The bacterial cell envelope is a complex structure composed of proteins, lipids, and other molecules that form the physical boundary between bacteria and the environment. In the gut microbiome, commensal bacteria play a fundamental role in maintaining intestinal homeostasis and modulating host physiology. Due to their strategic location, bacterial cell envelope components interact directly with the host immune system, intestinal cells, mucus, and other gut structures, leading to diverse biological effects. Even though beneficial bacteria produce effector molecules, the functional diversity of these molecules, especially among gut bacteria, remains largely unknown. This review compiles current knowledge on the structure and function of bacterial cell envelope components, with a specific focus on gut microbiome commensals and their role in host interactions. It highlights key effector molecules, their immunological recognizers, and the downstream mechanisms involved in such microbial-host interactions, in addition to stating important knowledge gaps. Identifying and characterizing these molecular interactions is a crucial step toward developing novel biotherapeutic targets, particularly for inflammatory bowel diseases and other gut-related disorders. By reviewing current findings and outlining key research deficits, this review deepens our understanding of how bacterial cell envelope components shape host-microbe interactions.

Probiotics are live microorganisms that have been widely investigated for their association with beneficial host outcomes, particularly in the context of gut-associated microbial communities. Despite extensive literature, the probiotic effects are recognized as strain-specific and highly context-dependent, which limits the identification of universal genetic determinants of probiosis. In this study, we present a machine learning-derived genomic dataset generated from comparative analyses of bacterial genomes belonging to taxa frequently reported as probiotics and reference gut-associated bacteria. Using pangenomic analysis combined with supervised machine learning approaches, including Random Forest, Support Vector Machine, and Logistic Regression, we extracted discriminative genomic features from large-scale genome data. The resulting dataset comprises 1,072 non-redundant protein-coding sequences, accompanied by gene presence-absence matrices and functional annotations. These features should not be interpreted as causal determinants of probiotic functionality, but rather as genomic patterns associated with bacterial taxa commonly used as probiotics, which may also reflect taxonomic and ecological signatures. All data and scripts used in this study are publicly available through an open-access repository, providing a reusable resource for exploratory analyses, comparative genomics, and methodological benchmarking in probiogenomics and microbial genomics. The final data, hereby called ProbioSML, is currently available on https://doi.org/10.5281/zenodo.14181443.

Faecalibacterium species are keystone commensals of the human gut, contributing to intestinal homeostasis, immune modulation, and epithelial health. However, their extreme sensitivity to oxygen and reactive oxygen species renders them highly vulnerable during inflammatory conditions, severely limiting their therapeutic application. Understanding the molecular mechanisms underlying their oxidative stress responses is therefore critical for harnessing these bacteria as next-generation probiotics to restore gut health. In this study, we investigated oxidative stress responses in Faecalibacterium duncaniae A2-165 using comprehensive proteomic and membrane fatty acid profiling. We demonstrated that increasing hydrogen peroxide (H₂O₂) concentrations extend the lag phase of growth and affect survival during the first hour of exposure, notably altering the redox potential. Exposure to H₂O₂ triggered a remodeling of the proteome, including detoxification systems, metal transporters, DNA repair systems, transcriptional regulators, and enzymes involved in cobalamin biosynthesis. Complementary RT-qPCR analyses revealed coordinated and time-dependent transcriptional activation of genes involved in oxidative stress response. Remarkably, cobalamin supplementation enhanced bacterial growth, mitigated H₂O₂-induced stress, and lowered superoxide levels in F. duncaniae, highlighting its direct antioxidant activity. By analyzing membrane fatty acid profiles, we showed that cobalamin preserves membrane fluidity, counteracting oxidative stress induced by H₂O₂ in F. duncaniae. These findings reveal the multifaceted strategies employed by F. duncaniae to withstand oxidative stress and provide a foundation for future efforts to optimize its production at industrial scales and its therapeutic potential as a next-generation probiotic.

