Biology subjects

Audrain, B.

Publications and source records attributed to Audrain, B..

3 recordsLinked to original sources

bioRxiv2026

Cross hybridization Inference for Phylogenetic Resolution (CIPHR)-FISH enables microbiome imaging with strain level taxonomic resolution

The spatial organization of microbial communities is a critical determinant of host-microbe interactions, yet species-level mapping remains challenging due to high 16S rRNA sequence homology and spectral crosstalk in multiplexed fluorescence in situ hybridization (FISH). To address this challenge, we developed Cross-hybridization Inference for Phylogenetic Resolution (CIPHR)-FISH, a pipeline that integrates strategic probe design with supervised machine learning. CIPHR-FISH transforms probe cross-hybridization and spectral overlap, traditionally viewed as experimental noise, into informative molecular signatures. Using a gnotobiotic zebrafish model colonized with a defined mix of 10 zebrafish bacterial strains, we trained a support vector machine (SVM) on empirical hybridization patterns from pure bacterial cultures. CIPHR-FISH achieved 99.2 % macro-averaged accuracy, significantly outperforming standard linear unmixing (62.5 %), and successfully discriminated strains with 99.7% sequence homology. Applying this tool to gnotobiotic zebrafish larvae revealed distinct biogeographies: the intestinal bulb hosted highly structured, multi-layered polymicrobial aggregates, while the skin exhibited sparse, uniformly dispersed individual bacterial cells. Notably, we observed significant inter-individual variation in spatial community structure that was obscured by traditional bulk 16S rRNA sequencing. CIPHR-FISH provides a robust, scalable framework for high-resolution spatial biology by converting the limitations of molecular labeling into a rich data source for taxonomic classification. This approach enables the quantification of micro-scale ecological and stochastic forces that shape the microbiome across hosts.

microbiology↗

bioRxiv2026

Coupling metabolic enhancement to plasmid spread enables programmable antimicrobial control

The rise of multidrug-resistant pathogens underscores the need for precise antimicrobial strategies that extend beyond conventional antibiotics. Conjugation-based approaches offer a powerful yet underexploited means of delivering targeted genetic interventions directly within microbial communities. In this work, we combined selective killing modules with rationally optimized conjugative vectors to target antibiotic-resistant pathogens and clinically relevant antimicrobial resistance plasmids. First, we engineered and validated toxin-intein modules, programmable cassettes that restrict toxic activity to highly specific regulatory contexts. Specifically, we developed and validated modules targeting Shigella spp., Salmonella enterica, and bacteria carrying the resistance plasmid pOXA-48, demonstrating a tunable system capable of selective activity at both the species and strain levels. To identify the most effective delivery platform, we compared mobilizable and conjugative systems and found that, in vitro, conjugative plasmids consistently outperformed mobilizable ones by approximately one order of magnitude. To further optimize delivery, we streamlined the broad-host-range plasmid RP4 and enhanced its functionality by incorporating either the metabolic fos locus, which confers a fitness advantage to cells carrying the delivery vehicle; a type IV pilus operon that promotes mating-pair stabilization and enables efficient conjugation in liquid environments; or both features combined. Using these engineered RP4 derivatives, we integrated the toxin-intein module targeting pOXA-48 and evaluated its performance in complex microbial communities. In this setting, the RP4 variant carrying both the fos locus and the type IV pilus operon effectively blocked the spread of pOXA-48. Together, this work advances the use of conjugative plasmids as robust and programmable platforms to combat antibiotic resistance and enable microbiome engineering. Beyond introducing highly specific antimicrobial modules and a new generation of optimized conjugative vectors, our results identify ecological competitiveness and plasmid transfer dynamics as critical determinants of the success of such interventions.

microbiology↗

bioRxiv2023

Bridges instead of boats? The Mla system of diderm Firmicute Veillonella parvula reveals an ancestral transenvelope core of phospholipid trafficking

Despite extensive characterisation of envelope biogenesis systems in diderm bacteria, glycerophospholipid (GPL) trafficking remains poorly understood, and has only been studied in a handful of model species. Within the Proteobacteria, the maintenance of lipid asymmetry (Mla) system facilitates retrograde GPL trafficking via six proteins, MlaA-F. GPLs are extracted from the outer leaflet of the outer membrane by the lipoprotein MlaA which associates with porin trimers, then shipped through the periplasmic space by the chaperone MlaC, which finally delivers GPLs to the inner membrane complex formed by MlaBDEF. Here, we investigate GPL trafficking in Veillonella parvula, a diderm member of the Firmicutes which encodes an Mla system devoid of MlaA and MlaC. V. parvula {Delta}mla mutants display phenotypes characteristic of disrupted lipid asymmetry such as hypervesiculation and detergent hypersensitivity, and lipid content analysis from outer membrane vesicles reveals an enrichment for the major lipid component phosphatidylethanolamine. Interestingly, suppressor analysis identifies mutations in tamB that rescue detergent hypersensitivity and hypervesiculation of {Delta}mla strains, supporting the involvement of these two systems in antagonistic GPL trafficking functions across diverse bacterial lineages. A combination of structural modeling and subcellular localisation assays shows that MlaDVp is longer than in classical diderm models and forms a transenvelope bridge, encoding both an inner membrane-localised MCE domain and an outer membrane {beta}-barrel. These results strongly suggest that V. parvula possesses a minimal Mla system for GPL trafficking, replacing the need for chaperones and outer membrane lipoproteins by directly connecting the two membranes. Finally, phylogenomic analysis indicates that this MlaEFD self-contained architecture is widely distributed in diderm bacteria and most likely represents the ancestral functional core of the Mla system, which subsequently increased in complexity in Proteobacteria and closely related phyla following the emergence of MlaABC. Our work broadens the diversity of current models of GPL trafficking in diderm bacteria, challenging the paradigm set by classical models and shedding light on the evolution of a crucial system in the biogenesis and maintenance of the bacterial outer membrane.

microbiology↗