Chemoproteomic profiling of substrate specificity in gut microbiota-associated bile salt hydrolases.

The gut microbiome possesses numerous biochemical enzymes that biosynthesize metabolites that impact human health. Bile acids comprise a diverse collection of metabolites that have important roles in metabolism and immunity. The gut microbiota-associated enzyme that is responsible for the gateway reaction in bile acid metabolism is bile salt hydrolase (BSH), which controls the host's overall bile acid pool. Despite the critical role of these enzymes, the ability to profile their activities and substrate preferences remains challenging due to the complexity of the gut microbiota, whose metaproteome includes an immense diversity of protein classes. Using a systems biochemistry approach employing activity-based probes, we have identified gut microbiota-associated BSHs that exhibit distinct substrate preferences, revealing that different microbes contribute to the diversity of the host bile acid pool. We envision that this chemoproteomic approach will reveal how secondary bile acid metabolism controlled by BSHs contributes to the etiology of various inflammatory diseases.

Coarse-grained modeling and dynamics tracking of nanoparticles diffusion in human gut mucus.

The gastrointestinal (GI) tract's mucus layer serves as a critical barrier and a mediator in drug nanoparticle delivery. The mucus layer's diverse molecular structures and spatial complexity complicates the mechanistic study of the diffusion dynamics of particulate materials. In response, we developed a bi-component coarse-grained mucus model, specifically tailored for the colorectal cancer environment, that contained the two most abundant glycoproteins in GI mucus: Muc2 and Muc5AC. This model demonstrated the effects of molecular composition and concentration on mucus pore size, a key determinant in the permeability of nanoparticles. Using this computational model, we investigated the diffusion rate of polyethylene glycol (PEG) coated nanoparticles, a widely used muco-penetrating nanoparticle. We validated our model with experimentally characterized mucus pore sizes and the diffusional coefficients of PEG-coated nanoparticles in the mucus collected from cultured human colorectal goblet cells. Machine learning fingerprints were then employed to provide a mechanistic understanding of nanoparticle diffusional behavior. We found that larger nanoparticles tended to be trapped in mucus over longer durations but exhibited more ballistic diffusion over shorter time spans. Through these discoveries, our model provides a promising platform to study pharmacokinetics in the GI mucus layer.

Keystone pathobionts associated with colorectal cancer promote oncogenic reprograming.

Fusobacterium nucleatum (Fn) and enterotoxigenic Bacteroides fragilis (ETBF) are two pathobionts consistently enriched in the gut microbiomes of patients with colorectal cancer (CRC) compared to healthy counterparts and frequently observed for their direct association within tumors. Although several molecular mechanisms have been identified that directly link these organisms to features of CRC in specific cell types, their specific effects on the epithelium and local immune compartment are not well-understood. To fill this gap, we leveraged single-cell RNA sequencing (scRNA-seq) on wildtype mice and mouse model of CRC. We find that Fn and ETBF exacerbate cancer-like transcriptional phenotypes in transit-amplifying and mature enterocytes in a mouse model of CRC. We also observed increased T cells in the pathobiont-exposed mice, but these pathobiont-specific differences observed in wildtype mice were abrogated in the mouse model of CRC. Although there are similarities in the responses provoked by each organism, we find pathobiont-specific effects in Myc-signaling and fatty acid metabolism. These findings support a role for Fn and ETBF in potentiating tumorigenesis via the induction of a cancer stem cell-like transit-amplifying and enterocyte population and the disruption of CTL cytotoxic function.

scMicrobe PTA: Near Complete Genomes from Single Bacterial Cells.

Microbial genomes produced by single-cell amplification are largely incomplete. Here, we show that primary template amplification (PTA), a novel single-cell amplification technique, generated nearly complete genomes from three bacterial isolate species. Furthermore, taxonomically diverse genomes recovered from aquatic and soil microbiomes using PTA had a median completeness of 81%, whereas genomes from standard amplification approaches were usually <30% complete. PTA-derived genomes also included more associated viruses and biosynthetic gene clusters.

Microbial transmission in the social microbiome and host health and disease.