BackgroundFaecalibacterium duncaniae A2-165 is a rod-shaped, non-motile, and Extremely Sensitive to Oxygen microorganism, belonging to one of the most abundant genera in the human gut microbiome. A decreased abundance of Faecalibacterium species is correlated with Inflammatory Bowel Diseases (IBDs), highlighting this genus as a marker of gut health and a promising Next-Generation Probiotic. While the anti-inflammatory effects of F. duncaniae A2-165 are well known, current studies lack a global protein-level understanding of this species' metabolism. To address this issue, this study investigated the proteome of F. duncaniae A2-165 during the stationary phase using a label-free LC-MS/MS proteomics approach. Results We quantified 1,280 proteins in total, corresponding to 44.7% of the in silico predicted proteome, with a clear distinction between insoluble and soluble protein abundances. The subcellular localization predictions for the quantified proteins identified 802 cytoplasmic proteins, 265 membrane proteins, six extracellular proteins, eight cell wall proteins, and 199 proteins with unknown localization. Functional analysis of the differentially abundant proteins between insoluble and soluble fractions showed a predominance of transporter proteins in the insoluble fraction. At the same time, the metabolism of amino acids, carbohydrates, and nucleotides was predominant in the soluble fraction. Further analysis of enriched pathways for each fraction showed that energy metabolism, carbon cycle, and amino acid metabolism were enriched in the soluble fraction. In contrast, ABC transporters, quorum sensing, and oxidative phosphorylation were enriched in the insoluble fraction. We identified proteins associated with anti-inflammatory effects, notably the key butyrate-production protein ButCoAT, the MAM protein and its ABC transporter, and shikimate pathway enzymes. Conclusion This study characterized, for the first time, the F. duncaniae A2-165 proteome in the stationary phase, profiling the subproteomes of soluble and insoluble fractions and identifying key proteins involved in F. duncaniaeA2-165 metabolism at the protein level. The results presented here could provide new insights into the study of F. duncaniae A2-165.

The genus Faecalibacterium is one of the major butyrate-producing bacteria in the human gut. Due to its production of beneficial metabolites and reduced populations in diseased patients, it is regarded as a potential biomarker of healthy gut microbiota. Strains Collection Nationale de Cultures de Microorganismes (CNCM) I-4540 and CNCM I-4541 were isolated from faeces of a healthy elderly and a healthy adult respectively. In this study, we conducted a taxonomic analysis of these strains. Phylogenetic analyses based on 16S rRNA gene and rpoA gene sequences revealed that the two strains belong to the genus Faecalibacterium. A core gene phylogenetic tree further supported their phylogenetic placement. CNCM I-4540 shared an average nucleotide identity (ANI) value of 96.7% and a digital DNA-DNA hybridization (dDDH) value of 74.3% with Faecalibacterium taiwanense HLW78T, indicating that CNCM I-4540 should be classified as F. taiwanense. In contrast, CNCM I-4541 did not exhibit ANI or dDDH values above the species threshold for any known species within the genus Faecalibacterium. These findings indicate that CNCM I-4541 represents a novel species within the genus Faecalibacterium, for which the name Faecalibacterium langellae sp. nov. is proposed. The type strain is CNCM I-4541T (=CIP 112513T=JCM 39552T).

IL22 is a cytokine with many immunoregulatory effects. It also promotes mucus production, cell proliferation and wound healing, making it a key component of intestinal health. An increasing body of studies highlight the potential of IL22 as a therapeutic drug against NAFLD through the induction of antimicrobial peptides, especially of the Reg3 lectins family, and the regulation of gut microbiota. However, adverse reactions to this molecule in long term administration are little-studied. In this study, we gave daily doses of recombinant L. lactis delivering DNA vaccine for REG3A and IL-22 production to mice fed with a butter-based high fat diet (milk-fat diet or MFD). We aim to compare the effects of REG3A and IL-22 expression by the epithelial cells on the development of obesity-related conditions and the composition of the microbiota. We show that they have opposite effects on fat accumulation in the liver and insulin resistance development despite inducing a similar shift in fecal microbiota composition. Here, we provide evidence that daily long term administration of IL22 can aggravate NAFLD by mechanisms independent from gut microbiota.

