Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors

Abstract

The fungal pathogen Candida albicans has a multilayered cell wall composed of an outer layer of proteins glycosylated with N- or O-linked mannosyl residues and an inner skeletal layer of beta-glucans and chitin. We demonstrate that cytokine production by human mononuclear cells or murine macrophages was markedly reduced when stimulated by C. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediated by mannose receptor binding to N-linked mannosyl residues and by TLR4 binding to O-linked mannosyl residues. Residual cytokine production was mediated by recognition of beta-glucan by the dectin-1/TLR2 receptor complex. C. albicans mutants with a cell wall defective in mannosyl residues were less virulent in experimental disseminated candidiasis and elicited reduced cytokine production in vivo. We concluded that recognition of C. albicans by monocytes/macrophages is mediated by 3 recognition systems of differing importance, each of which senses specific layers of the C. albicans cell wall.

Figure 1. Cell wall morphology in the C. albicans strains used in this study.

(A–E) TEM micrographs. (A) Wild-type strain NGY152 [CAI-4 plus CIp10 vector]. (B) och1 null (strain NGY357; ref. 26) or doxycycline-regulated conditional (strain NGY361; ref. 29) mutants, which are defective in the branched outer N-linked mannosyl chains. (C) mnt1 mnt2 mutant (strain NGY337; ref. 27), which lacks 4 terminal O-linked α_1,2-mannosyl residues. (D) pmr1 mutant (strain NGY355; ref. 26), which has gross defects in mannosylation, characterized by absence of phosphomannan and reduced O-linked and N-linked glycans. (E) mnn4 mutant (strain CDH15; ref. 28), which lacks phosphomannan. Scale bar: 100 nm. (F and G) Structure of the N- (F) and O-linked (G) glycans and the site of action of deleted gene products. Man, mannosyl; β-GlcNAc, β N-acetylglucosamine.

Figure 2. The role of mannan for the cytokine stimulation by C. albicans .

(A) Human MNCs or peritoneal macrophages from C57BL/6J mice were stimulated for 24 hours at 37°C with 50 μg/ml purified mannan from C. albicans. (B) Peritoneal macrophages from wild-type mice and mice deficient in MyD88, TLR2, or TLR4 were stimulated with mannan. TNF and IL-6 concentrations were measured in the supernatants by specific RIA and ELISA, respectively. Results are pooled data from 2 separate experiments with a total of 10 mice per group. (C) Purified mannan was preincubated for 1 hour at 37°C with human MNCs before stimulation with 1 × 10⁶ yeast cells/ml. Supernatants were collected after additional incubation for 24 hours, and TNF and IL-6 were measured. (D) Human MNCs were stimulated for 24 hours at 37°C with whole cells of either wild-type C. albicans (strain NGY152; ref. 26), a mutant defective in the Golgi transporter pmr1 (strain NGY355; ref. 26), or a control strain in which a wild-type copy of PMR1 was introduced into the pmr1 mutant (strain NGY356; ref. 26). TNF and IL-6 concentrations were assessed by RIA and ELISA, respectively. Results (mean ± SD) are pooled triplicate data from 2 separate experiments with a total of 8 volunteers per group. *P < 0.05 versus wild-type.

Figure 3. The role of N- and O-linked mannosyl residues for cytokine stimulation by C. albicans .

MNCs were stimulated for various time points with the various C. albicans strains: the wild-type parent NGY152 strain; the och1 mutant (strain NGY357; ref. 29), defective in N-linked mannan; the mnt1 mnt2 mutant (strain NGY337; ref. 27), defective in O-linked mannan; and the mnn4 mutant (strain CDH15; ref. 28), defective in phosphomannan. (A and C) C. albicans concentration-dependent stimulation curves for TNF (A) and IFN-γ (C) after stimulation for 24 hours. (B and D) Time-dependent stimulation curves for the 2 cytokines when MNCs were stimulated with the various C. albicans strains. Results (mean ± SD) are pooled triplicate data from 2 separate experiments with a total of 8 volunteers per group. *P < 0.05; **P < 0.01 versus wild-type.

