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Mycobiota and diet-derived fungal xenosiderophores promote Salmonella gastrointestinal colonization

Nature Microbiology volume 7, pages 2025–2038 (2022)Cite this article

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Abstract

The fungal gut microbiota (mycobiota) has been implicated in diseases that disturb gut homeostasis, such as inflammatory bowel disease. However, little is known about functional relationships between bacteria and fungi in the gut during infectious colitis. Here we investigated the role of fungal metabolites during infection with the intestinal pathogen Salmonella enterica serovar Typhimurium, a major cause of gastroenteritis worldwide. We found that, in the gut lumen, both the mycobiota and fungi present in the diet can be a source of siderophores, small molecules that scavenge iron from the host. The ability to use fungal siderophores, such as ferrichrome and coprogen, conferred a competitive growth advantage to Salmonella strains expressing the fungal siderophore receptors FhuA or FhuE in vitro and in a mouse model. Our study highlights the role of inter-kingdom cross-feeding between fungi and Salmonella and elucidates an additional function of the gut mycobiota, revealing the importance of these understudied members of the gut ecosystem during bacterial infection.

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Fig. 1: Salmonella is able to utilize purified fungal siderophores.
Fig. 2: Intestinal fungi can be a source of fungal siderophores.
Fig. 3: Dietary changes profoundly alter gut mycobiome composition.
Fig. 4: Mouse and human diets can contain fungal siderophores that promote Salmonella growth.
Fig. 5: Fungal siderophores confer a competitive advantage to Salmonella in vivo.

Data availability

Raw sequence reads have been deposited at NCBI Sequence Read Archive under project PRJNA769709, and the UNITE (v.8.2) database78 was used for all microbiome analysis. Data are additionally available at https://github.com/BehnsenLab/Siderophores. Data used to generate figures are available as supplementary tables and as source data files published alongside this manuscript. Source data are provided with this paper.

Code availability

Original code used has been deposited at https://github.com/BehnsenLab/Siderophores.

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Acknowledgements

We thank all the members of the Behnsen lab for technical assistance, helpful discussion and critical reading of the manuscript. D. Kiani, K. Perfecto, K. Jaswal and O. A. Todd assisted with the husbandry of germ-free mice. This work was supported by UIC Institutional Startup Funds to J.B. and grants from the National Institute of Health (R01DK093426 to D.M.U. and R01AI143641 to J.B.). Illustrations were created at Biorender.com.

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Illinois Chicago, Chicago, IL, USA

    William Santus, Amisha P. Rana, Jason R. Devlin, Kaitlyn A. Kiernan, Carol C. Jacob & Judith Behnsen

  2. Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA, USA

    Joshua Tjokrosurjo

  3. Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    David M. Underhill

  4. F. Widjaja Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    David M. Underhill

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  1. William Santus
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Contributions

J.B. conceived and administrated the study and was the primary supervisor. W.S. additionally supervised parts of the study and was responsible for execution and analysis of the majority of the experimental work, including all mouse experiments, and most of the in vitro experiments. J.B., J.R.D., K.A.K., C.C.J. and J.T. performed select bacterial growth curves, and J.B., K.A.K., J.R.D. and J.T. generated select Salmonella strains. W.S., A.P.R., K.A.K., J.R.D. and J.B. were involved in the husbandry of germ-free mice. D.M.U. was responsible for microbiome sequencing, and D.M.U. and A.P.R. analysed microbiome data and generated custom scripts. W.S. wrote the original draft of the manuscript. J.B., W.S. and D.M.U. revised the draft with input from all authors.

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Correspondence to Judith Behnsen.

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Extended data

Extended Data Fig. 1 Purified fungal siderophores promote Salmonella growth.

