Drauch, V. | Search bioRxiv

bioRxiv2026

Novel pESI-encoded autotransporter adhesin PeaP of epidemic Salmonella strains mediates adhesion, atypical biofilm formation, and poultry colonization

Salmonella enterica serovar Infantis (SIN) has rapidly become the dominant serovar in poultry worldwide, a success largely linked to the acquisition of the 285 kb megaplasmid pESI. While pESI-encoded antibiotic-resistance and iron-uptake systems are well characterized, pESI-mediated adhesion mechanisms remain poorly understood. Here we identify a novel pESI-encoded monomeric autotransporter adhesin, designated PeaP (pESI-encoded autotransporter protein), and demonstrate its pivotal role in atypical biofilm formation, interference with motility, and colonization of the chicken host. Biofilm assays revealed that pESI-harboring strain SIN 119944 forms robust biofilms at 37 {degrees}C and 42 {degrees}C, temperatures at which CsgD-dependent biofilm formation is negligible. Deletion of csgD did not impair this phenotype, whereas deletion of peaP abolished high-temperature biofilm development and restored motility to wild-type levels. Proteomic profiling of sessile versus planktonic cells highlighted PeaP as the most abundant pESI-derived protein in the biofilm fraction. AlphaFold-based modelling and negative-stain transmission electron microscopy showed that PeaP comprises a C-terminal {beta}-barrel and a 1,500 aa passenger domain with three tandem repeats, projecting filamentous appendages [~]37-nm from the outer membrane. Antibody blockade of PeaP reduced surface adhesion >6-fold, confirming its adhesive function. In an infection model of 2 day-old chicken, the peaP mutant displayed significantly lower colonization, indicating PeaP-mediated adhesion in vivo. Collectively, pESI-positive SIN deploys PeaP for CsgD-independent, temperature-tolerant biofilm formation and enhanced gastrointestinal colonization, providing a mechanistic basis for the epidemic spread of this multidrug-resistant pathogen in poultry.

microbiology↗

bioRxiv2025

Micro-scale spatial metagenomics: revealing high-resolution spatial biogeography of gut microbiomes

Spatial organisation is a fundamental yet poorly resolved aspect of gut microbial ecology. Conventional shotgun metagenomics provides rich functional information but relies on homogenised, macro-scale samples that obscure the micron-scale distributions critical for understanding microbial community dynamics. Here, we introduce Micro-Scale Spatial Metagenomics (MSSM), a new methodological framework that couples laser micro-dissection of tissue sections, ultra-low-input library preparation, and genome-resolved bioinformatics to reconstruct microbial communities from intestinal microsamples measuring as little as [~]500 {micro}m{superscript 2} ({approx}100 bacterial cells). We describe a fully optimised laboratory and computational pipeline that enables quantitative, strain-resolved, and functionally informed spatial profiling directly from intact gut tissue. Using chicken intestinal samples, we validated MSSM through combinatorial single-cell fluorescence in situ hybridisation (FISH) imaging and comparisons with macro-scale metagenomics, demonstrating its robustness and accuracy. MSSM captured fine-scale heterogeneity in taxonomic and functional composition across intestinal cryosections, hinting at spatially structured assemblages and segregation of metabolic capacities. Strain-level analyses uncovered coexisting Lawsonibacter lineages exhibiting distinct spatial distributions and host-specific occurrence patterns, while SNP-level microdiversity analyses showed that genetically coherent clonal populations cluster at spatial scales below [~]200 {micro}m. By enabling shotgun metagenomics at micron resolution, MSSM closes a longstanding methodological gap and provides a scalable platform for studying microbial ecosystems in situ. This approach unlocks a previously inaccessible view of microbial biogeography, offering new opportunities to investigate host-microbe and microbe-microbe interactions, and the spatial principles governing gut ecosystems. Significance statementUnderstanding how microbial communities are organised in space is essential to explaining their ecological and functional roles, yet microbiome research still relies overwhelmingly on bulk, spatially averaged measurements. We introduce micro-scale spatial metagenomics (MSSM), the first method that brings shotgun metagenomics to the microscale, enabling direct measurement of functional and taxonomic variation across regions containing as few as [~]100 cells. Unlike existing spatial approaches, MSSM reconstructs complete genomes and resolves strain-level diversity within intact tissue, allowing researchers to map metabolic potential, microdiversity, and community structure in situ. By coupling high-resolution sequencing with spatial context, MSSM reveals a previously inaccessible layer of microbial organisation, transforming how host-associated ecosystems can be studied.

microbiology↗