Activation of HIF-1α and LL-37 by commensal bacteria inhibits Candida albicans colonization

Journal name:
Nature Medicine
Volume:
21,
Pages:
808–814
Year published:
DOI:
doi:10.1038/nm.3871
Received
Accepted
Published online

Abstract

Candida albicans colonization is required for invasive disease1, 2, 3. Unlike humans, adult mice with mature intact gut microbiota are resistant to C. albicans gastrointestinal (GI) colonization2, 4, but the factors that promote C. albicans colonization resistance are unknown. Here we demonstrate that commensal anaerobic bacteria—specifically clostridial Firmicutes (clusters IV and XIVa) and Bacteroidetes—are critical for maintaining C. albicans colonization resistance in mice. Using Bacteroides thetaiotamicron as a model organism, we find that hypoxia-inducible factor-1α (HIF-1α), a transcription factor important for activating innate immune effectors, and the antimicrobial peptide LL-37 (CRAMP in mice) are key determinants of C. albicans colonization resistance. Although antibiotic treatment enables C. albicans colonization, pharmacologic activation of colonic Hif1a induces CRAMP expression and results in a significant reduction of C. albicans GI colonization and a 50% decrease in mortality from invasive disease. In the setting of antibiotics, Hif1a and Camp (which encodes CRAMP) are required for B. thetaiotamicron–induced protection against C. albicans colonization of the gut. Thus, modulating C. albicans GI colonization by activation of gut mucosal immune effectors may represent a novel therapeutic approach for preventing invasive fungal disease in humans.

At a glance

Figures

  1. Gastrointestinal colonization levels of Candida spp. in antibiotic-treated adult, germ-free adult mice and in infant to adolescent mice.
    Figure 1: Gastrointestinal colonization levels of Candida spp. in antibiotic-treated adult, germ-free adult mice and in infant to adolescent mice.

    CA SC5314 colonization levels in (a) adult C57/BL6 mice (Harlan) pretreated with STR, PCN, PS, clindamycin (C), metronidazole (M) or without antibiotics (sterile water) and then orally gavaged with CA. Mice continued on antibiotic water throughout the experiment; n = 8 per group. (b) Non-antibiotic-treated adult germ-free mice (C57/BL6); n = 4 per group. (c) Non-antibiotic-treated P14, P28 and P42 mice (C57BL/6, Harlan); n = 4 per group. (d,e) Colonization levels of other CA strains (d) and other Candida species (e). Adult C3H/HeN mice (Harlan) pretreated with or without antibiotics in the drinking water for 5 d, then orally gavaged with CA (d) or other Candida species (e). Mice continued on antibiotic water throughout the experiment; n = 4 per group. Candida levels were measured every 7 d (ac) or 21 d (d,e) after oral gavage. In all graphs, points represent results from individual animals. Horizontal lines with bars represent the median with interquartile range.

  2. Clostridial Firmicutes and Bacteroidetes promote C. albicans GI colonization resistance.
    Figure 2: Clostridial Firmicutes and Bacteroidetes promote C. albicans GI colonization resistance.

    (a) CA colonization levels of mice pretreated with antibiotics and orally gavaged with CA. CA levels measured 7 d after oral gavage; n = 8 per group. (b) Relative abundance of bacterial phyla as determined by 16S rRNA sequencing of fecal specimens collected from C3H/HeN mice treated with sterile water, STR, PCN or PS; n = 3 per group. (c) Bacterial group qPCR (copies per gram of feces) performed on fecal genomic DNA collected from mice treated with sterile water or oral antibiotics. n = 4 per group. (d,e) CA colonization levels (d) and bacterial group qPCR (e) in PS-treated CA-colonized mice after cessation of the oral antibiotic (discontinued on day 0). CA levels and bacterial group qPCR measured every 7 d; n = 4–5 per group. (f) Bacterial (black circles) and CA (red triangles) levels in antibiotic-treated mice colonized with CA and gavaged with one bacterial commensal species. Colonization levels measured 14 d after bacterial gavage; n = 8 per group. (g,h) Bacterial (black circles) and CA (red triangles) levels in mice colonized with CA and then gavaged with B. producta (g) or B. thetaiotamicron (h). Colonization levels measured at specified time points after bacterial gavage; n = 4 per group. For all experiments, points represent results from individual animals. Horizontal lines represent the median with interquartertile range (a,d,f–h). Bars represent the mean ± s.e.m. (c,e); statistical analysis by Mann-Whitney test, *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant.

