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Dutton, R. J.

Publications and source records attributed to Dutton, R. J..

11 recordsLinked to original sources

Enabling predictive modeling of molecular connections between fermented foods and human inflammation through a paired dataset of cell-based models and multi-omics approaches

Fermented foods are recognized for their rich microbial diversity and bioactive metabolites, which have recently been linked to anti-inflammatory effects and increased gut microbiome diversity. Despite extensive research on fermented foods including large-scale metagenomic surveys and metabolite characterization, a comprehensive mechanistic understanding of how diverse fermented foods, their microbes, and resulting metabolites interact with human biological pathways remains limited. Here, we systematically profiled over 100 commercially available fermented foods for their potential to prevent inflammation using a human cell-based model. We then generated bulk RNA sequencing of the human cells under these treatment conditions, along with metagenomic sequencing and metabolomics of the fermented foods used in the assays. By generating sample-matched multi-omics data, our work aims to lay the scientific groundwork that will enable scientists to generate predictive models and testable hypotheses about the molecular mechanisms underlying the anti-inflammatory effects of fermented foods. This open-source resource comprising all raw data and parsed intermediary files allows the community to begin elucidating the molecular mechanisms between fermented foods and human biology, paving the way for new, scientifically informed strategies for the user of fermented foods as functional foods.

microbiology↗

Leveraging publicly available datasets and machine learning approaches for predicting the health benefits of fermented foods

Fermented foods are an ancient, near universal component of human dietary culture and are increasingly recognized for their health benefits. Bioactive peptides and biosynthetic gene clusters (BGCs) produced by microbes during fermentation have been shown to be key mediators of human health benefits, such as ACE inhibitors and antibacterial bacteriocins. To broadly map this potential, we leveraged the growing abundance of publicly available fermented food datasets alongside recent advances in machine learning models for bioactivity prediction. We collected, curated, and re-analyzed multiple publicly available multi-omics datasets from diverse fermented foods, enabling cross-food comparisons of microbial and molecular profiles. Our analyses include profiling a curated database of [~]1,300 species-representative genomes across hundreds of fermented food metagenomes, predicting genome-encoded BGCs and peptides from 11,500 bacterial genomes, and applying machine learning classification models to predict the bioactivity of thousands of genome-encoded and peptidomics-detected peptides. These models predict 17 different bioactivities, providing novel, testable predictions for downstream experimental characterization. Most importantly, all curated resources, underlying computational tools, and resulting datasets are publicly available to the community, with an emphasis on creating tools and resources that are user-friendly and empower the community to generate more efficient and predictive models for future fermented foods research. A descriptive list of all generated resources is available below, with all computational tools available on GitHub at https://github.com/MicrocosmFoods and all raw files and datasets available on Zenodo at https://zenodo.org/communities/microcosmfoods/. ResourcesO_LIDataset with [~]13,500 genomes from [~]3,000 different samples representing 150 foods and 50 countries C_LIO_LISpecies-representative dataset with [~]1,300 genomes based on 95% ANI, with full functional annotations available as an explorable collection on SeqHub C_LIO_LIStrain-level representative dataset with [~]4,300 genomes based on 99% ANI, available as a free and publicly available narrative on KBase for users to run their own analyses with C_LIO_LIWorkflows for profiling metagenomic samples, performing functional annotation on bacterial genomes, and predicting peptide bioactivity using ML models C_LIO_LIPredicted biosynthetic gene clusters and peptides for [~]11,500 bacterial genomes C_LIO_LIPredicted bioactivities of peptides from [~]11,500 bacterial genomes and 5 proteomics datasets of fermented foods C_LI

microbiology↗

O-antigen biosynthesis mediates evolutionary trade-offs within a simple community

Diverse populations of bacteriophages infect and co-evolve with their bacterial hosts. Although host recognition and infection occurs within microbiomes, the molecular mechanisms underlying host-phage interactions within a community context remain poorly studied. The biofilms (rinds) of aged cheeses contain taxonomically diverse microbial communities that follow reproducible growth patterns and can be manipulated under laboratory conditions. In this study, we use cheese as a model for studying phage-microbe interactions by identifying and characterising a tractable host-phage pair co-occurring within a model Brie community. We isolated novel bacteriophage TS33 that kills Hafnia sp. JB232 (hereafter Hafnia), a member of the model community. TS33 is easily propagated in the lab and naturally co-occurs in the cheese with the Brie community, rendering it a prime candidate for the study of host-phage interactions. We performed growth assays of the Hafnia, TS33 and the fungal community members, Geotrichum candidum and Penicillium camemberti. Employing Random Barcode Transposon Sequencing (RB-TnSeq) experiments, we identified candidate host factors that contribute to TS33 infectivity, many of which are critical to the integrity of bacterial O-antigen. Notably, disruption of these genes results in decreased susceptibility to infection by phage TS33, while simultaneously exhibiting a significant negative effect on the fitness of Hafnia in the presence of the fungi. Therefore, O-antigen mutations may have pleiotropic effects on the interactions between Hafnia and the rest of the Brie community. Ongoing and future studies aim to unearth the molecular mechanisms by which the O-antigen of Hafnia mediates its interactions with its viral and fungal partners.

