Aspergillus fumigatus conidial surface-associated proteome reveals factors for fungal evasion and host immunity modulation

Abstract

Aspergillus fumigatus causes aspergillosis and relies on asexual spores (conidia) for initiating host infection. There is scarce information about A. fumigatus proteins involved in fungal evasion and host immunity modulation. Here we analysed the conidial surface proteome of A. fumigatus, two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis, as well as pathogenic Aspergillus lentulus, to identify such proteins. After identifying 62 proteins exclusively detected on the A. fumigatus conidial surface, we assessed null mutants for 42 genes encoding these proteins. Deletion of 33 of these genes altered susceptibility to macrophage, epithelial cells and cytokine production. Notably, a gene that encodes a putative glycosylasparaginase, modulating levels of the host proinflammatory cytokine IL-1β, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins are important for evasion and modulation of the immune response at the onset of fungal infection.

Extended data Fig. 1 |. Phenotypic comparison between A. fumigatus and A. fischeri isolates (n=8).

Statistical Multivariate analyses of fungal phenotypes. a. Principal component analysis (PCA) based on the measure of different phenotypes. b. Boxplots comparing twelve statistically different phenotypes and nine non statistically different phenotypes of A. fumigatus and A. fischeri. Statistical differences are shown for two-sided Welch t-test (p-values: *, <0.05; **, <0.01; and ***, <0.001). The median and the first and third quartiles of the distribution are represented in the box, with whiskers extending ±1.5 × interquartile range, considering points outside as outliers.

Extended data Fig. 2 |. Proteins identified by trypsin-shaving in resting and swolllen conidia of the four Aspergillus species included in this work.

a. Number (#) of proteins (n=3 biological repetitions). The # of proteins represent the selected results of the product of the total number of identified peptide sequences (peptide spectrum matches) for a specific protein divided by the number of aminoacids multiplied by the coverage (the percent coverage calculated by dividing the number of amino acids in all found peptides by the total number of amino acids in the entire protein sequence, and divided by 100) (PSMs/AAs)*(Cov/100) > 0.001). b. Venn diagrams ilustrating the intersection of proteins identified by trypsin shaving of resting and swollen conidia of Aspergillus spp. strains.

Extended data Fig. 3 |. Hydrophobicity of A. fumigatus conidia.

Distribution of conidia of A. fumigatus wild-type A1163 and deleted mutant strains in a 1:1 water-oil (tributyrin) interface.

Extended data Fig. 4 |. A. fumigatus surface-associated proteins are involved in host-pathogen interactions.

a. Conidia survival of deleted mutants in murine RAW 264.7 macrophages at 6 hpi expressed as fold change against A1163 wild-type strain. b. Conidia internalization in A549 epithelial cells at 3 hpi expressed as fold change against A1163 (wild-type) strain. c. Simple linear regression models of a. and b. datasets (no correlation). Thresholds of 30 %-fold change above (red) and below (green) the wild-type strain (A1163) condition were established for statistical significance testing. d. Damage to A549 epithelial cells as measured by specific ⁵¹Cr release. e and f. A. fumigatus wild-type and mutant conidia eliciting cytokines IL-1β and TNF-α production by BMDMs. The results are given as the average of three independent biological repetitions and are expressed as average ± standard deviation. Statistical analysis was performed using two-sided One-way ANOVA with Dunnettś multiple comparisons test for comparisons to the wild-type strain (A1163) (*p < 0.05).

Extended data Fig. 5 |. Validation of A. fumigatus mutants.

a. Diagnostic PCR and b. Southern blot for Aspergillus fumigatus ΔaspA mutants. Both PCR and Southern blot experiments were repeated twice and those figures are representative images of the experiments.