Faecalibacterium has recently garnered attention for its potential health implications. To better understand its role, we developed and assessed real-time PCR assays for detecting and quantifying various Faecalibacterium species in human stool samples from both healthy individuals and Crohn's disease patients, either in flare or remission. The assays targeted the Microbial Anti-inflammatory Molecule (MAM) genes, which encode MAM proteins. These assays demonstrated 100% species-specificity using strains from six Faecalibacterium species: Faecalibacterium prausnitzii, Faecalibacterium taiwanense, Faecalibacterium duncaniae, Faecalibacterium longum, Faecalibacterium hattori, and Faecalibacterium CNCM4541. They also showed high sensitivity with detection limits of 10^5 bacteria per gram of sample. In healthy individuals, the different Faecalibacterium species varied in abundance. F. taiwanense, F. duncaniae, and F. longum were the most prevalent, around 10^10 bacteria/g of stool. In contrast, F. hattori and CNCM4541 were less abundant, with 10^7 bacteria/g. Despite its low abundance, F. hattori was present in all healthy subjects, while CNCM4541 was detected in only 50% of them. Notably, F. taiwanense, F. duncaniae, and F. longum were found in all healthy individuals. In Crohn's disease patients, both in flare and remission, a decrease in Faecalibacterium species was observed, with no recovery in remission. The most abundant species in Crohn's disease patients were F. prausnitzii and F. duncaniae, around 10^7 bacteria/g, while F. longum, F. hattori, and F. taiwanense were present at lower levels (10^6 bacteria/g), and CNCM4541 was no longer detected. Interestingly, F. prausnitzii showed a smaller decrease in abundance compared with other species. Moreover, F. prausnitzii was significantly more prevalent in patients in remission than in those in flare, suggesting that it may be more resistant to inflammation. These findings highlight the importance of accurately characterizing and quantifying Faecalibacterium species to better understand their role in health and disease.

La dysbiose du microbiote désigne un déséquilibre ou une perturbation de la composition et de la fonction normales des bactéries présentes dans le microbiote intestinal. De plus en plus de données scientifiques montrent qu’elle est associée à des maladies métaboliques telles que l’obésité, le diabète de type 2 (DT2) ou la stéatose hépatique non alcoolique. En particulier, certaines bactéries du microbiote intestinal sont impliquées dans l’augmentation de l’appétit et de l’absorption d’énergie par l’hôte, renforçant le stockage des graisses, et contribuant à l’inflammation chronique de l’intestin. L’enjeu est donc de mieux comprendre le rôle du microbiote dans les maladies métaboliques pour pouvoir y pallier par de nouvelles stratégies thérapeutiques.

MAM (Microbial-Anti-Inflammatory Molecule) is a key effector protein with anti-inflammatory properties in Faecalibacterium duncaniae, a critical human gut microbiota species. Despite its importance, MAM function and molecular features remain poorly understood. This study elucidates MAM’s physiological importance by examining its cellular localization, secretion dynamics, and structural organization. Mass spectrometry and immunogold labeling confirmed MAM as the most abundant protein in the cell envelope and the second most abundant in the overall proteome, localizing it to the bacterial surface. Bioinformatic and in silico analyses suggest that MAM contains an N-terminal leader peptide with motifs recognized by a Peptidase-domain-Containing ABC Transporter (PCAT), enabling cargo transport to the cell envelope. After N-terminal excision, the cargo protein could be transported to the cell envelope via this PCAT, where it could assemble into a hexameric structure, as revealed by docking and AlphaFold3 modeling. Such results were supported by electron microscopy showing a lattice-like organization on the bacterial surface. This work introduces a novel discussion about the singular organization of the F. duncaniae cell envelope, having MAM as a key component for the bacteria, supporting the understanding of the unique biology of F. duncaniae and its potential as a next-generation probiotic or live biotherapeutic.

