Quotation: DuMez-Kornegay RN, Baker LS, Morris AJ, DeLoach WLM, Dowen RH (2024) Kombucha Tea-associated microbes rework host metabolic pathways to suppress lipid accumulation. PLoS Genet 20(3):
e1011003.
https://doi.org/10.1371/journal.pgen.1011003
Editor: Sean P. Curran, College of Southern California, UNITED STATES
Acquired: October 4, 2023; Accepted: February 22, 2024; Revealed: March 28, 2024
Copyright: © 2024 DuMez-Kornegay et al. That is an open entry article distributed below the phrases of the Creative Commons Attribution License, which allows unrestricted use, distribution, and replica in any medium, offered the unique writer and supply are credited.
Knowledge Availability: All related information are inside the paper and its Supporting Information recordsdata. The entire genome sequencing information can be found on the Sequencing Learn Archive (PRJNA1044129). Uncooked and processed mRNA-Seq information have been deposited in GEO (GSE236037).
Funding: This work was supported by NIGMS grant T32GM007092 to R.N.D., NCCIH grant F31AT012138 to R.N.D., and NIGMS grant R35GM137985 to R.H.D. The funders had no function in examine design, information assortment and evaluation, determination to publish, or preparation of the manuscript.
Competing pursuits: The authors have declared that no competing pursuits exist.
Introduction
Because the discovery of antibiotics, people have been efficiently eliminating microbes to remedy infections and sterilize our environments, however this nonspecific method to eradicate pathogenic microbes has additionally made it more and more evident simply how a lot we depend on interactions with commensal microbes to stay wholesome. Antibiotic use, western diets, a sedentary life-style, and plenty of illness states can set off dysbiosis, or a discount in microbial variety, which has been linked to metabolic syndromes, persistent irritation, and psychological well being problems [1–3]. For instance, C. difficile colitis can come up from antibiotic use and a subsequent lack of microbial variety within the intestine, leading to extreme gastrointestinal signs and probably dying [4]. Consumption of probiotics, or reside microbes related to well being advantages, can promote, or keep, a wholesome intestine microbiome whereas supplying the host with essential microbially-derived metabolites [5–7]. Understanding the molecular mechanisms underlying the host response to microbes, notably probiotics [8], is crucial for his or her incorporation into complementary well being care approaches.
Kombucha tea (KT) is a semi-sweet, fermented beverage that’s broadly consumed as a practical meals (i.e., offering well being advantages past dietary worth) and comprises probiotic microbes which have been presupposed to confer well being advantages, together with reducing blood stress, safety in opposition to metabolic illness, improved hepatoprotective exercise (i.e., safety in opposition to liver toxins), and anticancer results [9–13]. These probiotic microbes embody members of the Acetobacter, Lactobacillus, and Komagataeibacter genera [14,15]. Whereas a few of these well being advantages have begun to be examined in animal fashions, together with the flexibility of KT to ameliorate diabetic signs or restrict weight achieve in grownup mice [16–19], the mechanistic underpinnings of those phenotypes haven’t been rigorously investigated. Furthermore, the interactions between the microbes in Kombucha Tea, which embody each bacterial and yeast species, and the host stay utterly unexplored. As a result of this beverage comprises reside probiotic microbes and is broadly consumed below the largely unsubstantiated declare that it confers well being advantages, it’s crucial to realize mechanistic perception into the host physiological and mobile response to KT consumption.
The impression of particular person probiotic microbes, or on this case the small group of Kombucha-associated microbes, on human physiology is tough to deconvolute as people devour a fancy weight-reduction plan, have trillions of microbes colonizing their intestine, and mechanistic investigation of host-microbe interactions just isn’t possible in human topics. Subsequently, use of animal fashions is important to analyze how probiotic consumption influences host physiological processes. Caenorhabditis elegans has been broadly used to analyze mechanisms of metabolic regulation and the way nutrient sensing pathways govern organismal homeostasis [20,21]. C. elegans can also be an rising mannequin for finding out the impression of the intestine microbiome on host physiology [22,23]. Axenic preparation of C. elegans cultures renders these bacterivore animals microbe-free on the onset of life, permitting for full experimental management over which microbes are consumed throughout their lifetime (i.e., animals are germ-free earlier than encountering their microbial meals supply). Moreover, microbes that escape mechanical disruption throughout feeding can robustly colonize the intestinal lumen [22–24]. Thus, the straightforward digestive tract of C. elegans is successfully colonized by micro organism which can be offered as a meals supply, making it an excellent system to interrogate the host metabolic response to consumption of particular microbes. Certainly, earlier research have efficiently used C. elegans to analyze how particular person species of microbes, together with probiotics, can elicit physiological modifications by rewiring conserved genetic pathways [25–31].
Right here, we use C. elegans to analyze whether or not intestinal colonization with Kombucha-associated microbial species (KT microbes or KTMs) rewires host metabolism. We developed a reproducible methodology to tradition animals on lawns of KT microbes consisting of microbes present in all business and homebrewed KTs (i.e., micro organism from the Acetobacter and Komagataeibacter genera and a yeast species). We discovered that animals feeding advert libitum on KT microbes accumulate considerably much less fats than animals consuming both an E. coli weight-reduction plan, any of the person three KT-associated microbial species, or a easy non-fermenting combine of those three species. Moreover, our information counsel that KT consumption reduces fats storage by modulating host lipid metabolism pathways slightly than limiting caloric consumption. To realize perception into the mechanisms that underlie this discount in lipid ranges, we carried out a transcriptomic evaluation of KT microbe-fed animals, which revealed {that a} class of lysosomal lipases that perform in lipophagy was up-regulated and {that a} essential enzyme in triglyceride synthesis was down-regulated in response to KT microbes. Our outcomes counsel that Kombucha Tea consumption could alter lipid droplet dynamics by selling their degradation through lipophagy, whereas concurrently limiting lipid droplet growth by way of down-regulation of triglyceride synthesis. This investigation lays essential groundwork to deconvolute the molecular mechanisms which will underlie the purported well being advantages of KT utilizing a genetically tractable animal mannequin.
Outcomes
Rearing C. elegans on a garden of KT microbes leads to reproducible colonization of the intestine
Small batch brewing of KT is a serial fermentation course of during which the microbially-generated biofilm and a small quantity of absolutely fermented liquid tradition are transferred to a recent preparation of sucrose media, which then ferments for at the very least every week previous to consumption. This conventional methodology of brewing KT leads to a dynamic microbial group and pH shift over the course of fermentation (pH decreases from 7 to ~4). Contamination by environmental microbes is proscribed since these species are outcompeted by the core KT microbes (KTMs) because the pH drops [13,32–34]. Moreover, development of the protecting pellicular biofilm, colloquially known as a SCOBY (symbiotic tradition of micro organism and yeast), by the KTMs reduces outdoors contamination [35]. To analyze the physiological and metabolic results of Kombucha Tea consumption utilizing a genetic mannequin system, we first sought to determine a reproducible methodology to ship KTMs to C. elegans animals through feeding on our commonplace agar-based nematode development media (NGM), which don’t comprise any antibiotics or antifungals. We discovered that seeding KTMs which can be actively rising in a KT homebrew onto NGM plates is enough to generate a garden of microbes that expands in inhabitants and produces a biofilm over the course of 4 days (S1A and S1B Fig).
To realize a greater understanding of the microbial group dynamics in our KT tradition and to evaluate our skill to recapitulate the KT microbial group on NGM plates, we carried out 16S rDNA sequencing of the fermenting KT tradition and the KTMs washed from NGM plates remoted from three consultant brew cycles (S1 Table). After six days of fermentation, the microbial communities within the tradition and on NGM plates have been related and have been dominated by the anticipated set of Kombucha-associated microbes (i.e., Acetobacter and Komagataeibacter species), that are important elements of all business or homebrewed KTs (Figs 1A, 1B and S1C–S1E) [36]. Notably, the KT tradition microbial group remained related by way of day 12 of fermentation; nevertheless, the group on NGM plates was now not dominated by the anticipated KTMs at day 12, which can be as a result of growth of environmental microbes (Fig 1A and 1B). Thus, we solely used NGM plates between days 4–8 after KTM seeding for our subsequent experiments. Establishing this methodology to reproducibly tradition the KT microbial group on NGM plates was important to leveraging C. elegans as a mannequin to review the host response to KT consumption.
Fig 1. Consumption of Kombucha-associated microbes (KTMs) doesn’t impression feeding conduct and leads to strong intestine colonization of C. elegans animals.
(A) 16S rDNA sequencing of historically cultured KTMs, the biofilm of an actively fermenting Kombucha Tea, or KTMs grown on NGM agar plates. The frequency of recognized KTMs (inexperienced) is plotted relative to environmental microbial contaminants (grey) for 3 organic replicates throughout twelve days (d1-d12). (B) A Principal Part Evaluation of the weighted unifrac beta variety derived from the 16S rDNA sequencing revealed similarity between d5 plates and the d6 tradition, however divergence of the d12 plates. (C) Pictures (scale bar, 500 μm) and (D) quantification (imply ± SEM) of day 1 grownup animals on lawns of the indicated microbes (72 hr timepoint in contrast, ***, P<0.001, ns, not vital, one-way ANOVA). (E) Measurements of pumping charges for day 1 adults consuming every microbial meals supply (imply ± SD, ns, not vital, one-way ANOVA). (F) Quantification of the microbial CFUs from animals consuming every weight-reduction plan reveals KTMs colonize the intestine at increased ranges in comparison with the management diets (imply ± SEM, ****, P<0.0001, one-way ANOVA, three organic replicates, 10 animals per replicate). (G) Consultant scanning electron microscopy pictures of KTMs within the intestinal lumen (black arrows level to the intestinal microvilli and inexperienced arrow heads point out intact KTMs; left scale bar, 2 μm; proper scale bars, 1 μm). Expanded information for panel A could be present in S1 Table and the uncooked information underlying panels D, E, and F could be present in S1 Data.
Utilizing this standardized methodology of KTM culturing, we subsequent sought to find out if populations of C. elegans may very well be reared on a weight-reduction plan solely consisting of KTMs. Provided that KTMs are a mixture of microbial and yeast species, we first performed an avoidance assay to evaluate whether or not C. elegans animals would stay on or flee from the garden, which is a typical response to a pathogen [37–40]. Importantly, animals remained on the KTM microbial garden all through improvement and into maturity at ranges much like the E. coli controls (Fig 1C and 1D), indicating that C. elegans animals could be efficiently reared on KT microbes. These comparisons, in addition to all our subsequent characterizations of the KTMs, have been performed together with two commonplace laboratory E. coli diets (OP50 and HT115 [41]) and a pressure of the bacterium Acetobacter tropicalis that we remoted from our KT tradition. A. tropicalis is a serious constituent of all KTs and produces vitamin B12 amongst different bioactive molecules present in KTs [14,15,34,35,42–45]. OP50 and HT115 E. coli strains modulate C. elegans physiology in another way, which could be partially attributed to variations in vitamin B12 ranges [28,46–48]. Apparently, when offered with a selection of diets animals didn’t choose the KTM garden (S2A and S2B Fig). This conduct was constant throughout totally different C. elegans wild isolates and different Caenorhabditis species (S2C–S2I Fig), suggesting that these animals are both interested in the E. coli or are repelled by a part of the KTM tradition. Although animals appear to want different meals sources, animals provided solely KTMs don’t flee the garden, demonstrating that C. elegans could be reliably reared on a KTM-exclusive weight-reduction plan utilizing commonplace advert libitum feeding strategies.
