(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Kombucha Tea-associated microbes remodel host metabolic pathways to suppress lipid accumulation [1]

['Rachel N. Dumez-Kornegay', 'Curriculum In Genetics', 'Molecular Biology', 'University Of North Carolina At Chapel Hill', 'Chapel Hill', 'North Carolina', 'United States Of America', 'Lillian S. Baker', 'Department Of Biology', 'The University Of North Carolina At Chapel Hill']

Date: 2024-04

The popularity of the ancient, probiotic-rich beverage Kombucha Tea (KT) has surged in part due to its purported health benefits, which include protection against metabolic diseases; however, these claims have not been rigorously tested and the mechanisms underlying host response to the probiotics in KT are unknown. Here, we establish a reproducible method to maintain C. elegans on a diet exclusively consisting of Kombucha Tea-associated microbes (KTM), which mirrors the microbial community found in the fermenting culture. KT microbes robustly colonize the gut of KTM-fed animals and confer normal development and fecundity. Intriguingly, animals consuming KTMs display a marked reduction in total lipid stores and lipid droplet size. We find that the reduced fat accumulation phenotype is not due to impaired nutrient absorption, but rather it is sustained by a programed metabolic response in the intestine of the host. KTM consumption triggers widespread transcriptional changes within core lipid metabolism pathways, including upregulation of a suite of lysosomal lipase genes that are induced during lipophagy. The elevated lysosomal lipase activity, coupled with a decrease in lipid droplet biogenesis, is partially required for the reduction in host lipid content. We propose that KTM consumption stimulates a fasting-like response in the C. elegans intestine by rewiring transcriptional programs to promote lipid utilization. Our results provide mechanistic insight into how the probiotics in Kombucha Tea reshape host metabolism and how this popular beverage may impact human metabolism.

Kombucha is a popular fermented tea that has been purported to have many human health benefits, including protection against metabolic diseases like diabetes and obesity. These health benefits are thought to be conferred by the probiotic microbes found in Kombucha Tea, which includes both bacterial and yeast species, that may be able to colonize the human intestine and alter host physiology. The mechanisms by which the Kombucha Tea-associated probiotic microorganisms (KTMs) impact host physiology are largely unknown. Using the nematode Caenorhabditis elegans as an animal model system to study the host physiological response to KTMs, we show that KTMs colonize the C. elegans intestine and impart widespread changes in the expression of evolutionarily conserved lipid metabolism genes, resulting in reduced fat levels in the host. The host metabolic response to actively fermenting KTMs requires an increase in proteins that break down lipids paired with a reduction in a protein that builds triglycerides, which mirrors the events that occur during fasting. These findings are consistent with the reported human health benefits of Kombucha Tea and provide new insights into the host response to Kombucha-associated microbes, which could inform the use of Kombucha in complementary health care approaches in the future.

Here, we use C. elegans to investigate whether intestinal colonization with Kombucha-associated microbial species (KT microbes or KTMs) rewires host metabolism. We developed a reproducible method to culture animals on lawns of KT microbes consisting of microbes found in all commercial and homebrewed KTs (i.e., bacteria from the Acetobacter and Komagataeibacter genera and a yeast species). We found that animals feeding ad libitum on KT microbes accumulate significantly less fat than animals consuming either an E. coli diet, any of the individual three KT-associated microbial species, or a simple non-fermenting mix of these three species. Furthermore, our data suggest that KT consumption reduces fat storage by modulating host lipid metabolism pathways rather than restricting caloric intake. To gain insight into the mechanisms that underlie this reduction in lipid levels, we performed a transcriptomic analysis of KT microbe-fed animals, which revealed that a class of lysosomal lipases that function in lipophagy was up-regulated and that a crucial enzyme in triglyceride synthesis was down-regulated in response to KT microbes. Our results suggest that Kombucha Tea consumption may alter lipid droplet dynamics by promoting their degradation via lipophagy, while simultaneously restricting lipid droplet expansion through down-regulation of triglyceride synthesis. This investigation lays crucial groundwork to deconvolute the molecular mechanisms that may underlie the purported health benefits of KT using a genetically tractable animal model.

