogen peroxide-stimulated cells and find that they remain well below the affinity required for substantial NUDT9H occupancy. Together, our findings indicate that high-affinity binding to the MHR1/2 domain is sufficient to drive TRPM2 activation under physiological conditions, whereas the NUDT9H domain likely contributes to maintaining the structural integrity of the channel rather than directly mediating ligand-dependent activation. These results provide a quantitative framework for understanding ligand-dependent TRPM2 regulation in cells.

biophysics↗

Personalized microbiotas (counter-)select for antibiotic resistant strains

Antibiotic resistant pathogens are an increasing public health threat, as development of novel therapeutics is outpaced by resistance emergence and dissemination. Approaches to slow down or even revert antibiotic resistance are necessary to maintain efficacy of both existing and new antibiotics. Such approaches exploit the fitness cost of resistance elements, but have largely relied on assessing this cost in laboratory conditions that poorly reflect the native context in which pathogens reside. Here we present a method that allows to investigate the influence of personalized human gut microbiota compositions on the competitive fitness of antibiotic resistant pathogens. Using fecal matter-derived microbiomes we identified a specific community that selected for a carbapenem-resistant Klebsiella pneumoniae strain. This selective advantage was due to mutations arising in a LacI-type transcriptional regulator, GlyR, which upregulated expression of the downstream glycoporin GlyP, causing the effect. By deconvoluting the microbiome composition, we identified a focal E. coli strain as a central driver of the selection, which was further modulated by other microbiota members. We further demonstrate that the selective advantage was due to carbohydrate competition, and in particular for glycerol-containing compounds. Importantly, glyR mutations are under strong positive but conditional selection in clinical K. pneumoniae isolates. This implies a reduced competitiveness in other environments, which we experimentally validated in vitro. Overall, this study offers a path to identify microbiome-specific interactions that modulate the competitiveness of antibiotic resistant pathogens.

microbiology↗

Gut microbiota within-host evolution enforces colonization resistance against enteric infection

Limited resource availability in the gut promotes competitive interactions between bacteria, which drive adaptive within-host evolution (1-3). While adaptive evolution of bacterial communities has been increasingly studied in the recent years (4-7), its functional implications for host physiology remain unknown. Here, we show that within-host evolution of the human commensal Enterococcus faecalis boosts colonization resistance to enteric Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. During gut colonization, E. faecalis evolves the ability to metabolize fructoselysine, an abundant Amadori rearrangement product generated by thermal food processing. The depletion of this diet-derived nutrient prevents S. Typhimurium colonization by restricting an essential resource. This protective mechanism was conserved across independent mouse colonies and arises via diverse evolutionary trajectories, including nucleotide polymorphisms, gene amplifications, and a horizontal gene transfer event. Additionally, analysis of E. faecalis isolates from human infants revealed that adaptation to fructoselysine availability occurs in a diet-dependent manner. Isolates from infants fed with fructoselysine-rich formula were able to utilize fructoselysine, whereas those from infants fed with fructoselysine-poor breast milk were not. Conclusively, our results identify an inherent microbiome-driven self-healing mechanism, wherein bacterial evolution restores colonization resistance against enteric pathogens through evolved nutrient depletion. Understanding these evolutionary dynamics will inform microbiome-targeted approaches to prevent and treat infectious diseases by harnessing adaptive bacterial metabolism.

microbiology↗

Metabolic specialization structures gut bacterial niches and drives colorectal cancer progression