Extended data Fig. 6 |. A. fumigatus ΔaspA mutant has reduced viability in the presence of alveolar macrophages and can modulate IL-1β, IL-6, IL-18, but not TNF-α.

a to d. ΔaspA conidia modulate cytokine production upon exposure of the wild-type and ΔaspA mutants to alveolar macrophages for 24 h. e. ΔaspA mutants have reduced viability by the BMDMs when compared to the wild-type strain. a to d. Heterologously expressed AspA can modulate IL-1β, IL-6, IL-18, and TNF-α levels production by alveolar macrophages. Denatured AspA has decreased cytokine production in alveolar macrophages. The statistical analysis was performed using a two-sided One-way ANOVA (Tukey’s test) for multiple comparisons. The results graphic shows the value of five to ten biological replicates ± standard deviation, with adjusted (adj.) P values. **adj. 0,0015, ****adj. P < 0.0001. ns not significant.

Extended data Fig. 7 |. Gating procedure used to quantify the CD45+ CD11b+ Ly6G+ and CD45+ CD11b+ Ly6G/Ly6C+ triple-positive neutrophil populations.

Arrows indicate the gate selected and carried forward for the next analysis. After gating for single cells (a.) and live cells (b. and c.), the resulting population was gated for CD45+ myeloid cells (d.), followed by gating for Ly6G+ CD11b+ (e.) and for Ly6G/Ly6C+ CD11b+ double-positive cells (f.) within the CD45+ population. The results graphic shows the percentage of CD45+ CD11b+ Ly6G+ positive cells in mice infected with 5× 10⁸ conidia of A. fumigatus wild-type or ΔaspA-1 strains (eight biological replicates ± standard deviation) (g. and h.). The p-values for the comparison between the groups were calculated using non-parametric t-test (two-tailed) with Mann-Whitney correction (p > 0.05).

Extended data Fig. 8 |. Heterologously expressed AspA.

a. SDS polyacrilamide gel showing the purified heterologously expressed AspA in Pichia pastoris. Ap, raw extract; fractions 12, 14, 62, and 63. b. IL-1β and TNF-α production in the presence of AspA treated or not with Peptide:N-glycosidase F (PNGaseF). The statistical analysis was performed using a two-sided One-way ANOVA (Bonferroni’s test) for multiple comparisons. Data are presented as values from three biological experiments ± standard deviation, *p < 0.01, ****p < 0.0001 vs glycosyl-asp boiled. ns. not significant.

Fig. 1 |. A. fumigatus has increased virulence and triggers higher cytokine production by BMDMs than the other three species.

a, Phylogeny of Aspergillus section Fumigati representative species constructed from concatenation analysis of a 5,215-gene data matrix. Branch lengths correspond to nucleotide substitutions per site. Adapted from ref. . b, Survival curves for A. fumigatus, A. fischeri, A. oerlinghausenensis and A. lentulus in a chemotherapeutic murine model of IPA. log-rank (Mantel–Cox) test, n = 5 mice per group in 2 independent experiments, *P < 0.0001 vs A. fumigatus group. c, Phagocytic index calculated using the number of conidia that had been phagocytosed for each macrophage (each point plotted is a cell). Fifty cells were counted for each data set in 2 independent experiments. The median, and the first and third quartiles of the distribution are represented in the box, with whiskers extending ±1.5× interquartile range, considering points outside as outliers. Statistical analysis was performed using two-sided one-way ANOVA (Bonferroni’s test) for multiple comparisons. Line at mean, *P < 0.01, **P < 0.0001 vs A. fumigatus group. d, Conidia viability. BMDM cells were seeded at a density of 10⁶ cells per ml and challenged with A. fumigatus conidia at an MOI of 1:10. After 24 h of incubation, media were removed and the cell suspensions were seeded in Sabouraud dextrose agar media. After 24 h of growth, the number of c.f.u.s ml⁻¹ was evaluated. Data calculated using two-sided two-way ANOVA and subsequent Dunnett’s multiple comparison test. Data are presented as mean ± s.d. from 3 biological replicates; ****P < 0.0001 versus A. fumigatus. e, Percentage of acidified phagolysosomes (PLs) containing conidia. Data represent means ± s.d. of 3 biological replicates. P values were calculated using two-sided one-way ANOVA with Bonferroni’s multiple comparisons test. NS, not significant. f–i, Cytokine production by BMDMs after infection with A. fumigatus, A. fischeri, A. oerlinghausenensis and A. lentulus conidia (MOI 1:10). Two-sided one-way ANOVA with subsequent Bonferroni’s multiple comparison tests is shown for ELISAs. Data are presented as mean ± s.d. from 2 biological triplicates, with adjusted P values. **P_adj < 0.0010, ****P_adj < 0.0001 vs A. fumigatus. ND, no data. j, Conidia internalization in A549 epithelial cells at 3 h post infection. Endocytosed conidia per HPF. Statistical analysis was performed using two-sided one-way ANOVA (Bonferroni’s test) for multiple comparisons. Data are presented as mean ± s.d. from 3 biological duplicates. **P_adj < 0.0014. k, Damage to A549 epithelial cells as measured by specific ⁵¹Cr release. Statistical analysis was performed using two-sided one-way ANOVA (Bonferroni’s test) for multiple comparisons. Data are presented as mean ± s.d. from 3 biological duplicates. *P_adj < 0.0265, ****P_adj < 0.0001.