To check whether or not KTMs altered feeding conduct, which could lead to diminished caloric consumption, we measured pumping charges (i.e., the speed at which animals’ pharyngeal muscle contracts to consumption meals) of particular person animals consuming KTMs or management meals sources. We discovered no vital distinction in pumping charges of animals consuming KTMs in comparison with any of the opposite meals sources (Fig 1E), suggesting feeding conduct just isn’t altered on KTM lawns. Lastly, we assessed whether or not KTMs colonize the intestinal lumen of C. elegans animals, as can be predicted for these probiotic microbes within the human gastrointestinal tract. After rearing animals on totally different diets, we eliminated floor microbes, extracted the intestinal microbes, and quantified the colony forming items (CFUs) current. Animals consuming KTMs contained at the very least 5 instances extra CFUs than animals consuming another weight-reduction plan, indicating that KTMs robustly colonize the C. elegans intestine (Fig 1F). To additional examine this intestinal colonization, we used scanning electron microscopy to picture the gut of animals consuming KTMs and located intact microbial cells current within the intestinal lumen (Fig 1G). Collectively, these outcomes display that C. elegans animals could be efficiently reared on a KTM-exclusive weight-reduction plan, which intently mirrors the microbial group discovered within the KT tradition, leading to strong KTM colonization of the intestine.
Animals consuming Kombucha microbes exhibit diminished fats accumulation
Dietary elements, together with these produced by probiotic microbes, can play a considerable function in modulating host metabolism, together with lipid storage and lipolysis [49–51]. Constantly, C. elegans metabolism is remarkably delicate to variations in microbial diets, as even extremely related strains of E. coli promote markedly totally different ranges of fats content material [28,29,41]. Given the purported metabolic advantages of KT in people, together with decreased danger of weight problems [9–13], we reasoned that consumption of KTMs could impression lipid ranges in C. elegans. Nearly all of fats in C. elegans animals is saved in intestinal epithelial cells inside lipid droplets within the type of triglycerides (TAGs), with smaller lipid deposits discovered within the hypodermis and germline [52]. Utilizing the well-established lipophilic dyes Oil Crimson O and Nile Crimson, which each stain impartial lipids, we examined the fats content material of animals consuming KTMs and management microbes [52,53]. Animals consuming KT microbes gathered considerably much less fats than animals consuming different meals sources, together with A. tropicalis, which is especially noteworthy provided that A. tropicalis is probably the most ample microbial species in KT (Fig 2A–2D). These tendencies continued throughout and after the reproductive interval (S3A Fig), suggesting that KTMs limit host lipid accumulation all through replica and through the getting old course of. Importantly, the KTM-fed animals efficiently commit a major proportion of their somatic fats shops to the germline and growing embryos at maturity (Fig 2C), suggesting that reproductive packages aren’t impaired regardless of the general discount in lipid ranges. The lower in Oil Crimson O and Nile Crimson staining means that animals consuming KTMs could have diminished TAG ranges in comparison with animals on management diets. Subsequently, we used a biochemical assay to quantify the full quantity of TAGs in populations of animals fed every weight-reduction plan [54,55]. In step with our earlier observations, animals consuming KTMs had an ~85% or ~90% lower in TAG ranges in comparison with animals consuming E. coli OP50 or A. tropicalis, respectively (Fig 2E). Collectively, these information clearly display that animals consuming KT microbes accumulate much less fats than E. coli-fed animals and that probably the most ample microbe in KT, A. tropicalis, just isn’t enough to recapitulate this phenotype. This discovering is especially related to human well being, as KT consumption has been proven to limit weight achieve and alleviate diabetic signs to the same diploma as metformin in rodent fashions [16–19].
Fig 2. KTMs limit lipid accumulation within the host.
(A) Consultant pictures (scale bar, 500 μm) and (B) quantification (imply ± SD, ****, P<0.0001, one-way ANOVA) of day 1 adults stained with Oil Crimson O. (C) Consultant fluorescence pictures (scale bar, 500 μm) and (D) quantification (imply ± SD, ****, P<0.0001, one-way ANOVA) of day 1 adults stained with Nile Crimson. (E) Biochemical quantification of the triglycerides (TAGs per animal) in animals consuming every meals supply (imply ± SEM, ***, P<0.001, *, P<0.05, ns, not vital, one-way ANOVA). (F) Consultant fluorescence pictures of DHS-3::GFP (dhs, dehydrogenase, short chain) at intestinal lipid droplets in animals consuming the indicated microbial diets (scale bar, 5 μm). (G) Lipid droplet dimension measurements with every datapoint representing the typical intestinal lipid droplet diameter for a single animal (imply ± SD, ****, P<0.0001, one-way ANOVA). (H) Lipid droplet density measurements with every datapoint representing the variety of lipid droplets per μm2 for a single animal (imply ± SD, ****, P<0.0001, *, P<0.05, ns, not vital, one-way ANOVA). Uncooked information underlying panels B, D, E, G, and H could be present in S2 Data.
Provided that the main web site of lipid storage in C. elegans is in intestinal lipid droplets (LD), we hypothesized that LD dimension or abundance could also be impacted within the gut of KTM-fed animals. Making the most of a transgenic pressure that expresses the LD-residing DHS-3::GFP protein (dhs, dehydrogenase, short chain), we measured LD abundance and dimension in intestinal cells of animals fed every weight-reduction plan. Each lipid droplet dimension and abundance have been dramatically diminished in animals consuming KTMs relative to E. coli- or A. tropicalis-fed animals (Figs 2F–2H and S3B). Collectively, these outcomes counsel that regulation of lipid droplet synthesis or stability could account for the diminished lipid accumulation that we noticed in KTM-fed animals.
KTM consumption accelerates development charges and doesn’t considerably alter fecundity
Totally different microbial diets can have a profound impression on C. elegans development fee and fecundity [29,30]. A KTM weight-reduction plan may limit developmental fee or alter reproductive packages. Furthermore, diminished nutrient absorption stemming from a KTM weight-reduction plan may lead to caloric restriction and diminished lipid accumulation. Certainly, genetic or dietary fashions of caloric restriction trigger animals to develop extra slowly, to build up much less intestinal fats, and to have a delayed reproductive interval that finally leads to much less progeny manufacturing [28,56–58].
Subsequently, we sought to find out whether or not animals consuming KTMs exhibit slower developmental charges and smaller brood sizes than animals consuming both an E. coli or A. tropicalis weight-reduction plan. To analyze variations in developmental fee, we employed a transgenic pressure expressing a GFP-PEST protein below the management of the mlt-10 promoter (Pmlt-10::GFP-PEST), which is particularly expressed throughout every of the 4 molt phases, leading to 4 peaks of GFP fluorescence all through improvement (Fig 3A). The PEST amino acid sequence ensures fast GFP turnover by proteolytic degradation and permits for exact temporal analyses. Animals consuming KTMs molt at the same, if not an accelerated fee relative to animals on the management meals sources (Fig 3A–3C), clearly indicating that KTM consumption doesn’t lower developmental fee. To realize a extra complete view of animal improvement throughout KTM consumption, we carried out mRNA sequencing (mRNA-Seq) of grownup animals consuming E. coli, A. tropicalis, or KTMs. Upon inspection of two,229 genes beforehand related to C. elegans improvement [29], we noticed only a few gene expression variations between KTM-fed animals and people fed management diets (Fig 3D–3F), suggesting that the KTM-fed inhabitants reaches maturity synchronously. Collectively, these outcomes counsel that animals consuming KT microbes exhibit wild-type improvement.
Fig 3. Developmental timing is accelerated and reproductive output is just modestly diminished throughout KTM feeding, suggesting that caloric consumption just isn’t restricted by KTM consumption.
(A-C) Profiles of Pmlt-10::GFP-PEST expression all through improvement after dropping synchronized L1s on the indicated microbes. The reporter is expressed solely through the larval molts (proven in grey in A). A single consultant experiment is displayed in panels A-C. (D-F) Scatter plots evaluating the expression of two,229 developmental genes as decided by mRNA-Seq (RPKM, reads per kilobase of transcript per million mapped reads). A linear regression evaluation and the corresponding R2 worth is reported for every comparability. (G) The frequency (imply ± SEM) of wild-type N2 and eat-2(ad465) people on the indicated developmental phases after 48 hours of development on advert libitum KTM, advert libitum E. coli, or caloric restriction E. coli (108 or 109 CFUs/mL) plates (****, P<0.0001, chi-squared take a look at). (H) Brood sizes of wild-type animals reared on the totally different diets (imply ± SD, ***, P<0.001, *, P<0.05, one-way ANOVA). (I) A plot of progeny manufacturing for every day through the reproductive interval demonstrating that KTM-fed animals exhibit the same egg laying fee in comparison with E. coli OP50-fed animals. (J) Normalized vit-2 gene expression values (RPKM, reads per kilobase of transcript per million mapped reads; imply ± SEM, *, P<0.05, T-test) and (Okay) quantification of VIT-2::GFP fluorescence in early embryos (imply ± SD, ***, P<0.001, T-test) from animals consuming an E. coli OP50 or KTM weight-reduction plan. (L-N) Scatter plots and a linear regression evaluation (R2 worth reported) evaluating the expression of two,367 replica genes as decided by mRNA-Seq. Uncooked information underlying panels A-N could be present in S3 Data.
Caloric restriction has a profound impression on C. elegans physiology, together with diminished developmental fee [58]. The eat-2 mutant is a genetic mannequin of caloric restriction, as lack of eat-2 leads to impaired pharyngeal pumping and diminished nutrient consumption [58]. Decreasing nutrient availability (i.e., E. coli OP50 lawns with concentrations ≤ 109 CFU/ml) gives a second efficient methodology of caloric restriction [59]. Subsequently, to additional consider whether or not animals consuming KTMs are calorically restricted (CR), we performed developmental fee assays with wild-type and eat-2 mutant animals consuming advert libitum E. coli lawns, CR E. coli lawns (108–109 CFU/ml), or our commonplace advert libitum lawns of KTMs. This evaluation revealed that each wild-type and eat-2 animals exhibited accelerated developmental charges when consuming KTMs in comparison with the E. coli OP50 weight-reduction plan (Fig 3G). Importantly, eat-2 animals confirmed diminished developmental charges on CR E. coli lawns relative to advert libitum E. coli lawns, indicating that the results of the eat-2 mutation are additional enhanced by extra caloric restriction; nevertheless, KTM-feeding partially suppressed the developmental defects of the eat-2 mutation (Fig 3G). These information display that KTM consumption doesn’t mimic the results of restricted caloric consumption.
