Zinc Restriction Alters the Functional Potential of the Gut Microbiome

Zinc Restriction Alters Composition of the Gut Microbiome

Background and Design

Abstract Altered Gene Expression in

Intestines of Zinc Restricted Fish

Summary

Restriction of Dietary Zinc and its Impact on the Structure and Function of the Gut Microbiome of Adult Zebrafish

Christopher A. Gaulke1, Laura M. Beaver4, Carrie L. Barton3, Carmen P. Wong4, Robert L. Tanguay3, Emily Ho4, Thomas J. Sharpton1,2

Growing evidence suggests that the microbiome plays an important role in establishing and maintaining gastrointestinal homeostasis. Changes in diet are known to have rapid and profound impacts on microbiome structure. However, less is known about how micronutrients contribute to the structure and function of the gut microbiome and how this in turn can influence host health. Zinc is an essential micronutrient that plays a vital role in normal growth and development, protein synthesis and immune function. Zinc is also essential for the growth of most microorganisms and the host microbiota competes for zinc in the gastrointestinal tract. We examined the effects of limiting dietary zinc concentrations on the host microbiome structure and function using whole-genome shotgun and 16S amplicon sequencing for adult zebrafish fed a zinc deficient defined diet (n=15), zinc adequate defined diet (n =15), and a zinc adequate commercially available zebrafish chow (n=15). While the animals receiving the zinc adequate diets harbored microbiomes similar in taxonomic composition and abundance, significant differences in taxonomic abundance and composition were observed between the microbiome of fish receiving the zinc adequate and zinc deficient diets. The abundance of the phyla Fusobacteria and Tenericutes were significantly increased and Proteobacteria decreased in the animals receiving the zinc deficient diet when compared to those receiving zinc adequate diets (p <0.05). Similar observations were made at lower taxonomic and OTU level analysis. The functional capacity of the gut microbiomes of fish receiving zinc deficient and zinc adequate diets was then quantified using shotgun metagenomics. Zebrafish intestinal gene expression was quantified to examine potential relationships between altered microbiome function and host physiology. These experiments provide novel insights into the effects of nutrient limitation on gut microbiome structure and function and expand our understanding of how these shifts may be involved in altered host physiology.

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1. Zinc restriction is associated with altered gut community composition in zebrafish. 2. Marginal zinc intake was associated with increased abundance of microbial genes involved in glycan degradation and multidrug resistance. 3. Fish fed zinc deficient diets have increased expression of genes involved in cell death, zinc transport, and epithelial development. 4. Mice and fish may respond similarly to zinc restriction.

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Figure 1. Experimental timeline. Forty-five 5D line zebrafish were fed one of three diets 1) a standard lab diet (Gemma Micro 300; LDC), 2) a defined diet with sufficient levels of zinc (DDC), and 3) a defined diet with deficient levels of zinc (CZMD). Two months after hatch fish were randomly assigned to a diet and tank (n=5/tank; three tanks/diet). A Fecal samples was collected 7.5 months after the switch to diet. Eight months after the diet switch another fecal sample was collected, fish were necropsied, and the intestinal tract was extracted.

Figure 2. Diagram of fecal collection in zebrafish. Fish were isolated in spawning tanks (1 fish/tank) and housed overnight. The design of the spawning tanks allowed fecal pellets to fall to the bottom of tanks where they could not be consumed by fish. Feces was collected the following morning and stored at -20˚C until processing.

Table 1. Diet Composition.

Diet Protein(%) Fat(%) Carbs(%) Fiber(%)Vitamins&Minerals(%)

Zinc(mg/kg)

LDC 59 14 9.5 0.2 17.3 130DDC 49.5 12 26.5 3 9 33CZMD 49.5 12 26.5 3 9 12.46

Figure 3. Zinc restriction is associated with altered microbial community structure. A) 16S rRNA community analysis workflow. The V4 region of the 16S rRNA gene was amplified and sequenced (250bp PE) on an Illumina miseq. The resulting reads were demultiplex and quality filtered using ea-utils and analyzed in QIIME. B) Non-metric multidimensional scaling plot of OTU abundances across diets. C) The top five most abundant phyla across diets. D-F) Phyla level abundance plots across diets. * p < 0.05, ** p < 0.01.

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Figure 4. Zinc restriction is associated with altered functional potential of zebrafish gut microbial communities. A) Metagenomics workflow. Extracted DNA from zebrafish fecal samples was used to construct and sequence 21 Nextera XT libraries (n=7/diet). Sequence reads were quality filtered with shotcleaner, annotated with shotmap, and subjected to pathway analysis. B) Non-metric multidimensional scaling plot of protein family abundances across diets.

Acknowledgements

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Glycan DegradationFigure 5.Zinc deficiency is associated with altered microbiome functional potential. A) A heat map of KEGG orthologous protein families that are significantly differentially abundant across zinc sufficient and zinc deficient diet (Kruskal-Wallis with Mann-Whitney U-Test post hoc). Only the protein families that significantly differed in abundance between CZMD and DDC and between CZMD and LDC diets were included in the heat map. A colored bar above the heat map indicates the diet of each sample. B) Cumulative relative pathway abundance of glycan degradation across all diets. C) Cumulative module relative abundance of multidrug resistance across all diets. p < 0.05 for pathway analysis.

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B Figure 6. Low dietary zinc results in altered gut gene expression profiles. A) Workflow of gene expression analysis. Extracted RNA was used to generate PrepX RNA libraries and sequenced on an Illumina HiSeq 3000 (150bp PE). Sequencing reads were quality filtered and trimmed using ea-utils, aligned to the zebrafish genome using TopHat, and differential gene expression was quantified using DESeq2. B) A principle components analysis plot of gene expression across different diets.

Figure 7. Gene enrichment analysis indicates zinc deficiency is associated with altered homeostasis and increased cell turnover. A) A heat map of altered gene expression between zinc deficient and zinc adequate fish. The colored bar above the heat map indicates the group identity of the each sample. Only genes with a 2-fold change or greater were included in the heat map. B) GO term enrichment analysis of up (red bars) and down (green bars) regulated genes (CZMD vs DDC).

cell death

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cellular homeostasis

response to bacterium

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Oregon State University 1Department of Microbiology, 2Department of Statistics, 3Department of Environmental and Molecular Toxicology, 4Department of Nutrition and Exercise Sciences

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We would like to thank the employees of the Sinnhuber Aquatic Research Laboratory at Oregon State University for their assistance with zebrafish husbandry. We also thank members of the Center for Genome Research and Biocomputing at Oregon State University for their assistance in sequencing libraries and for their assistance with the computational infrastructure. Funding for this work was made available by a NIEHS supported Environmental Health Sciences Center P30-ES000210 and a pilot grant to T.J.S., institutional funds to T.J.S, and NIEHS funds to E.H R21-ES023937.

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Figure 8. Venn diagram of differentially abundant KOs in fish and in mice. Co-housed C57Bl/6 mice were fed zinc adequate or zinc restricted diets similar to those fed to fish. Shotgun metagenome libraries were constructed from DNA extracted from mouse fecal pellets, sequenced, and analyzed as in Figure 4. Differentially abundant protein families in mice were compared to those observed in fish. Approximately 20% of differentially abundantprotein families are shared between fish and mice.

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