Fig. 2 |. Comparative trypsin-shaving proteomics to study the surface-associated proteome surfome of A. fumigatus.

a, Representative images of 3 different experiments showing A. fumigatus, A. fischeri, A. oerlinghausenensis and A. lentulus conidia at time 0 and 4 h post incubation at 37 °C (early germination) in liquid potato dextrose (PD) media and radial growth (10⁴ conidia were displayed at the centre of MM plates and incubated for 5 days at 37 °C; radial diameters shown in Supplementary Table 8). b, Trypsin-shaving-based proteomic analysis workflow. Proteins obtained from trypsin-treated resting and swollen conidia were analysed by LC–MS/MS. Created with BioRender.com. c, Sixty-two uniquely detected proteins constitute the A. fumigatus conidial surfome. The Venn diagram illustrates the intersection of proteins identified by trypsin shaving of resting and swollen conidia of Aspergillus spp. strains. The conidial surfome of A. fumigatus is composed of 62 uniquely detected proteins (in red). Functional categorization of the 62 proteins that comprise A. fumigatus surfome according to their description in FungiDB is also shown. d, Heat map of the mRNA accumulation (FPKM, fragments per kilobase of transcript per million fragments mapped) during conidial germination of 62 genes encoding the A. fumigatus surfome proteins. RNAseq database according to ref. . e, Growth phenotypes of the wild-type A1160 strain (in blue) and deletion strains grown for 72 h at 37 °C or 44 °C in solid MM and MM supplemented with Congo red (CR, 10 μg ml⁻¹) or hydrogen peroxide (H₂O₂, 1.5 mM). The results are the average of 3 biological repetitions. f, Germination rates of the wild-type A1160 strain (in blue) and deletion strains. Only those hits below or above the 30% fold-change threshold and statistically different from the parental strain A1160 are displayed in the figure. Results are the average of 3 biological repetitions. g, Adhesion, biofilm formation measured by CV assay, of resting (○) and swollen (Δ) conidia for all mutant strains and A1160 (in blue). The results are the average of 8 biological repetitions. h, In vivo adhesion assay with A549 pulmonary cells. i, Detection of chitin (CFW), N-acetylglucosamine (GlcNAc) (WGA) and β-(1,3)-glucan (Dectin) contents on the conidial surface of all mutant strains and A1160 (in blue) in resting (○) and swollen (Δ) conidia. The results are the average of 8 biological repetitions. Only hits that share the same significant phenotype (lower and higher detection in green and red, respectively) in both stages are highlighted in the figure. In the data in e–i, we have considered only the mutants that have 30% increase or decrease compared with the parental strain (A1163). Statistical analysis was performed using two-sided one-way ANOVA (Dunnett’s test) for multiple comparisons. The selected mutants were significantly different from the A1163 wild type with P ≤ 0.05.