Reproductive output (i.e., brood dimension) of C. elegans is modulated by weight-reduction plan, presumably by way of the tuning of reproductive packages on the transcriptional stage [29,60]. Subsequently, we measured the brood sizes of animals consuming KTMs and management diets, discovering that the typical brood dimension of animals consuming KTMs was solely modestly decrease than these consuming E. coli OP50 (Figs 3H and S4; 295 versus 240, P<0.05). Moreover, we discovered that animals consuming KTMs lay their eggs at the same fee relative to E. coli-fed animals (Figs 3I and S4). In distinction, calorically restricted animals, similar to eat-2 mutants, have prolonged egg laying durations, as much as 12 days, and have considerably diminished brood sizes, with eat-2 mutants averaging 100–175 progeny [28,57]. Thus, the ~20% discount in fertility for KTM-fed animals is inconsistent with the extra extreme discount in brood dimension of CR animals. It may, nevertheless, be according to impaired maternal provisioning of lipid-rich yolk to oocytes from intestinal fats shops, a course of termed vitellogenesis. Thus, we subsequent examined the mRNA ranges of vit-2, which encodes a vitellogenin protein that mediates the intestine-to-oocyte transport of lipids, discovering that vit-2 ranges are elevated in animals fed a KTM weight-reduction plan in comparison with E. coli-fed animals (Fig 3J). Constantly, vitellogenin protein ranges, which we measured in early embryos (previous to the 44-cell stage) utilizing an endogenously tagged VIT-2::GFP protein, have been additionally elevated in KTM-fed animals (Fig 3K), additional substantiating that KTM consumption doesn’t impair maternal lipid provisioning. Lastly, we inspected the expression of two,367 genes implicated in replica [29], discovering that KTM consumption doesn’t broadly alter reproductive gene expression packages relative to manage diets (Fig 3L–3N). Collectively, our outcomes point out that reproductive packages aren’t dramatically altered in animals consuming KTMs. This discovering, together with the statement that animals consuming KTMs exhibit wild-type developmental charges, is according to the rivalry that caloric consumption just isn’t impaired throughout KTM consumption and substantiates C. elegans as mannequin to analyze the impression of Kombucha-associated microbes on host metabolic pathways.
Lengthy-term KTM co-culturing is required to rework host metabolism
Sequencing of commercially obtainable and non-commercial-small-batch KTs has revealed {that a} reproducible set of core microbes are present in KT [14,15,34,36,61]. These embody micro organism within the Acetobacter, Komagataeibacter, Gluconacetobacter, Gluconobacter, and Lactobacillus genera, in addition to yeast within the Brettanomyces, Zygosaccharomyces, Candida, Dekkera, Lachancea, and Schizosaccharomyces genera. Moreover, Huang and colleagues not too long ago established a minimal KT microbiome that recapitulates key features of historically brewed KT based mostly on the factors that this microbial combine may (1) coexist as in KT, (2) produce a KT-like biochemical composition, and (3) construct a pellicle. Intriguingly, whatever the ratio of bacteria-to-yeast on the onset of fermentation, by day 6 this ratio stabilizes with comparatively equal illustration of every species whatever the focus of the microbial species mixed [36].
Provided that KTMs robustly colonize the C. elegans intestine and that feeding animals the recognized dominant KT microbe, A. tropicalis, fails to recapitulate the host response to KTMs, we sought to establish extra microbes from our KT tradition that may colonize the gut of animals after KTM consumption. Isolation of those species would facilitate the creation of a KTM tradition consisting of a minimal microbiome core, which can be enough to confer metabolic phenotypes in C. elegans animals. Our preliminary extraction of intestinal microbes from KTM-fed animals (Fig 1F) remoted a bacterial species, Acetobacter tropicalis, and a yeast species, of both the Zygosaccharomyces or Brettanomyces genera, which we recognized by 16S and 18S rDNA sequencing, respectively (S5A and S5B Fig). Whereas these microbes signify two of the species generally present in KT, they don’t represent a minimal KT tradition as a result of they can not type a pellicle [36]. Subsequently, we sought to isolate the cellulose-producing species from our tradition that’s chargeable for constructing the pellicle. We eliminated a small piece of the biofilm from our KT tradition and used a mixture of enzymatic digestion (driselase) and mechanical disruption (sonication) to dislodge the micro organism from the cellulose matrix. The cellulose-producing bacterium was remoted on mannitol agar plates containing Calcofluor White, which stains cellulose and chitin and fluoresces below ultraviolet gentle [62–64]. This technique resulted within the isolation of a further KT microbe that was recognized as a member of the Komagataeibacter genus by 16S rDNA sequencing (S5C Fig).
To realize extra genetic details about our individually remoted KT microbes, we carried out brief learn complete genome sequencing of the genomic DNA. Subsequently, the Kraken algorithm [65], a bioinformatic pipeline for metagenomic classification, was used to find out the approximate taxonomy of our particular person KT microbes. Based mostly on these taxonomical classifications, in addition to a compiled listing of beforehand printed KT-associated microbes, we aligned our KT microbe sequences to a number of obtainable reference genomes to realize species stage info (Fig 4A and S2 Table). This technique recognized our KT microbes as Komagataeibacter rhaeticus (98.76.% alignment fee to pressure ENS_9a1a), Acetobacter tropicalis (87.55% alignment fee to pressure NBRC101654), and Zygosaccharomyces bailli (86.88% alignment fee to pressure CLIB213) [66].
Fig 4. The host lipid depletion response is restricted to actively fermenting KTM cultures and isn’t conferred by particular person microbes or a non-fermenting combination.
(A) Purification and complete genome sequencing of the microbes from our Kombucha tradition resulted in species-level identification of the core KTMs. (B) A schematic of the preparation and supply strategies for the three KT-derived diets (orange, A. tropicalis; tan, Okay. rhaeticus; grey, Z. bailii; d, days). Candy tea media consists of a black and inexperienced tea combine with 5% cane sugar that has been filter sterilized. KTM cultures are maintained through serial fermentation, whereas the KTM-M and KTM-FM are de novo cultures. (C) Quantification of Oil Crimson O staining of day 1 adults fed the indicated diets (imply ± SD, ****, P<0.0001, one-way ANOVA). A Z. bailii weight-reduction plan doesn’t assist animal improvement. (D) Consultant fluorescence pictures (scale bars, 5 μm) and (E) quantification of lipid droplet diameter (imply ± SD, n = 10 people, ****, P<0.0001, unpaired T-test) within the intestines of DHS-3::GFP transgenic animals. The KTM lipid droplet picture and dimension measurements proven in (D-E) are additionally displayed in Fig 2F and 2G, as all these samples have been processed in parallel. (F-G) Quantification of Oil Crimson O staining of day 1 adults fed the indicated KT diets (imply ± SD, ****, P<0.0001, ns, not vital, one-way ANOVA). (F) Animals consuming KTM-FM have related lipid ranges as KTM-fed animals, whereas (G) KTM-M have to be co-cultured for at the very least 14 days to limit host lipid accumulation. (H) The experimental design to check whether or not the KTM tradition supernatant is required for host lipid depletion. (I) Quantification of Oil Crimson O staining of day 1 adults following a weight-reduction plan of KTMs, KTMs washed extensively with 5% sucrose, or KTM-M (imply ± SD, ****, P<0.0001, ns, not vital, one-way ANOVA). (J) Quantification of Oil Crimson O staining of day 1 grownup animals consuming E. coli, KTMs, and Z. bailii with or with out lifeless E. coli supplementation as an inert nutrient supply (imply ± SD, ****, P<0.0001, ns, not vital, one-way ANOVA). (Okay) Consultant DIC and fluorescence pictures of the gut after feeding the indicated diets supplemented with C1-BODIPY-C12 (stars point out the intestinal lumen and arrowheads point out the intestinal epithelial cells; scale bars, 10 μm). Expanded information for panel A could be present in S2 Table and the uncooked information underlying panels C, E, F, G, I, and J could be present in S4 Data.
The isolation and identification of the dominant KT microbes from our tradition allowed us to additional examine how consumption of the person KT microbes, or mixtures of microbes, alter C. elegans lipid metabolism (Fig 4B). We initially fed the person KT microbes to animals, discovering that diets of A. tropicalis or Okay. rhaeticus promoted lipid accumulation at ranges much like E. coli-fed animals, whereas a weight-reduction plan of the yeast species Z. bailli didn’t assist animal improvement (Figs 4C and S6A–S6D). Surprisingly, rising the focus of KTMs current within the garden fivefold (5x KTM) additional diminished lipid ranges in comparison with our commonplace KTM garden (S6A–S6C Fig), indicating that a rise within the microbial focus, which seemingly leads to extra obtainable vitamins, doesn’t enhance host lipid accumulation.
We then hypothesized {that a} combination of Okay. rhaeticus, Z. bailii, and A. tropicalis would signify the minimal core of KT microbes, which when co-cultured would ferment sucrose, construct a pellicle, and produce a biochemical composition much like Kombucha tea. Subsequently, we mixed the three KT microbe isolates in filter sterilized KT media (i.e., steeped black and inexperienced tea containing ~5% sucrose) and allowed them to ferment for a number of weeks till a pellicle was fashioned. We check with this de novo KT as KTM-Fermented Combine or “KTM-FM” (Fig 4B). To evaluate the flexibility of our KTM-FM tradition to change host lipid metabolism, we carried out Oil Crimson O staining on animals consuming KTM-FM or a easy non-fermenting mixture of the three KT microbes (known as KTM-Combine, abbreviated “KTM-M”, Fig 4B). Intriguingly, we discovered that the KTM-M weight-reduction plan didn’t scale back lipid accumulation, lipid droplet dimension, or lipid droplet abundance within the host (Figs 4C–4E and S6E); nevertheless, consumption of KTM-FM diminished lipid ranges to the same diploma as the unique KTM weight-reduction plan (Fig 4F). Importantly, neither the KTM-M nor the KTM-FM weight-reduction plan impaired developmental or behavioral packages (S6F–S6I Fig). These outcomes counsel that long-term fermentation is important for the host metabolic response to KT consumption. Moreover, the statement {that a} non-fermented mixture of KT microbes fails to limit host lipid accumulation additional helps our conclusion that KTM-fed animals aren’t calorically restricted.
To higher perceive the significance of fermentation time, we fed animals KTM-FM cultures after totally different lengths of fermentation and measured lipid ranges utilizing Oil Crimson O staining. Animals that have been fed KTM-FMs with fermentation instances lower than one week had elevated lipid ranges; nevertheless, KTM-FMs fermented for two weeks or extra promoted the depletion of host lipids (Fig 4G). Moreover, elimination of the absolutely fermented KTM supernatant adopted by repeated washes of the KTMs with a 5% sucrose answer previous to seeding the NGM plates didn’t alter host lipid accumulation in response to KTMs (Fig 4H and 4I), suggesting that the small molecules within the inexperienced and black tea could also be dispensable for conferring host lipid phenotypes. This end result, nevertheless, doesn’t rule out the likelihood that the tea-derived metabolites are essential for establishing the symbiotic Kombucha tradition. Collectively, these information argue that KT microbes should type a longtime group to reconfigure host lipid metabolism pathways.
Though we noticed colonization of C. elegans intestine with A. tropicalis (Fig 1F), it’s unclear whether or not the opposite KT isolates, Okay. rhaeticus or Z. bailii, are ingested by animals. To visualise these microbes within the intestine of reside animals, we stained animals fed E. coli, Okay. rhaeticus, or KTMs with Calcofluor White, which selectively stains the polysaccharides in chitin and cellulose. We noticed the cellulose-producing microbe Okay. rhaeticus, which helps animal improvement, within the intestinal lumen (S6J Fig), suggesting that Okay. rhaeticus micro organism can colonize the intestine whereas synthesizing cellulose. Surprisingly, we additionally noticed chitin-producing yeast cells within the intestinal lumen, indicating that Z. bailii could be consumed by animals on the grownup stage (S6K and S6L Fig). Importantly, these outcomes are according to the presumption that each one three of KT microbes (Z. bailii, Okay. rhaeticus, and A. tropicalis) remoted from our KT tradition can escape mechanical disruption within the pharynx and could be discovered within the intestinal lumen of C. elegans. To additional assess the flexibility of the KT microbes to colonize the intestine, we quantified the intestinal lumen dimension of animals reared on the E. coli OP50, KTM, and KTM-M diets. Utilizing animals expressing ERM-1::GFP, which localizes to the apical floor of intestinal cells and facilitates luminal measurements, we discovered that people consuming a KTM weight-reduction plan had an elevated intestinal lumen diameter in comparison with animals consuming E. coli OP50 however not the KTM-M weight-reduction plan, suggesting that any weight-reduction plan containing KT microbes stimulates intestinal bloating (S6M Fig).