The impact of individual probiotic microbes, or in this case the small community of Kombucha-associated microbes, on human physiology is difficult to deconvolute as humans consume a complex diet, have trillions of microbes colonizing their gut, and mechanistic investigation of host-microbe interactions is not feasible in human subjects. Therefore, use of animal models is essential to investigate how probiotic consumption influences host physiological processes. Caenorhabditis elegans has been widely used to investigate mechanisms of metabolic regulation and how nutrient sensing pathways govern organismal homeostasis [ 20 , 21 ]. C. elegans is also an emerging model for studying the impact of the gut microbiome on host physiology [ 22 , 23 ]. Axenic preparation of C. elegans cultures renders these bacterivore animals microbe-free at the onset of life, allowing for complete experimental control over which microbes are consumed during their lifetime (i.e., animals are germ-free before encountering their microbial food source). Additionally, microbes that escape mechanical disruption during feeding can robustly colonize the intestinal lumen [ 22 – 24 ]. Thus, the simple digestive tract of C. elegans is effectively colonized by bacteria that are provided as a food source, making it an ideal system to interrogate the host metabolic response to consumption of specific microbes. Indeed, previous studies have successfully used C. elegans to investigate how individual species of microbes, including probiotics, can elicit physiological changes by rewiring conserved genetic pathways [ 25 – 31 ].

Kombucha tea (KT) is a semi-sweet, fermented beverage that is widely consumed as a functional food (i.e., providing health benefits beyond nutritional value) and contains probiotic microbes that have been purported to confer health benefits, including lowering blood pressure, protection against metabolic disease, improved hepatoprotective activity (i.e., protection against liver toxins), and anticancer effects [ 9 – 13 ]. These probiotic microbes include members of the Acetobacter, Lactobacillus, and Komagataeibacter genera [ 14 , 15 ]. While some of these health benefits have begun to be tested in animal models, including the ability of KT to ameliorate diabetic symptoms or limit weight gain in adult mice [ 16 – 19 ], the mechanistic underpinnings of these phenotypes have not been rigorously investigated. Moreover, the interactions between the microbes in Kombucha Tea, which include both bacterial and yeast species, and the host remain completely unexplored. Because this beverage contains live probiotic microbes and is widely consumed under the largely unsubstantiated claim that it confers health benefits, it is imperative to gain mechanistic insight into the host physiological and cellular response to KT consumption.

Since the discovery of antibiotics, humans have been successfully eliminating microbes to cure infections and sterilize our environments, but this nonspecific approach to eliminate pathogenic microbes has also made it increasingly evident just how much we rely on interactions with commensal microbes to remain healthy. Antibiotic use, western diets, a sedentary lifestyle, and many disease states can trigger dysbiosis, or a reduction in microbial diversity, which has been linked to metabolic syndromes, chronic inflammation, and mental health disorders [ 1 – 3 ]. For example, C. difficile colitis can arise from antibiotic use and a subsequent loss of microbial diversity in the gut, resulting in severe gastrointestinal symptoms and potentially death [ 4 ]. Consumption of probiotics, or live microbes associated with health benefits, can promote, or maintain, a healthy gut microbiome while supplying the host with crucial microbially-derived metabolites [ 5 – 7 ]. Understanding the molecular mechanisms underlying the host response to microbes, particularly probiotics [ 8 ], is critical for their incorporation into complementary health care approaches.