Despite the established association between the gut microbiome and colorectal cancer (CRC), the functional distinction between microbial passengers and drivers of CRC progression remains unresolved. Here, we collected stool, blood, as well as paired tumor, and normal mucosa tissues from seventy-seven CRC patients to characterize the systemic and localized impact of the gut microbiome on early- and late-stage CRC. By deep shotgun metagenomic sequencing, we identified distinct bacterial species and functions residing in tumor versus normal mucosa, highlighting an enrichment of oral-associated bacteria in tumor tissues. Several of these species remained undetected in the stool microbiome analysis. We further combined bacterial culturing with untargeted metabolomics of bacteria enriched in tumor and normal mucosa tissues, revealing distinct clusters of metabolic potential. Functional testing of multiple members from one cluster comprising both tumor- and mucosa-enriched species revealed Leptotrichia wadei as a pro-tumorigenic bacterium in a murine CRC model. Single-nucleus RNA sequencing and in vitro experiments further demonstrated that L. wadei and its secretome induces M2 macrophage polarization to promote tumor growth. Overall, our study shows that metabolic specialization structures microbial colonization niches, while species-specific metabolic outputs identify functional drivers of CRC progression, and uncovers L. wadei as an oncogenic bacterium in CRC.

microbiology↗

putzig safeguards genome integrity by contributing to the Piwi-mediated repression of transposon activity in the female germline of Drosophila in a two-tiered fashion

Genome integrity in the germline is jeopardized by the activity of transposable elements (TEs). Transposon activity is kept in check by the Piwi-piRNA pathway, which silences TEs post-transcriptionally in the cytoplasm as well as co-transcriptionally in the nucleus. piRNAs derive from long precursor transcripts originating at piRNA-loci by non-conventional transcription. They are processed in the cytoplasm and loaded into Piwi-piRNA complexes that then enter the nucleus to bind to target RNAs and direct local heterochromatin formation at TE loci, involving several downstream effectors. In this work, we have analyzed the role of Putzig (Pzg) in Piwi-mediated TE silencing in the female germline. Pzg has multi-facetted roles during Drosophila development and is important for DNA integrity, germ cell differentiation and survival. Here, we provide evidence for a two-tier activity of Pzg by serving as a hub for various Piwi-pathway members. Firstly, Pzg assists transcription initiation at piRNA clusters by coupling the Trf2-Moonshiner transcription initiation complex to the Rhino-Deadlock-Cutoff complex, thereby promoting formation of piRNA-precursor transcripts. Secondly, in the context of co-transcriptional gene silencing, Pzg licences heterochromatin formation by linking the Piwi-piRNA silencing machinery and the histone demethylase Lsd1, involved in promoter inactivation.

genetics↗

Multi-omics and biophysical phosphoproteomics upon BRAF inhibition uncover functional networks of BRAFV600E-driven signaling

Dysregulated kinase activity drives oncogenic signaling, disrupts cellular homeostasis, and promotes tumour progression. The BRAFV600E mutation constitutively activates the MAPK pathway and is a key therapeutic target in melanoma and other cancers, but the functional relevance of most downstream phosphorylation events and mechanisms of drug resistance remain unclear. To address this, a global multi-omic model of BRAF inhibition response was established in BRAFV600E-mutant cells by integrating time-resolved and biophysical phosphoproteomics, transcriptomics, and thermal proteome profiling. Ultradeep phosphoproteomics revealed extensive phosphorylation changes upon BRAF inhibitor treatment, while biophysical phosphoproteomics identified phosphorylation events linked to altered protein solubility and subcellular localization, suggesting changes in nucleic acid interactions and nuclear reorganisation. Network-based integration of these datasets prioritized functionally relevant phosphorylation sites and kinases. Experimental validation identified CDK9, CLK3, and TNIK as critical regulators of BRAFV600E signaling and candidate targets for combinatorial inhibition capable of re-sensitising resistant cells. The transcription factor ETV3 emerged as a previously unrecognised effector of BRAF signaling. Biophysical proteomics data confirmed that ETV3 phosphorylation modulates DNA-binding, while functional assays combining knockdown, metabolomics, and drug screening demonstrated its role in coordinating transcriptional and metabolic adaptations to BRAF inhibition. This study provides a systems-level framework linking phosphorylation dynamics to protein function and phenotype, identifies ETV3 as a new node in oncogenic BRAF signaling, and illustrates how integrated, site-resolved models can reveal mechanisms of kinase-driven oncogenesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=43 SRC="FIGDIR/small/704793v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1df1401org.highwire.dtl.DTLVardef@9a77a5org.highwire.dtl.DTLVardef@124f819org.highwire.dtl.DTLVardef@1c6b57_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LITime- and cell type-resolved phosphoproteomics maps BRAF inhibition dynamics C_LIO_LIBiophysical phosphoproteomics, combining quantitative phosphoproteomics with solubility profiling or nuclear fractionation, reveals phosphorylation-driven changes of protein solubility and localization C_LIO_LIIntegration of abundance and biophysical phosphoproteomics data identifies functionally relevant phosphorylation events of BRAFV600E signaling C_LIO_LINetwork integration of multimodal phosphoproteomic, transcriptomic and thermal proteome profiling data links signaling to protein function and cellular phenotypes C_LIO_LIBiophysical evidence improves models and identifies non-canonical kinases driving BRAF signaling as well as novel downstream regulators such as ETV3 C_LIO_LIFollow-up experiments reveal a ETV3-GLUT3-mediated metabolic adaptation in BRAFV600E cells C_LI