The presence of Z. bailii within the intestine, which can contribute to intestinal bloating, raised the presumably that the yeast (or the opposite KT microbes) could limit nutrient absorption, leading to caloric restriction. Subsequently, we supplemented KTM and Z. bailii diets with warmth killed E. coli OP50 and assessed lipid ranges utilizing Oil Crimson O staining. Whereas E. coli supplementation had little impression on the flexibility of the KTM weight-reduction plan to limit host lipid accumulation, supplementation to a Z. bailii weight-reduction plan supported animal improvement and promoted lipid accumulation regardless of the presence of the yeast (Fig 4J). Subsequent, we assessed nutrient absorption in KTM-fed animals by supplementing the KTM garden with the very important dye C1-BODIPY-C12, which is consumed with the meals and might readily cross the intestinal apical membrane. Following a three-hour pulse of BODIPY, animals consuming E. coli OP50, KTM, and KTM-M all have detectable ranges of BODIPY of their intestinal epithelial cells (Figs 4K and S7). Since animals consuming KTMs have only a few, small lipid droplets the BODIPY staining was diffusely distributed all through the intestinal cells; nevertheless, within the E. coli OP50 and KTM-M-fed animals the dye localized to intestinal lipid droplets and lysosome-related organelles [53,67]. Collectively, these findings are according to our earlier observations that animals consuming KT microbes, both individually or together, aren’t impaired of their skill to soak up vitamins; however slightly, the KTM weight-reduction plan seemingly restricts lipid accumulation by modulating host metabolic pathways.
An intestinally pushed metabolic response to KTM consumption
KTM-fed animals bear regular improvement and present no detectable impairment in nutrient absorption, but retailer markedly much less lipids than management animals, together with these fed the KTM-M weight-reduction plan. Whereas our transcriptomics urged that the expression of genes concerned in improvement or replica have been constant throughout diets, we hypothesized that the expression of metabolic genes could also be particularly altered by KTM consumption. Subsequently, we carried out extra analyses of our mRNA-Seq information derived from day one grownup animals consuming both KTM, KTM-M, A. tropicalis, or the 2 E. coli diets to analyze if particular metabolic packages are altered by these diets. A PCA evaluation revealed that the transcriptomes of animals fed the identical weight-reduction plan cluster, with the transcriptomes of animals fed KTM, KTM-M, and A. tropicalis distinctly clustering other than the transcriptomes of the E. coli-fed animals (Fig 5A), indicating that there’s at the very least some commonality between the transcriptional responses of animals consuming any of the KT-associated diets that’s totally different from E. coli diets. To eradicate the opportunity of transgenerational epigenetic results of the KTM weight-reduction plan, we in contrast the transcriptomes of animals fed KTMs for one technology to animals subjected to 5 generations of the KTM weight-reduction plan, discovering no vital distinction between these transcriptomes (Figs 5A and S8A).
Fig 5. Host lipid metabolism gene expression is modulated by KTM consumption.
(A) A Principal Part Evaluation of the normalized mRNA-Seq information for the indicated diets (1G, KTM feeding for one technology; 5G, KTM feeding for 5 consecutive generations previous to assortment). (B) The overlap of the differentially expressed genes, decided relative to E. coli OP50, between every meals supply. (C) A Gene Ontology enrichment evaluation carried out on the 295 genes which can be uniquely differentially expressed in animals consuming KTMs. (D) Enrichment for differential expression of genes which can be expressed within the indicated tissues (noticed/anticipated, hypergeometric P values reported). Values <1 point out that genes expressed within the indicated tissue kind have a tendency to not be differentially expressed (under-enriched), whereas values >1 point out tissues the place differential expression is extra frequent than anticipated by random likelihood (over-enriched). (E) A scatter plot and linear regression (R2 = 0.9556) of the RPMK values for five,676 metabolism-related genes (the genes of curiosity are indicated with arrows). (F) A schematic and gene expression heatmap (Log2 fold change values relative to E. coli OP50) for the indicated lipid metabolism genes for every weight-reduction plan (bins from left to proper: KTM, A. tropicalis, KTM-M, E. coli HT115). Uncooked information underlying panels A-F could be present in S5 Data and S4 Desk.
Deeper investigation of our mRNA-Seq information revealed that every KT-associated weight-reduction plan did certainly lead to some stage of differential gene expression in comparison with the E. coli OP50 weight-reduction plan (A. tropicalis, 3,952 genes; KTM, 1,237 genes; KTM-M, 1,007 genes; 1% FDR; Figs 5B and S8B–S8F). Intriguingly, 295 genes have been distinctive to the KTM weight-reduction plan (Fig 5B). Altered expression of those KTM-unique genes may very well be a serious driver of the diminished lipid ranges that we noticed particularly within the KTM-fed animals. A gene ontology (GO) enrichment evaluation [68] of the KTM-unique genes revealed an enrichment for genes annotated to have practical roles in lipid metabolism (Fig 5C). Since misexpression of core metabolic genes alters longevity and stress resistance pathways [69,70], we queried whether or not these similar genes have been additionally misexpressed in animals with diminished ranges of DAF-2 (i.e., the insulin receptor), which ends up in elevated stress resistance, improved healthspan, and prolonged lifespan [71,72]. Certainly, depletion of DAF-2 in numerous tissues [72], together with the gut, leads to transcriptional modifications which can be according to these seen in KTM-fed animals (S8G–S8I Fig). Collectively, these information counsel that consumption of fermenting KT microbes could rework host lipid metabolism and stress resilience pathways to limit fats accumulation and enhance healthspan.
In C. elegans, the gut capabilities as the first hub for nutrient absorption, lipid storage, and metabolic regulation [52]. Our transcriptome information indicated that genes concerned in lipid metabolism are modulated by KTM consumption, prompting us to analyze whether or not the host transcriptional response to KTMs happens within the gut. Utilizing beforehand established gene expression information for the main tissues, we queried whether or not every set of diet-induced differentially expressed genes have been enriched for a selected tissue [73,74]. We discovered that in response to KTM consumption there was a hanging enrichment for differential expression of intestinal genes, in addition to a depletion of neuronal and germline genes (Fig 5D). These information point out that whereas genes expressed within the gut are generally differentially expressed in animals consuming KTMs, genes expressed in different tissue varieties have a tendency to not be differentially expressed in KTM-fed animals.
To establish candidate genes that could be chargeable for the metabolic results of KTM consumption, we analyzed the expression ranges of 5,676 genes which can be annotated to perform in metabolism [29]. This revealed that a number of genes recognized to perform in lipid biology have altered expression in KTM-fed animals (Fig 5E and 5F). These included down-regulated genes that act within the β-oxidation of lipids (acdh-1, acdh-2), fatty acid desaturation (fat-5, fat-6, fat-7), or triglyceride synthesis (dgat-2), in addition to up-regulated genes that act in lipolysis (lipl-1, lipl-2, lipl-3). These information counsel that expression of particular lipid metabolism genes within the gut is modulated by KTM consumption. Constantly, intestinal expression of a GFP-based transcriptional reporter for the acdh-1 gene, which encodes a conserved acyl-CoA dehydrogenase that catabolizes brief chain fatty acids and department chained amino acids, was diminished when animals have been fed a KTM weight-reduction plan (S8J and S8K Fig). Collectively, our outcomes counsel that transcriptional regulation of metabolic genes could, at the very least partially, underlie the discount in intestinal lipids that we noticed in KTM-fed animals.
KTM consumption restricts lipid accumulation by regulating lipid droplet dynamics
Coordination of intestinal lipid shops is ruled by each transcriptional and post-translational mechanisms that dynamically alter lipid droplets in response to exterior indicators. Growth of LDs is carried out through de novo lipogenesis and the motion of acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes, which catalyze the ultimate step in TAG synthesis [75,76]. In distinction, lipases and lipophagy, a selective LD autophagy pathway, limit LD dimension and quantity, respectively, and promote lipid catabolism [77–81]. Provided that KTM-fed animals show a discount in lipid ranges and lipid droplet dimension, we reasoned that the expression of triglyceride lipases could also be induced in response to KTMs; nevertheless, we discovered that expression of the adipocyte triglyceride lipase gene (atgl-1/ATGL), which encodes a LD-associated and starvation-responsive TAG lipase [52,82], and the hormone-sensitive lipase gene (hosl-1/HSL), which encodes a hormone-responsive TAG lipase, aren’t altered by KTM feeding (Fig 6A and 6B). We then inspected the expression of the remaining lipase genes inside our mRNA-Seq information (Fig 5F), discovering that three ATGL-like lipase genes (i.e., lipl-1, lipl-2, and lipl-3) have been markedly up-regulated in KTM-fed animals relative to these consuming the E. coli or KTM-M diets (Fig 6C–6E). Apparently, lipl-1,2,3 gene expression is understood to extend upon fasting and the encoded proteins all localize to the lysosomes within the gut the place they break down LD-associated TAGs through lipophagy [83]. In step with these observations, expression of a single-copy Plipl-1::mCherry transcriptional reporter was particularly induced within the gut in response to KTMs in comparison with the opposite meals sources (Figs 6F and S9A). Up-regulation of the lipl-1,2,3 lysosomal lipase genes, in addition to the concomitant discount in TAGs, means that KTM-fed animals could expertise a fasting-like state even within the presence of enough nutrient availability.
Fig 6. KTM consumption stimulates host lipid catabolism and impairs TAG synthesis.
(A-E) Normalized gene expression values (RPKM, reads per kilobase of transcript per million mapped reads; imply ± SEM, ****, P<0.0001, *, P<0.05, ns, not vital, one-way ANOVA) for the indicated lipase genes. (F) Consultant pictures of animals expressing a Plipl-1::mCherry transcriptional reporter upon consumption of the indicated diets (white arrow heads level to the gut; scale bars, 500μm). (G) Quantification of Oil Crimson O stained intestinal lipids in day 1 grownup wild-type N2 and lipl mutant animals after consumption of the KTM-M (left group) or KTM (proper group) diets (imply ± SD, ****, P<0.0001, ***, P<0.001, **, P<0. 01, ns, not vital, one-way ANOVA). Knowledge are proven for the next mutants: lipl-1(tm1954) lipl-2(ttTi14801) in circles, lipl-1(rhd279) lipl-2(rhd282) in triangles, lipl-1(tm1954) lipl-2(ttTi14801) lipl-3(tm4498) in diamonds, and lipl-1(rhd279) lipl-2(rhd282) lipl-3(tm4498) in hexagons. (H) Consultant pictures (scale bars, 5 μm) and (I) lipid droplet density measurements (imply ± SD, ****, P<0.0001, ns, not vital, one-way ANOVA) of DHS-3::GFP-containing lipid droplets in wild-type N2 and lipl-1(tm1954) lipl-2(ttTi14801) lipl-3(tm4498) mutant animals. (J) Normalized gene expression values for the TAG synthesis gene dgat-2 (imply ± SEM, ****, P<0.0001, one-way ANOVA). (Okay) Quantification of Oil Crimson O staining of intestinal lipids in wild-type N2 and DGAT-2::GFP transgenic animals, which constitutively overexpress DGAT-2 within the gut (dgat-2 OE; imply ± SD, ****, P<0.0001, T-test). (L) A mannequin of KTM modulation of host lipid metabolism pathways displaying 1) the induction of the lysosomal lipases which can be important to lipophagy and a pair of) the down-regulation of the TAG synthesis gene dgat-2 thereby limiting lipid droplet initiation/growth. Uncooked information underlying panels A-E, G, and I-Okay could be present in S6 Data.