Although social interactions are known to drive pathogen transmission, the contributions of socially transmissible host-associated mutualists and commensals to host health and disease remain poorly explored. We use the concept of the social microbiome-the microbial metacommunity of a social network of hosts-to analyze the implications of social microbial transmission for host health and disease. We investigate the contributions of socially transmissible microbes to both eco-evolutionary microbiome community processes (colonization resistance, the evolution of virulence, and reactions to ecological disturbance) and microbial transmission-based processes (transmission of microbes with metabolic and immune effects, inter-specific transmission, transmission of antibiotic-resistant microbes, and transmission of viruses). We consider the implications of social microbial transmission for communicable and non-communicable diseases and evaluate the importance of a socially transmissible component underlying canonically non-communicable diseases. The social transmission of mutualists and commensals may play a significant, under-appreciated role in the social determinants of health and may act as a hidden force in social evolution.

Prevotella copri variants among a single host diverge in sphingolipid production.

Sphingolipids serve as vital structural and signaling components of the cell membranes in both eukaryotes and prokaryotes. Within the gut microbiome, Bacteroides species have been identified as major producers of sphingolipids, and Bacteroides-produced sphingolipids have been shown to be modulators of host immune and metabolic functions. While Bacteroides species are a prominent feature of the gut microbiomes of populations living in industrialized countries, Prevotella copri, a member of the same phyla, albeit a different family, is the dominant feature across the remainder of the global population, although their sphingolipid-producing capabilities have not been as thoroughly investigated. To fill this gap, we examined the genomes of over 60 diverse isolates of P. copri and identified several key enzymes involved in sphingolipid synthesis in P. copri. Combining bioorthogonal labeling and liquid chromatography-mass spectrometry (LC-MS) based lipidomics, we functionally characterized the first step in P. copri de novo sphingolipid synthesis in addition to profiling the sphingolipidomes of P. copri strains, identifying key enzymes that may play roles in producing a diverse set of P. copri sphingolipids. Given the limited genetic engineering tools amenable for use in P. copri, our approach takes advantage of comparative genomics and phenotypic profiling to explore sphingolipid production in these understudied, yet highly prevalent, organisms.IMPORTANCESphingolipids are important signaling molecules for maintaining metabolic and immune homeostasis in the host. These lipids are also produced by gut commensals, most notably by Bacteroides species. Despite the global prevalence of Prevotella copri in gut microbiomes of individuals, little is known about the types of sphingolipids they produce and whether they are similar in composition and structure to those produced by Bacteroides. Given the varied associations of P. copri with diverse sphingolipid-related health outcomes, such as rheumatoid arthritis and glucose intolerance, it is important to first characterize the specific sphingolipids produced by individual strains of P. copri and to identify the genes involved in their pathways of production. This characterization of P. copri-derived sphingolipids provides further insight into how bacterial sphingolipid production can serve as a mechanism for microbial modulation of host phenotypes.

Precision run-on sequencing (PRO-seq) for microbiome transcriptomics.

Bacteria respond to environmental stimuli through precise regulation of transcription initiation and elongation. Bulk RNA sequencing primarily characterizes mature transcripts, so to identify actively transcribed loci we need to capture RNA polymerase (RNAP) complexed with nascent RNA. However, such capture methods have only previously been applied to culturable, genetically tractable organisms such as E. coli and B. subtilis. Here we apply precision run-on sequencing (PRO-seq) to profile nascent transcription in cultured E. coli and diverse uncultured bacteria. We demonstrate that PRO-seq can characterize the transcription of small, structured, or post-transcriptionally modified RNAs, which are often absent from bulk RNA-seq libraries. Applying PRO-seq to the human microbiome highlights taxon-specific RNAP pause motifs and pause-site distributions across non-coding RNA loci that reflect structure-coincident pausing. We also uncover concurrent transcription and cleavage of CRISPR guide RNAs and transfer RNAs. We demonstrate the utility of PRO-seq for exploring transcriptional dynamics in diverse microbial communities.

Effects of long-term high dose aspartame on body mass, bone strength, femoral geometry, and microbiota composition in a young and aged cohort of male and female mice.

