FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection

The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia.


Introduction
Aspergillus fumigatus (A. fumigatus) is an ubiquitous opportunistic pathogen that causes invasive and often fatal lung infection, particularly in immunocompromised patients [1]. Aspergillus fumigatus produces small hydrophobic conidia that are easily inhaled into the lungs and require robust host defense mechanisms to prevent infection. The mechanisms of clearance of conidia from the lung are incompletely understood but phagocytosis by macrophages is known to be important [2][3][4][5]. Macrophages express Dectin-1, a C-type lectin that recognizes β-1-3 glucan on the surface of A. fumigatus conidia. Although the amount of surface accessible β-1-3 glucan is low on resting conidia, it is much higher in swollen conidia that appear early during germination and infection [6]. Binding of β-glucan by Dectin-1 promotes macrophage killing of A. fumigatus conidia, and other macrophage receptors, such as the mannose receptor and toll-like receptors (TLR) -2 and -4, cooperate in this killing effect [7,8]. Notably, however, the phagocytosis of A. fumigatus conidia by macrophages is incompletely blocked by inhibitors of Dectin-1, mannose receptor, and TLR-2/4 [9], which means that macrophages must employ additional mechanisms to phagocytose and kill A. fumigatus.
Many microorganisms use lectins as adhesins to interact with host glycoproteins. For example, Pseudomonas, Burkholderia, Ralstonia, and Chromobacterium bacteria all express adhesins that include galactophilic and fucophilic lectins that initiate bacterial adherence to glycan receptors on host cells and tissues [10][11][12][13][14]. In addition, fungi such as Aleuria aurantia, A. oryzae, and A. fumigatus all express fucophilic lectins [15][16][17][18] with multiple fucose binding sites [11,19]. For example, A. fumigatus lectin (AFL, also known as FleA) exists as a dimer with 12 fucose-binding sites available for strong multivalent interactions with fucosylated structures [18]. As a result, FleA has unusually high binding affinity for fucosylated structures [18,20], but its role in fungal biology is unknown. Fucosylated glycans are abundant in plants and animals, and FleA expression by fungi may help them bind plant or animal tissues, as has been found for fucose binding lectins in bacteria [11,21]. Aspergillus fumigatus conidia enter the human host via inhalation, and fucosylated proteins in the lung that could bind conidial FleA include mucins in the airway mucus gel and multiple glycoproteins in the glycocalyx of macrophages [22][23][24]. Fucose in different linkages is a common carbohydrate structure in gel-forming mucins [22] such as MUC5AC and MUC5B [23], and recent studies in transgenic mice have revealed the essential role of gel-forming mucins in host defense against lung infection [25]. In addition, membrane-tethered mucins (such as MUC1 and MUC4) function as receptors in epithelial cells [26] and macrophages [24] and it is possible that they may function as adhesins for A. fumigatus. To determine the role of FleA in the pathogenesis of A. fumigatus pneumonia, we studied the behavior of recombinant FleA and fleA-deficient conidia in multiple complimentary functional assays, including mucin binding assays, macrophage binding assays, and a mouse model of A. fumigatus pneumonia.

Phylogenetic analysis of FleA
We generated a phylogenetic tree to illustrate the fungal genera and species that contain a putative FleA ortholog. The tree shows that FleA is not widespread in the Kingdom Fungi and is found in several pathogenic species of fungi. FleA is present in the genomes of only a few Aspergillus species, including A. fumigatus, A. flavus, A. oryzae and A. calidoustus (S1 Fig). FleA is also present in other human pathogens including dermatophytes (Trichophyton, Microsporum and Arthroderma), entomopathogenic fungi (Metarhizium, Ophiocordyceps), a nematode pathogen (Arthrobotrys oligospora), several plant pathogens (Penicillium expansum, Marssonina, Magnaporthiopsis, Ceratocystis, Gaumannomyces, Rhizoctonia) and Trichoderma spp. (pathogenic on other fungi).

