scholarly journals Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts

2020 ◽  
Author(s):  
Leonor García-Bayona ◽  
Michael J. Coyne ◽  
Noam Hantman ◽  
Paula Montero-Llopis ◽  
Salena Von ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Stress Responses ◽  
Fluorescent Proteins ◽  
Gut Bacteria ◽  
Mucus Layer ◽  
Human Gut ◽  
Terminal Electron Acceptor ◽  
Cellular Processes ◽  
Dynamic Type ◽  
Gut Symbionts

AbstractMechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut “anaerobes” are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen (1), with oxygen available to bacteria growing at the mucus layer. Using a combination of analyses, we show that Bacteroides species are metabolically and energetically robust and do not mount stress responses in the presence of 0.10 - 0.14% oxygen, defined as nanaerobic conditions (2). Taking advantage of this metabolic capability, we show that nanaerobic growth provides sufficient oxygen for the maturation of oxygen-requiring fluorescent proteins in Bacteroides species. Type strains of four different Bacteroides species show bright GFP fluorescence when grown nanaerobically versus anaerobically. We compared four different red fluorescent proteins and found that mKate2 yields high fluorescence intensity in our assay. We show that GFP-tagged proteins can be localized in nanaerobically growing bacteria. In addition, we used time-lapse fluorescence microscopy to image dynamic Type VI secretion system processes in metabolically active B. fragilis. The ability to visualize fluorescently-labeled Bacteroides and fluorescently-linked proteins in actively growing nanaerobic gut symbionts ushers in a new age of imaging analyses in these bacteria.SignificanceDespite many recent technological advances to study the human gut microbiota, we still lack a facile system to image dynamic cellular processes in most abundant gut species due to the requirement of oxygen for chromophore maturation of commonly used fluorescent proteins. Here, we took advantage of the ability of anaerobes of the gut microbiota to respire aerobically and grow robustly at 0.10– 0.14% oxygen. This physiologic concentration of oxygen is sufficient for fluorescent proteins to mature, allowing for visualization of biological processes never before imaged in these bacteria. This advance will allow for numerous types of analyses in actively-growing “nanaerobic” gut bacteria including subcellular protein localizations, single-cell analyses, biofilm imaging, and protein interactions with other microbes and the host.

scholarly journals Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts

2020 ◽  
Vol 117 (39) ◽  
pp. 24484-24493
Author(s):  
Leonor García-Bayona ◽  
Michael J. Coyne ◽  
Noam Hantman ◽  
Paula Montero-Llopis ◽  
Salena S. Von ◽  
...  
Keyword(s):  
Stress Responses ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Time Lapse ◽  
Mucus Layer ◽  
Type Vi Secretion System ◽  
Terminal Electron Acceptor ◽  
Cellular Processes ◽  
Dynamic Type ◽  
Gut Symbionts

Mechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut “anaerobes” are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen [L. Albenberg et al., Gastroenterology 147, 1055–1063.e8 (2014)], with oxygen available to bacteria growing at the mucus layer. Here, we show that Bacteroides species are metabolically and energetically robust and do not mount stress responses in the presence of 0.10 to 0.14% oxygen, defined as nanaerobic conditions [A. D. Baughn, M. H. Malamy, Nature 427, 441–444 (2004)]. Taking advantage of this metabolic capability, we show that nanaerobic growth provides sufficient oxygen for the maturation of oxygen-requiring fluorescent proteins in Bacteroides species. Type strains of four different Bacteroides species show bright GFP fluorescence when grown nanaerobically versus anaerobically. We compared four different red fluorescent proteins and found that mKate2 yields the highest red fluorescence intensity in our assay. We show that GFP-tagged proteins can be localized in nanaerobically growing bacteria. In addition, we used time-lapse fluorescence microscopy to image dynamic type VI secretion system processes in metabolically active Bacteroides fragilis. The ability to visualize fluorescently labeled Bacteroides and fluorescently linked proteins in actively growing nanaerobic gut symbionts ushers in an age of imaging analyses not previously possible in these bacteria.