microbiology↗

Metabolomics of bacterial-fungal pairwise interactions reveal conserved molecular mechanisms

Bacterial-fungal interactions (BFIs) can shape the structure of microbial communities, but the small molecules mediating these BFIs are often understudied. We explored various optimization steps for our microbial culture and chemical extraction protocols for bacterial-fungal co-cultures, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that metabolomic profiles are mainly comprised of fungi derived features, indicating that fungi are the key contributors to small molecule mediated BFIs. LC-inductively coupled plasma MS (LC-ICP-MS) and MS/MS based dereplication using database searching revealed the presence of several known fungal specialized metabolites and structurally related analogues in these extracts, including siderophores such as desferrichrome, desferricoprogen, and palmitoylcoprogen. Among these analogues, a novel putative coprogen analogue possessing a terminal carboxylic acid motif was identified from Scopulariopsis spp. JB370, a common cheese rind fungus, and its structure was elucidated via MS/MS fragmentation. Based on these findings, filamentous fungal species appear to be capable of producing multiple siderophores with potentially different biological roles (i.e. various affinities for different forms of iron). These findings highlight that fungal species are important contributors to microbiomes via their production of abundant specialized metabolites and their role in complex communities should continue to be a priority.

microbiology↗

Longitudinal, Multi-platform Metagenomics Yields a High-quality Genomic Catalog and Guides an In Vitro Model for Cheese Communities

Microbiomes are intricately intertwined with human health, geochemical cycles and food production. While many microbiomes of interest are highly complex and experimentally intractable, cheese rind microbiomes have proven powerful model systems for the study of microbial interactions. To provide a more comprehensive view of the genomic potential and temporal dynamics of cheese rind communities, we combine longitudinal, multi-platform metagenomics of three ripening washed-rind cheeses with whole genome sequencing of community isolates. Sequencing-based approaches revealed a highly reproducible microbial succession in each cheese, co-existence of closely related Psychrobacter species, and enabled the prediction of plasmid and phage diversity and their host associations. Combined with culture-based approaches, we established a genomic catalog and a paired 16-member in vitro washed rind cheese system. The combination of multi-platform metagenomic time-series data and an in vitro model provides a rich resource for further investigation of cheese rind microbiomes both computationally and experimentally. ImportanceMetagenome sequencing can provide great insights into microbiome composition and function and help researchers develop testable hypotheses. Model microbiomes, such as those composed of cheese rind bacteria and fungi, then allow the testing of these hypotheses in a controlled manner. Here, we first generate an extensive longitudinal metagenomic dataset. This dataset reveals successional dynamics, yields a phyla-spanning bacterial genomic catalog, associates mobile genetic elements with their hosts and provides insights into functional enrichment of Psychrobacter in the cheese environment. Next, we show that members of the washed-rind cheese microbiome lend themselves to in vitro community reconstruction. This paired metagenomic data and in vitro system can thus be used as a platform for generating and testing hypotheses related to the dynamics within, and functions associated with, cheese rind microbiomes.

microbiology↗

Higher-order interactions shape microbial interactions as microbial community complexity increases

Non-pairwise interactions, or higher-order interactions (HOIs), in microbial communities have been claimed to explain the emergent features in microbiomes. Yet, the re-organization of microbial interactions between pairwise cultures and larger communities remains largely unexplored from a molecular perspective but is central to our understanding and further manipulation of microbial communities. Here, we used a bottom-up approach to investigate microbial interaction mechanisms from pairwise cultures up to 4-species communities from a simple microbiome (Hafnia alvei, Geotrichum candidum, Pencillium camemberti and Escherichia coli). Specifically, we characterized the interaction landscape for each species combination involving E. coli by identifying E. colis interaction-associated genes using an RB-TnSeq-based interaction assay. We observed a deep reorganization of the interaction-associated genes, with very few 2-species interactions conserved all the way up to a 4-species community and the emergence of multiple HOIs. We further used a quantitative genetics strategy to decipher how 2-species interactions were quantitatively conserved in higher community compositions. Epistasis-based analysis revealed that, of the interactions that are conserved at all levels of complexity, 82% follow an additive pattern. Altogether, we demonstrate the complex architecture of microbial interactions even within a simple microbiome, and provide a mechanistic and molecular explanation of HOIs.