Background: Recent reassessment of the safety of aspartame has prompted increased evaluation of its effect on the health of a range of tissues. The gut microbiome is altered by oral aspartame. One prior study suggested that changes in the microbiome caused by aspartame could influence the strength of bone in young skeletally developing mice. Here we ask how aspartame influences bone in mice of different age and sex. Objective: The objective of this study was to determine the effect of aspartame on the bone strength and gut microbiota of young and aged mice. Methods: Male and female C57Bl/6J mice were untreated or treated with a high dose of aspartame in their drinking water from 1 month of age until 4 (young cohort; n = 80) or 22 months (aged cohort; n = 52). Results: In aged males, mice treated with aspartame had greater body mass, whole bone strength, and femoral geometry relative to untreated. Specifically, in aged males, aspartame led to 9% increase in body mass (p < 0.001), 22% increase in whole bone strength (p = 0.006), and 17% increase in section modulus (p < 0.001) relative to untreated mice. Aged males and females receiving aspartame had a different microbiota than untreated mice and a decreased abundance of Odoribacter. No differences in body mass, whole bone strength, or femoral geometry were associated with aspartame dosing in young males or young or aged females. Conclusions: Aspartame treated aged males had greater whole bone strength and the effect appeared to be explained by greater body mass. Aspartame treatment did not alter whole bone strength in young males or young or aged females despite the aspartame having a similar effect on the microbiota of both aged males and females.

Human-gut bacterial protein-protein interactions: understudied but impactful to human health.

The human gut microbiome is associated with a wide range of diseases; yet, the mechanisms these microbes use to influence human health are not fully understood. Protein-protein interactions (PPIs) are increasingly identified as a potential mechanism by which gut microbiota influence their human hosts. Similar to some PPIs observed in pathogens, many disease-relevant human-gut bacterial PPIs function by interacting with components of the immune system or the gut barrier. Here, we highlight recent advances in these two areas. It is our opinion that there is a vastly unexplored network of human-gut bacterial PPIs that contribute to the prevention or pathogenesis of various diseases and that future research is warranted to expand PPI discovery.

Clinically relevant antibiotic resistance genes are linked to a limited set of taxa within gut microbiome worldwide.

The acquisition of antimicrobial resistance (AR) genes has rendered important pathogens nearly or fully unresponsive to antibiotics. It has been suggested that pathogens acquire AR traits from the gut microbiota, which collectively serve as a global reservoir for AR genes conferring resistance to all classes of antibiotics. However, only a subset of AR genes confers resistance to clinically relevant antibiotics, and, although these AR gene profiles are well-characterized for common pathogens, less is known about their taxonomic associations and transfer potential within diverse members of the gut microbiota. We examined a collection of 14,850 human metagenomes and 1666 environmental metagenomes from 33 countries, in addition to nearly 600,000 isolate genomes, to gain insight into the global prevalence and taxonomic range of clinically relevant AR genes. We find that several of the most concerning AR genes, such as those encoding the cephalosporinase CTX-M and carbapenemases KPC, IMP, NDM, and VIM, remain taxonomically restricted to Proteobacteria. Even cfiA, the most common carbapenemase gene within the human gut microbiome, remains tightly restricted to Bacteroides, despite being found on a mobilizable plasmid. We confirmed these findings in gut microbiome samples from India, Honduras, Pakistan, and Vietnam, using a high-sensitivity single-cell fusion PCR approach. Focusing on a set of genes encoding carbapenemases and cephalosporinases, thus far restricted to Bacteroides species, we find that few mutations are required for efficacy in a different phylum, raising the question of why these genes have not spread more widely. Overall, these data suggest that globally prevalent, clinically relevant AR genes have not yet established themselves across diverse commensal gut microbiota.

Spatial Mapping of Mobile Genetic Elements and their Cognate Hosts in Complex Microbiomes.

The frequent exchange of mobile genetic elements (MGEs) between bacteria accelerates the spread of functional traits, including antimicrobial resistance, within the human microbiome. Yet, progress in understanding these intricate processes has been hindered by the lack of tools to map the spatial spread of MGEs in complex microbial communities, and to associate MGEs to their bacterial hosts. To overcome this challenge, we present an imaging approach that pairs single molecule DNA Fluorescence In Situ Hybridization (FISH) with multiplexed ribosomal RNA FISH, thereby enabling the simultaneous visualization of both MGEs and host bacterial taxa. We used this methodology to spatially map bacteriophage and antimicrobial resistance (AMR) plasmids in human oral biofilms, and we studied the heterogeneity in their spatial distributions and demonstrated the ability to identify their host taxa. Our data revealed distinct clusters of both AMR plasmids and prophage, coinciding with densely packed regions of host bacteria in the biofilm. These results suggest the existence of specialized niches that maintain MGEs within the community, possibly acting as local hotspots for horizontal gene transfer. The methods introduced here can help advance the study of MGE ecology and address pressing questions regarding antimicrobial resistance and phage therapy.