Motivation: Understanding interactions from microbiome data is a central aspect in microbial ecology, as it provides insights into ecosystem stability, disease mechanisms, and can be used to design synthetic communities. Current network inference tools reconstruct global networks from co-abundance data, which means they capture the overall correlation structure for the entire set of taxa considered. These approaches are computationally intensive and suboptimal when the focus is on the local neighborhood of specific taxa of interest. Results: We introduce NeighborFinder, a local network inference method that enables the targeted discovery of direct neighbors around a species of interest. Using cross-validated multiple linear regression with ℓ1 penalty and microbiome specific filters, our approach infers interpretable species-centered interactions, with F1 score ≥ 0.95 on simulated cohorts ranging from 250 to 1000 samples. This method is well-suited for large metagenomic datasets and is particularly valuable for exploratory studies where the targeted hypotheses outweigh the need for global community structure. The approach complements existing methods by being a biologically intuitive and computationally efficient. Availability and Implementation: The R package is freely available on GitHub: https://github.com/ metagenopolis/NeighborFinder. The data and source code used to calculate performances and produce the use case example in this paper can be found respectively at: https://doi.org/10.57745/UPITJ0 and https://doi.org/10.57745/HJLWW4. ### Competing Interest Statement The authors have declared no competing interest. Carnot Institute Qualiment, https://ror.org/032k75171, #20 CARN 0026 01 Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-11-DPBS-0001

Microbial anti-inflammatory molecule (MAM) is a key effector of the next-generation probiotic Faecalibacterium duncaniae A2-165, a species whose depletion in the gut microbiota is strongly linked to inflammatory bowel disease (IBD) and other conditions. Despite its importance, the direct anti-inflammatory effects of purified MAM have never been evaluated in vitro or in intestinal inflammation models. Prior studies have relied on bacterial supernatants, synthetic peptides, or DNA delivery systems, each with inherent limitations. In this study, we produced and purified recombinant MAM (R-MAM) under denaturing conditions and, for the first time, demonstrated its direct anti-inflammatory activity in vitro and its protective effects in a colitis murine model. Despite numerous attempts, we were not able to obtain a non-aggregated R-MAM. Therefore, we can assume that the R-MAM used here is partly or totally denatured. Nevertheless, in vitro assays with human intestinal epithelial cells (HT-29) and peripheral blood mononuclear cells (PBMCs) confirmed the ability of MAM to induce an anti-inflammatory cytokine profile. In addition, in a DNBS-induced colitis model, oral administration of R-MAM significantly prevented weight loss and reduced colon weight and thickness, key macroscopic indicators of inflammation. These findings provide a critical validation step for the therapeutic potential of MAM in intestinal inflammation, despite its purification under denaturing conditions. Future studies should focus on optimizing protein stability and conformational integrity to increase its therapeutic potential as a biotherapeutic agent.