To evaluate whether or not lysosomal lipases are required for the host response to KT, we used Oil Crimson O staining to find out the degrees of intestinal lipids in beforehand generated lipl mutants [83,84]. We discovered that lipid ranges have been elevated in lipl-1(tm1954); lipl-2(ttTi14801) double mutants and lipl-1(tm1954); lipl-2(ttTi14801); lipl-3(tm4498) triple mutants relative to wild-type animals upon KTM consumption (Fig 6G). We additionally generated putative loss-of-function nonsense mutations within the lipl-1 and lipl-2 genes utilizing CRISPR/Cas-9, crossed these alleles to the prevailing lipl-3(tm4498) mutant [83], and carried out Oil Crimson O staining of the ensuing triple mutant. In step with our preliminary observations, simultaneous lack of lipl-1,2 or lipl-1,2,3 elevated lipid ranges in KTM-fed animals (Fig 6G). Because the LIPL-1,2,3 proteins localize to lysosomes and catabolize LD-associated TAGs, we reasoned that LD dimension or abundance could also be altered in lipl-1,2,3 mutants consuming KTMs. Subsequently, we crossed the DHS-3::GFP reporter into the lipl-1(tm1954); lipl-2(ttTi14801); lipl-3(tm4498) triple mutant and measured intestinal LDs. Triple mutant animals fed the KTM weight-reduction plan, however not the KTM-M weight-reduction plan, had extra LDs in comparison with wild-type animals; nevertheless, the LD dimension was related between wild-type and mutant animals (Figs 6H–6I and S9B), suggesting that the LIPL-1,2,3 proteins promote LD degradation, however not LD shrinking, in KTM-fed animals. Collectively, these outcomes point out that up-regulation of the lipl-1,2,3 lysosomal lipases in response to Kombucha Tea consumption partially governs the host metabolic response to KTMs and facilitates lipid catabolism.
Along with induction of lipid catabolism pathways, Kombucha-associated microbes could impair TAG accumulation or LD growth. To analyze this additional utilizing our mRNA-Seq information, we in contrast the expression ranges of genes which can be recognized to perform in LD synthesis or growth for animals fed E. coli OP50, KTMs, or the KTM-M [75,85]. Though ranges of seipin (seip-1), lipin (lpin-1), and acs-22/FATP4 (a long-chain fatty acid transporter and acyl-CoA synthetase enzyme) weren’t altered in response to KTM feeding, the dgat-2/DGAT2 gene was dramatically and particularly down-regulated upon KTM consumption (Figs 6J and S9C–S9E), suggesting that TAG synthesis could also be impaired in these animals. To check whether or not down-regulation of dgat-2 restricts lipid accumulation in KTM-fed animals, we employed a pressure that expresses dgat-2 below the management of a constitutive intestinal promoter (Pvha-6::GFP::dgat-2), which isn’t predicted to reply to KTM consumption. Certainly, constitutive expression of dgat-2 partially suppressed the KTM-dependent depletion of intestinal lipid shops (Fig 6K). Collectively, these outcomes assist a mannequin the place the concomitant down-regulation of dgat-2 and up-regulation of the lysosomal lipase genes limits TAG synthesis whereas selling LD breakdown, which collectively restricts intestinal lipid accumulation in response to Kombucha consumption (Fig 6L).
Dialogue
The primary information of Kombucha Tea consumption could be traced to historical China the place it was included into frequent medical practices [86]. Whereas its recognition has expanded all through historical past, a latest surge in worldwide consumption makes it one of the vital standard probiotic-containing fermented drinks, with its quite a few purported human well being advantages being a serious contributor to its recognition [86]. Regardless of this lengthy historical past and widespread anecdotal proof that it improves metabolic well being [9–13], little is understood about whether or not Kombucha Tea consumption alters host metabolism and, if that’s the case, by which mechanisms this may occasionally happen. To analyze Kombucha Tea’s motion in an animal mannequin system, we established a reproducible methodology to ship a weight-reduction plan of KT-associated microbes (KTM) to C. elegans although commonplace advert libitum feeding practices. Supply of KTMs by feeding helps regular C. elegans improvement and fecundity, and importantly, leads to strong KTM colonization of the intestinal lumen. Our examine is the primary to leverage a well-established animal mannequin system to elucidate the molecular mechanisms of Kombucha Tea motion within the host.
Right here, we display that animals consuming KTMs are markedly devoid of lipids relative to animals fed different microbial diets, as decided by Oil Crimson O and Nile Crimson staining, biochemical triglyceride measurements, and dimension calculations of intestinal lipid droplets. Collectively, our outcomes counsel that KTM consumption stimulates a fasting-like state in C. elegans that’s distinct from conventional fashions of caloric restriction. Certainly, there are a number of strains of proof that argue that KTM-fed animals aren’t experiencing caloric restriction, together with 1) KTM feeding helps an elevated fee of improvement for each wild-type and calorically restricted animals (i.e., eat-2 mutants), 2) KTM-fed animals are fertile (i.e., they exhibit almost regular brood sizes, reproductive lifespans, and expression of replica genes), 3) the person KT microbes (A. tropicalis, Okay. rhaeticus, and Z. bailli supplemented with lifeless E. coli), in addition to a easy combination of the three microbes (KTM-M), fail to deplete host lipid shops, and 4) supplementation of KTMs with extra vitamins, both lifeless E. coli or increased concentrations of KTMs, didn’t enhance lipid accumulation. Importantly, calorically restricted animals have extreme development and fertility defects [28,56–59], phenotypes which can be inconsistent with these produced by KTM consumption. Lastly, we discovered that host lipid utilization was maintained after washing the concentrated KT microbes with naïve, sucrose-only media previous to plating, supporting the speculation that the bioactive molecule(s) chargeable for altering host lipid metabolism are intrinsic to the KTM microbes slightly than discovered within the cell-free, fermented tea supernatant. Identification of those KTM-derived metabolites will probably be essential to realize perception into the molecular mechanisms of KT motion.
To realize a complete view of the host metabolic response to Kombucha, we carried out mRNA sequencing of animals consuming KTMs. Whereas expression of developmental or replica genes have been globally unchanged, expression of quite a few lipid metabolism genes have been particularly altered in response to KTMs, with a robust enrichment for genes recognized to perform within the gut. These embody gene merchandise that perform in varied features of lipid biology, together with β-oxidation of lipids (acdh-1 and acdh-2), fatty acid desaturation (fat-5 and fat-7), triglyceride synthesis (dgat-2), and lipolysis (lipl-1, lipl-2, and lipl-3). The stearoyl-CoA desaturase genes, particularly fat-5 and fat-7, have been down-regulated in KTM-fed animals. This discovering is notable because the C. elegans desaturases have lipid substrate preferences, and thus, differential expression of particular person fats genes may end up in alterations within the abundance of particular monounsaturated or polyunsaturated fatty acids [87]. FAT-5, which desaturates palmitic acid (16:0) to generate palmitoleic acid (16:1n-7), is transcriptionally down-regulated in KTM-fed animals, presumably leading to a lower in palmitoleic acid and enhance in palmitic acid or different unsaturated fatty acids which can be derived from palmitic acid. Particular modifications within the abundance of monounsaturated or polyunsaturated fatty acids could contribute to the fasting-like state displayed by KTM-fed animals; nevertheless, lipidomic research, paired with fatty acid supplementation experiments and genetic analyses, will probably be wanted to resolve the function of the C. elegans desaturases in mediating the host response to KTM consumption.
On this examine, we centered on three intestinal ATGL-like lipase genes lipl-1, lipl-2, and lipl-3 that have been particularly upregulated in KTM-fed animals, whereas the opposite 5 lipl genes, in addition to the lipid droplet lipase genes atgl-1 and hosl-1, remained unchanged. These findings argue that Kombucha consumption triggers a selected catabolic response to limit lipid accumulation. The lipl-1,2,3 genes encode three, seemingly redundantly appearing lysosomal lipases that perform in lipophagy-mediated break down of LD-associated TAGs [83]. Right here, we display that the lipl-1,2,3 genes are partially required for KTM-mediated lipid catabolism, suggesting that lipophagy is induced by KTM consumption. Lipophagy, which is a selective type of autophagy that targets lipid droplet TAGs to liberate free fatty acids for additional catabolism, is important for lipid homeostasis and survival in instances of low nutrient availability or throughout states of fasting. Along with these situations, homeostatic pathways can dynamically govern lipophagy induction below totally different nutrient- and stress-related situations (i.e., fed, fasted, and oxidative stress states) [80]. For instance, lipl-3 transcription could be ruled by the interaction between the DAF-16/FOXO, PHA-4/FoxA, and HLH-30/TFEB transcription components in particular contexts [80]. We suggest that KTM consumption stimulates a fasting-like state in C. elegans to advertise lipid utilization through lipophagy; nevertheless, future research will probably be wanted to dissect the exact molecular mechanisms that result in lipophagy induction in response to KTMs. It’s notable {that a} latest examine by Xu and colleagues [19] in rodents lends substantial physiological proof supporting the well being claims made concerning human KT consumption, together with safety in opposition to weight problems and Kind 2 Diabetes, that are illness states which can be generally related to impaired lipid utilization or dyslipidemia [81,88–90]. Our discovery that C. elegans animals consuming a KTM weight-reduction plan could have elevated ranges of lipophagy, and probably a broader autophagy-driven metabolic reprograming, is according to these claims and means that future research deconvoluting the host response to Kombucha consumption on the molecular stage will present perception into how Kombucha Tea could alter human metabolism.
Our mRNA-Seq information additionally revealed that dgat-2, which encodes an acyl-CoA:diacylglycerol acyltransferase (DGAT) enzyme, is dramatically down-regulated in response to KTM consumption. The DGAT enzymes catalyze the synthesis of triglycerides from diacylglycerol and a fatty acyl-CoA, leading to TAG manufacturing and the growth of lipid droplets. Constitutive over-expression of dgat-2 elevated lipid accumulation in animals consuming KTMs, suggesting that down-regulation of dgat-2 expression, and consequently diminished TAG synthesis, could also be a part of the programmed host response to KT. Notably, induction of dgat-2 in C. elegans helps the growth of LDs in response to the pathogen Stenotrophomonas maltophilia [91], suggesting that dgat-2 expression could also be dynamically regulated by nutrient sensing or innate immunity pathways to manipulate lipid storage ranges inside LDs. It’s doable that dgat-2 expression is managed by the identical signaling networks that management the expression of the LIPL-1,2,3 lysosomal lipases, which collectively limit the buildup of lipids throughout KTM feeding. This might additionally clarify why lack of lipl-1,2,3 will increase LD abundance however not dimension, as dgat-2 seemingly stays down-regulated in these animals following KTM consumption.