Results

Animals consuming Kombucha microbes exhibit reduced fat accumulation Dietary components, including those produced by probiotic microbes, can play a substantial role in modulating host metabolism, including lipid storage and lipolysis [49–51]. Consistently, C. elegans metabolism is remarkably sensitive to differences in microbial diets, as even highly similar strains of E. coli promote markedly different levels of fat content [28,29,41]. Given the purported metabolic benefits of KT in humans, including decreased risk of obesity [9–13], we reasoned that consumption of KTMs may impact lipid levels in C. elegans. The majority of fat in C. elegans animals is stored in intestinal epithelial cells within lipid droplets in the form of triglycerides (TAGs), with smaller lipid deposits found in the hypodermis and germline [52]. Using the well-established lipophilic dyes Oil Red O and Nile Red, which both stain neutral lipids, we examined the fat content of animals consuming KTMs and control microbes [52,53]. Animals consuming KT microbes accumulated significantly less fat than animals consuming other food sources, including A. tropicalis, which is particularly noteworthy given that A. tropicalis is the most abundant microbial species in KT (Fig 2A–2D). These trends continued during and after the reproductive period (S3A Fig), suggesting that KTMs restrict host lipid accumulation throughout reproduction and during the aging process. Importantly, the KTM-fed animals successfully commit a significant proportion of their somatic fat stores to the germline and developing embryos at adulthood (Fig 2C), suggesting that reproductive programs are not impaired despite the overall reduction in lipid levels. The decrease in Oil Red O and Nile Red staining suggests that animals consuming KTMs may have reduced TAG levels compared to animals on control diets. Therefore, we used a biochemical assay to quantify the total amount of TAGs in populations of animals fed each diet [54,55]. Consistent with our previous observations, animals consuming KTMs had an ~85% or ~90% decrease in TAG levels compared to animals consuming E. coli OP50 or A. tropicalis, respectively (Fig 2E). Together, these data clearly demonstrate that animals consuming KT microbes accumulate less fat than E. coli-fed animals and that the most abundant microbe in KT, A. tropicalis, is not sufficient to recapitulate this phenotype. This finding is particularly relevant to human health, as KT consumption has been shown to restrict weight gain and alleviate diabetic symptoms to a similar degree as metformin in rodent models [16–19]. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. KTMs restrict lipid accumulation in the host. (A) Representative images (scale bar, 500 μm) and (B) quantification (mean ± SD, ****, P<0.0001, one-way ANOVA) of day 1 adults stained with Oil Red O. (C) Representative fluorescence images (scale bar, 500 μm) and (D) quantification (mean ± SD, ****, P<0.0001, one-way ANOVA) of day 1 adults stained with Nile Red. (E) Biochemical quantification of the triglycerides (TAGs per animal) in animals consuming each food source (mean ± SEM, ***, P<0.001, *, P<0.05, ns, not significant, one-way ANOVA). (F) Representative fluorescence images 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 size measurements with each datapoint representing the average intestinal lipid droplet diameter for a single animal (mean ± SD, ****, P<0.0001, one-way ANOVA). (H) Lipid droplet density measurements with each datapoint representing the number of lipid droplets per μm2 for a single animal (mean ± SD, ****, P<0.0001, *, P<0.05, ns, not significant, one-way ANOVA). Raw data underlying panels B, D, E, G, and H can be found in S2 Data. https://doi.org/10.1371/journal.pgen.1011003.g002 Given that the major site of lipid storage in C. elegans is in intestinal lipid droplets (LD), we hypothesized that LD size or abundance may be impacted in the intestine of KTM-fed animals. Taking advantage of a transgenic strain that expresses the LD-residing DHS-3::GFP protein (dhs, dehydrogenase, short chain), we measured LD abundance and size in intestinal cells of animals fed each diet. Both lipid droplet size and abundance were dramatically reduced in animals consuming KTMs relative to E. coli- or A. tropicalis-fed animals (Figs 2F–2H and S3B). Together, these results suggest that regulation of lipid droplet synthesis or stability may account for the reduced lipid accumulation that we observed in KTM-fed animals.

