Albumin orchestrates a natural host defense mechanism against mucormycosis

Abstract

Mucormycosis is an emerging, life-threatening human infection caused by fungi of the order Mucorales. Metabolic disorders uniquely predispose an ever-expanding group of patients to mucormycosis via poorly understood mechanisms. Therefore, it is highly likely that uncharacterized host metabolic effectors confer protective immunity against mucormycosis. Here, we uncover a master regulatory role of albumin in host defense against Mucorales through the modulation of the fungal pathogenicity program. Our initial studies identified severe hypoalbuminemia as a prominent metabolic abnormality and a biomarker of poor outcome in independent cohorts of mucormycosis patients. Strikingly, we found that purified albumin selectively inhibits Mucorales growth among a range of human pathogens, and albumin-deficient mice display susceptibility specifically to mucormycosis. The antifungal activity of albumin is mediated by the release of bound free fatty acids (FFAs). Importantly, albumin prevents FFA oxidation, which results in loss of their antifungal properties. A high degree of FFA oxidation is found in the sera of patients with mucormycosis. Physiologically, albumin-bound FFAs blocks the expression of the mycotoxin mucoricin and renders Mucorales avirulent in vivo. Overall, we discovered a novel host defense mechanism that directs the pathogen to suppress its growth and the expression of virulence factors in response to unfavorable metabolic cues regulated by albumin. These findings have major implications for the pathogenesis and management of mucormycosis.

Keywords: Albumin; Free fatty acids; Mucorales; Rhizopus; fungi; host defense; mucoricin; mucormycosis; serum.

Figure 1. Specialized activity of albumin against Mucorales.

(a) Serum albumin levels on the time of diagnosis in contemporaneously matched controls (n = 33), patients with pulmonary aspergillosis (n = 50) and patients with mucormycosis (n = 97). ****p < 0.0001, one-way ANOVA and Tukey’s multiple comparisons post hoc test. (b) Kaplan–Meier survival curves of mucormycosis patients with severe hypoalbuminemia (≤ 2.5 g/dL) compared with those of other mucormycosis patients from clinical cohorts in the USA (n = 81), India (n = 95) and France (n = 26). The nonparametric log-rank (Mantel–Cox) test was used to calculate differences between survival curves. (c, d)Correlation between serum albumin levels and R. delemar growth in human sera obtained from patients with (c) cirrhotic disease (n = 18) or (d)hematologic malignancies (n = 20). (e) Assessment of the inhibitory effects of albumin depletion from human serum obtained from healthy individuals on R. delemar(n = 15) and A. fumigatus growth (n = 6). ****p < 0.0001, Wilcoxon matched-pairs signed rank test. (f) Human serum and albumin-rich eluted fragments were analysed via Coomassie blue staining. (g) Length of germinating R. delemar spores cultured in medium or medium supplemented with albumin isolated from human serum, as assessed by time lapse microscopy ****p < 0.0001, multiple unpaired t tests. (h)Representative fluorescence images of Calcofluor White-labelled R. delemar spores cultured for 8 h in regular media or media supplemented with albumin. Scale bar, 50 mm. (i)Inhibition of R. delemar growth by increasing concentrations of human serum albumin (HSA) or bovine serum albumin (BSA). (j) Quantification of the growth of different Mucorales species and other bacterial and fungal human pathogens cultured in regular media or media supplemented with albumin.

Figure 2. Physiological FFAs mediate the antifungal activity of albumin against Mucorales.

(a) Outline of generation of flow-through and isolation of albumin from human serum following filtration with a 3 kDa molecular weight cut-off centrifugal filter (upper panel). Representative images of R. delemar spores cultured for 5 h in medium alone or medium supplemented with isolated human serum albumin or albumin flow through (lower panel). Scale bar, 50 mm. (b) Lipidomic analysis of FFAs of isolated human serum albumin and albumin flow through, obtained as described in a. (c) Antifungal activity of major serum FFAs against R. delemar. Red-striped areas designate physiological FFA serum concentrations. (d) Inhibitory effect of increasing concentrations of various short- to long-chain FFAs on R. delemar growth. (e)(Left) Representative images of R. delemar spores cultured for 5 h in mediasupplemented with isolated human serum albumin or charcoal-stripped isolated human albumin. Scale bar, 50 mm. (Right) Inhibitory effects of BSA, FFA-free BSA and charcoal-stripped BSA on R. delemar growth. ****p < 0.0001, one-way ANOVA and Tukey’s multiple comparisons post hoc test. (f) Inhibitory effects of charcoal-stripped BSA, oleic acid or charcoal-stripped BSA loaded with oleic acid on R. delemar growth. ****p < 0.0001, one-way ANOVA and Tukey’s multiple comparisons post hoc test. (g) Representative fluorescence images of CW-labelled R. delemar spores cultured for 5 h in the presence of FITC-labelled albumin. Scale. Scale bar, 20 mm.