Just lately, it has turn into more and more evident that C. elegans provides a robust system to analyze potential human probiotic microbes to realize perception into their mechanisms of motion and to establish potential human well being advantages [23,25,26,31,92–94]. Our examine establishes a rigorous, reproducible, and broadly relevant system that leverages the genetic tractability of C. elegans to interrogate the physiological and mechanistic host response to probiotic microbes. Whereas that is an thrilling proposition, it’s crucial to notice that this work, as with different research performed utilizing C. elegans as a mannequin to analyze human-probiotic interactions, just isn’t instantly translatable to human well being outcomes and provides no medical recommendation or context for human Kombucha Tea consumption. We additionally acknowledge that the origin of this now standard fermented beverage has deep roots in historical Chinese language medical practices and was created by a tradition totally different from our personal. Subsequently, we wish to make it explicitly clear that we don’t make judgements, conclusions or claims concerning Kombucha Tea’s use in any human medical practices or its leisure consumption. Our findings do, nevertheless, provide thrilling insights into doable mechanisms of KT microbe-mediated host metabolic reprogramming and lays the inspiration for future research in mammalian mannequin methods that might deconvolute the organic underpinnings of Kombucha Tea’s potential well being advantages.
Supplies and strategies
C. elegans strains and upkeep
All Caenorhabditis strains have been maintained at 20°C on Nematode Progress Media (NGM) agar plates containing E. coli OP50 as beforehand described [95]. A full listing of the strains used on this examine are proven in S3 Table. All C. elegans strains have been well-fed for at the very least three generations earlier than use in experiments. Except in any other case acknowledged, eggs have been harvested from gravid adults reared on E. coli OP50 by bleaching and animals have been synchronized to the L1 stage by incubating the eggs in a single day at room temperature. Preparation of L1 animals by bleaching was required to stop E. coli contamination of Kombucha NGM plates within the following technology.
The Plipl-1::mCherry transgenic pressure was constructed utilizing Mos1-mediated Single Copy Insertion (mosSCI). The lipl-1 promoter (1,228 bp; chromosome V: 12,918,779–12,920,006; WS288) was amplified by PCR and fused to mCherry::unc-54 3’UTR in pCFJ151 by Gibson Meeting. The ensuing plasmid, pRD172[Plipl-1::mCherry::unc-54 3’UTR + cb-unc-119(+)], was microinjected into EG6699 to isolate the single-copy integrant rhdSi53[Plipl-1::mCherry::unc-54 3’UTR + cb-unc-119(+)] as beforehand described [96]. The lipl-1(rhd279[A391*]) and lipl-2(rhd282[A423*]) nonsense alleles have been generated by CRISPR/Cas9 gene enhancing. Briefly, single-stranded oligonucleotide HR donor molecules and the Cas9::crRNA:tracrRNA complexes (crRNA sequence: 5’-UAGAGAACUUCUACUCAAAA-3’) have been microinjected into the germline of wild-type animals as beforehand described [97]. The HR donor sequence included a brand new XbaI lower web site which allowed for genotyping through PCR adopted by restriction digest previous to Sanger sequencing. The transcriptional reporter strains rhdSi53[Plipl-1::mCherry::unc-54 3’UTR + cb-unc-119(+)] and wwIs24[Pacdh-1::GFP + cb-unc-119(+)] have been imaged with a Nikon SMZ-18 stereo microscope outfitted with a DS-Qi2 monochrome digital camera at 10X zoom.
Kombucha brewing
Kombucha was brewed utilizing a serial fermentation methodology tailored from a homebrewing Kombucha equipment (The Kombucha Store). Ultrapure water (1L) was boiled for 3 minutes, faraway from the warmth, and dried tea leaves (2.5 g of Assam Black Tea and a pair of.5 g Inexperienced Tea) have been steeped for five minutes utilizing an infuser. After elimination of the tea infuser, 128 g of granulated cane sugar (Domino) was dissolved within the tea and the answer was poured right into a clear 5L glass brewing jar earlier than 3L of chilled ultrapure water was added. As soon as the answer cooled to beneath 30°C, the SCOBY and ~500 mL of the earlier fermented Kombucha broth was added to the brew jar and a decent weave muslin fabric was affixed to the jar opening to restrict contamination throughout fermentation. The jar was then positioned in oblique daylight at room temperature (between 24–28°C) and allowed to ferment for no less than 8 days earlier than a brand new tradition was began, which allowed for full fermentation and a pH of ~4.
NGM Kombucha plates
For the one microbial diets, NGM plates have been seeded with both E. coli strains (OP50 or HT115) after ~16 hours of development at 25°C or with A. tropicalis, Okay. rhaeticus, or Z. bailii grown for at the very least 3 days at 25°C. The E. coli strains have been grown in 25 mL of LB with shaking (250 rpm) whereas A. tropicalis, Okay. rhaeticus, and Z. bailii have been grown in 25 mL of mannitol development media (5 g Yeast Extract, 3 g Peptone, and 25 g Mannitol in 1 L) supplemented with 1% D-glucose and 1% glycerol with shaking (250 rpm). The strains have been concentrated through centrifugation at 4,000 rcf for five minutes adopted by resuspension in 5 mL of the suitable media earlier than seeding on NGM plates. To calculate the microbial focus of meals sources, OD600 readings have been taken adopted by serial dilution and CFU quantification.
To organize KTM NGM agar plates, 50 mL of the Kombucha Tea tradition on day 2 or 3 of fermentation was eliminated and the microbes have been concentrated through centrifugation for five minutes at 4,000 rcf. The supernatant was eliminated leaving 5 mL to resuspend the pelleted KTMs. Following resuspension through vortexing, 300 μL or 2 mL of concentrated KT was added to the center of a 6cm or 10cm NMG plate, respectively. For 5x KTM plates, 250 mL of tradition was concentrated to five mL. Plates have been allowed to mature for 4 days at room temperature earlier than being utilized in experiments. The KTM-M NGM plates have been ready by first individually rising up 20 mL cultures of A. tropicalis, Okay. rhaeticus, and Z. bailii. The microbes have been then concentrated by centrifugation, resuspended with filter sterilized tea media (2.5 g of Assam Black Tea, 2.5 g Inexperienced Tea, 128 g of granulated cane sugar, 1L of water), mixed right into a single tradition, washed with sterilized tea media, reconcentrated by centrifugation, resuspended in 5 mL of the supernatant, seeded onto NGM plates, and incubated for 4 days at room temperature. To substantiate that the filter sterilized tea media was free from microbes and/or spores, the sterilized tea media was plated on NGM plates and monitored for development over 14 days, which resulted in plates freed from microbial development.
Equally, microbes have been grown independently, harvested, and mixed in sterilized tea media to generate the small-scale KTM-FM cultures, which have been maintained in 50 mL conical tubes with loosely tapped lids at room temperature. At totally different timepoints, 30 mL was faraway from the tradition and changed with 30 mL of recent sterilized tea media. The next day 25 mL was faraway from the tradition, concentrated by centrifugation, seeded onto NGM plates, and incubated at room temperature for 4 days prior to make use of. An extended-term, established KTM-FM tradition was began similarly, however the tradition was fermented in a 500 mL graduated cylinder coated in a cheese fabric, which was serially fermented over time by eradicating 50 mL of fermented tradition (used for plates) previous to the addition of fifty mL of recent sterilized tea media.
16S rDNA sequencing of Kombucha tradition and plates
The Kombucha Tea tradition was initiated and KTMs have been seeded onto plates as described above. For the day 1 tradition timepoint, 1 mL of 10x concentrated Kombucha was subjected to additional centrifugation at 16,000 krcf for 10 minutes, the supernatant was eliminated, and the pellet was flash frozen in liquid nitrogen. The KTM plates have been ready for 16S sequencing on the similar time utilizing 10x concentrated Kombucha. For the following tradition sampling, 10 mL of KT was collected and the KTMs have been harvested by centrifugation. For KTM plate samples, the microbes have been faraway from NGM plates at totally different timepoints utilizing a cell scrapper and have been collected into 1 mL of UltraPure DNase/RNase free water, concentrated by centrifugation, and frozen. All 16S rDNA sequencing was carried out by the UNC Microbiome Core on an Illumina MiSeq instrument (PE 250). The information evaluation was carried out on 32,000–95,000 uncooked reads per pattern utilizing Qiime2 [98].
Garden avoidance assay
Roughly 50 synchronized L1 animals have been dropped outdoors of every microbial garden and the variety of animals on every garden was counted at 48, 72, and 96 hours later. The proportion of animals off the garden was calculated as Noff garden/Ncomplete for every timepoint. Every organic replicate was averaged from three technical replicates and the info have been plotted because the imply ± SEM utilizing Prism 9. An abnormal one-way ANOVA adopted by Sidak’s a number of comparisons take a look at was used to calculate statistical significance between teams.
Meals selection assay
NGM plates have been seeded in 4 quadrants, every with 30 μL of one of many 4 meals sources (E. coli OP50, HT115, A. tropicalis, and KTMs). Roughly 50 synchronized L1 animals have been dropped in the midst of the plate and the fraction of animals on the totally different microbial lawns was counted 48 hours later.
Pumping fee measurements
Animals have been grown on every weight-reduction plan from synchronized L1s and the pumping fee of 15 day one grownup animals was manually counted utilizing a Nikon SMZ800N Stereo microscope. The variety of pharyngeal contractions over a one-minute span was counted and information have been plotted because the imply ± SD utilizing Prism 9. A one-way ANOVA adopted by Tukey’s a number of comparisons take a look at was used to calculate statistical significance between teams.
Intestine colonization assay
Measurement of the bacterial hundreds in C. elegans animals after consumption of every weight-reduction plan was carried out as beforehand described [99]. Briefly, ~150 animals have been grown from synchronized L1s on every weight-reduction plan to maturity and ~30 animals have been picked to an empty plate for half-hour to attenuate bacterial switch from garden. Ten animals have been hand-picked to M9 media containing 100 μg/mL levamisole, allowed to settle, and have been washed 3 times with M9 media containing levamisole and gentamicin (100 μg/mL). Animals have been lysed in 250 μL 1% Triton X-100 utilizing thirty 1.5 mm sterile zirconium oxide beads (Subsequent Advance) with an electrical benchtop homogenizer (BioSpec Mini-beadbeater). The 1.5 mL tubes have been shaken twice for 90 sec earlier than serial dilution of the lysates and plating onto commonplace NGM plates. The CFUs/animal values have been calculated as described [99]. Knowledge have been plotted utilizing Prism 9 and the statistical significance between meals sources was decided by one-way ANOVA adopted by Sidak’s a number of comparisons take a look at.
SEM imaging of the C. elegans Gut
Day 1 grownup animals have been mounted with 2% paraformaldehyde in 150 mM sodium phosphate buffer (PB, pH 7.4) at room temperature and saved at 4°C. Samples have been washed 3 instances with PB, adopted by 3 water rinses, dehydrated utilizing an ethanol gradient (30%, 50%, 75%, 100%, 100%, 100%), washed with two hexamethyldisilazane (HMDS) exchanges, and allowed to dry in HMDS. Dried animals have been brushed onto double-sided carbon adhesive mounted to a 13 mm aluminum stub and a scalpel was used to slice the C. elegans animals open by drawing the blade upward although the physique of the animal whereas they have been adhered to the adhesive. Mounted samples have been then sputter coated with 5 nm of gold-palladium (60 Au:40 Pd, Cressington Sputter Coater 208HR, mannequin 8000–220, Ted Pella Inc). Pictures have been taken utilizing a Zeiss Supra 25 FESEM working at 5 kV, utilizing the InLens detector, ~7 mm working distance, and 30 μm aperture (Carl Zeiss SMT Inc) at 5,000X and 15,000X zoom.