systems biology↗

CRISPR-enabled genetic screens identify synthetic lethal targets across frequently altered cancer drivers

Synthetic lethality (SL) provides a treatment paradigm for targeting cancer with alterations in driver genes that are not conventionally druggable, including loss-of-function (LoF) mutations in tumor suppressor genes and gain-of-function (GoF) alterations in oncogenes. We undertook a series of genome-wide CRISPR screens using functionally validated isogenic cell lines and also conducted a large-scale SL analysis using data from the cancer dependency map (DepMap). We charted SL interactions across 15 genetic alterations characteristic of diseases with high incidence and unmet clinical need: FBXW7, CCNE1, CDK12, ARID1A, KMT2D, DNMT3A, TET2, KEAP1, STK11, IDH1, SF3B1, SRSF2, U2AF1, chromosome 18q loss, and chromosome 13q loss. We show validation of several SL interactions between tractable targets with cancer drivers, including ARID1A and the hexosamine biosynthetic pathway aminotransferase GFPT1, STK11 with CAMK protein kinase family members including MARK2, FBXW7 and the CDK1 regulatory kinase PKMYT1, and CCNE1 amplification and the anaphase promoting complex or cyclosome (APC/C). In summary, this study offers a rich resource of genetic interactions across cancer drivers enabling the discovery of new biological insights and drug targets for future therapeutic development.

cancer biology↗

Linking interpersonal differences in gut microbiota composition and drug biotransformation activity

Individuals vary widely in their responses to drugs, and growing evidence implicates the gut microbiome as a contributor to this variability. While prior studies show that gut bacteria can metabolize drugs, how differences in microbial community composition influence drug metabolism remains poorly understood. Here, we characterize the biotransformation of 271 drugs by 89 gut microbial communities derived from human donors and preclinical animal models. Over 90% of tested drugs were metabolized by at least one microbiome. We identified 66 drugs exhibiting highly variable metabolism across human-derived microbiomes and several drugs whose biotransformation differed markedly between human and animal microbiomes. To enable prediction of microbiota-mediated drug metabolism, we developed and compared multiple modeling approaches based on metagenomic data. These results, together with the provided data and analytical resources contribute to a better understanding of microbiome-drug interactions and support their future integration into drug discovery, personalized prescription, and therapeutic drug monitoring.

