Human Gut Bacteria Research Articles

Engineering an artificial catch bond using mechanical anisotropy

Catch bonds are a rare class of protein-protein interactions where the bond lifetime increases under an external pulling force. Here, we report how modification of anchor geometry generates catch bonding behavior for the mechanostable Dockerin G:Cohesin E (DocG:CohE) adhesion complex found on human gut bacteria. Using AFM single-molecule force spectroscopy in combination with bioorthogonal click chemistry, we mechanically dissociate the complex using five precisely controlled anchor geometries. When tension is applied between residue #13 on CohE and the N-terminus of DocG, the complex behaves as a two-state catch bond, while in all other tested pulling geometries, including the native configuration, it behaves as a slip bond. We use a kinetic Monte Carlo model with experimentally derived parameters to simulate rupture force and lifetime distributions, achieving strong agreement with experiments. Single-molecule FRET measurements further demonstrate that the complex does not exhibit dual binding mode behavior at equilibrium but unbinds along multiple pathways under force. Together, these results show how mechanical anisotropy and anchor point selection can be used to engineer artificial catch bonds.

Complete genome sequence of the novel virulent phage PMBT24 infecting Enterocloster bolteae from the human gut

PMBT24, the first reported virulent bacteriophage infecting the anaerobic human gut bacterium Enterocloster bolteae strain MBT-21, was isolated from a municipal sewage sample and its genome was sequenced and analysed. Transmission electron microscopy revealed a phage with an icosahedral head and a long, non-contractile tail. The circularly permutated, 99,962-bp dsDNA genome of the pac-type phage has a mol% G + C content of 32.1 and comprises 173 putative ORFs. Using amino acid sequence-based phylogeny, phage PMBT24 showed similarity to other, hitherto non-published phage genomes in the databases. Our data suggested phage PMBT24 to present the type phage of a novel genus (proposed name Kielvirus) and novel family of phages (proposed name Kielviridae).

Diindoles produced from commensal microbiota metabolites function as endogenous CAR/Nr1i3 ligands

Numerous studies have demonstrated the correlation between human gut bacteria and host physiology, mediated primarily via nuclear receptors (NRs). Despite this body of work, the systematic identification and characterization of microbe-derived ligands that regulate NRs remain a considerable challenge. In this study, we discover a series of diindole molecules produced from commensal bacteria metabolites that act as specific agonists for the orphan constitutive androstane receptor (CAR). Using various biophysical analyses we show that their nanomolar affinities are comparable to those of synthetic CAR agonists, and that they can activate both rodent and human CAR orthologues, which established synthetic agonists cannot. We also find that the diindoles, diindolylmethane (DIM) and diindolylethane (DIE) selectively up-regulate bona fide CAR target genes in primary human hepatocytes and mouse liver without causing significant side effects. These findings provide new insights into the complex interplay between the gut microbiome and host physiology, as well as new tools for disease treatment.

Carotenoid productivity in human intestinal bacteria Eubacterium limosum and Leuconostoc mesenteroides with functional analysis of their carotenoid biosynthesis genes

The human intestinal microbiota that comprise over 1,000 species thrive in dark and anaerobic environments. They are recognized for the production of diverse low-molecular-weight metabolites crucial to human health and diseases. Carotenoids, low-molecular-weight pigments known for their antioxidative activity, are delivered to humans through oral intake. However, it remains unclear whether human intestinal bacteria biosynthesize carotenoids as part of the in-situ microbiota. In this study, we investigated carotenoid synthesis genes in various human gut and probiotic bacteria. As a result, novel candidates, the crtM and crtN genes, were identified in the carbon monoxide-utilizing gut anaerobe Eubacterium limosum and the lactic acid bacterium Leuconostoc mesenteroides subsp. mesenteroides. These gene candidates were isolated, introduced into Escherichia coli, which synthesized a carotenoid substrate, and cultured aerobically. Structural analysis of the resulting carotenoids revealed that the crtM and crtN gene candidates of E. limosum and L. mesenteroides mediate the production of 4,4′-diaponeurosporene through 15-cis-4,4′-diapophytoene. Evaluation of the crtE-homologous genes in these bacteria indicated their non-functionality for C40-carotenoid production. E. limosum and L. mesenteroides, along with the known carotenogenic lactic acid bacterium Lactiplantibacillus plantarum, were observed to produce no carotenoids under strictly anaerobic conditions. The two lactic acid bacteria synthesized detectable levels of 4,4′-diaponeurosporene under semi-aerobic conditions. The findings highlight that the obligate anaerobe E. limosum retains aerobically functional C30-carotenoid biosynthesis genes, potentially with no immediate self-utility, suggesting an evolutionary direction in carotenoid biosynthesis. (229 words)