Introduction Les spondylarthrites (SpA) se caractérisent par des inflammations ostéoarticulaires affectant le squelette axial et périphérique fréquemment associé à des manifestations extra-articulaires, dont les maladies inflammatoires de l’intestin (MICI). De nombreux arguments sont en faveur du rôle potentiel d’un déséquilibre du microbiote intestinal ou dysbiose dans les SpA comme cela a été démontré dans les MICI. Au cours des SpA, la dysbiose se caractérise par une augmentation au premier plan de l’abondance relative de Ruminococcus gnavus corrélée avec l’activité de la maladie. Dans cette étude, notre objectif était d’isoler des souches de R. gnavus de patients atteints de SpA et des témoins sains pour les comparer entre elles par des analyses génétiques et fonctionnelles. Nous avons notamment évalué si les souches de R. gnavus des patients atteints de SpA avaient davantage de propriétés toxiques et pro-inflammatoires que celles des témoins sains. Patients et méthodes Des colonies de R. gnavus ont été isolées en chambre de Freiter à partir de biopsies coliques et des selles de patients atteints de SpA et de témoins sains. Les colonies ont été séquencées par « next generation sequencing » (NGS). Des expériences d’aérotolérance ont été réalisées pour tester la sélection de R. gnavus par l’environnement pro-inflammatoire de l’intestin au cours de la SpA. Les fonctions pro-inflammatoires de ces souches ont été évaluées par leur capacité à induire la mortalité et la production de « tumor necrosis factor » (TNF) dans des monocytes isolés de patients atteints de SpA. Résultats Le taux de succès d’isolement des souches de R. gnavus a été le plus élevé à partir des biopsies coliques de patients SpA et significativement plus que chez les témoins sains (13/17, 76 % vs. 4/17, 24 % ; p=0,002). Le séquençage nous a permis d’identifier 48 souches différentes provenant de 19 patients atteints de SpA et de 4 témoins sains. Le classement phylogénétique a mis en évidence deux clades distincts, l’un enrichi en souches de patients, l’autre en souches de témoins sains. Sur le plan fonctionnel, l’aérotolérance n’était pas significativement différente entre les souches des patients et de témoins. En revanche, les souches de patients se sont révélées plus toxiques et pro-inflammatoires sur les monocytes que celles des témoins sains. Conclusion L’isolement des souches de R. gnavus a été plus fréquent chez les patients atteints de SpA que chez des témoins sains, en accord avec une plus grande abondance de cette bactérie commensale chez les patients. De plus, les souches de R. gnavus isolées chez les patients atteints de SpA se sont révélées plus toxiques et pro-inflammatoires in vitro que celles de témoins sains. L’étude phylogénétique indique que ces différences de comportement pourraient être en rapport avec des particularités génétiques qui restent à préciser.

The gut-lung axis is critical during viral respiratory infections such as influenza. Gut dysbiosis during infection translates into a massive drop of microbially produced short-chain fatty acids (SCFAs). Among them, butyrate is important during influenza suggesting that microbiome-based therapeutics targeting butyrate might hold promises. The butyrate-producing bacterium Faecalibacterium duncaniae (formerly referred to as F. prausnitzii) is an emerging probiotic with several health-promoting characteristics. To investigate the potential effects of F. duncaniae on influenza outcomes, mice were gavaged with live F. duncaniae (A2-165 or I-4574 strains) five days before infection. Supplementation of F. duncaniae was associated with less severe disease, a lower pulmonary viral load, and lower levels of lung inflammation. F. duncaniae supplementation impacted on gut dysbiosis induced by infection, as assessed by 16S rRNA sequencing. Interestingly, F. duncaniae administration was associated with a recovery in levels of SCFAs (including butyrate) in infected animals. The live form of F. duncaniae was more potent that the pasteurized form in improving influenza outcomes. Lastly, F. duncaniae partially protected against secondary (systemic) bacterial infection. We conclude that F. duncaniae might serve as a novel next generation probiotic against acute viral respiratory diseases.

Probiotics are live microorganisms that, when administered in adequate amounts, can bring health benefits to the host. Most of these organisms are found naturally in the human gastrointestinal tract. Escherichia coli strains Nissle 1917 (EcN), and CEC15 have shown beneficial effects in murine models of intestinal inflammation, such as colitis and mucositis. The present study evaluated the effects as postbiotic of heat-inactivated and cell-free supernatant preparations of EcN and CEC15 in attenuating 5-fluorouracil (5-FU)-induced intestinal mucositis in mice and compared them with the probiotic effects of the live preparations. BALB/c mice were fed, by daily gavage, with 1010 CFU of live or inactivated bacteria or with 300 µL of cell-free supernatant for 12 days. On the 10th day, all animals, except for the control group, received an intraperitoneal injection of 5-FU (300 mg/kg). After 72 h of 5-FU administration, animals were euthanized, and the ileum and blood were collected for analysis. Treatments with live and heat-inactivated CEC15 mitigated weight loss, preserved intestinal length, reduced histological damage, maintained goblet cells, decreased neutrophil infiltration, and modulated expression of inflammatory and barrier genes when compared to 5-FU mucositis controls. EcN showed more limited effects. CEC15 upregulated mRNA expression of the mucin MUC2 and tight junction protein TJP1. CEC15 demonstrated protective effects against 5-FU-induced mucositis, whether administered with live, heat-inactivated, or cell-free supernatant. This suggests that CEC15 mediates a protective response via secreted metabolites and does not require viability. The postbiotic forms of CEC15 present advantages for use in immunocompromised patients. This study elucidates the anti-inflammatory and barrier-protective effects of CEC15 against intestinal mucositis.