Design, Synthesis and Bioactivity of Core 1 O-glycan and its Derivative on Human Gut Microbiota

2019 ◽  
Vol 16 (12) ◽  
pp. 1348-1353
Author(s):  
Huanhuan Qu ◽  
Baixue Li ◽  
Jingyi Yang ◽  
Huaiwen Liang ◽  
Meixia Li ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Initial Step ◽  
Building Blocks ◽  
Glycan Structure ◽  
Human Gut ◽  
Design Synthesis ◽  
The Core ◽  
Human Gut Microbiota ◽  
Biochemical Studies ◽  
Gut Symbionts

Background: Disaccharide core 1 (Galβ1-3GalNAc) is a common O-glycan structure in nature. Biochemical studies have confirmed that the formation of the core 1 structure is an important initial step in O-glycan biosynthesis and it is of great importance for human body. Objective: Our study will provide meaningful and useful sights for O-glycan synthesis and their bioassay. And all the synthetic glycosides would be used as intermediate building blocks in the scheme developed for oligosaccharide construction. Methods: In this article, we firstly used chemical procedures to prepare core 1 and its derivative, and a novel disaccharide was efficiently synthesized. The structures of the synthesized compounds were elucidated and confirmed by 1H NMR, 13C NMR and MS. Then we employed three human gut symbionts belonging to Bacteroidetes, a predominantphyla in the distal gut, as models to study the bioactivity of core 1 and its derivative on human gut microbiota. Results: According to our results, both core 1 and derivative could support the growth of B. fragilis, especially the core 1 derivative, while failed to support the growth of B. thetaiotaomicron and B. ovatus. Conclusion: This suggested that the B. fragilis might have the specificity glycohydrolase to cut the glycosidic bond for acquiring monosaccharide.

scholarly journals If you eat it, or secrete it, they will grow: the expanding list of nutrients utilized by human gut bacteria

2020 ◽  
Author(s):  
Robert W.P. Glowacki ◽  
Eric C. Martens
Keyword(s):  
Synthetic Biology ◽  
Nutrient Utilization ◽  
Gut Bacteria ◽  
Maillard Reaction Products ◽  
Reaction Products ◽  
Human Gut ◽  
Host Diet ◽  
Health And Disease ◽  
Gut Symbionts ◽  
Polysaccharide Utilization Loci

In order to persist, successful bacterial inhabitants of the human gut need to adapt to changing nutrient conditions, which are influenced by host diet and a variety of other factors. For members of the Bacteroidetes and several other phyla, this has resulted in diversification of a variety of enzyme-based systems that equip them to sense and utilize carbohydrate-based nutrients from host, diet, and bacterial origin. In this review, we focus first on human gut Bacteroides and describe recent findings regarding polysaccharide utilization loci (PULs) and the mechanisms of the multi-protein systems they encode, including their regulation and the expanding diversity of substrates that they target. Next, we highlight previously understudied substrates such as monosaccharides, nucleosides, and Maillard reaction products that can also affect the gut microbiota by feeding symbionts that possess specific systems for their metabolism. Since some pathogens preferentially utilize these nutrients, they may represent nutrient niches competed for by commensals and pathogens. Finally, we address recent work to describe nutrient acquisition mechanisms in other important gut species such as those belonging to the Gram-positive anaerobic phyla Actinobacteria and Firmicutes, as well as the Proteobacteria. Because gut bacteria contribute to many aspects of health and disease, we showcase advances in the field of synthetic biology, which seeks to engineer novel, diet-controlled nutrient utilization pathways within gut symbionts to create rationally designed live therapeutics.

scholarly journals Special Issue: Gut Bacteria-Mucus Interaction

2019 ◽  
Vol 7 (1) ◽  
pp. 6 ◽  
Author(s):  
Nathalie Juge
Keyword(s):  
Gastrointestinal Tract ◽  
Gut Microbiota ◽  
Critical Role ◽  
Gut Bacteria ◽  
Mucus Layer ◽  
Special Issue

The mucus layer covering the gastrointestinal tract plays a critical role in maintaining a homeostatic relationship with our gut microbiota. [...]

scholarly journals Dissecting the collateral damage of antibiotics on gut microbes

2020 ◽  
Author(s):  
Lisa Maier ◽  
Camille V. Goemans ◽  
Mihaela Pruteanu ◽  
Jakob Wirbel ◽  
Michael Kuhn ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Gut Bacteria ◽  
Commensal Bacteria ◽  
Strong Impact ◽  
Collateral Damage ◽  
Microbiota Composition ◽  
Human Gut ◽  
Gut Microbes ◽  
Species Specific ◽  
Gut Microbiota Composition

