Biology subjects

Castagner, B.

Publications and source records attributed to Castagner, B..

9 recordsLinked to original sources

bioRxiv2026

Discovery of a hydrolysis operon in Lachnospiraceae provides a genetic basis to understanding castalagin metabolism by the human microbiome in a clinical study.

Gut microbes metabolize the ellagitannin castalagin to ellagic acid and urolithins, a process associated with health effects for the host. Despite the use of the castalagin-rich camu camu extracts in clinical trials, the genes and taxa responsible for castalagin hydrolysis remain elusive. We used multi-omics to discover a castalagin associated hydrolase (cah) operon sparsely distributed in Lachnospiraceae. Analysis of fecal samples from a camu camu supplementation clinical trial demonstrates functional redundancy with prevalence of cah and other ellagic acid metabolism genes in different taxa explaining the prevalence of metabolites. Employing genome mining strategies, we discovered and validated ellagitannin metabolism genes in uncharacterized and uncultured species. We further identified flavogallonic acid as a prevalent metabolite of castalagin in human fecal samples. Collectively, this work provides a genetic basis for castalagin metabolism to urolithins and flavogallonic acid end-metabolites and sets the stage to study the role of metabolism in clinical trials.

microbiology↗

bioRxiv2026

Mistrafficking of KCC2 promotes hyperexcitability in hippocampal circuitry in a murine model of Christianson Syndrome

How endosomal trafficking shapes inhibitory neurotransmission remains poorly understood, despite both processes being linked independently to epilepsy and neurodevelopmental disease. Christianson syndrome (CS), an X-linked neurodevelopmental disorder, represents a potential tractable condition to investigate such coupled processes. CS is caused by loss-of-function mutations in the SLC9A6 gene which encodes the organellar (Na+, K+)/H+ exchanger NHE6 isoform whose loss overacidifies recycling endosomes and disrupts cargo delivery. Prior work has emphasized NHE6's role in excitatory neurons and glia; whether endosomal dysfunction reshapes inhibitory circuits and thereby drives the early treatment-resistant epilepsy that defines CS has not been addressed. Using Nhe6-/Y mice, we identify a previously unrecognized endosomal-to-inhibitory axis: NHE6 is required for the surface delivery and stability of the neuron-specific potassium-chloride co-transporter KCC2 (encoded by SLC12A5) that sets the driving force for GABAergic inhibition. Loss of NHE6 produces developmental downregulation and mistargeting of KCC2 in hippocampal neurons, impaired chloride homeostasis, and circuit-level hyperexcitability in response to a subthreshold convulsant challenge. These results recast CS epilepsy not as a downstream consequence of excitatory dysfunction but as a primary failure of inhibitory development driven by endosomal mistrafficking. More broadly, they establish endosomal pH regulation as a determinant of KCC2 biology; a node implicated across genetic and acquired epilepsies. Pharmacologically restoring KCC2 function therefore offers a mechanism-based therapeutic strategy for CS, and likely for the broader class of disorders in which endosomal trafficking and inhibitory imbalance converge.

neuroscience↗

bioRxiv2026

The Microbiota Dictates Vendor-Derived Differences in a Murine Clostridioides difficile Infection Model

Clostridioides difficile infection (CDI) is the leading cause of healthcare-associated infectious diarrhea and remains a major burden to healthcare systems worldwide. The development of novel therapeutics for CDI requires robust and reproducible preclinical models. However, the microbiota has emerged as a major source of variability in animal studies. Here, we found that genetically similar mice obtained from two commercial vendors, Jackson Laboratory (JAX) and Charles River Laboratories (CRL), exhibited marked differences in susceptibility to CDI, with JAX mice developing fulminant disease and CRL mice remaining resistant. Using full-length 16S rRNA gene sequencing, we show that JAX and CRL mice harboured distinct gut microbiota, and that cohousing susceptible JAX mice with resistant CRL mice was sufficient to shift the JAX microbiota toward the CRL community structure and confer resistance to CDI. Differential abundance analysis identified taxa distinguishing resistant and susceptible mice, providing candidates for future mechanistic investigation. These findings demonstrate that vendor-derived variation in the gut microbiota drives differential susceptibility to CDI in mice, and that this phenotype is transferable via cohousing, highlighting the importance of accounting for the microbiota when designing and interpreting animal models of infectious disease.

microbiology↗

bioRxiv2026

Early life γδT cell activation enforces intestinal barrier integrity during intergenerational C. difficile colonization

