Commensal Fungi Recapitulate the Protective Benefits of Intestinal Bacteria

Abstract

Commensal intestinal microbes are collectively beneficial in preventing local tissue injury and augmenting systemic antimicrobial immunity. However, given the near-exclusive focus on bacterial species in establishing these protective benefits, the contributions of other types of commensal microbes remain poorly defined. Here, we show that commensal fungi can functionally replace intestinal bacteria by conferring protection against injury to mucosal tissues and positively calibrating the responsiveness of circulating immune cells. Susceptibility to colitis and influenza A virus infection occurring upon commensal bacteria eradication is efficiently overturned by mono-colonization with either Candida albicans or Saccharomyces cerevisiae. The protective benefits of commensal fungi are mediated by mannans, a highly conserved component of fungal cell walls, since intestinal stimulation with this moiety alone overrides disease susceptibility in mice depleted of commensal bacteria. Thus, commensal enteric fungi safeguard local and systemic immunity by providing tonic microbial stimulation that can functionally replace intestinal bacteria.

Keywords: Candida albicans; Saccharomyces cerevisiae; commensal microbes; dextran sodium sulfate; fungal immunity; influenza A virus; mannans.

Figure 1. Candida albicans intestinal mono-colonization bypasses the protective necessity of commensal enteric bacteria

(A) Recoverable bacterial colony forming units (CFUs) from feces of mice after supplementing the drinking water with an antibiotic cocktail (ABX) containing ampicillin, gentamicin, metronidazole, neomycin, vancomycin, compared with no-antibiotic treated conventional (CNV) controls housed under specific-pathogen free conditions. (B) Bacterial-specific 16S rDNA qPCR of feces for mice described in panel (A) normalized to conventional (CNV) controls housed under specific-pathogen free conditions. (C) Recoverable fungal CFUs in the feces (left) and each intestinal segment (right) for mice inoculated with C. albicans (CA) and maintained on ABX treatment for 14 days (ABX+CA), compared to ABX treated controls without CA administration (ABX). (D) Weight gain after C. albicans inoculation among antibiotic treated mice (ABX+CA) compared with ABX only controls. (E) Fungal-specific internal transcribed spacer (ITS-1) rDNA (left) and eukaryotic 18S rDNA (right) qPCR of feces for mice described in panel (C) normalized to germ-free (GF) controls. (E) Percent survival after DSS supplementation in the drinking water (for six days) for mice described in panels (A,C). (F) Percent survival after influenza A PR8-OVA (Flu A) intranasal infection (6 × 10⁴ PFUs) for mice described in panels (A,C). (G) Total number of Flu A-specific K^bOVA tetramer⁺ CD8 T cells (left), and IFN-γ⁺ CD8 T cells after in vitro OVA_257–264 peptide stimulation (right), from lungs nine days after Flu A infection for mice described in panels (A,C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by Log-rank (Mantel-Cox) test (F,G) or nonparametric Kruskal-Wallis test with Dunn’s correction (H). Data are representative of at least two independent experiments each with similar results. Bars, mean ± s.e.m. L.o.D., limit of detection. See also Figures S1–3.

Figure 2. Saccharomyces cerevisiae intestinal mono-colonization overturns disease susceptibility induced by commensal bacteria depletion

(A) Recoverable fungal colony forming units (CFUs) in the feces of specific-pathogen free mice administered S. cerevisiae (SC) and maintained on drinking water supplemented with an antibiotic cocktail containing ampicillin, gentamicin, metronidazole, neomycin, vancomycin for 14 days (ABX+CA), compared with antibiotic treated controls without SC inoculation (ABX). (B) Percent survival after DSS supplementation in the drinking water (for six days) for mice described in panel (A). (C) Colon length after DSS treatment (for six days) for mice described in panel (A). (D) Percent survival after influenza A PR8-OVA (Flu A) intranasal infection (6 × 10⁴ PFUs) for mice described in panel (A). (E) Total number of Flu A-specific K^bOVA tetramer⁺ CD8 T cells (left), and IFN-γ⁺ CD8 T cells after in vitro OVA_257–264 peptide stimulation (right), from lungs nine days after Flu A infection for mice described in panel (A). *P < 0.05, **P < 0.01, ***P < 0.001, by ****P < 0.0001, Log-rank (Mantel-Cox) test (B,D) or unpaired t-test with Welch’s correction (C,E). Data are representative of at least two independent experiments each with similar results. Bars, mean ± s.e.m. L.o.D., limit of detection.

Figure 3. Persistent fungal intestinal colonization is required for maintaining their protective benefits

(A) Fungal colony forming units (CFUs) in the feces with or without supplementing fluconazole (FLUC) to specific-pathogen free mice previously administered C. albicans (CA) and maintained on drinking water supplemented with an antibiotic cocktail (ABX) containing ampicillin, gentamicin, metronidazole, neomycin, and vancomycin. (B) Percent survival after DSS supplementation in the drinking water (for six days) for mice described in panel (A). (C) Percent survival after influenza A PR8-OVA (Flu A) intranasal infection (6 × 10⁴ PFUs) for mice described in panel (A). *P < 0.05, **P < 0.001, by Log-rank (Mantel-Cox) test. Data are representative of at least two independent experiments each with similar results. Bars, mean ± s.e.m. L.o.D., limit of detection.

Figure 4. Protective benefits of commensal fungi are mediated by mannans and through TLR4 dependent pathways

(A) Percent survival after DSS supplementation (for six days) among specific-pathogen free mice maintained on drinking water supplemented with an antibiotic cocktail (ABX) containing ampicillin, gentamicin, metronidazole, neomycin, vancomycin, and administered C. albicans (CA) three days after initiating antibiotic treatment, or intrarectally administered mannan, curdlan, zymosan or saline (PBS) every other day starting one day prior to DSS challenge. (B) NF-kB expression induced among RAW-blue macrophages after incubation with or without mannan in the presence or absence of the indicated neutralizing antibodies. (C) Percent survival after DSS supplementation (for six days) among conventional (CNV) dectin-1 deficient mice housed in specific-pathogen free conditions, administered antibiotics (ABX), or C. albicans (CA) inoculated three days after initiating antibiotic treatment (ABX+CA). (D) Percent survival after DSS supplementation (for six days) among conventional (CNV) TLR4 deficient mice housed in specific-pathogen free conditions, administered antibiotics (ABX), or C. albicans (CA) inoculated three days after initiating antibiotic treatment (ABX+CA). *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA with Holm-Sidak’s correction (B) or Log-rank (Mantel-Cox) test (C,D). Data are representative of at least two independent experiments each with similar results. Bars, mean ± s.e.m. See also Figure S4.