KTM consumption accelerates growth rates and does not substantially alter fecundity Different microbial diets can have a profound impact on C. elegans growth rate and fecundity [29,30]. A KTM diet could restrict developmental rate or alter reproductive programs. Moreover, reduced nutrient absorption stemming from a KTM diet could result in caloric restriction and reduced lipid accumulation. Indeed, genetic or nutritional models of caloric restriction cause animals to develop more slowly, to accumulate less intestinal fat, and to have a delayed reproductive period that ultimately results in less progeny production [28,56–58]. Therefore, we sought to determine whether animals consuming KTMs exhibit slower developmental rates and smaller brood sizes than animals consuming either an E. coli or A. tropicalis diet. To investigate variations in developmental rate, we employed a transgenic strain expressing a GFP-PEST protein under the control of the mlt-10 promoter (Pmlt-10::GFP-PEST), which is specifically expressed during each of the four molt stages, resulting in four peaks of GFP fluorescence throughout development (Fig 3A). The PEST amino acid sequence ensures rapid GFP turnover by proteolytic degradation and allows for precise temporal analyses. Animals consuming KTMs molt at a similar, if not an accelerated rate relative to animals on the control food sources (Fig 3A–3C), clearly indicating that KTM consumption does not decrease developmental rate. To gain a more comprehensive view of animal development during KTM consumption, we performed mRNA sequencing (mRNA-Seq) of adult animals consuming E. coli, A. tropicalis, or KTMs. Upon inspection of 2,229 genes previously associated with C. elegans development [29], we observed very few gene expression differences between KTM-fed animals and those fed control diets (Fig 3D–3F), suggesting that the KTM-fed population reaches adulthood synchronously. Together, these results suggest that animals consuming KT microbes exhibit wild-type development. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Developmental timing is accelerated and reproductive output is only modestly reduced during KTM feeding, suggesting that caloric intake is not restricted by KTM consumption. (A-C) Profiles of Pmlt-10::GFP-PEST expression throughout development after dropping synchronized L1s on the indicated microbes. The reporter is expressed exclusively during the larval molts (shown in gray in A). A single representative experiment is displayed in panels A-C. (D-F) Scatter plots comparing the expression of 2,229 developmental genes as determined by mRNA-Seq (RPKM, reads per kilobase of transcript per million mapped reads). A linear regression analysis and the corresponding R2 value is reported for each comparison. (G) The frequency (mean ± SEM) of wild-type N2 and eat-2(ad465) individuals at the indicated developmental stages after 48 hours of growth on ad libitum KTM, ad libitum E. coli, or caloric restriction E. coli (108 or 109 CFUs/mL) plates (****, P<0.0001, chi-squared test). (H) Brood sizes of wild-type animals reared on the different diets (mean ± SD, ***, P<0.001, *, P<0.05, one-way ANOVA). (I) A plot of progeny production for each day during the reproductive period demonstrating that KTM-fed animals exhibit a similar egg laying rate compared to E. coli OP50-fed animals. (J) Normalized vit-2 gene expression values (RPKM, reads per kilobase of transcript per million mapped reads; mean ± SEM, *, P<0.05, T-test) and (K) quantification of VIT-2::GFP fluorescence in early embryos (mean ± SD, ***, P<0.001, T-test) from animals consuming an E. coli OP50 or KTM diet. (L-N) Scatter plots and a linear regression analysis (R2 value reported) comparing