microbiology↗

Gut bacteria generate prodrugs in situ increasing systemic drug exposure

The human gut microbiome has been increasingly recognized as a metabolic compartment that influences health and therapeutic responses. While recent studies have cataloged diverse sets of drug biotransformation reactions as a result of microbial metabolism1-3, the pharmacological relevance of the resulting drug metabolites remains mostly uncharacterized. We investigated whether certain microbial drug metabolites can act as functional prodrugs. By systematically mining 871 putative drug metabolites produced by gut bacteria1, we identified microbial drug metabolites with key physicochemical properties of prodrugs. Using bacterial culturing, a genetic gain-of-function screen coupled to mass spectrometry and comparative genomics, we uncover that Bacteroidota bacteria encode conserved methyltransferases that can generate prodrugs through carboxyl methylation. As a case in point, we demonstrate that bezafibrate gets converted to a prodrug by a distinct bacterial enzyme, increasing epithelial drug permeability in vitro and systemic drug exposure in vivo in a gnotobiotic mouse model. Additionally, we tested 170 structurally and clinically diverse drugs, and demonstrate that microbial drug-prodrug conversion is a common result of gut bacterial drug biotransformation. Altogether, our findings suggest a novel mechanism of how the gut microbiota influences an individuals pharmacokinetics, and may generally cause interpersonal differences in microbiota-host metabolic interactions.

microbiology↗

Vitamin B2 Production by Vaginal Lactobacilli Promotes Symbiosis

The human vaginal microbiome, particularly with lactobacilli as the main inhabitants, plays a key role in maintaining womens health. While lactic acid-mediated pathogen exclusion is well known, broader metabolic functions of vaginal lactobacilli remain underexplored. In this study, we analyzed the vaginal microbiome and metabolome of 258 healthy women from the Isala program. Using targeted metabolomics analysis, we detected a high prevalence with strong interpersonal differences of most B-vitamins, their precursors, and vitamin A in the vaginal microenvironment. Riboflavin (B2) and biotin (B7) showed strong associations with Lactobacillus crispatus and Limosilactobacillus sp. Comparative genomics, phenotypic assays, and in vivo metatranscriptomic data (VIRGO2) collectively confirmed riboflavin biosynthesis by these taxa. Using a riboflavin overproducing Lim. reuteri as a functional model, we showed that microbially derived riboflavin and its pathway intermediates are transported across the vaginal epithelium and modulate host redox balance, cytokine production, and activation of mucosal-associated invariant T (MAIT) cells via induction of MR1 (Major histocompatibility complex, class I-related protein receptor), revealing a potential immunometabolic interface between the vaginal microbiota and its host.

microbiology↗

A toolkit for transposon libraries and functional genomics in intestinal Bacteroidales

Members of the order Bacteroidales include some of the most prevalent and abundant bacterial species in the healthy human gut microbiota. Yet, most of the functions encoded in their genomes remain poorly characterized, limiting our understanding of the different roles they play in the human gut microbiome. Towards addressing this gap, we developed tools and methods for genome-wide transposon mutagenesis in Bacteroidales, including broad-range transposon vectors with several antibiotic selection markers, a dual conjugation-cloning donor strain, and protocols for convenient library generation in liquid media. We then created saturated, barcoded, insertion mutant libraries in the type strains of three key representatives of the main genera within Bacteroidales: Bacteroides uniformis (ATCC 8492), Phocaeicola vulgatus (ATCC 8482) and Parabacteroides merdae (ATCC 43184). Based on the dense transposon insertion profiles and a workflow for comparing essentialomes across species, we identified 275 core essential genes shared across the three species, and 163 species-specific essential genes, some of which could be explained by functional redundancy and alternative metabolic pathways. We further identified essential non-protein coding elements and essential protein domains with known and unknown functions. Finally, using insertion directionality bias, we could map potential toxic modalities in the three genomes, including toxin-antitoxin pairs, mobile elements encoding toxic products and enzymes leading to toxic metabolic intermediates. Overall, the tools, workflows and genome-wide resources reported here expand the experimental repertoire for characterizing genes in key bacteria of the human gut microbiome, and pave the way for the establishment of similar genetic toolkits for other gut bacteria.

microbiology↗