FleA mediates binding of A. fumigatus conidia to airway mucin in a fucose dependent manner
To determine if A. fumigatus conidia can bind to mucin glycans in a FleA-dependent manner, we coated microtiter plates with mucins that we purified from the induced sputum of healthy subjects, and we used labeled recombinant FleA to quantify FleA-mucin binding. We found that FleA binds strongly to airway mucin and that this binding is inhibited by fucose ( Fig 1A). No inhibition was observed with galactose ( Fig 1A). To examine the specificity of FleA for the different linkage forms of fucose found naturally on mucins, we synthesized disaccharides with fucose linked to glucose in α1,2, α1,3, α1,4, or α1,6 positions. These fucose-glucose compounds all strongly inhibited FleA binding to mucin in a dose dependent manner (Fig 1B).

Generation of ΔfleA A. fumigatus conidia
To further characterize fleA in A. fumigatus and examine the functional role of FleA binding to mucin, we created reagents to allow us to make quantitative measurements of A. fumigatus binding to mucin and to dissect the specific binding role of FleA. To facilitate visualization of the conidia, we created strains of A. fumigatus expressing nuclear GFP by transformation of AF293.1 and AF293.6 strains with the plasmid pJMP51 resulting in histone 2A fused GFP prototrophic (TJMP131.5) and auxotrophic (TGJF5.3) strains, respectively. TGJF5.3 was further modified and transformed to prototrophy using a disruption cassette targeting disruption of the fleA locus (Fig 2A) and multiple ΔfleA deletion transformants were identified. Confirmation of fleA deletion was demonstrated by Southern and northern blotting (Fig 2B and 2C). Despite repeated attempts to complement the ΔfleA deletion strains, we encountered problems such as phenotypic abnormalities in spore size and shape, which precluded generation of reliable complement reagents. Hence the mucin binding studies described below were repeated with several ΔfleA deletions to confirm the role of FleA as an A. fumigatus fucose binding protein. The mucin binding studies were also repeated using a novel synthetic glycomimetic inhibitor of FleA as an alternative experimental approach. To assess localization of FleA in conidia, we made a C-terminal RFP tagged fleA mutant in A. fumigatus by modifying the native fleA locus using a disruption cassette similar to that used to delete the fleA ORF (Fig 2D and 2E).

Generation of ΔfleA A. flavus conidia
Multiple ΔfleA deletion A. flavus strains were generated using the same methods described for A. fumigatus above and as illustrated in S2 Fig. FleA is present on resting and swollen A. fumigatus conidia Protein levels of FleA on A. fumigatus conidia and hyphae have not been characterized. We found that RFP-tagged FleA is present on A. fumigatus conidia when fleA-rfp is expressed under its endogenous promoter (Fig 3A), and we used two different strains of FleA-RFP tagged conidia to quantify FleA levels over time by fluorescence microscopy (Fig 3B). FleA fluorescence was high on resting conidia (with some variation among conidia), low in swollen conidia, and largely absent in hyphae (Fig 3B and 3C). Time-lapse microscope images of germinating conidia show that FleA begins to decrease by 21 hours and is almost entirely absent by 27 hours (Fig 3C and S1 Movie). These results were confirmed in extracts from resting and swollen conidia and from hyphae that were analyzed for FleA protein by western blot (Fig 3D). We also investigated the possibility that FleA was secreted by conidia, even though FleA lacks a canonical secretion signal peptide according to the Signal P 4.0 server [27]. Analysis of proteins in concentrated culture supernatant of A. fumigatus at various developmental timepoints revealed that resting conidia shed significant amounts of FleA, whereas conditioned media from swollen conidia and hyphal culture supernatant did not have detectable FleA (Fig 3D).

Binding of A. fumigatus conidia to airway mucins and phagocytosis by macrophages is FleA dependent
To examine if binding of A. fumigatus conidia to airway mucins is FleA dependent, we first developed a binding assay to determine the specific binding role of FleA. For the conidia-binding assay, we coated mucins onto chamber slides and used confocal microscopy to image binding of conidia. As shown in Fig 4A and 4B, the binding of three independent ΔfleA deletion mutant strains (TGJF 6.7, 6.8 and 6.13) to mucin is much weaker than the binding of WT conidia. These data demonstrate that A. fumigatus conidia bind to mucin in a FleA-dependent manner. After confirming that all 3 ΔfleA mutant strains showed identical patterns in mucin binding assays, we chose the A. fumigatus strain TGJF6.7 for all further functional studies.