Dual membrane-spanning anti-sigma factors regulate vesiculation in Bacteroides thetaiotaomicron

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are mediated in part by Outer Membrane Vesicles (OMVs). Here, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron (Bt). We identified a family of Dual membrane-spanning anti-sigma factors (Dma) that control OMV biogenesis. We conducted molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, modulates OMV production by controlling the activity of the ECF21 family sigma factor, Das1, and its downstream regulon. Dma1 has a previously uncharacterized domain organization that enables Dma1 to span both the inner and outer membrane of Bt. Phylogenetic analyses reveal that this common feature of the Dma family is restricted to the phylum Bacteroidota. This study provides mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

Dynamics of bacterial recombination in the human gut microbiome.

Horizontal gene transfer (HGT) is a ubiquitous force in microbial evolution. Previous work has shown that the human gut is a hotspot for gene transfer between species, but the more subtle exchange of variation within species-also known as recombination-remains poorly characterized in this ecosystem. Here, we show that the genetic structure of the human gut microbiome provides an opportunity to measure recent recombination events from sequenced fecal samples, enabling quantitative comparisons across diverse commensal species that inhabit a common environment. By analyzing recent recombination events in the core genomes of 29 human gut bacteria, we observed widespread heterogeneities in the rates and lengths of transferred fragments, which are difficult to explain by existing models of ecological isolation or homology-dependent recombination rates. We also show that natural selection helps facilitate the spread of genetic variants across strain backgrounds, both within individual hosts and across the broader population. These results shed light on the dynamics of in situ recombination, which can strongly constrain the adaptability of gut microbial communities.

High-throughput transcriptomics of 409 bacteria-drug pairs reveals drivers of gut microbiota perturbation.

Many drugs can perturb the gut microbiome, potentially leading to negative health consequences. However, mechanisms of most microorganism-drug responses have not been elucidated at the genetic level. Using high-throughput bacterial transcriptomics, we systematically characterized the gene expression profiles of prevalent human gut bacteria exposed to the most frequently prescribed orally administered pharmaceuticals. Across >400 drug-microorganism pairs, significant and reproducible transcriptional responses were observed, including pathways involved in multidrug resistance, metabolite transport, tartrate metabolism and riboflavin biosynthesis. Importantly, we discovered that statin-mediated upregulation of the AcrAB-TolC efflux pump in Bacteroidales species enhances microbial sensitivity to vitamin A and secondary bile acids. Moreover, gut bacteria carrying acrAB-tolC genes are depleted in patients taking simvastatin, suggesting that drug-efflux interactions generate collateral toxicity that depletes pump-containing microorganisms from patient microbiomes. This study provides a resource to further understand the drivers of drug-mediated microbiota shifts for better informed clinical interventions.

Polyethylene Terephthalate Hydrolases in Human Gut Microbiota and Their Implications for Human Health.

Polyethylene terephthalate (PET), primarily utilized for food and beverage packaging, consistently finds its way into the human gut, thereby exerting adverse effects on human health. PET hydrolases, critical for the degradation of PET, have been predominantly sourced from environmental microbial communities. Given the fact that the human gut harbors a vast and intricate consortium of microorganisms, inquiry into the presence of potential PET hydrolases within the human gut microbiota becomes imperative. In this investigation, we meticulously screened 22,156 homologous sequences that could potentially encode PET hydrolases using the hidden Markov model (HMM) paradigm, drawing from 4984 cultivated genomes of healthy human gut bacteria. Subsequently, we methodically validated the hydrolytic efficacy of five selected candidate PET hydrolases on both PET films and powders composed of micro-plastics (MPs). Notably, our study also unveiled the influence of both diverse PET MP powders and their resultant hydrolysates on the modulation of cytokine expression in macrophages. In summary, our research underscores the ubiquitous prevalence and considerable potential of the human gut microbiota in PET hydrolysis. Furthermore, our study significantly contributes to the holistic evaluation of the potential health hazards posed by PET MPs to human well-being.