AbstractAntibiotics are used for fighting pathogens, but also target our commensal bacteria as a side effect, disturbing the gut microbiota composition and causing dysbiosis and disease1-3. Despite this well-known collateral damage, the activity spectrum of the different antibiotic classes on gut bacteria remains poorly characterized. Having monitored the activities of >1,000 marketed drugs on 38 representative species of the healthy human gut microbiome4, we here characterize further the 144 antibiotics therein, representing all major classes. We determined >800 Minimal Inhibitory Concentrations (MICs) and extended the antibiotic profiling to 10 additional species to validate these results and link to available data on antibiotic breakpoints for gut microbes. Antibiotic classes exhibited distinct inhibition spectra, including generation-dependent effects by quinolones and phylogeny-independence by β-lactams. Macrolides and tetracyclines, two prototypic classes of bacteriostatic protein synthesis inhibitors, inhibited almost all commensals tested. We established that both kill different subsets of prevalent commensal bacteria, and cause cell lysis in specific cases. This species-specific activity challenges the long-standing divide of antibiotics into bactericidal and bacteriostatic, and provides a possible explanation for the strong impact of macrolides on the gut microbiota composition in animals5-8 and humans9-11. To mitigate the collateral damage of macrolides and tetracyclines on gut commensals, we exploited the fact that drug combinations have species-specific outcomes in bacteria12 and sought marketed drugs, which could antagonize the activity of these antibiotics in abundant gut commensal species. By screening >1,000 drugs, we identified several such antidotes capable of protecting gut species from these antibiotics without compromising their activity against relevant pathogens. Altogether, this study broadens our understanding of antibiotic action on gut commensals, uncovers a previously unappreciated and broad bactericidal effect of prototypical bacteriostatic antibiotics on gut bacteria, and opens avenues for preventing the collateral damage caused by antibiotics on human gut commensals.

scholarly journals Bacterial Atlas of Mouse Gut Microbiota

2021 ◽  
Author(s):  
Mengqi Chu ◽  
Xiaobo Zhang
Keyword(s):  
Complex System ◽  
Gut Microbiota ◽  
Bacterial Communities ◽  
Large Scale ◽  
Gut Bacteria ◽  
Human Gut ◽  
The Core ◽  
Human Gut Microbiota ◽  
Human Immunity ◽  
Mouse Gut Microbiota

Abstract Background: Mouse model is one of of the most widely used animal models for exploring the roles of human gut microbiota, a complex system involving in human immunity and metabolism. However, the structure of mouse gut bacterial community has not been explored at a large scale. To address this concern, the diversity and composition of the gut bacteria of 600 mice was characterized in this study. Results: The results showed that the bacteria belonging to 8 genera were found in the gut microbiota of all mouse individuals, indicating that the 8 bacteria were the core bacteria of mouse gut microbiota. The dominant genera of the mouse gut bacteria contained 15 bacterial genera. It was found that the bacteria in the gut microbiota were mainly involved in host’s metabolisms via the collaborations between the gut bacteria. The further analysis demonstrated that the composition of mouse gut microbiota was similar to that of human gut microbiota. Conclusion: Our study presented a bacterial atlas of mouse gut microbiota, providing a solid basis for investing the bacterial communities of mouse gut microbiota.

scholarly journals Prominent members of the human gut microbiota express endo-acting O-glycanases to initiate mucin breakdown

2019 ◽  
Author(s):  
Lucy I. Crouch ◽  
Marcelo V. Liberato ◽  
Paulina A. Urbanowicz ◽  
Arnaud Baslé ◽  
Christopher A. Lamb ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Current Model ◽  
Initial Step ◽  
Gut Bacteria ◽  
Glycoside Hydrolases ◽  
Gut Health ◽  
New Paradigm ◽  
Human Gut ◽  
Peptide Backbone ◽  
Human Gut Microbiota

AbstractThe human gut microbiota (HGM) are closely associated with health, development and disease. The thick intestinal mucus layer, especially in the colon, is the key barrier between the contents of the lumen and the epithelial cells, providing protection against infiltration by the microbiota as well potential pathogens. The upper layer of the colonic mucus is a niche for a subset of the microbiota which utilise the mucin glycoproteins as a nutrient source and mucin grazing by the microbiota appears to play a key role in maintaining barrier function as well as community stability. Despite the importance of mucin breakdown for gut health, the mechanisms by which gut bacteria access this complex glycoprotein are not well understood. The current model for mucin degradation involves exclusively exo-acting glycosidases that sequentially trim monosaccharides from the termini of the glycan chains to eventually allow access to the mucin peptide backbone by proteases. However, this model is in direct contrast to the Sus paradigm of glycan breakdown used by the Bacteroidetes which involves extracellular cleavage of glycans by surface located endo-acting enzymes prior to import of the oligosaccharide products. Here we describe the discovery and characterisation of endo-acting family 16 glycoside hydrolases (GH16s) from prominent mucin degrading gut bacteria that specifically target the oligosaccharide side chains of intestinal mucins from both animals and humans. These endo-acting O-glycanases display β1,4-glactosidase activity and in several cases are surface located indicating they are involved in the initial step in mucin breakdown. The data suggest a new paradigm for mucin breakdown by the microbiota and the endo-mucinases provide a potential tool to explore changes that occur in mucin structure in intestinal disorders such as inflammatory bowel disease and colon cancer.