Figure 3. Albumin protects FFAs from oxidation and the subsequent loss of inhibitory activity against Mucorales.

(a) Relative concentrations of oxidized FFAs analysed in the sera of matched controls (n = 27), patients with invasive aspergillosis (n = 6) and patients with mucormycosis (n = 18). ****p < 0.0001, one-way ANOVA and Tukey’s multiple comparisons post hoc test. (b) Relative concentrations of oxidized FFAs analysed in the serum of healthy controls (n = 21), cirrhotic patients (n = 18) and patients with hematologic malignancies (n = 20). **p = 0.0038, ****p < 0.0001, one-way ANOVA and Tukey’s multiple comparisons post hoc test. (c, d) Correlation of the percentage of oxidized serum FFAs with (c) albumin levels and (d) the inhibitory activity of sera from cirrhotic patients (n = 18) or patients with hematologic malignancies (n = 20) against R. delemar. A simple linear regression test was used to determine deviation from zero. (e)Inhibitory effects of increasing concentrations of oleic acid and oxidized oleic acid on R. delemar growth. (f) Representative flow cytometry analysis of Nile Red fluorescence intensity, which is indicative of FFA uptake by R. delemar spores cultured in medium, medium supplemented with oleic acid or medium supplemented with oxidized oleic acid (g)(Left) Representative fluorescent (upper panel) and bright field (lower panel) images of R. delemarspores stained with Nile Red as in f. Scale bar, 20 mm. (Right) Cumulative data of Nile Red mean fluorescence intensity (MFI) in R. delemar spores. ****p < 0.0001, one-way ANOVA and Tukey’s multiple comparisons post hoc test. (h) Serum lipids were isolated from healthy individuals and subjected to oxidation. The inhibitory effects of serum lipids and oxidized serum lipids on R. delemar growth were assessed (n = 13). ***p = 0.0002, Wilcoxon matched-pairs signed rank test. (i) GC–MS analysis of nonoxidized oleic acid (OA) content in OA and BSA-conjugated OA before and after microwave oxidation. **p = 0.0041, ****p < 0.0001, one-way ANOVA and Tukey’s multiple comparisons post hoc test. (j) Representative images of R. delemar spores cultured for 5 h in mediasupplemented with oxidized OA and oxidized BSA-conjugated OA (left panel). Scale bar, 100 mm. (Right) Length of germinating R. delemar spores cultured in mediasupplemented with oxidized OA and oxidized BSA-conjugated OA. ****p < 0.0001, Mann-Whitney test. (k) Inhibitory effect of mock-treated and glycosylated BSA on R. delemar growth. *p = 0.0286, Mann-Whitney test.

Figure 4. Albumin-bound FFAs target Mucorales pathogenicity by inhibiting the expression of mucoricin.