the expression of 2,367 reproduction genes as determined by mRNA-Seq. Raw data underlying panels A-N can be found in S3 Data. https://doi.org/10.1371/journal.pgen.1011003.g003 Caloric restriction has a profound impact on C. elegans physiology, including reduced developmental rate [58]. The eat-2 mutant is a genetic model of caloric restriction, as loss of eat-2 results in impaired pharyngeal pumping and reduced nutrient intake [58]. Reducing nutrient availability (i.e., E. coli OP50 lawns with concentrations ≤ 109 CFU/ml) provides a second effective method of caloric restriction [59]. Therefore, to further evaluate whether animals consuming KTMs are calorically restricted (CR), we conducted developmental rate assays with wild-type and eat-2 mutant animals consuming ad libitum E. coli lawns, CR E. coli lawns (108–109 CFU/ml), or our standard ad libitum lawns of KTMs. This analysis revealed that both wild-type and eat-2 animals exhibited accelerated developmental rates when consuming KTMs compared to the E. coli OP50 diet (Fig 3G). Importantly, eat-2 animals showed reduced developmental rates on CR E. coli lawns relative to ad libitum E. coli lawns, indicating that the effects of the eat-2 mutation are further enhanced by additional caloric restriction; however, KTM-feeding partially suppressed the developmental defects of the eat-2 mutation (Fig 3G). These data demonstrate that KTM consumption does not mimic the effects of restricted caloric intake. Reproductive output (i.e., brood size) of C. elegans is modulated by diet, possibly through the tuning of reproductive programs at the transcriptional level [29,60]. Therefore, we measured the brood sizes of animals consuming KTMs and control diets, finding that the average brood size of animals consuming KTMs was only modestly lower than those consuming E. coli OP50 (Figs 3H and S4; 295 versus 240, P<0.05). Additionally, we found that animals consuming KTMs lay their eggs at a similar rate relative to E. coli-fed animals (Figs 3I and S4). In contrast, calorically restricted animals, such as eat-2 mutants, have extended egg laying periods, up to 12 days, and have substantially reduced brood sizes, with eat-2 mutants averaging 100–175 progeny [28,57]. Thus, the ~20% reduction in fertility for KTM-fed animals is inconsistent with the more severe reduction in brood size of CR animals. It could, however, be consistent with impaired maternal provisioning of lipid-rich yolk to oocytes from intestinal fat stores, a process termed vitellogenesis. Thus, we next examined the mRNA levels of vit-2, which encodes a vitellogenin protein that mediates the intestine-to-oocyte transport of lipids, finding that vit-2 levels are increased in animals fed a KTM diet compared to E. coli-fed animals (Fig 3J). Consistently, vitellogenin protein levels, which we measured in early embryos (prior to the 44-cell stage) using an endogenously tagged VIT-2::GFP protein, were also elevated in KTM-fed animals (Fig 3K), further substantiating that KTM consumption does not impair maternal lipid provisioning. Finally, we inspected the expression of 2,367 genes implicated in reproduction [29], finding that KTM consumption does not broadly alter reproductive gene expression programs relative to control diets (Fig 3L–3N). Together, our results indicate that reproductive programs are not dramatically altered in animals consuming KTMs. This finding, along with the observation that animals consuming KTMs exhibit wild-type developmental rates, is consistent with the contention that caloric intake is not impaired during KTM consumption and substantiates C. elegans as model to investigate the impact of Kombucha-associated microbes on host metabolic pathways.