scholarly journals Extensive transfer of genes for edible seaweed digestion from marine to human gut bacteria

2020 ◽  
Author(s):  
Nicholas A. Pudlo ◽  
Gabriel Vasconcelos Pereira ◽  
Jaagni Parnami ◽  
Melissa Cid ◽  
Stephanie Markert ◽  
...  
Keyword(s):  
Future Generation ◽  
Gut Bacteria ◽  
Mobile Dna ◽  
Terrestrial Plants ◽  
Human Gut ◽  
Edible Seaweed ◽  
Edible Macroalgae ◽  
Dna Elements ◽  
Gut Symbionts ◽  
Global Exchange

SummaryHumans harbor numerous species of colonic bacteria that digest the fiber polysaccharides in commonly consumed terrestrial plants. More recently in history, regional populations have consumed edible macroalgae seaweeds containing unique polysaccharides. It remains unclear how extensively gut bacteria have adapted to digest these nutrients and use these abilities to colonize microbiomes around the world, especially outside Asia. Here, we show that the ability of gut bacteria to digest seaweed polysaccharides is more pervasive than previously appreciated. Using culture-based approaches, we show that known Bacteroides genes involved in seaweed degradation have mobilized into many members of this genus. We also identify several previously unknown examples of marine bacteria-derived genes, and their corresponding mobile DNA elements, that are involved in degrading seaweed polysaccharides. Some of these genes reside in gut-resident, Gram-positive Firmicutes, for which phylogenetic analysis suggests an origin in the Epulopiscium gut symbionts of marine fishes. Our results are important for understanding the metabolic plasticity of the human gut microbiome, the global exchange of genes in the context of dietary selective pressures and identifying new functions that can be introduced or engineered to design and fill orthogonal niches for a future generation of engineered probiotics.

scholarly journals Metabolic and enzymatic elucidation of cooperative degradation of red seaweed agarose by two human gut bacteria

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Eun Ju Yun ◽  
Sora Yu ◽  
Na Jung Park ◽  
Yoonho Cho ◽  
Na Ree Han ◽  
...  
Keyword(s):  
Bifidobacterium Longum ◽  
Gut Bacteria ◽  
Specific Gene ◽  
Red Seaweed ◽  
Human Gut ◽  
Metabolic Cooperation ◽  
Red Seaweeds ◽  
Metabolic Intermediates ◽  
Gut Symbionts ◽  
Related Enzyme

AbstractVarious health beneficial outcomes associated with red seaweeds, especially their polysaccharides, have been claimed, but the molecular pathway of how red seaweed polysaccharides are degraded and utilized by cooperative actions of human gut bacteria has not been elucidated. Here, we investigated the enzymatic and metabolic cooperation between two human gut symbionts, Bacteroides plebeius and Bifidobacterium longum ssp. infantis, with regard to the degradation of agarose, the main carbohydrate of red seaweed. More specifically, B. plebeius initially decomposed agarose into agarotriose by the actions of the enzymes belonging to glycoside hydrolase (GH) families 16 and 117 (i.e., BpGH16A and BpGH117) located in the polysaccharide utilization locus, a specific gene cluster for red seaweed carbohydrates. Then, B. infantis extracted energy from agarotriose by the actions of two agarolytic β-galactosidases (i.e., Bga42A and Bga2A) and produced neoagarobiose. B. plebeius ultimately acted on neoagarobiose by BpGH117, resulting in the production of 3,6-anhydro-l-galactose, a monomeric sugar possessing anti-inflammatory activity. Our discovery of the cooperative actions of the two human gut symbionts on agarose degradation and the identification of the related enzyme genes and metabolic intermediates generated during the metabolic processes provide a molecular basis for agarose degradation by gut bacteria.

scholarly journals A benzoxazole derivative as an inhibitor of anaerobic choline metabolism by human gut microbiota

2020 ◽  
Vol 11 (12) ◽  
pp. 1402-1412
Author(s):  
Moustafa T. Gabr ◽  
David Machalz ◽  
Szymon Pach ◽  
Gerhard Wolber
Keyword(s):  
Gut Microbiota ◽  
Metabolic Pathways ◽  
Therapeutic Targets ◽  
Gut Bacteria ◽  
Human Diseases ◽  
Human Gut ◽  
Human Gut Microbiota ◽  
Choline Metabolism

Metabolic pathways mediated by human gut bacteria have emerged as potential therapeutic targets because of their association with the pathophysiology of various human diseases.

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