Dietary- and host-derived metabolites are used by diverse gut bacteria for anaerobic respiration.

Respiratory reductases enable microorganisms to use molecules present in anaerobic ecosystems as energy-generating respiratory electron acceptors. Here we identify three taxonomically distinct families of human gut bacteria (Burkholderiaceae, Eggerthellaceae and Erysipelotrichaceae) that encode large arsenals of tens to hundreds of respiratory-like reductases per genome. Screening species from each family (Sutterella wadsworthensis, Eggerthella lenta and Holdemania filiformis), we discover 22 metabolites used as respiratory electron acceptors in a species-specific manner. Identified reactions transform multiple classes of dietary- and host-derived metabolites, including bioactive molecules resveratrol and itaconate. Products of identified respiratory metabolisms highlight poorly characterized compounds, such as the itaconate-derived 2-methylsuccinate. Reductase substrate profiling defines enzyme-substrate pairs and reveals a complex picture of reductase evolution, providing evidence that reductases with specificities for related cinnamate substrates independently emerged at least four times. These studies thus establish an exceptionally versatile form of anaerobic respiration that directly links microbial energy metabolism to the gut metabolome.

O02 Impact of dietary habits on AMR development in human gut bacteria: insights from a hollow fibre infection model

Abstract Background As efforts to reduce antibiotic exposure through medical prescriptions have made significant progress, the potential consequences of residual antibiotics in animal-based food sources have been somewhat overlooked. The question of whether chronic exposure to antibiotic remnants in human diets could lead to the development of antimicrobial resistance (AMR) in human gut bacteria remains largely unanswered. This research aimed to address this gap by utilizing the hollow fibre infection model (HFIM) to investigate whether routine consumption of antibiotics from an omnivorous diet contributes to the emergence of resistance in a previously antibiotic-susceptible strain of E. coli isolated from the human gut. Methods To select the antibiotic mixtures for our study, we conducted a comprehensive diet survey involving 131 participants. Additionally, we employed LC-MS to quantify antibiotic concentrations in the most commonly consumed animal-based food products. We rigorously analysed triplicates of 34 food and beverage items, encompassing beef, chicken, pork, fish, and dairy, to detect and measure the concentrations of 10 specific antibiotics. Our findings revealed the presence of low levels of nine antibiotics across the samples, with amoxicillin and trimethoprim being the most frequently detected antibiotics. Alarmingly, 12 out of 34 products exceeded the acceptable daily intake levels for amoxicillin, ampicillin, and enrofloxacin. We further adapted the estimated daily intake formula to calculate antibiotic combinations and their intake from each meal. Subsequently, we subjected bacterial cells to the HFIM for 7 days, exposing them to three antibiotic ‘meals’ daily. We collected samples from the HFIM every 24 h to monitor changes in resistance phenotypes through MIC testing, as well as for subsequent transcriptomic analysis. Results Astonishingly, we observed the development of resistance to both individual antibiotics and combinations of antibiotics from the very first day, with resistance levels progressively increasing over the course of the week. Interestingly, the rate at which MICs increased was most rapid in the initial days, followed by a slower rate of increase while MICs continued to rise. This study provides compelling evidence that chronic exposure to low levels of antibiotics, akin to those found in animal-derived foods, can lead to rapid resistance development across multiple antibiotics within human gut bacteria. Further examination of samples from the HFIM through RT-PCR and WGS is warranted to elucidate the mechanisms underlying resistance development in the gut environment due to dietary factors. Conclusions Our research highlights the urgent need for greater scrutiny of antibiotic residues in the food chain and emphasizes the potential role of dietary habits in driving AMR development within human gut bacteria. Understanding the underlying mechanisms is crucial for devising effective strategies to mitigate this emerging public health concern.