Background Lactobacillus delbrueckii is a bacterium used in the dairy industry, improving dairy products' organoleptic characteristics and nutritional value. Preclinical studies have demonstrated the probiotic effects of these microorganisms. However, unlike other lactic acid bacteria species, few studies have explored L. delbrueckii strains, using a probiogenomics approach, about their benefits and safety for host health. Scope and approach This comprehensive review describes the main health effects and safety features of L. delbrueckii strains. Furthermore, it also identifies potential genetic factors and molecular mechanisms obtained through the probiogenomics approach, which can offer insights into this species' effects on host health and its safety level for biotechnological and therapeutic applications. Key findings and conclusions L. delbrueckii can tolerate industrial and gastrointestinal stressors, and this property could be enhanced by incorporating these microorganisms into dairy matrices. The biotherapeutic activity of L. delbrueckii has been extensively demonstrated in intestinal inflammation, enteric infections, and metabolic and psychological disorders. Many beneficial effects are elicited by components of the bacteria cell membrane or secreted molecules, such as cell surface proteins, exopolysaccharides, bioactive metabolites, and peptides. Furthermore, few plasmids were identified in L. delbrueckii, reducing the spread of antibiotic-resistance genes. Additionally, this species has a lower capacity to act as a pathogen. However, studies show a discrepancy between the phenotype and genotype data, which may be related to the gene expression level or regulation in specific environmental conditions. Therefore, further studies should be conducted to characterize the possible biological role of this species and identify the genetic markers linked to these processes.

Introduction S taphylococcus epidermidis is a commensal bacterium ubiquitously present on human skin. This species is considered as a key member of the healthy skin microbiota, involved in the defense against pathogens, modulating the immune system, and involved in wound repair. Simultaneously, S. epidermidis is the second cause of nosocomial infections and an overgrowth of S. epidermidis has been described in skin disorders such as atopic dermatitis. Diverse isolates of S. epidermidis co-exist on the skin. Elucidating the genetic and phenotypic specificities of these species in skin health and disease is key to better understand their role in various skin conditions. Additionally, the exact mechanisms by which commensals interact with host cells is partially understood. We hypothesized that S. epidermidis isolates identified from different skin origins could play distinct roles on skin differentiation and that these effects could be mediated by the aryl hydrocarbon receptor (AhR) pathway. Methods For this purpose, a library of 12 strains originated from healthy skin (non-hyperseborrheic (NH) and hyperseborrheic (H) skin types) and disease skin (atopic (AD) skin type) was characterized at the genomic and phenotypic levels. Results and discussion Here we showed that strains from atopic lesional skin alter the epidermis structure of a 3D reconstructed skin model whereas strains from NH healthy skin do not. All strains from NH healthy skin induced AhR/OVOL1 path and produced high quantities of indole metabolites in co-culture with NHEK; especially indole-3-aldehyde (IAld) and indole-3-lactic acid (ILA); while AD strains did not induce AhR/OVOL1 path but its inhibitor STAT6 and produced the lowest levels of indoles as compared to the other strains. As a consequence, strains from AD skin altered the differentiation markers FLG and DSG1. The results presented here, on a library of 12 strains, showed that S. epidermidis originated from NH healthy skin and atopic skin have opposite effects on the epidermal cohesion and structure and that these differences could be linked to their capacity to produce metabolites, which in turn could activate AHR pathway. Our results on a specific library of strains provide new insights into how S. epidermidis may interact with the skin to promote health or disease.