(a) Growth of Salmonella strains WT (n = 5), iroN (DMEM n = 5, DMEM + Lcn2 n = 6), iroN fhuA (DMEM n = 3, DMEM + Lcn2 n = 4), iroN fhuE (n = 3) at different time points (2, 5, 8 h) in DMEM/F12 + 10% FBS or DMEM/F12 + 10% FBS + 100 ng/ml lipocalin-2. (b) Growth of Salmonella strains WT, iroN, iroN fhuA or iroN fhuE in DMEM/F12 + 10% FBS + 100 nM coprogen (n = 3) or DMEM/F12 + 10% FBS + 100 nM ferrichrome (n = 3). (c) Growth of Salmonella strains WT, iroN fepA and iroN fepA fhuA fhuE in LB. Time points were taken at 2, 5, 8 and 24 h (n = 3). (d) Growth of Salmonella strains WT and fhuA fhuE at 1 h, 3 h, 5 h, 7 h post inoculation in co-culture in minimal medium (n = 3), minimal medium + 100 nM ferrichrome (n = 3), minimal medium + 100 ng/ml deferoxamine (n = 3). Statistics: 2way ANOVA with Tukey’s multiple comparisons test. Data represent geometric mean + /- geometric SD. Numerical p values are indicated within the figure.

Source data

Extended Data Fig. 2 Fungal siderophores from gut fungi promote Salmonella growth.

(a) Viability of fungi at 48 h in minimal medium measured as dry weight, mg/tube for A. niger and mouse isolate or CFU/mg for S. cerevisiae and M. furfur (n = 3). (b-d) Growth of Salmonella strains iroN fepA and iroN fepA fhuA fhuE at 8 h post inoculation in co-culture in minimal media with or without the addition of (b) live fungi (n = 4), (c) fungal supernatant (n = 3) or (d) fungal homogenate (n = 3). (e) Growth of Salmonella WT and Malassezia furfur in co-culture or alone in LB supplemented with 1% Tween40 and 0.2% oleic acid (n = 4). Data represent geometric mean and +/− geometric SD. Statistics: one-way ANOVA with Tukey’s multiple comparisons test. Numerical p values are indicated within the figure.

Source data

Extended Data Fig. 3 Diet composition affects composition of the bacterial microbiota.

(a) Principal component analysis of 16 S, left panel, and ITS, right panel, sequencing data of diet switch from chow LM485 to purified 50ppm diet (n = 5). (b) Relative abundance of selected bacterial genera prior and after diet change (n = 5) (c) Relative abundance of selected fungal genera prior and after diet change (n = 5). (b, c) Data represent mean +/− SD. Statistics: unpaired t test. Numerical p values are indicated within the figure.

Source data

Extended Data Fig. 4 Diet composition affects composition of the fungal microbiota.

(a) Principal component analysis of 16 S, left panel, and ITS, right panel, sequencing data of diet switch from chow LM485 to chow 2914 (n = 5). (b) Relative abundance of selected bacterial genera prior and after diet change (n = 5) (c) Relative abundance of selected fungal genera prior and after diet change (n = 5). (b, c) Data represent mean +/− SD. Statistics: unpaired t test. Numerical p values are indicated within the figure.

Source data

Extended Data Fig. 5 Fungal siderophores from mouse and human food promote Salmonella growth.

(a) Growth of Salmonella strains iroN fepA and iroN fepA fhuA fhuE at 8 h post inoculation in minimal medium with the addition of chow suspensions from chow 5066 (n = 7), chow LM485 (n = 7), purified 200ppm (n = 7) and purified 50ppm (n = 3) in aerobic shaking liquid culture. (b) Growth of Salmonella strains iroN fepA and iroN fepA fhuA fhuE at 8 h post inoculation in minimal medium with the addition of chow suspensions and ferrichrome (5 nM, 10 nM, 50 nM) (n = 3) (c, d) Growth of Salmonella strains iroN fepA and iroN fepA fhuA fhuE at 8 h post inoculation in minimal medium with the addition of chow suspensions in (c) aerobic non-shaking liquid culture (n = 3) and (d) anaerobic non-shaking liquid culture (n = 3). (e) Growth of Salmonella strains iroN fepA and iroN fepA fhuA fhuE at 8 h post inoculation in minimal medium with the addition of human food suspensions (minimal medium, Agaricus bisporus mushrooms, Quorn™, sourdough n = 3; beer, red wine, white bread, chicken, flour, lettuce, banana n = 4; blue cheese n = 5). Data are depicted as geometric mean +/− geometric SD. Statistics: Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Numerical p values are indicated within the figure.