microbiology↗

From iron to antibiotics: Identification of conserved bacterial-fungal interactions across diverse partners

Microbial interactions are major determinants in shaping microbiome structure and function. Although fungi are found across diverse microbiomes, the mechanisms through which fungi interact with other species remain largely uncharacterized. In this work, we explore the diversity of ways in which fungi can impact bacteria by characterizing interaction mechanisms across 16 different bacterial-fungal pairs, involving 8 different fungi and 2 bacteria (Escherichia coli and Pseudomonas psychrophila). Using random barcode transposon-site sequencing (RB-TnSeq), we identified a large number of bacterial genes and pathways important in fungal interaction contexts. Within each interaction, fungal partners elicit both antagonistic and beneficial effects. Using a panel of phylogenetically diverse fungi allowed us to identify interactions that were conserved across all species. Our data show that all fungi modulate the availability of iron and biotin, suggesting that these may represent conserved bacterial-fungal interactions. Several fungi also appear to produce previously uncharacterized antibiotic compounds. Generating a mutant in a master regulator of fungal secondary metabolite production showed that fungal metabolites are key shapers of bacterial fitness profiles during interactions. This work demonstrates a diversity of mechanisms through which fungi are able to interact with bacterial species. In addition to many species-specific effects, there appear to be conserved interaction mechanisms which may be important across microbiomes.

microbiology↗

Algorithmic Learning for Auto-deconvolution of GC-MS Data to Enable Molecular Networking within GNPS.

Gas chromatography-mass spectrometry (GC-MS) represents an analytical technique with significant practical societal impact. Spectral deconvolution is an essential step for interpreting GC-MS data. No public GC-MS repositories that also enable repository-scale analysis exist, in part because deconvolution requires significant user input. We therefore engineered a scalable machine learning workflow for the Global Natural Product Social Molecular Networking (GNPS) analysis platform to enable the mass spectrometry community to store, process, share, annotate, compare, and perform molecular networking of GC-MS data. The workflow performs auto-deconvolution of compound fragmentation patterns via unsupervised non-negative matrix factorization, using a Fast Fourier Transform-based strategy to overcome scalability limitations. We introduce a "balance score" that quantifies the reproducibility of fragmentation patterns across all samples. We demonstrate the utility of the platform with breathomics analysis applied to the early detection of oesophago-gastric cancer, and by creating the first molecular spatial map of the human volatilome.

bioinformatics↗

Native Electrospray-based Metabolomics Enables the Detection of Metal-binding Compounds

Metals are essential for the molecular machineries of life, and microbes have evolved a variety of small molecules to acquire, compete for, and utilize metals. Systematic methods for the discovery of metal-small molecule complexes from biological samples are limited. Here we describe a two-step native electrospray ionization mass spectrometry method, in which double-barrel post-column metal-infusion and pH adjustment is combined with ion identity molecular networking, a rule-based informatics workflow. This method can be used to identify metal-binding compounds in complex samples based on defined mass (m/z) offsets of ion features with the same chromatographic profiles. As this native metal metabolomics approach can be easily implemented on any liquid chromatography-based mass spectrometry system, this method has the potential to become a key strategy for elucidating and understanding the role of metal-binding molecules in biology.

biochemistry↗

Mobile-CRISPRi: Enabling Genetic Analysis of Diverse Bacteria

Introductory paragraphThe vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. CRISPRi, a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs (sgRNAs), has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to human. However, the difficulty of establishing effective CRISPRi systems in non-model bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish \"Mobile-CRISPRi\", a suite of CRISPRi systems that combine modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in Proteobacteria and Firmicutes at the individual gene scale by examining drug-gene synergies and at the library scale by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microbe interactions.

microbiology↗

Changes in the genetic requirements for microbial interactions with increasing community complexity

Microbial community structure and function rely on complex interactions whose underlying molecular mechanisms are poorly understood. To investigate these interactions in a simple microbiome, we introduced E. coli into an experimental community based on a cheese rind and identified the differences in E. colis genetic requirements for growth in interactive and non-interactive contexts using Random Barcode Transposon Sequencing (RB-TnSeq) and RNASeq. E. colis genetic requirements varied among pairwise growth conditions and between pairwise and community conditions. Our analysis points to mechanisms by which growth conditions change as a result of increasing community complexity and suggests that growth within a community relies on a combination of pairwise and higher order interactions. Our work provides a framework for using the model organism E. coli as a probe to investigate microbial interactions regardless of the genetic tractability of members of the studied ecosystem.

microbiology↗