Figure 4. Reintegration of the defective genes restores cytokine production.

(A) MNCs were stimulated with the parent NGY152 strain, the N-linked mannosyl-defective C. albicans strain (och1; strain NGY357; ref. 29), and the complemented reintegrant och1/och1/OCH1 strain (strain NGY358). (B) Stimulation was also performed with the conditional doxycycline-dependent mutant (pTET/och1; strain NGY361; ref. 29) in both the absence and the presence of doxycycline (doxy). (C) Comparison of the mnt1 mnt2 mutant (strain NGY337; ref. 27), defective in O-linked mannosyl residues, with the mnt1 mnt2 + MNT1 reintegrant strain (strain NGY335; ref. 27). After 24 hours’ stimulation at 37°C, supernatants were collected, and cytokines were determined by RIA or ELISA. Results (mean ± SD) are pooled triplicate data from 2 separate experiments with a total of 8 volunteers per group. *P < 0.05; **P < 0.01 versus wild-type.

Figure 5. Differential recognition of O - andN -linked mannosyl residues by TLR4 and MR.

(A) Human MNCs were stimulated with the various C. albicans strains — the parent NGY152 strain; the och1 mutant (strain NGY357; ref. 29), defective in N-linked mannosylation; and the mnt1 mnt2 mutant (strain NGY337; ref. 27), defective in O-linked mannosylation — in the presence of monoclonal antibodies against TLR4 or MR or a isotype-matched control antibody. After 24 hours’ stimulation at 37°C, supernatants were collected, and TNF concentration was measured by RIA. Results are pooled triplicate data from 2 separate experiments with a total of 8 volunteers per group. (B) Murine peritoneal macrophages from TLR4^+/+ C57BL/10J and TLR4^–/– ScCr mice were stimulated with the various C. albicans strains: NGY152, the och1 mutant (NGY357; ref. 29), and the mnt1 mnt2 mutant (NGY337; ref. 27). After 24 hours’ stimulation at 37°C, supernatants were collected, and TNF levels were determined by RIA. Results (mean ± SD) are pooled data from 2 separate experiments with a total of 10 mice per group. *P < 0.05 versus stimulation in the presence of control antibodies (A) or versus TLR4^+/+ mice (B).

Figure 6. The role of β-glucan/dectin-1 interaction for cytokine stimulation by C. albicans .

The role of β-glucan/dectin-1 interaction for C. albicans–induced TNF was investigated using 2 approaches with combinations of mutant C. albicans strains and receptor blockade: (A) stimulation with the och1 NGY357 strain (29) in TLR4^–/– mice and (B) stimulation with mnt1 mnt2 NGY337 strain (27) in the presence of anti-MR antibodies in human MNCs. In both situations, the signals induced by N-linked mannosyl/MR and O-linked mannosyl/TLR4 complexes were deficient. The residual cytokine production stimulated by C. albicans in these 2 experimental conditions was completely blocked by laminarin, a ligand of dectin-1. Results (mean ± SD) are pooled data from 2 separate experiments with a total of 10 mice per group (A) or 8 human volunteers (B). *P < 0.05; **P < 0.01; ***P < 0.001 versus wild-type.

Figure 7. The role of N-linked mannosyl residues in virulence and in vivo cytokine production of C. albicans .

(A) Survival of mice infected intravenously with 5 × 10⁶ CFU of wild-type C. albicans, the och1 strain, or the reintegrant control strain. (B) Fungal burden of C. albicans in the kidneys of mice infected intravenously with 1 × 10⁵ CFU of the strains described in A. (C) Cytokine levels on day 3 in the kidneys of mice infected with the strains described in A. Data are presented as means ± SD. Results are pooled data from 2 separate experiments with a total of 10 mice per group. *P < 0.05 versus wild-type.