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Extended Data Fig. 6 Siderophores from diet and mycobiota promote Salmonella gut colonization.

(a) mRNA expression of innate immune genes extracted from cecal tissue 96 h post infection with WT Salmonella or iroN fepA Salmonella. Expression is normalized to housekeeping gene Actb (Tnf, Ifng, Il17a, Il22 n = 8; Cxcl1 WT n = 7, iroN fepA n = 8). (b) Colonization measured as CFU/mg of Salmonella strains iroN fepA and iroN fepA fhuA in fecal samples of SPF mice collected at different time points post infection [24 h (n = 8), 48 h (n = 8), 72 h (n = 5), 96 h (n = 8)]. (c) Dissemination measured as CFU/mg of Salmonella strains iroN fepA and iroN fepA fhuA in peripheral organs of SPF mice collected at 96 h post infection [liver, spleen (n = 7), Peyer’s patches (PP) and mesenteric lymph nodes (MLN) (n = 8)]. (d) Competitive index of Salmonella strain iroN fepA over iroN fepA fhuA in organ homogenate liver, spleen (n = 7), Peyer’s patches (PP) and mesenteric lymph nodes (MLN) (n = 8) at 96 h. (e) Colonization measured as CFU/mg of Salmonella strains iroN fepA and iroN fepA fhuA fhuE in fecal samples of ASF mice collected at different time points post infection [24 h (chow n = 19, purified n = 20), 48 h (chow n = 19, purified n = 20), 72 h (chow = 15, purified n = 16), 96 h (chow n = 14, purified n = 16)]. (f) Competitive index (CI) of Salmonella strain iroN fepA over iroN fepA fhuA fhuE in fecal samples of ASF mice at 24 h, 48 h, 72 h, and 96 h. Each line represents CI development in one mouse (shown are mice with samples at more than 2 time points; chow n = 15, purified n = 16). (g) Slope of CI development in (f) (mice with samples at all time points; chow n = 14, purified n = 16). (h) CI of Salmonella strain iroN fepA over iroN fepA fhuA fhuE in fecal samples of ASF mice at 96 h compared to 24 h (mice with samples at both time points; chow n = 14, purified n = 16). (i) Colonization measured as CFU/mg of Salmonella strains iroN fepA and iroN fepA fhuA fhuE in fecal samples of ASF mice collected at 96 h post infection (chow n = 5, purified n = 6, purified + fungi n = 7). Data are depicted as geometric mean +/− geometric SD (a-c, e, i), box plot from 25th to 75th percentile, median, minimum and maximum (d), or median with 95% confidence interval (g, h). Statistics: For panel (a), (d), (g), and (h) one sample t test compared to no competitive advantage (CI = 1) was used. For panel (g) and (h) unpaired Mann-Whitney test was used. Numerical p values are indicated within the figure.

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Extended Data Fig. 7 Proposed model.

Early during Salmonella infection, before the onset of inflammation, Salmonella can acquire iron in the gut environment via secretion of siderophores enterobactin and salmochelin. Other sources of iron are siderophores present in the mouse diet and siderophores produced by the mycobiota. At later stages of infection, after the onset of inflammation, host protein lipocalin-2 binds enterobactin and prevents Salmonella to acquire iron via this siderophore, rendering fungal siderophores present in the diet and produced by the mycobiota more important for Salmonella growth in the murine gut.

Supplementary information

Reporting Summary

Supplementary Table

Relative abundances 16S and ITS, diet information and resources table.

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Santus, W., Rana, A.P., Devlin, J.R. et al. Mycobiota and diet-derived fungal xenosiderophores promote Salmonella gastrointestinal colonization. Nat Microbiol 7, 2025–2038 (2022). https://doi.org/10.1038/s41564-022-01267-w

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