Characterizing conjugative plasmids from an antibiotic-resistant dataset for use as broad-host delivery vectors.

Human microbiome engineering is increasingly proposed as a way to modulate health outcomes. However, one of the current limitations to engineering microbial communities in situ is delivery of a genetic payload for introducing or modifying genes. Indeed, there is a need to identify novel broad-host delivery vectors for microbiome engineering. Therefore, in this study, we characterized conjugative plasmids from a publicly available dataset of antibiotic-resistant isolate genomes in order to identify potential broad-host vectors for further applications. From the 199 closed genomes available in the CDC & FDA AR Isolate Bank, we identified 439 plasmids, of which 126 were predicted to be mobilizable and 206 conjugative. Various characteristics of the conjugative plasmids, such as size, replication origin, conjugation machinery, host defense mechanisms, and plasmid stability proteins, were analyzed to determine these plasmids' potential host-range. Following this analysis, we clustered plasmid sequences and chose 22 unique, broad-host range plasmids that would be suitable for use as delivery vectors. This novel set of plasmids will provide a valuable resource for engineering microbial communities.

Keystone pathobionts associated with colorectal cancer promote oncogenic reprograming.

Fusobacterium nucleatum (Fn) and enterotoxigenic Bacteroides fragilis (ETBF) are two pathobionts consistently enriched in the gut microbiomes of patients with colorectal cancer (CRC) compared to healthy counterparts and frequently observed for their direct association within tumors. Although several molecular mechanisms have been identified that directly link these organisms to features of CRC in specific cell types, their specific effects on the epithelium and local immune compartment are not well-understood. To fill this gap, we leveraged single-cell RNA sequencing (scRNA-seq) on wildtype mice and mouse model of CRC. We find that Fn and ETBF exacerbate cancer-like transcriptional phenotypes in transit-amplifying and mature enterocytes in a mouse model of CRC. We also observed increased T cells in the pathobiont-exposed mice, but these pathobiont-specific differences observed in wildtype mice were abrogated in the mouse model of CRC. Although there are similarities in the responses provoked by each organism, we find pathobiont-specific effects in Myc-signaling and fatty acid metabolism. These findings support a role for Fn and ETBF in potentiating tumorigenesis via the induction of a cancer stem cell-like transit-amplifying and enterocyte population and the disruption of CTL cytotoxic function.

Evolution of promoter-proximal pausing enabled a new layer of transcription control.

Promoter-proximal pausing of RNA polymerase II (Pol II) is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here we analyzed transcription in species across the tree of life. We found that unicellular eukaryotes display a slow acceleration of Pol II near transcription start sites. This proto-paused-like state transitioned to a longer, focused pause in derived metazoans which coincided with the evolution of new subunits in the NELF and 7SK complexes. Depletion of NELF reverts the mammalian focal pause to a proto-pause-like state and compromises transcriptional activation for a set of heat shock genes. Collectively, this work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve.

Epsilon toxin-producing Clostridium perfringens colonize the multiple sclerosis gut microbiome overcoming CNS immune privilege.

Multiple sclerosis (MS) is a complex disease of the CNS thought to require an environmental trigger. Gut dysbiosis is common in MS, but specific causative species are unknown. To address this knowledge gap, we used sensitive and quantitative PCR detection to show that people with MS were more likely to harbor and show a greater abundance of epsilon toxin-producing (ETX-producing) strains of C. perfringens within their gut microbiomes compared with individuals who are healthy controls (HCs). Isolates derived from patients with MS produced functional ETX and had a genetic architecture typical of highly conjugative plasmids. In the active immunization model of experimental autoimmune encephalomyelitis (EAE), where pertussis toxin (PTX) is used to overcome CNS immune privilege, ETX can substitute for PTX. In contrast to PTX-induced EAE, where inflammatory demyelination is largely restricted to the spinal cord, ETX-induced EAE caused demyelination in the corpus callosum, thalamus, cerebellum, brainstem, and spinal cord, more akin to the neuroanatomical lesion distribution seen in MS. CNS endothelial cell transcriptional profiles revealed ETX-induced genes that are known to play a role in overcoming CNS immune privilege. Together, these findings suggest that ETX-producing C. perfringens strains are biologically plausible pathogens in MS that trigger inflammatory demyelination in the context of circulating myelin autoreactive lymphocytes.