Early postnatal life is a highly dynamic period in which the intestine is inundated with billions of microbes that not only battle for colonization, but also strongly influence immune system function and long-term health. This brief period of development provides a window of opportunity to imprint resilience against disease, yet the intercellular dialogue that dictates tissue protection are only beginning to be discovered. By performing a kinetic analysis of immune cell activation in the colon of mice from early life to adulthood, we identified transient activation of a microbiota-dependent type 3 immune response during the weaning period. This response was characterized by a selective increase in IL-17 production by fetal-derived {gamma}{delta} T cells that occurred in an IL-1 receptor-dependent, cell-intrinsic manner. The subsequent differentiation of IL-10 producing Ror{gamma}t+ T regulatory cells extinguished IL-17 production by {gamma}{delta} T cells and prevented immunopathology. Microbial gain-of-function approaches determined that colonic {gamma}{delta} T cell activation occurred in response to passive acquisition of C. difficile at birth. In turn, IL-17 limited C. difficile growth and intestinal barrier breach by the commensal microbiota. Collectively, our results reveal how an agile immune cell network meets the demands of a maturing microbiota to support gut health during early life. Highlights- Colonic {gamma}{delta}17 T cells are activated during early life in a microbiota-specific manner - Fetal-derived {gamma}{delta}17 T cells require IL-1 signals for activation - Passive acquisition of C.difficile induces {gamma}{delta}17 T cell activation - IL-17 limits C. difficile growth and systemic dissemination of commensals

immunology↗

bioRxiv2025

The presence and induction of regioselective dehydroxylases dictate urolithin metabolism by Enterocloster species

Urolithins are a class of bioactive metabolites derived from the metabolism of dietary ellagitannins by the human gut microbiota. In the gut, urolithins are dehydroxylated regioselectively based on microbiota composition and activity. A single 9-hydroxy urolithin dehydroxylase (ucd) operon in gut resident Enterocloster species has been described to date; however, most enzymes in the urolithin metabolic pathway remain uncharacterized. Here, we investigate urolithin cross-feeding between members of the gut microbiota and discover a novel urolithin dehydroxylase in a subset of Enterocloster species. We show that urolithin intermediates, released by gut resident Gordonibacter species during ellagic acid metabolism, are dehydroxylated at both the 9- and 10-positions by E. asparagiformis, E. citroniae, and E. pacaense, but not E. bolteae. Using untargeted proteomics, we uncover a 10-hydroxy urolithin dehydroxylase operon, termed uxd, responsible for these species-specific differences in urolithin metabolism. By inducing uxd expression with diverse urolithins, we show that 9-hydroxy urolithins are required for uxd transcription and 10-position dehydroxylation. Collectively, this study reveals some of the genes, proteins, and substrate features underlying differences in urolithin metabolism by the human gut microbiota.

microbiology↗

bioRxiv2024

Clostridioides difficile Toxins Unhinged: Allosterically Switchable Network Orients β-flap

Allosteric proteins exhibit a functional response upon substrate binding far from the active site. Clostridioides difficile toxins use allosteric binding by an endogenous co-factor to orchestrate self-cleavage from within the target cell. This binding event induces a conformational shift, primarily effecting a lever-like "{beta}-flap" region, with two known orientations. We uncovered a mechanism for this allosteric transition using extensive atomistic MD simulations and computational and experimental mutagenesis. The mechanism relies on a switchable interaction network. The most prominent interaction pair is K600-E743, with K600 interactions explaining [~]70 % of the allosteric effect. Rather than gradually morphing between two end states, the interaction network adopts two mutually exclusive configurations in the active and inactive state. Similar switchable networks may explain allostery more broadly. This mechanism in particular could aid in drug development targeting the Clostridioides difficile toxins autoproteolysis.

molecular biology↗

bioRxiv2024

Identification of novel fructo-oligosaccharide bacterial consumers by pulse metatranscriptomics in a human stool sample