Calcofluor White Staining
Calcofluor White (or Fluorescent Brightener #28), which stains chitin and cellulose, was added to levamisole paralyzing answer at roughly 1 mg/mL. The animals fed totally different diets have been picked to agar pads containing levamisole and Calcofluor White, coated with a coverslip, and stained for 10 minutes. For Okay. rhaeticus and E. coli OP50 imaging (S6J Fig), animals have been imaged with a Leica DMI8 with an xLIGHT V3 confocal microscope with a spinning disk head (89 North) outfitted with a Hamamatsu ORCAFusion GENIII sCMOS digital camera utilizing a 63X oil goal (Plan-Apochromat, 1.4 NA). For imaging of the KTMs (S6L Fig), animals have been imaged with a Ti2 widefield microscope outfitted with a Hamamatsu ORCA-Fusion BT camara utilizing a 100X oil goal (Plan Apo λ). Importantly, the stain was vulnerable to fast photobleaching, and thus, areas of curiosity have been discovered utilizing the DIC channel and animals have been solely uncovered to the florescent gentle throughout picture acquisition. Nikon Components was used to denoise and deconvolute the KTM pictures and each units of pictures have been processed in Fiji v2.9.0 [100] to introduce pseudo coloring.
Oil Crimson O and Nile Crimson staining
Roughly 150 animals have been grown from synchronized L1s on NGM plates containing totally different meals sources for 72 hours at 20°C. Day 1 grownup animals have been washed off the plates in M9 media, allowed to decide on ice, washed 3 times with S-basal media, and stuck in 60% isopropanol. For Oil Crimson O staining, mounted animals have been handled with filtered 0.5% Oil Crimson O for 7 hours earlier than washing the animals with 0.01% Triton X-100 in S-basal as beforehand described [101]. For Nile Crimson staining, isopropanol-fixed animals have been stained for two hours with recent Nile Crimson/isopropanol answer (150 μL Nile Crimson inventory at 0.5 mg/mL per 1 mL of 40% isopropanol) [29]. For complete physique analyses, animals have been mounted on agar pads and imaged for Oil Crimson O staining at 3X zoom with a Nikon SMZ-18 Stereo microscope outfitted with a DS-Qi2 monochrome digital camera or for Nile Crimson staining at 4X zoom utilizing a Ti2 widefield microscope outfitted with a Hamamatsu ORCA-Fusion BT digital camera. Colour pictures of Oil Crimson O-stained animals have been obtained at 10X magnification utilizing the Ti2 widefield microscope outfitted with a Nikon DS-FI3 coloration camara. For evaluation of intestinal Oil Crimson O staining (Fig 6G and 6K), animals have been imaged at 10x with the Ti2 widefield microscope outfitted with a Hamamatsu ORCA-Fusion BT digital camera.
For quantification of Oil Crimson O staining, complete animals have been outlined utilizing Fiji and the typical grey worth (0 to 65,536) for every particular person was measured. The ensuing values have been subtracted from 65,536, which inverts the dimensions in order that strongly stained animals now have increased values. True background values are the unstained areas inside every animal; nevertheless, these areas are unimaginable to establish objectively. Thus, no background subtraction was carried out, which compresses the info to a small vary of values (55,000 to 65,000, which we report 5.5 to six.5). We discovered this method to be extremely reproducible. For the intestinal Oil Crimson O staining analyses (Fig 6G and 6K), the imply grey values have been calculated in a field (25×25 pixels) drawn inside the first two intestinal cells and the evaluation was carried out as described above. For Nile Crimson staining, common fluorescence intensities have been additionally measured utilizing Fiji and no background subtraction was carried out. All information have been plotted utilizing Prism 9 because the imply ± SD and a one-way ANOVA adopted by Tukey’s a number of comparisons statistical take a look at was carried out for every experiment. For every set of staining experiments, at the very least three organic replicates have been carried out and yielded related outcomes.
Quantification of Lipid Droplets
Dwell day 1 grownup ldrIs1[Pdhs-3::dhs-3::GFP] animals have been mounted on agar pads with levamisole and imaged with a Ti2 widefield microscope outfitted with a Hamamatsu ORCA-Fusion BT camara utilizing a 63X oil goal. Vibrant subject DIC and GFP pictures capturing the final two intestinal cell pairs have been imaged in 0.2 μm slices utilizing the identical settings throughout samples. Following acquisition, Nikon Components was used to pick a consultant slice in the midst of the stack for downstream evaluation utilizing Fiji. The DIC picture was used to stipulate the intestinal cell pair and the diameter of the lipid droplets have been measured by hand within the GFP channel utilizing the road device and ROI supervisor. Measurements have been collected from the final intestinal cell pair for 10 consultant animals for every meals supply and the typical lipid droplet measurements for every animal was plotted utilizing Prism 9. A one-way ANOVA adopted by Tukey’s a number of comparisons take a look at was used to match teams. Two impartial experiments have been performed with related outcomes.
Biochemical triglyceride measurements
Day 1 grownup animals consuming every meals supply have been harvested and processed as beforehand described [55]. A Triglyceride Assay Package was used to measure triglycerides per the manufactures’ directions (Abcam, ab65336). Three organic replicates have been carried out, the info have been plotted in Prism 9, and a one-way ANOVA adopted by Tukey’s a number of comparisons take a look at was carried out to match teams.
Molting assays
Between 1–8 synchronized mgIs49[Pmlt-10::GFP::PEST] L1s have been dropped into every properly of a 24-well plate containing NGM media and seeded with 20 μL of every meals supply (one plate per meals supply). Animals have been reared at 20°C and visualized by fluorescence microscopy each hour for 70 hours on a Nikon SMZ-18 Stereo microscope. At every time level, animals have been scored as inexperienced (molting) or nongreen (not molting). Wells with out animals have been censored. The fraction of animals molting for every timepoint was calculated and plotted with Prism 9. The experiments have been carried out at the very least twice (aside from KTM-M and KTM-FM) with related outcomes.
Developmental timing measurements
Wild-type N2 and eat-2(ad465) mutants have been grown to maturity and egg prepped as described above. The caloric restriction plates containing 108 or 109 CFUs/mL of E. coli OP50 have been ready as beforehand described [59]. Roughly 20 synchronized L1s have been dropped on every meals supply in technical triplicates and grown at 20°C. After precisely 48 hours the animals have been scored based mostly on vulva morphology as younger adults, L4 larval stage, or lower than L4 larval stage. The % of animals at every larval stage was calculated for 3 organic replicates and the info have been plotted because the imply ± SEM utilizing Prism 9.
Brood dimension measurements
Animals have been grown on their respective meals sources for 48 hours at which period 15 L4s have been singled to the corresponding meals supply and allowed to mature. The animals have been moved to recent plates each 24 hours for six days. Two days after the grownup hermaphrodite was moved to a brand new plate, the L3/L4 progeny have been counted and eliminated. The unhatched eggs weren’t counted. Complete progeny for every particular person hermaphrodite was plotted as imply ± SD utilizing Prism 9 and a one-way ANOVA take a look at with a Tukey’s a number of comparability correction was carried out. The common reproductive output per day was additionally calculated and an unpaired T-test was carried out to establish variations between these means.
VIT-2::GFP quantification
Animals expressing VIT-2::GFP at endogenous ranges (pressure BCN9071) have been grown to maturity, egg prepped, and hatched over evening at room temperature as described above. The starved L1s have been dropped on NGM plates seeded with their respective meals sources and grown for 72 hours at 20°C. Gravid day 1 grownup animals have been washed off the NGM plates and eggs have been liberated by bleaching. Following three washes with M9 media, embryos have been mounted on agar pads and imaged with a Ti2 widefield microscope outfitted with a Hamamatsu ORCA-Fusion BT camara utilizing a 20X goal. Vibrant subject DIC and GFP pictures have been captured and the fluorescent depth of 30 early-stage embryos (previous to the 44-cell stage) for every situation was measured utilizing Fiji. The imply fluorescent depth ± SD was plotted utilizing Prism 10 and statistical variations between the teams was calculated utilizing an unpaired T-test. Three impartial organic replicates have been carried out and yielded related outcomes.
Intestinal lumen measurements
Day 1 grownup ERM-1::GFP animals (pressure BOX213) consuming every weight-reduction plan have been mounted on agar pads with levamisole and imaged utilizing a Nikon SMZ-18 Stereo microscope outfitted with a DS-Qi2 monochrome digital camera. Bight subject and GFP pictures have been acquired for at the very least 10 people. Three measurements of the intestinal lumen diameter (positioned on the anterior gut, the vulva, and the posterior gut) have been carried out utilizing Fiji. For every particular person, a ratio of the lumen width relative to physique width was calculated at every of the three positions alongside the animal and the three values have been averaged. Ten people have been measured for every organic replicate and the info are reported because the imply ± SEM of three organic replicates. A one-way ANOVA take a look at with a Tukey’s a number of comparability correction was carried out in Prism 10.
BODIPY staining
C1-BODIPY-C12 (Thermo Fisher, D3823) was resuspended in DMSO to generate a ten mM inventory answer. This answer was diluted in S-basal media and overlaid onto the microbial garden to provide a ultimate focus of 10 μM inside the NGM plate. The plates have been allowed to dry for at the very least 1 hour in the dead of night earlier than day 1 grownup animals have been picked to the BODIPY plates. After 3 hours, animals have been mounted on agar pads with levamisole and imaged on a Ti2 widefield microscope outfitted with a Hamamatsu ORCA-Fusion BT camara utilizing a 40X oil goal. Vibrant subject DIC and GFP pictures have been captured for at the very least 20 animals at each the anterior and posterior sections of the gut. Consultant pictures displaying detectible ranges of BODIPY staining have been chosen from two impartial experiments and are displayed in Figs 4K and S7.
Complete genome sequencing and evaluation of Kombucha microbes
Mannitol development media supplemented with 1% D-glucose and 1% glycerol was inoculated with the KT related microbes and the cultures have been grown for 48 hrs at 25°C with shaking. The gDNA was remoted from cell pellets utilizing the Wizard Genomic DNA Purification equipment (Promega, A1120). Preparation and Illumina brief learn sequencing (PE 150) of DNA-Seq libraries was carried out by Novogene (Sacramento, CA). Initially, an unbiased metagenomic evaluation was carried out utilizing Kraken 2 [65] to establish candidate microbial species for every Kombucha-associated microbe. Subsequent, we downloaded the entire genome reference sequences for varied strains for every candidate species from the NCBI Genome database and mapped our reads in opposition to these reference genomes utilizing Bowtie 2 with the default settings [102]. The general alignment fee generated by the Bowtie 2 algorithm was reported. The entire genome sequencing information can be found on the Sequencing Learn Archive (PRJNA1044129).
mRNA sequencing
Wild-type N2 animals have been grown on 10 cm NGM agarose plates (1000 animals/plate) within the presence of their respective meals sources. Day 1 adults have been harvested, washed 3 instances in M9 buffer, and flash frozen. The whole RNA was remoted utilizing Trizol (Thermo Fisher), adopted by two rounds of chloroform extraction, RNA precipitation with isopropanol, and an 80% ethanol wash of the RNA pellet. In some circumstances, an RNA Clear & Concentrator-25 equipment (Zymo, R1017) was used to extend the purity of the pattern. The mRNA-Seq libraries have been ready and sequenced by Novogene (Illumina, PE 150). The information have been processed precisely as beforehand described [103]. RPKM values and identification of differentially expressed genes (1% FDR) have been calculated utilizing the DESeq2 algorithm [104], which could be present in S4 Table. Lists of developmental, replica, and metabolic genes have been beforehand described [29], and scatter plots displaying expression ranges of those genes have been generated utilizing the DESeq2 RPKM values. Heatmaps and PCA plots have been generated with the pheatmap [105] and tidyverse [106] R packages, respectively. All different plots displaying mRNA-Seq information have been made in Prism 9. Uncooked and processed mRNA-Seq information have been deposited in GEO (GSE236037).