Fig. 3 |. The A. fumigatus ΔaspA mutant has reduced viability in the presence of BMDMs and can modulate IL-1β production.

a, The ΔaspA mutants have increased engulfment and adherence, and reduced viability in the presence of BMDMs compared with the wild-type strain. Phagocytic index (left) was calculated using the number of conidia that had been phagocytosed for each macrophage, and adherence (right) was calculated using the number of conidia counted in each macrophage surface (each point plotted is a cell). Fifty cells were counted for each data set in two independent experiments, totalling 100 cells. The median, and the first and third quartiles of the distribution are represented in the box, with whiskers extending to ±1.5× the interquartile range, considering points outside as outliers. Statistical analysis was performed using two-sided one-way ANOVA (Bonferroni’s test) for multiple comparisons. *P_adj < 0.0131, **P_adj < 0.0037, ***P_adj < 0.0002 and ****P < 0.0001. b,c, BMDMs cells were challenged with A. fumigatus conidia at an MOI of 1:10. After 24 h of incubation, the cell suspensions were seeded and the number of c.f.u.s ml⁻¹ (b) was evaluated. Statistical analysis was performed using two-sided one-way ANOVA (Bonferroni’s test) for multiple comparisons. Data are presented as mean ± s.d. of 2 biological experiments with 3 replicates; ****P < 0.0001 vs A1163 wild-type strain. Fungal viability (c) upon 24 h exposure to BMDMs as measured by MTT and CFW accumulation. Statistical analysis was performed using two-sided one-way ANOVA (Bonferroni’s test) for multiple comparisons. Data are presented as mean ± s.d. of 5 independent biological repetitions. *P_adj < 0.0128, **P_adj < 0.0100 and ****P_adj < 0.0001 vs A1163 wild-type strain. d, UV-killed conidia show that ΔaspA conidia need to be alive to modulate IL-1β (left) and TNF-α (right) production. Statistical analysis was performed using two-sided one-way ANOVA (Tukey’s test) for multiple comparisons. Data are presented as mean ± s.d. of 4 independent biological repetitions. **P_adj < 0.0072, ***P_adj < 0.0007 and ****P_adj < 0.0001. e, ROS accumulation upon exposure of the wild type and ΔaspA mutants to BMDMs in the indicated periods of time. PMA was used as a positive control for ROS induction. Statistical analysis was performed using two-sided one-way ANOVA (Dunnett’s test) for multiple comparisons. Data are presented as mean ± s.d. of 3 independent biological repetitions. *P adj < 0,0267, **P adj < 0,0033. f, Percentage of PLs. Statistical analysis was performed using two-sided one-way ANOVA (Dunnet’s test) for multiple comparisons. Data are presented as mean ± s.d. of 3 independent biological repetitions. g,h, Transwell migration assays. Wild type and ΔaspA mutants were exposed to BMDMs, and IL-1β and TNF-α production were measured. ‘+’ and ‘−’ indicate when the Transwell insert was added or not to the well plate, respectively. Results are the mean ± s.d. of 3 independent biological repetitions. Statistical analysis was performed using two-sided two-way ANOVA (Bonferroni’s test) for multiple comparisons; ****P < 0.0001.

Fig. 4 |. Infection with A. fumigatus ΔaspA mutants results in decreased fungal burden in immunocompetent mice.

