Mucus-associated microbiota consortia: Insights from the advanced TIM-2muc model

Ruizenaar, S.; Lange, K.; Heckert, O.; Lelieveld, J.; Hrynevich, A.; Pacheco, D.; Verdurmen, L.; Chatterjee, M.

doi:10.1101/2025.07.16.665087

Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.07.16.665087

Mucus-associated microbiota consortia: Insights from the advanced TIM-2muc model

Ruizenaar, S.·Lange, K.·Heckert, O.·Lelieveld, J.·Hrynevich, A.·Pacheco, D.·Verdurmen, L.·Chatterjee, M.

View original source ↗Export metadata ↓

Abstract

The mucus layer lining the epithelium of the colon contains a microbiome that is distinct from the luminal microbiome; this is referred to as the mucus-associated microbiome (MAM). Recently, research on the MAM has gained momentum due to its significant role in immune and metabolic health. However, the study of the MAM is limited by conventional experimental techniques, as sampling this microbiome often requires invasive procedures, making in vivo research challenging. Additionally, obtaining samples from the MAM can compromise the lumen, complicating simultaneous studies of both the MAM and the lumen. To address these limitations, we present the TIM-2muc, an advanced version of the colonic in vitro model TIM-2, which includes a mucus compartment comprising both colonic mucus beads - Gut3Beads and an electrospun sheet coated with Gut3Gel. To validate whether the integrated mucus compartment behaves like the in vivo MAM, we examined the bacterial composition, alpha and beta diversity, and the MAMs composition compared to the lumen compartment of both the TIM-2muc and the conventional TIM-2 model. Our findings suggest that the MAM in the TIM-2muc model closely resembles its in vivo counterpart, thereby enhancing the physiological relevance of the luminal microbiome. TIM-2muc, an advanced TIM-2 model, presents a novel, precise, standardized, and non-invasive method for studying the MAM and LM simultaneously.

Explore related subjects

microbiology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Ruizenaar, S., Lange, K., Heckert, O., Lelieveld, J., Hrynevich, A., Pacheco, D., Verdurmen, L., Chatterjee, M.. 2025-07-16. Mucus-associated microbiota consortia: Insights from the advanced TIM-2muc model. https://doi.org/10.1101/2025.07.16.665087

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Matrix-controlled emergence of biofilm architecture shapes antimicrobial survival

Biofilms are structured microbial communities whose extracellular matrix is widely regarded as a basis of their protection against antimicrobial compounds. Yet how matrix production by individual bacteria gives rise to collective architecture and antimicrobial protection remains poorly understood. Here, we systematically varied expression of the master biofilm regulator csgD in Salmonella enterica and found that increasing matrix production reorganizes biofilms from dense, isotropic packings into sparse, nematically aligned communities by altering cell-cell interactions. By combining experimentally measured biofilm architectures with reaction-diffusion modeling, we show that these structural changes produce distinct patterns of antimicrobial killing, ranging from preferential killing near the liquid-biofilm interface to more uniform killing throughout the community. Consequently, increasing matrix production unexpectedly reduces antimicrobial survival by shifting the biofilm into different transport regimes, while strain-specific physiological differences further modulate antimicrobial depletion. Rather than acting as a passive barrier, EPS therefore shapes antimicrobial susceptibility by reorganizing biofilm architecture and its transport properties. EPS thus provides a physical link between molecular regulation, collective architecture and antimicrobial survival, providing a quantitative framework for understanding how cellular matrix production generates emergent biofilm function.

microbiology↗

bioRxiv2026

Mapping virulence-associated protein interaction networks reveals regulators of thermotolerance in Cryptococcus neoformans

Protein-protein interactions (PPIs) influence critical biological processes in pathogenic microorganisms, such as the human fungal pathogen, Cryptococcus neoformans. Fungal thermotolerance and stress response pathways are key virulence determinants that directly impact pathogen adaptation and survival and the infection process. To establish a comprehensive baseline of PPIs in C. neoformans and explore these interactions to infer functional roles for uncharacterized proteins, we applied size exclusion chromatography coupled with mass spectrometry to the secreted and cellular proteomes of the fungi. As a result, 216 and 1699 unique proteins were identified across 24 secretome and proteome fractions, respectively. The predicted secretome networks included expected proteins associated with vesicles and virulence, indicating a role in extracellular defense. Whereas the cryptococcal proteome highlighted interactions among proteins with defined roles in fungal virulence for protein stability and thermotolerance, including two previously uncharacterized proteins, CNAG_00287 and CNAG_05199, putatively involved in complex formation with heat-shock proteins (HSP). Based on sequence and structure homology, we propose that CNAG_00287 is a tetratricopeptide repeat-containing co-chaperone that modulates Hsp 70 activity and CNAG_05199 functions as a Hsp70. We validated the thermotolerance role of CNAG_00287 in heat-related stress, as its absence significantly impaired fungal growth in nutrient-limited media at 37 {degrees}C. Together, this work resolves virulence-associated PPIs within C. neoformans and reveals new molecular regulators of thermotolerance that underpin fungal pathogenicity.

microbiology↗

bioRxiv2026

Environmental filtering and host identity collectively shape root-associated microbiomes of Ericaceae and ectomycorrhizal plants in fumarole fields

Background Symbiosis with microbes is a key strategy that has enabled plants to colonize extreme environments. Since the benefits conferred by root-associated microbes depend on both environmental conditions and host-microbe combinations, plant adaptation to harsh environments is closely linked to the assembly of root microbial communities. Understanding how environmental and host filtering jointly shape these communities is therefore fundamental to elucidating the mechanisms underlying plant adaptation to extreme environments. Results In this study, we investigated the differentiation of root-associated prokaryotic and fungal communities and individual operational taxonomic units (OTUs) across two contrasting habitats surrounding fumaroles, solfatara-field and forest-edge habitats, and six dominant Ericaceae and ectomycorrhizal plant taxa. Prokaryotic and fungal OTUs rarely exhibited strong preferences for both habitat and host identity. Instead, many of prokaryotic and fungal OTUs specialized to one of these niches, collectively generating root microbial communities differentiated by both factors. Nonetheless, striking specializations in habitat and host niches were observed in the fungal family Hyaloscyphaceae (Helotiales). To gain insight into the evolutionary basis of microbial specialization, we examined phylogenetic signals in preference phenotypes. The resulting weak phylogenetic signals in these preference phenotypes further suggest that this fungal clade has undergone substantial ecological divergence. Conclusion Overall, our findings indicate that root-associated microbial communities in extreme environments are assembled through the accumulation of microbial taxa specialized to either habitat or host, and that strong ecological specialization in fungi can arise with little phylogenetic constraint.

microbiology↗