Supporting info
S1 Fig. The phylogenetic profile of the KTMs on NGM plates is much like that of the KT tradition.
(A) Pictures of NGM worm plates seeded with a KTM garden. The preparation begins at day 0 when a brand new KT brew cycle is initiated, the microbes are seeded on day 1, and incubated at room temperature to day 5 earlier than the KTM plates are used. (B) Consultant pictures of KT brews at day 1 and day 7 of fermentation. The KTMs are extracted from the tradition at day 1 and plated. (C) A complete view of 16S rDNA sequencing outcomes of the KT microbes from fermenting Kombucha tradition, seeded NGM plates, or the pellicular biofilm from the Kombucha tradition. The plot reveals the frequency of every species (8 most ample microbes displayed; a whole listing could be present in S1 Table). (D) A plot of Religion’s phylogenetic variety index displaying the distinction in α-diversity between the indicated samples (**, p<0.01, one-way ANOVA). (E) The Pielou Evenness Range Index, measuring the microbial variety and species richness within the indicated samples (ns, not vital, one-way ANOVA). Uncooked information underlying panels C-E could be present in S7 Data.
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S2 Fig. Worms select different diets over a KTM weight-reduction plan.
(A) A schematic depicting the meals selection assay. (B) The portion of wild-type N2 animals on the L4 stage on every meals supply 48 hours after dropping L1s (n>200/trial, 3 organic replicates). (C-E) Meals selection assays for the N2, MY10, and JU1212 C. elegans strains scored on the L4 stage (48h publish L1 drop, n>150/trial, 3 organic replicates). (F-I) The portion of L4 stage worms on every meals supply at 48h publish L1 drop for the N2 C. elegans, PB2801 C. brenneri, AF16 C. briggsae, and PB4641 C. remanei strains (n>75/trial, 3 organic replicates). All meals selection information are plotted because the imply ± SEM. All meals selection assays embody n>150 animals per replicate and the info are plotted because the imply ± SEM (****, P<0.0001, ***, P<0.001, **, P<0.01, *, P<0.05, ns, not vital; one-way ANOVA). Uncooked information underlying panels B-I could be present in S8 Data.
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S3 Fig. Host lipid distributions throughout replica and throughout people.
(A) Quantification of day 3 and day 5 adults stained with Oil Crimson O (imply ± SD, ****, P<0.0001, one-way ANOVA). (B) Measurements of particular person lipid droplet sizes measured throughout ten people consuming E. coli OP50, KTMs, or KTM-Combine (imply ± SD, n = 10 animals/trial, 2 organic replicates). The distribution of lipid droplet sizes is analogous throughout people fed the identical weight-reduction plan. Uncooked information underlying panels A and B could be present in S9 Data.
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S4 Fig. Common progeny per day.
A desk displaying the typical progeny laid per day of the reproductive interval demonstrates that KTM-fed animals exhibit the same egg laying fee relative to E. coli OP50-fed animals (imply, ****, P<0.0001, ***, P<0.001, **, P<0. 01, *, P<0.05, ns, not vital, T-test). Uncooked information underlying the determine could be present in S10 Data.
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S5 Fig. rDNA sequencing identifies candidate KTMs.
Outcomes from 16S rDNA sequencing of the remoted bacterial KTMs point out that (A) A. tropicalis and (C) a member of the Komagataeibacter genus are elements of our Kombucha tradition. (B) Sequencing of the ITS area of the KTM yeast isolate revealed that the pressure belongs to the Brettanomyces or Zygosaccharomyces genus. Uncooked information underlying panels A-C could be present in S1 Table.
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S6 Fig. Deconvolution of Kombucha Tea facilitates the creation of fermenting and non-fermenting mixes of KTM.
(A-B) Measurements of the microbial concentrations in every of the indicated microbial mixes or single microbial cultures (imply ± SEM, ****, P<0.0001, one-way ANOVA). (C) Oil Crimson O staining of day 1 grownup animals fed an E. coli OP50, KTM-Combine, or KTMs weight-reduction plan, in addition to a 5X concentrated model of the KTM weight-reduction plan (imply ± SD, ****, P<0.0001, one-way ANOVA). Rising the focus of KTMs decreases lipid storage. (D) Consultant pictures of animals on and off a garden of Z. bailii yeast 72 hours publish L1 drop, which reveals that animals fail to develop when consuming a Z. bailii weight-reduction plan (worms are indicated with white arrow heads; scale bar, 500 μm). (E) Lipid droplet density measurements with every datapoint representing the variety of lipid droplets per μm2 for the final two intestinal cells of animals consuming a KTM or KTM-M weight-reduction plan (the KTM information are additionally proven in Fig 2H; imply ± SD, **, P<0.01, T-test). (F) A scatter plot evaluating the expression of two,229 developmental genes in animals fed E. coli versus KTM-M as decided by mRNA-Seq (RPKM, reads per kilobase of transcript per million mapped reads). A linear regression evaluation and the corresponding R2 worth is reported. (G) A selection assay displaying the portion of wild-type N2 animals on the L4 stage on the indicated meals sources 48 hours after dropping L1s (n>200/trial, 3 organic replicates; imply ± SEM, ****, P<0.0001, *, P<0.05, ns, not vital, one-way ANOVA). (H-I) The developmental fee of animals expressing a Pmlt-10::GFP-PEST reporter when fed a KTM, KTM-Combine, or a KTM-FM weight-reduction plan. Synchronized L1 worms have been reared at 20°C for ~72 hours and scored hourly. (J) Consultant pictures of animals consuming Okay. rhaeticus or E. coli OP50 after staining with Calcofluor White, which selectively labels intestinal microbes producing chitin or cellulose (white arrow heads point out the intestinal lumen; scale bars, 10 μm). (Okay) Consultant brightfield DIC pictures displaying yeast cells within the gut of animals consuming KTMs and yeast cells on the slide (grey arrow heads point out yeast cells, magnified inset picture proven for readability, scale bar 5 μm). (L) Consultant pictures of animals consuming KTMs after staining with Calcofluor White (grey arrow heads within the inset point out yeast cells; scale bars, 5 μm). (M) Intestinal lumen width measurements of animals consuming the E. coli OP50, KTM, and KTM-M diets. Knowledge are reported because the % of the physique width taken up by the intestinal lumen (imply± SEM, **, P<0.01, ns, not vital, one-way ANOVA). Uncooked information underlying panels A-C, E-I, and M could be present in S11 Data.
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S7 Fig. BODIPY lipids are absorbed into intestinal cells of KTM-fed animals.
Consultant DIC and fluorescence pictures displaying C1-BODIPY-C12 absorption into the intestinal epithelial cells of animals feeding on an E. coli OP50, KTM, or KTM-M weight-reduction plan. The pink stars point out BODIPY remaining within the intestinal lumen, the pink arrowheads level to partial BODIPY absorption into the intestinal cells, white stars point out a scarcity of BODIPY remaining within the intestinal lumen, and white arrowheads level to completely stained cells which have absorbed BODIPY (scale bars, 10 μm).
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S8 Fig. KTM consumption leads to widespread modifications in gene expression.
(A) A scatter plot and linear regression evaluation evaluating the expression of all genes in animals fed KTMs for one technology (1G) or for 5 generations (5G), suggesting that pervasive transgenerational epigenetic regulation of gene expression by KTMs is unlikely. (B-F) Volcano plots displaying the differentially expressed genes for the indicated samples relative to the E. coli OP50 pattern. (G) Enrichment (noticed/anticipated, hypergeometric P values reported) for differentially expressed genes frequent between KTM-fed animals and animals depleted of DAF-2::AID within the indicated tissues utilizing the auxin degron system [72]. Values >1 point out over-enrichment, or that the identical genes are usually in another way expressed in each animals consuming KTMs and animals depleted of DAF-2 in comparison with random likelihood. The overlap between differentially expressed genes which can be both (H) up-regulated or (I) down-regulated in animals consuming KTMs and animals depleted DAF-2::AID within the gut (hypergeometric P values are proven). (J) Consultant fluorescent pictures (scale bar, 500 μm) and (Okay) quantification of the acyl-CoA dehydrogenase Pacdh-1::GFP reporter on the indicated microbial diets (n = 40, imply ± SD, ****, P<0.0001, ns, not vital, one-way ANOVA). Uncooked information underlying panels A-I and Okay could be present in S12 Data.
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S9 Fig. Expression of the lipl-1 gene is modulated within the gut upon KTM consumption, however the lysosomal lipases genes lipl-1,2,3 aren’t required to limit lipid droplet dimension.
(A) Quantification of the expression ranges of the lysosomal lipase Plipl-1::mCherry reporter in animals grown on E. coli OP50, KTM, and KTM-M (n>200, imply ± SD, ****, P<0.0001, one-way ANOVA). (B) Lipid droplet dimension measurements in wild-type N2 and lipl-1(tm1954) lipl-2(ttTi14801) lipl-3(tm4498) mutant animals with every datapoint representing the typical intestinal lipid droplet diameter for a single animal (imply ± SD, ***, P<0.001, ns, not vital, one-way ANOVA). (C-E) Normalized gene expression values for the indicated TAG synthesis genes (imply ± SEM, ***, P<0.001, *, P<0.05, ns, not vital, one-way ANOVA). Uncooked information underlying panels A-E could be present in S13 Data.
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S1 Table. An Excel spreadsheet containing the taxonomy report from 16S rDNA sequencing.
Proven are particular person sequencing outcomes for organic replicates of the Kombucha Tea cultures, the Kombucha Tea biofilm (one replicate), and Kombucha Tea-associated microbes remoted from C. elegans NGM plates.
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S4 Table. The DESeq2 outputs from the mRNA-Seq evaluation.
An Excel spreadsheet containing, in separate tabs, gene counts (RPKM, reads per kilobase of transcript per million mapped reads) for all genes, in addition to the differential gene expression requires the next comparisons: E. coli OP50 vs. E. coli HT115, E. coli OP50 vs. Acetobacter tropicalis, E. coli OP50 vs. KTM, and E. coli OP50 vs. KTM-M.
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S3 Data. Excel spreadsheet containing, in separate tabs, the numerical information underlying Fig 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M and 3N.
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S12 Data. Excel spreadsheet containing, in separate tabs, the numerical information underlying S8A, S8B, S8C, S8D, S8E, S8F, S8G, S8H, S8I and S8K Fig.
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