  3. B. theta induces Hif1a and Camp in mouse colons.
    Figure 3: B. theta induces Hif1a and Camp in mouse colons.

    (a,b) Hif1a (a) and Camp (b) mRNA expression in colons resected from CA colonization–resistant mice (treated with water or STR) and CA colonization–susceptible mice (treated with PCN or PS); n = 4 per group. (c,d) mRNA expression of HIF1A (c, left), Hif1a (c, right), CAMP (d, left) or Camp (d, right) measured in cultured human colonocytes exposed or not exposed to CA (c,d left) and colons of antibiotic-treated mice with or without CA colonization (c,d right); n = 4. (e,f) Hif1a (e) or Camp (f) mRNA expression measured in the colons of antibiotic treated mice ± oral gavage with commensal bacteria; n = 4 per group. (g) Representative western blot of CRAMP expression from distal colon extracts of wild-type (WT), Camp-knockout (KO) and antibiotic-treated mice ± oral gavage with commensal bacteria. Synthetic CRAMP peptide (5 ng, amino acids 135–173, 4.419 kDa) was used as a positive control. Camp-KO protein extracts were used as a negative control. Actin was used as a loading control. &, nonspecific band from mouse colon protein extracts. (h) Normalized CRAMP expression. Densitometric values obtained for CRAMP immunoblots were normalized to the optical densities of corresponding immunoblots for actin; n = 4 per group. WT, wild-type. (i,j) Hif1a (i) and Camp (j) mRNA expression measured in the distal colons of germ-free mice colonized with CA, theta (BT) or theta and CA. For all experiments, n = 4 per group. All data shown are means ± s.e.m. Statistical analysis by Mann-Whitney test. *P < 0.05; **P < 0.01. NS, not significant.

  4. L-mimosine-dependent activation of HIf1a and Camp in vivo decreases C. albicans GI colonization and dissemination.
    Figure 4: L-mimosine–dependent activation of HIf1a and Camp in vivo decreases C. albicans GI colonization and dissemination.

    (a,b) C. albicans GI colonization levels in Hif1afl/fl and Hif1afl/flVil-Cre+ (a), or Camp-knockout (KO) (b) mice treated with antibiotics, colonized with CA and then treated or untreated with L-mimosine; n = 8 per group. Bars represent the mean ± s.e.m. (c,d) Survival curves of Hif1afl/fl and Hif1afl/flVil-Cre+ (c) or Camp-KO (d) mice treated with antibiotic water, colonized with CA, treated or untreated with L-mimosine for 5 d, and then given cyclophosphamide (Cy). L-Mimosine treatment continued for an additional 7 d after the first cyclophosphamide dose; n = 8 per group. (e,f) Hif1a (e) and Camp (f) mRNA levels in colons of Hif1afl/fl, Hif1afl/flVil-Cre+ and Camp-KO mice treated or untreated with L-mimosine; n = 4. All data shown are means ± s.e.m. Assays were performed in triplicate. (g) C. albicans (red triangles) and B. theta (black circles) GI colonization levels in antibiotic-treated Hif1afl/fl, Hif1afl/flVil-Cre+ and Camp-KO mice; n = 6 for Hif1afl/fl and Camp-KO mice; n = 5 for Hif1afl/flVil-Cre+ mice. Black circles (B. theta) and red triangles (CA) represent results from individual animals. Horizontal lines represent the median with interquartertile range. Statistical analysis by Mann-Whitney test (a,b,eg) or log-rank test (c,d). *P < 0.05; **P < 0.01; NS, not significant.

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Author information

Affiliations

  1. Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas.

    • Di Fan,
    • Laura A Coughlin,
    • Megan M Neubauer,
    • Tiffany R Simms-Waldrip &
    • Andrew Y Koh
  2. Department of Clinical Science, University of Texas Southwestern Medical Center, Dallas, Texas.

    • Jiwoong Kim,
    • Min Soo Kim,
    • Xiaowei Zhan &
    • Yang Xie
  3. Center for Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, Texas.

    • Xiaowei Zhan &
    • Lora V Hooper
  4. Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas.

    • Yang Xie &
    • Andrew Y Koh
  5. Department of Immunology, University of Texas Southwestern Medical Center, Dallas, Texas.

    • Lora V Hooper
  6. The Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas.

    • Lora V Hooper
  7. Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas.

    • Lora V Hooper &
    • Andrew Y Koh

Contributions

A.Y.K. and L.V.H. conceived and designed the experiments. L.V.H. provided gnotobiotic mice and mucosal immunology instruction and support. A.Y.K., D.F., L.A.C., T.R.S.-W. and M.M.N. performed the experiments. A.Y.K., J.K., M.K., X.Z. and Y.X. conducted microbial profiling and statistical analysis. A.Y.K. and L.V.H. analyzed the data. A.Y.K. wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

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