Effect of Millet Polyphenol-Rich Extracts on Human Fecal Bacteria and Dextran Sulfate Sodium-Induced Human Intestinal Epithelial Cell Derangements

ABSTRACT There is a rejuvenated interest in functional food research on polyphenols due to their gut microbial modulation and capacity to alleviate clinical conditions. In this study, polyphenol-rich extracts (PREs) from Finger millet (FM) and Kodo millet (KM) were assessed for modulating effects on selected human gut bacteria during fecal batch fermentation along with the protective effects on Dextran Sulfate Sodium (DSS)-induced colonic aberrations in vitro. Fecal batch fermentation of FM-PRE and KM-PRE enhanced the abundance of selected beneficial bacteria, such as Bifidobacterium spp. Lactobacillus spp. and the production of SCFAs. Catechin and naringin were the main phenolic fractions of fermented FM-PRE and KM-PRE. Moreover, the millet polyphenols protected the intestinal cells against DSS-induced intestinal barrier dysfunction by reducing inflammatory cytokines, TEER by ~40%, FITC-dextran fluorescence and upregulating the expression of tight junction genes, signifying improved gut functions. The study also imply that FM and KM polyphenols may inhibit intestinal inflammation.

Glycoside-metabolizing oxidoreductase D3dgpA from human gut bacterium.

The Gfo/Idh/MocA family enzyme DgpA was known to catalyze the regiospecific oxidation of puerarin to 3"-oxo-puerarin in the presence of 3-oxo-glucose. Here, we discovered that D3dgpA, dgpA cloned from the human gut bacterium Dorea sp. MRG-IFC3, catalyzed the regiospecific oxidation of various C-/O-glycosides, including puerarin, in the presence of methyl β-D-3-oxo-glucopyranoside. While C-glycosides were converted to 3"- and 2"-oxo-products by D3dgpA, O-glycosides resulted in the formation of aglycones and hexose enediolone from the 3"-oxo-products. From DFT calculations, it was found that isomerization of 3"-oxo-puerarin to 2"-oxo-puerarin required a small activation energy of 9.86 kcal/mol, and the O-glycosidic bond cleavage of 3"-oxo-products was also thermodynamically favored with a small activation energy of 3.49 kcal/mol. In addition, the reaction mechanism of D3dgpA was discussed in comparison to those of Gfo/Idh/MocA and GMC family enzymes. The robust reactivity of D3dgpA was proposed as a new general route for derivatization of glycosides.

A Blautia producta specific gFET-based aptasensor for quantitative monitoring of microbiome quality.

The use of health-relevant bacteria originating from human microbiomes for the control or therapy of diseases, including neurodegenerative disorders or diabetes, is currently gaining increasing importance in medicine. Directed and successful engineering of microbiomes via probiotic supplementation requires subtle, precise as well as, more importantly, easy, fast and convenient monitoring of its success, e.g., in patients' gut. Based on a previously described polyclonal SELEX aptamer library evolved against the human gut bacterium Blautia producta, we finally isolated three individual aptamers that proved their performance concerning affinity, specificity and robustness in reliably labeling the target bacterium and in combination with "contaminating" control bacteria. Using biofunctionalization molecules on gFETs, we could specifically quantify 101-106 cells per mL, retrace their number in mixtures and determine aptamer Kd-values around 2 nM. These measurements were possible even in the context of a real human stool sample. Our results qualify gFETs in combination with BL2, BL7 and BL8 aptamers as a promising foundation for the construction of respective sensing devices, which will open new avenues towards developing an intended monitoring technique for probiotic therapy and microbiome engineering approaches.

A toolbox for genetic manipulation in intestinal Clostridium symbiosum

Gut microbes are closely related with human health, but remain much to learn. Clostridium symbiosum is a conditionally pathogenic human gut bacterium and regarded as a potential biomarker for early diagnosis of intestinal tumors. However, the absence of an efficient toolbox that allows diverse genetic manipulations of this bacterium limits its in-depth studies. Here, we obtained the complete genome sequence of C. symbiosum ATCC 14940, a representative strain of C. symbiosum. On this basis, we further developed a series of genetic manipulation methods for this bacterium. Firstly, following the identification of a functional replicon pBP1 in C. symbiosum ATCC 14940, a highly efficient conjugative DNA transfer method was established, enabling the rapid introduction of exogenous plasmids into cells. Next, we constructed a dual-plasmid CRISPR/Cas12a system for genome editing in this bacterium, reaching over 60 % repression for most of the chosen genes as well as efficient deletion (>90 %) of three target genes. Finally, this toolbox was used for the identification of crucial functional genes, involving growth, synthesis of important metabolites, and virulence of C. symbiosum ATCC 14940. Our work has effectively established and optimized genome editing methods in intestinal C. symbiosum, thereby providing strong support for further basic and application research in this bacterium.