Dietary fibres influence the composition of the human gut microbiota and directly contribute to its downstream effects on host health. As more research supports the use of glycans as prebiotics for therapeutic applications, the need to identify the gut bacteria that metabolize glycans of interest increases. Fructooligosaccharide (FOS) is a common diet-derived glycan that is fermented by the gut microbiota and has been used as a prebiotic. Despite being well studied, we do not yet have a complete picture of all FOS-consuming gut bacterial taxa. To identify new bacterial consumers, we used a short exposure of microbial communities in a stool sample to FOS or galactomannan as the sole carbon source to induce glycan metabolism genes. We then performed metatranscriptomics, paired with whole metagenomic sequencing (WMS), and 16S amplicon sequencing. The short incubation induced genes involved in carbohydrate metabolism, like carbohydrate-active enzymes (CAZymes), including glycoside hydrolase family 32 genes, which hydrolyze fructan polysaccharides like FOS and inulin. Interestingly, FOS metabolism transcripts were notably overexpressed in Blautia species not previously reported to be fructan consumers. We therefore validated the ability of different Blautia species to ferment fructans by monitoring their growth and fermentation in defined media. This pulse metatranscriptomics approach is a useful method to find novel consumers of prebiotics and increase our understanding of prebiotic metabolism by CAZymes in the gut microbiota. SignificanceComplex carbohydrates are key contributors to the composition of the human gut microbiota and play an essential role in the microbiotas effects on host health. Understanding which bacteria consume complex carbohydrates, or glycans, provides a mechanistic link between dietary prebiotics and their beneficial health effects, an essential step for their therapeutic application. Here, we used a pulse metatranscriptomics pipeline to identify bacterial consumers based on glycan metabolism induction in a human stool sample. We identified novel consumers of FOS among Blautia species, expanding our understanding of this well-known glycan. Our approach can be applied to identify consumers of understudied glycans and expand our prebiotic repertoire. It can also be used to study prebiotic glycans directly in stool samples in distinct patient populations to help delineate the prebiotic mechanism.

microbiology↗

bioRxiv2024

Prevalent human gut Enterocloster spp. produce urolithin A from its dietary precursor using a molybdenum-dependent dehydroxylase

Urolithin A (uroA) is a polyphenol derived from the multi-step metabolism of dietary ellagitannins by the human gut microbiota that can affect host health by stimulating mitophagy. Most individuals harbor a microbiota capable of uroA production; however, the mechanisms underlying the dehydroxylation of its catechol-containing dietary precursor (uroC) are unknown. Here, we use a combination of untargeted bacterial transcriptomics, proteomics, and comparative genomics to uncover an inducible uroC dehydroxylase (ucd) operon in Enterocloster spp. We show that Enterocloster spp. are sensitive to iron chelation by uroC, and dehydroxylation to uroA rescues growth by disrupting the iron-binding catechol. Importantly, only microbiota samples actively transcribing ucd could produce uroA, establishing ucd-containing Enterocloster spp. as keystone urolithin metabolizers. Overall, this work identifies Enterocloster spp. and the ucd operon as main contributors to uroA production and establishes a multi-omics framework to further our mechanistic understanding of polyphenol metabolism by the human gut microbiota.

microbiology↗

bioRxiv2019

Dual delivery of nucleic acids and PEGylated-bisphosphonates via calcium phosphate nanoparticles

Despite many years of research and a few success stories with gene therapeutics, efficient and safe DNA delivery remains a major bottleneck for the clinical translation of gene-based therapies. Gene transfection with calcium phosphate (CaP) nanoparticles brings the advantages of low toxicity, high DNA entrapment efficiency and good endosomal escape properties. The macroscale aggregation of CaP nanoparticles can be easily prevented through surface coating with bisphosphonate conjugates. Bisphosphonates, such as alendronate, recently showed promising anticancer effects. However, their poor cellular permeability and preferential bone accumulation hamper their full application in chemotherapy. Here, we investigated the dual delivery of plasmid DNA and alendronate using CaP nanoparticles, with the goal to facilitate cellular internalization of both compounds and potentially achieve a combined pharmacological effect on the same or different cell lines. A pH-sensitive poly(ethylene glycol)-alendronate conjugate was synthetized and used to formulate stable plasmid DNA-loaded CaP nanoparticles. These particles displayed good transfection efficiency in cancer cells and a strong cytotoxic effect on macrophages. The in vivo transfection efficiency, however, remained low, calling for an improvement of the system, possibly with respect to the extent of particle uptake and their physical stability.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC=\"FIGDIR/small/621102v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (29K):\norg.highwire.dtl.DTLVardef@5f5d56org.highwire.dtl.DTLVardef@e8bc5eorg.highwire.dtl.DTLVardef@1a6f000org.highwire.dtl.DTLVardef@d690d8_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