An intestinally driven metabolic response to KTM consumption KTM-fed animals undergo normal development and show no detectable impairment in nutrient absorption, yet store markedly less lipids than control animals, including those fed the KTM-M diet. While our transcriptomics suggested that the expression of genes involved in development or reproduction were consistent across diets, we hypothesized that the expression of metabolic genes may be specifically altered by KTM consumption. Therefore, we performed additional analyses of our mRNA-Seq data derived from day one adult animals consuming either KTM, KTM-M, A. tropicalis, or the two E. coli diets to investigate if specific metabolic programs are altered by these diets. A PCA analysis revealed that the transcriptomes of animals fed the same diet cluster, with the transcriptomes of animals fed KTM, KTM-M, and A. tropicalis distinctly clustering apart from the transcriptomes of the E. coli-fed animals (Fig 5A), indicating that there is at least some commonality between the transcriptional responses of animals consuming any of the KT-associated diets that is different from E. coli diets. To eliminate the possibility of transgenerational epigenetic effects of the KTM diet, we compared the transcriptomes of animals fed KTMs for one generation to animals subjected to five generations of the KTM diet, finding no significant difference between these transcriptomes (Figs 5A and S8A). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 5. Host lipid metabolism gene expression is modulated by KTM consumption. (A) A Principal Component Analysis of the normalized mRNA-Seq data for the indicated diets (1G, KTM feeding for one generation; 5G, KTM feeding for five consecutive generations prior to collection). (B) The overlap of the differentially expressed genes, determined relative to E. coli OP50, between each food source. (C) A Gene Ontology enrichment analysis performed on the 295 genes that are uniquely differentially expressed in animals consuming KTMs. (D) Enrichment for differential expression of genes that are expressed in the indicated tissues (observed/expected, hypergeometric P values reported). Values <1 indicate that genes expressed in the indicated tissue type tend not to be differentially expressed (under-enriched), while values >1 indicate tissues where differential expression is more common than expected by random chance (over-enriched). (E) A scatter plot and linear regression (R2 = 0.9556) of the RPMK values for 5,676 metabolism-related genes (the genes of interest 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 each diet (boxes from left to right: KTM, A. tropicalis, KTM-M, E. coli HT115). Raw data underlying panels A-F can be found in S5 Data and S4 Table. https://doi.org/10.1371/journal.pgen.1011003.g005 Deeper investigation of our mRNA-Seq data revealed that each KT-associated diet did indeed result in some level of differential gene expression compared to the E. coli OP50 diet (A. tropicalis, 3,952 genes; KTM, 1,237 genes; KTM-M, 1,007 genes; 1% FDR; Figs 5B and S8B–S8F). Intriguingly, 295 genes were unique to the KTM diet (Fig 5B). Altered expression of these KTM-unique genes could be a major driver of the reduced lipid levels that we observed specifically in the KTM-fed animals. A gene ontology (GO) enrichment analysis [68] of the KTM-unique genes revealed an enrichment for genes annotated to have functional roles in lipid metabolism (Fig 5C). Since misexpression of core metabolic genes alters longevity and stress resistance pathways [69,70], we queried whether these same genes were also misexpressed in animals with reduced levels of DAF-2 (i.e., the insulin receptor), which results in increased stress resistance, improved healthspan, and extended lifespan [71,72]. Indeed, depletion of DAF-2 in different tissues [72], including the intestine, results in transcriptional changes that are consistent with those seen in KTM-fed animals (S8G–S8I Fig). Together, these data suggest that consumption of fermenting KT microbes may remodel host lipid metabolism and stress resilience pathways to restrict fat accumulation and improve healthspan. In C. elegans, the intestine functions as the primary hub for nutrient absorption, lipid storage, and metabolic regulation [52]. Our transcriptome data indicated that genes involved in lipid metabolism are modulated by KTM consumption, prompting us to investigate whether the host transcriptional response to KTMs occurs in the intestine. Using previously established gene expression data for the major tissues, we queried whether each set of diet-induced differentially expressed genes were enriched for a specific tissue [73,74]. We found that in response to KTM consumption there was a striking enrichment for differential expression of intestinal genes, as well as a depletion of neuronal and germline genes (Fig 5D). These data indicate that while genes expressed in the intestine are commonly differentially expressed in animals consuming KTMs, genes expressed in other tissue types tend not to be differentially expressed in KTM-fed animals. To identify candidate genes that may be responsible for the metabolic effects of KTM consumption, we analyzed the expression levels of 5,676 genes that are annotated to function in metabolism [29]. This revealed that several genes known to function in lipid biology have altered expression in KTM-fed animals (Fig 5E and 5F). These included down-regulated genes that act in the β-oxidation of lipids (acdh-1, acdh-2), fatty acid desaturation (fat-5, fat-6, fat-7), or triglyceride synthesis (dgat-2), as well as up-regulated genes that act in lipolysis (lipl-1, lipl-2, lipl-3). These data suggest that expression of specific lipid metabolism genes in the intestine is modulated by KTM consumption. Consistently, intestinal expression of a GFP-based transcriptional reporter for the acdh-1 gene, which encodes a conserved acyl-CoA dehydrogenase that catabolizes short chain fatty acids and branch chained amino acids, was reduced when animals were fed a KTM diet (S8J and S8K Fig). Together, our results suggest that transcriptional regulation of metabolic genes may, at least in part, underlie the reduction in intestinal lipids that we observed in KTM-fed animals.

[END]
---
[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011003

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.