Microbiome diversity protects against pathogens by nutrient blocking.

The human gut microbiome plays an important role in resisting colonization of the host by pathogens, but we lack the ability to predict which communities will be protective. We studied how human gut bacteria influence colonization of two major bacterial pathogens, both in vitro and in gnotobiotic mice. Whereas single species alone had negligible effects, colonization resistance greatly increased with community diversity. Moreover, this community-level resistance rested critically upon certain species being present. We explained these ecological patterns through the collective ability of resistant communities to consume nutrients that overlap with those used by the pathogen. Furthermore, we applied our findings to successfully predict communities that resist a novel target strain. Our work provides a reason why microbiome diversity is beneficial and suggests a route for the rational design of pathogen-resistant communities.

(1 → 3),(1 → 6) and (1 → 3)-β-D-glucan physico-chemical features drive their fermentation profile by the human gut microbiota

Mushroom polysaccharides consist of a unique set of polymers that arrive intact in the human large intestine becoming available for fermentation by resident gut bacteria with potential benefits to the host. Here we have obtained four glucans from two mushrooms (Pholiota nameko and Pleurotus pulmonarius) under different extraction conditions and their fermentation profile by human gut bacteria in vitro was evaluated. These glucans were isolated and characterized as (1 → 3),(1 → 6)-β-D-glucans varying in branching pattern and water-solubility. An aliquot of each (1 → 3),(1 → 6)-β-D-glucan was subjected to controlled smith degradation process in order to obtain a linear (1 → 3)-β-D-glucan from each fraction. The four β-D-glucans demonstrated different water solubilities and molar mass ranging from 2.2 × 105 g.mol−1 to 1.9 × 106 g.mol−1. In vitro fermentation of the glucans by human gut microbiota showed they induced different short chain fatty acid production (52.0–97.0 mM/50 mg carbohydrates), but an overall consistent high propionate amount (28.5–30.3 % of total short chain fatty acids produced). All glucans promoted Bacteroides uniformis, whereas Anaerostipes sp. and Bacteroides ovatus promotion was strongly driven by the β-D-glucans solubility and/or branching pattern, highlighting the importance of β-D-glucan discrete structures to their fermentation by the human gut microbiota.

In vitro digestion under simulated saliva, gastric and small intestinal conditions and fermentation of nicotinamide mononucleotide, and its effects on the gut microbiota

Nicotinamide Mononucleotide (NMN) is a derivative of vitamin B3, which plays a significant role in a plethora of metabolic reactions in the human body and is intricately associated with both immunity and metabolism. Nonetheless, in the intestine metabolic pathway of NMN and the relationship between NMN, gut microbiota, and SCFAs remain hitherto obscure. This study examined the digestion of NMN in simulated saliva, gastric, and small intestine environments, as well as exploring the interaction between NMN and human gut microbiota utilizing an in vitro fermentation model. NMN was progressively degraded into nicotinamide ribose (NR), nicotinamide (NAM), and ribose, with niacinate (NA) constituting the ultimate degradation product due to hydrolysis and metabolism by microbiota. NMN was ingested by human intestinal microbiota with a slower fermentation rate. As a result of NMN ingestion by human gut bacteria，the concentrations of propionate and butyrate increased by 88% and 23%, respectively, compared to the blank control group, the proliferation of beneficial gut bacteria (Bifidobacterium, Phascolarctobacterium, Faecalibacteriun, and Alistipes) significantly increased, while the proliferation of some harmful bacteria (Sutterella, Desulfovibrio and Pseudomonas) drastically declined. These findings illustrated the metabolic processes of NMN in the intestine, elaborating the relationship between NMN, SCFAs and gut microbiota. NMN might be a potential prebiotic to improve intestinal health.