Structural insight into protein-protein interactions between intestinal microbiome and host.

Protein-protein interactions between the microbiome and host organism play an important role in shaping host health. These host-modulating proteins have therapeutic potential in treating microbiome-linked disorders such as inflammatory bowel disease and obesity. Structural analysis of interacting proteins provides highly mechanistic insight into the domains driving these interactions and the resulting influence on host cell processes. Here, we briefly review recent publication of microbiome protein structures involved in host binding interactions, the effects of these interactions on host physiology, and the need for further study to increase the ability to detect proteins with therapeutic potential.

Collective effects of human genomic variation on microbiome function.

Studies of the impact of host genetics on gut microbiome composition have mainly focused on the impact of individual single nucleotide polymorphisms (SNPs) on gut microbiome composition, without considering their collective impact or the specific functions of the microbiome. To assess the aggregate role of human genetics on the gut microbiome composition and function, we apply sparse canonical correlation analysis (sCCA), a flexible, multivariate data integration method. A critical attribute of metagenome data is its sparsity, and here we propose application of a Tweedie distribution to accommodate this. We use the TwinsUK cohort to analyze the gut microbiomes and human variants of 250 individuals. Sparse CCA, or sCCA, identified SNPs in microbiome-associated metabolic traits (BMI, blood pressure) and microbiome-associated disorders (type 2 diabetes, some neurological disorders) and certain cancers. Both common and rare microbial functions such as secretion system proteins or antibiotic resistance were found to be associated with host genetics. sCCA applied to microbial species abundances found known associations such as Bifidobacteria species, as well as novel associations. Despite our small sample size, our method can identify not only previously known associations, but novel ones as well. Overall, we present a new and flexible framework for examining host-microbiome genetic interactions, and we provide a new dimension to the current debate around the role of human genetics on the gut microbiome.

Biophysical determinants of biofilm formation in the gut.

The gastrointestinal (GI) tract harbors the most complex microbial ecosystem in the human body. The mucosal layer that covers the GI tract serves as a polymer-based defensive barrier that prevents the microbiome from reaching the epithelium and disseminating inside the body. Colonization of the mucus may result in the formation of structured polymicrobial communities or biofilms, a hallmark in pathologies such as colorectal cancer, inflammatory bowel disease, and chronic gut wounds. However, the mechanisms by which multispecies biofilms establish on the gut mucosa is unknown. Whether mucus-associated biofilms exist as part of a healthy mucosal barrier is still debated. Here, we discuss the impact that diet and microbial-derived polymers has on mucus structure and microcolony formation and highlight relevant biophysical forces that further shape nascent biofilms.

Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR.

The horizonal transfer of plasmid-encoded genes allows bacteria to adapt to constantly shifting environmental pressures, bestowing functional advantages to their bacterial hosts such as antibiotic resistance, metal resistance, virulence factors, and polysaccharide utilization. However, common molecular methods such as short- and long-read sequencing of microbiomes cannot associate extrachromosomal plasmids with the genome of the host bacterium. Alternative methods to link plasmids to host bacteria are either laborious, expensive, or prone to contamination. Here we present the One-step Isolation and Lysis PCR (OIL-PCR) method, which molecularly links plasmid-encoded genes with the bacterial 16S rRNA gene via fusion PCR performed within an emulsion. After validating this method, we apply it to identify the bacterial hosts of three clinically relevant beta-lactamases within the gut microbiomes of neutropenic patients, as they are particularly vulnerable multidrug-resistant infections. We successfully detect the known association of a multi-drug resistant plasmid with Klebsiella pneumoniae, as well as the novel associations of two low-abundance genera, Romboutsia and Agathobacter. Further investigation with OIL-PCR confirmed that our detection of Romboutsia is due to its physical association with Klebsiella as opposed to directly harboring the beta-lactamase genes. Here we put forth a robust, accessible, and high-throughput platform for sensitively surveying the bacterial hosts of mobile genes, as well as detecting physical bacterial associations such as those occurring within biofilms and complex microbial communities.

The comings and goings of the healthy human gut microbiota.

SNP-level analysis of 5,000+ metagenomic samples enabled Hildebrand and colleagues (2021) to identify human gut microbiota by their dispersal strategies in this issue of Cell Host & Microbe. This brings us a step closer in understanding how microbial communities are formed and maintained and also hints at ways in which microbiomes can be manipulated to improve human health.