Burckhardt, J. C. | bioRxiv Science

bioRxiv2022

Gut commensal Enterocloster species host inoviruses that are secreted in vitro and in vivo.

BackgroundBacteriophages in the family Inoviridae, or inoviruses, are under-characterized phages previously implicated in bacterial pathogenesis by contributing to biofilm formation, immune evasion, and toxin secretion. Unlike most bacteriophages, inoviruses do not lyse their host cells to release new progeny virions; rather, they encode a secretion system that actively pumps them out of the bacterial cell. To date, no inovirus associated with the human gut microbiome has been isolated or characterized. ResultsIn this study, we utilized in silico, in vitro and in vivo methods to detect inoviruses in bacterial members of the gut microbiota. By screening a representative genome library of gut commensals, we detected inovirus prophages in Enterocloster spp. and we confirmed the secretion of inovirus particles in in vitro cultures of these organisms using imaging and qPCR. To assess how the gut abiotic environment, bacterial physiology, and inovirus secretion may be linked, we deployed a tripartite in vitro assay that progressively evaluated growth dynamics of the bacteria, biofilm formation, and inovirus secretion in the presence of changing osmotic environments. Counter to other inovirus-producing bacteria, inovirus production was not correlated with biofilm formation in Enterocloster spp. Instead, the Enterocloster strains inoviruses had heterogeneous responses to changing osmolality levels relevant to gut physiology. Notably, increasing osmolality induced inovirus secretion in a strain-dependent manner. We confirmed inovirus secretion in a gnotobiotic mouse model inoculated with individual Enterocloster strains in vivo in unperturbed conditions. Furthermore, consistent with our in vitro observations, inovirus secretion was regulated by a changed osmotic environment in the gut due to osmotic laxatives. ConclusionIn this study, we report on the detection and characterization of novel inoviruses from gut commensals in the Enterocloster genus. Together, our results demonstrate that human gut-associated bacteria can secrete inoviruses and begin to elucidate the environmental niche filled by inoviruses in commensal bacteria.

microbiology↗

bioRxiv2022

Single-strain behavior predicts responses to environmental pH and osmolality in the gut microbiota.

Changes to gut environmental factors such as pH and osmolality due to disease or drugs correlate with major shifts in microbiome composition; however, we currently cannot predict which species can tolerate such changes or how the community will be affected. Here, we assessed the growth of 92 representative human gut bacterial strains spanning 28 families across multiple pH values and osmolalities in vitro. The ability to grow in extreme pH or osmolality conditions correlated with the availability of known stress response genes in many cases, but not all, indicating that novel pathways may participate in protecting against acid or osmotic stresses. Machine learning analysis uncovered genes or subsystems that are predictive of differential tolerance in either acid or osmotic stress. For osmotic stress, we corroborated the increased abundance of these genes in vivo during osmotic perturbation. The growth of specific taxa in limiting conditions in isolation in vitro correlated with survival in complex communities in vitro and in an in vivo mouse model of diet-induced intestinal acidification. Our data show that in vitro stress tolerance results are generalizable and that physical parameters may supersede interspecies interactions in determining the relative abundance of community members. Importantly, we provide an extensive resource for predicting shifts in microbial composition and gene abundance in complex perturbations. Furthermore, this work highlights the physical environment as a major driver of bacterial composition and the importance of performing physical measurements in animal and clinical studies to elucidate the drivers of shifts in microbiota abundance. Significance StatementChanges in pH and particle concentration (osmolality) commonly result from gut disease or the ingestion of common drugs, causing changes in bacterial growth and microbiota composition within the intestine. Thus far, the effects of physical parameters on the growth of intestinal bacterial taxa have not been well documented in the context of predicting microbiota community composition. To address this gap, we examined the growth of 92 bacterial species under varying pH and osmolality conditions. We found that physical parameters are key predictors of bacterial abundance in individual-strain cultures and in complex bacterial communities. Moreover, our results identified specific genes and pathways that are predictive of growth in specific environments. Together, these findings can aid in determining the effectiveness of microbiota therapies in gut environments subjected to various perturbations.

microbiology↗