a, Fungal burden of the wild type and ΔaspA mutants in a murine chemotherapeutic mouse infection model. b, Immunocompetent mouse infection model. Statistical analysis was performed using two-sided one-way ANOVA (Dunnett’s test) for multiple comparisons. Data are presented as values from n = 10 mice per group in two independent experiments. Results are the mean ± s.d. of 2 independent biological repetitions. c–h, Cytokine production (TNF-α, IL-1β, IL-18, IL-12, INF-γ and IL-6) in the immunocompetent mouse model infected with wild type and ΔaspA mutants. i, Chemokine production (CXCL-1) in the immunocompetent mouse model infected with wild type and ΔaspA mutants. The results graphic shows the mean ± s.d of 3–5 technical replicates. Statistical analysis was performed using two-sided one-way ANOVA (Dunnett’s test) for multiple comparisons. For a–i, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs the wild-type group. j,k, Fluorescence activated cell sorting (FACS) analysis of interstitial and alveolar neutrophils in mice infected with A. fumigatus wild-type and ΔaspA-1 strains. A representative cytometry dot plot of CD11b⁺Ly6G⁺/Ly6C⁺ cells gated from the CD45⁺ population is shown from mice in each group. Data are presented as mean ± s.d of n = 4 mice per group in 2 independent experiments. The P values for the comparison between the groups were calculated using two-sided non-parametric t-test (two-tailed) with Mann–Whitney correction (P > 0.05).

Fig. 5 |. Heterologously expressed AspA modulates IL-1β and TNF-α levels.

a, Viability of BMDMs exposed for 24 h at 37 °C to different concentrations of AspA and measured by LDH activity or b, exposed to DMSO, 0.2 or 2 μg AspA:GFP, and measured by XTT assay. In a, statistical analysis was performed using two-sided one-way ANOVA (Bonferroni’s test) for multiple comparisons. Data are presented as mean ± s.d. of 3 biological experiments; **P < 0.0001. In b, statistical analysis was performed using two-sided one-way ANOVA (Bonferroni’s test) for multiple comparisons. Data are presented as mean ± s.d. of 5 biological experiments. *P_adj < 0.0148, ****P_adj < 0.0001 vs DMSO. c,d, Increasing concentrations of AspA modulate IL-1β and TNF-α production by BMDMs. Statistical analysis was performed using two-sided one-way ANOVA (Bonferroni’s test) for multiple comparisons. Data are presented as mean ± s.d. of 4 biological experiments; *P < 0.05, **P < 0.01, ****P < 0.0001. e, A. fumigatus AspA asparaginase activity. Data are presented as mean ± s.d. of 6 biological experiments; two-sided non-parametric t-test (two-tailed) was used for the statistical analysis; ****P < 0.0001. f,g, Denatured AspA triggers less IL-1β and TNF-α levels in BMDMs. Statistical analysis was performed using two-sided one-way ANOVA (Bonferroni’s test) for multiple comparisons. Data are presented as mean ± s.d. of 3 biological experiments; *P < 0.05, ***P < 0.001 and ****P < 0.0001. h, Conidial viability of BMDMs exposed or not to 2 μg AspA for 8 h and subsequent addition of conidia or 2 μg AspA together with conidia, evaluated after 32 and 24 h. Results are the average of 3 biological repetitions and are expressed as individual data points ±s.d. Statistical analysis was performed using two-sided one-way ANOVA (Dunnett’s test) for multiple comparisons; *P < 0.05.

Fig. 6 |. Proteomic profiling of BMDMs exposed to AspA.

a, Principal component analysis distribution of 3 biological repetitions of BMDM and BMDM proteins exposed to AspA. b, Heat map of protein abundance of BMDM and BMDM exposed to AspA. c,d, Enriched categorization of BMDM and BMDM proteins exposed to AspA. Blue and pumpkin colours represent down- and up-expressed categories, respectively. All categories and P values are described in Supplementary Table 15. e, Mouse functional protein association network based on 15 selected proteins that are modulated in the BMDMs in the presence of AspA. Each node represents a protein that is differentially expressed in the BMDMs upon exposure to AspA. Each edge represents a functional protein association retrieved from the STRING (https://string-db.org) server (medium confidence threshold of 0.4 for the interaction score), and node sizes represent the number of edges connected to the node. Proteins were annotated on the basis of https://www.genecards.org and are: SIGLEC1, Msr1, Csf1r, TfrC, PtprC, Hcls1, Lsp1, Lamp2, Ctss, Ncf4, Colt1, CtsA, CtsB, CtsZ and Ncf1.