Biochemical investigations of polyphenol degradation enzymes in the phototrophic bacterium Rubrivivax gelatinosus.

Phloroglucinol (1,3,5-trihydroxybenzene) is an important intermediate in the degradation of flavonoids and tannins by anaerobic bacteria. Recent studies have shed light on the enzymatic mechanism of phloroglucinol degradation in butyrate-forming anaerobic bacteria, including environmental and intestinal bacteria such as Clostridium and Flavonifractor sp. Phloroglucinol degradation gene clusters have also been identified in other metabolically diverse bacteria, although the polyphenol metabolism of these microorganisms remain largely unexplored. Here, we describe biochemical studies of polyphenol degradation enzymes found in the purple non-sulfur bacterium Rubrivivax gelatinosus IL144, an anaerobic photoheterotroph reported to utilize diverse organic compounds as carbon sources for growth. In addition to the phloroglucinol reductase and dihydrophloroglucinol cyclohydrolase that catalyze phloroglucinol degradation, we characterize a Mn2+-dependent phloretin hydrolase that catalyzes the cleavage of phloretin into phloroglucinol and phloretic acid. We also report a Mn2+-dependent decarboxylase (DeC) that catalyzes the reversible decarboxylation of 2,4,6-trihydroxybenzoate to form phloroglucinol. A bioinformatics search led to the identification of DeC homologs in diverse soil and gut bacteria, and biochemical studies of a DeC homolog from the human gut bacterium Flavonifractor plautii demonstrated that it is also a 2,4,6-trihydroxybenzoate decarboxylase. Our study expands the range of enzymatic mechanisms for phloroglucinol formation, and provides further biochemical insight into polyphenol metabolism in the anaerobic biosphere.

In Vitro Fermentation Characteristics of Fungal Polysaccharides Derived from Wolfiporia cocos and Their Effect on Human Fecal Microbiota.

Gut microbiota has been described as a new 'organ' that interferes with host physiology by its metabolites produced from the utilization and biotransformation of undigested food components. Fu Ling (FL), the sclerotia of fungi Wolfiporia cocos, contains β-glucan, which is a known natural polysaccharide with strong medicinal efficacy. This study endeavors to evaluate the fermentability of FL and polysaccharides extracted from its sclerotia. An in vitro fermentation of structurally characterized FL and its β-glucan by human fecal microbiota was conducted. Total bacterial count, pH change, short-chain fatty acid profile and microbiota profile were assessed post-fermentation. FL containing over 70% of β-(1 → 3) and (1 → 6)-glucans with a low degree of branching of 0.24 could enhance acetic acid (a major microbial metabolite) production. Both FL and its extracted β-glucan had similar modulation on microbial composition. They enriched Phascolarctobacterium faecium, Bacteroides dorei and Parabacteroides distasonis, all of which are shown to possess anti-inflammatory effects. FL polysaccharide can be utilized as a natural whole food for its potential health benefits to human gut bacteria.

Amino acid auxotrophies in human gut bacteria are linked to higher microbiome diversity and long-term stability

Amino acid auxotrophies are prevalent among bacteria. They can govern ecological dynamics in microbial communities and indicate metabolic cross-feeding interactions among coexisting genotypes. Despite the ecological importance of auxotrophies, their distribution and impact on the diversity and function of the human gut microbiome remain poorly understood. This study performed the first systematic analysis of the distribution of amino acid auxotrophies in the human gut microbiome using a combined metabolomic, metagenomic, and metabolic modeling approach. Results showed that amino acid auxotrophies are ubiquitous in the colon microbiome, with tryptophan auxotrophy being the most common. Auxotrophy frequencies were higher for those amino acids that are also essential to the human host. Moreover, a higher overall abundance of auxotrophies was associated with greater microbiome diversity and stability, and the distribution of auxotrophs was found to be related to the human host’s metabolome, including trimethylamine oxide, small aromatic acids, and secondary bile acids. Thus, our results suggest that amino acid auxotrophies are important factors contributing to microbiome ecology and host-microbiome metabolic interactions.