(a) Outline of intratracheal (i.t.) instillation of dormant or swollen R. delemar spores. Swollen spores were generated via R. delemar culture in medium (control-swollen) or medium supplemented with 4.5 g/dL BSA (albumin-swollen) for 3 h. (b) Survival analysis of C57BL/6 mice infected i.t. with 2.5×10⁶ dormant (n = 6), control-swollen (n = 18) or albumin-swollen (n = 12) R. delemar spores as in a. The nonparametric log-rank (Mantel-Cox) test was used to calculate differences between survival curves. (c) Histopathology (H&E staining) of representative lung sections from mice at 0 h and 6 h post infection with control-swollen and albumin-swollen R. delemar spores as described in a. Black arrowheads designate fungal spores. Scale bar, 100 mm. (d) Histopathology (left: active caspase 3; middle: H&E; right: GMS staining) of representative lung sections from mice on day 1 post infection with control-swollen (upper panel) and albumin-swollen (lower panel) R. delemar spores as in a. Scale bar, 100 mm. (e) Outline of RNA-sequencing (RNA-seq) analysis of dormant and swollen R. delemar spores. Swollen spores were generated via R. delemar culture in medium (control-swollen) or medium supplemented with BSA(albumin-swollen) for 3 h and 6 h. (f) Differential mRNA expression of R. delemar virulence-related genes following incubation in medium containing albumin (+albumin) or without albumin (medium) for 6 hours. The log (base 2)-transformed RPKM values that were normalized across all 6 samples are plotted. Red indicates high gene expression; blue indicates low expression. Each column represents an individual sample. (g) Representative confocal images of mucoricin expression in CFW-labelled, control- andalbumin-swollen R. delemar spores after 3 h of culture. Scale bar, 10 mm. (h) Representative confocalimages of mucoricin expression in CFW-labelled, control- and FFA-swollen R. delemar spores. Scale bar, 10 mm. (i) Cumulative data of mucoricin expression in control and albumin-swollen R. delemar sporesacquired in g. ****p < 0.0001, Mann-Whitney test. (j) Cumulative data of mucoricin expression in control- and FFA-swollen R. delemar spores acquired in h. ****p < 0.0001, Mann-Whitney test. (k) Survivalanalysis of C57BL/6 mice infected i.t. with 2.5×10⁶ swollen control-RNAi (n = 14) or mucoricin-RNAi (n = 15) R. delemar spores. The nonparametric log-rank (Mantel-Cox) test was used to calculate differencesbetween survival curves.

Figure 5. Albumin knockout (KO) mice display selective susceptibility to mucormycosis.

(a) (Left panel) Survival analysis of WT (Alb^+/+) and Albumin-KO (Alb^−/−) mice infected intravenously with 1×10⁵ dormant R. delemar spores (disseminated infection, n = 24–27). (Right panel) Survival analysis of neutropenic Alb^+/+ and Alb^−/− mice infected i.t. with 1×106 dormant R. delemar spores (pulmonary infection, n = 24). (b) Survival analysis of neutropenic Alb^+/+ and Alb^−/− mice infected i.t. with 1×10⁶ dormant A. fumigatus spores (pulmonary infection, n = 11). (c) (Left panel) Outline of prophylactic and therapeutic administration models of FFA-free HSA in pulmonary mucormycosis. (Right panel) Survival analysis of neutropenic Alb^−/− mice, either untreated or administered FFA-free HSA prior to infection (left; prophylactic model, n = 10–13) or upon i.t. infection (right; therapeutic model, n = 10–11) with 1×10⁶ dormant R. delemar spores. The nonparametric log-rank (Mantel-Cox) test was used to calculate differences between survival curves in a-c. (d) Histopathology (upper panel: H&E; lower panel: GMS staining) of representative lung sections from neutropenic Alb^+/+ and Alb^−/− mice on day 3 of infection with R. delemar spores. Scale bar, 100 mm. (e) (Left panel) Representative fluorescence images of mucoricin expression in lung sections from neutropenic Alb^+/+ and Alb^−/− mice on day 3 of infection with R. delemar spores. Scale bar, 100 mm. (Right panel) Cumulative data of mucoricin expression in lung sections from neutropenic Alb^+/+ and Alb^−/− mice on day 3 of infection with R. delemar spores. ****p < 0.0001, Mann-Whitney test. (f) Inhibitory effect of mouse serum from Alb^+/+ and Alb+ mice on R. delemar growth (n = 8). ***p = 0.0006, Mann-Whitney test. (g) Representative images of R. delemar spores cultured for 5 h in BALF from Alb^+/+ and Alb^−/− mice. (h) Inhibitory effect of mouse BALF obtained from Alb^+/+ and Alb^−/− mice on R. delemar growth (n = 8). ***p = 0.0007, Mann-Whitney test. (i) Inhibitory effect of mouse serum from Alb^−/− mice and Alb^−/− mice prophylactically supplemented with FFA-free HSA, as in c, on R. delemar growth (n = 4). *p = 0.0286, Mann-Whitney test. (j) Inhibitory effect of isolated mouse serum lipids from Alb^+/+ and Alb^−/− mice on R. delemar growth (n = 7). ***p = 0.0005, one-way ANOVA and Tukey’s multiple comparisons post hoc test. (k) Concentrations of non-oxidized and oxidized FFAs in the serum of Alb^+/+ (left panel) and Alb^−/− (right panel) mice (n = 6). *p = 0.0313, Wilcoxon matched-pairs signed rank test.