Binding of A. flavus conidia to airway mucins is FleA dependent
To determine if FleA is also required for binding of A. flavus conidia to mucin, we examined the binding of WT and ΔfleA A. flavus conidia in our chamber slide assay. As shown in Fig 4C, the binding of three independent ΔfleA deletion mutant strains of A. flavus (TFYL 62.1, 62.2 and 62.3) to mucin is much weaker than the binding of WT conidia.
Fucosylated glycoproteins are not restricted to mucin glycoproteins. Macrophages also express multiple fucosylated proteins on their cell surface, including membrane-tethered mucins [24]. We therefore explored whether fleA expression by A. fumigatus might mediate binding or phagocytosis by macrophages. We first explored binding of recombinant FleA to macrophages by flow cytometry and showed that FleA binds very strongly to the surface of RAW264.7 mouse macrophages and primary human alveolar macrophages in a fucose-dependent manner (Fig 5A and 5B). Next, we investigated phagocytosis of A. fumigatus conidia to RAW264.7 mouse macrophages. Using confocal microscopy, we found that A. fumigatus WT conidia bound to RAW264.7 cells and that many are internalized/phagocytosed ( Fig 5C). Notably, the internalization of ΔfleA conidia was 50% less than that of WT (Fig 5C and 5D). We repeated these experiments in alveolar macrophages isolated from bronchoalveolar lavage from healthy human subjects. Similar to data from the RAW264.7 cells, we found that A. fumigatus conidia are internalized and phagocytosed by human macrophages and that ΔfleA conidia show a reduction in phagocytosis of 40% compared to WT (Fig 5E and 5F). Thus, fucosylated structures on lung macrophages act as receptors for A. fumigatus FleA, resulting in enhanced phagocytosis of conidia.  To rule out the possibility that deletion of FleA causes changes to β-glucan in the fungal cell wall that could be affecting binding and phagocytosis, we used FACS to examine the binding of a β-glucan ligand (Dectin-1) to ΔfleA and WT conidia. We found no significant differences in the affinity of recombinant biotinylated Dectin-1 for ΔfleA and WT conidia in these experiments ( Fig 5G). We also coated fluorescent microspheres with FleA to create a simplified model of conidia and found that FleA causes a significant and fucose-dependent increase in binding and phagocytosis of microspheres by RAW264.7 cells ( Fig 5H).

(2E)-hexenyl α-L-fucopyranoside is a potent functional inhibitor of FleA
To provide additional evidence that binding of A. fumigatus conidia to mucins and alveolar macrophages is FleA-dependent, we synthesized a library of modified fucopyranoside structures using methods we previously described for inhibitors of the FimH lectin in E. coli [28] (Fig 6). To screen for the relative potency of different fucopyranosides, we used the FleAmucin binding assay. In this way, we found that (2E)-hexenyl α-L-fucopyranoside (2EHex) (Fig 6A) inhibits FleA with marked (nanomolar) potency ( Fig 6B). In contrast to its potent inhibition of FleA binding to mucin, 2EHex does not show potent inhibition of PA-IIL (a fucose-binding lectin from Pseudomonas aeruginosa) binding to mucin (Fig 6C). We next tested the effects of 2EHex and fucose on interactions between A. fumigatus conidia and mucins and macrophages. We found that both 2EHex and fucose decreased the mucin binding of WT A. fumigatus conidia to the levels observed with ΔfleA conidia (Fig 6D). 2EHex also decreased the phagocytosis of WT A. fumigatus conidia by RAW264.7 cells and primary human macrophages to the levels observed with the ΔfleA conidia (Fig 6E and 6F). These inhibition studies confirm that binding of A. fumigatus conidia to mucins and macrophages requires the fucose binding activity of FleA.

FleA loss increases lung infection and lung injury by A. fumigatus
Based on our data that FleA is required for binding and phagocytosis of conidia by macrophages, we hypothesized that ΔfleA conidia might evade phagocyte killing leading to increased lung infection compared to WT. To test this hypothesis, we infected immunocompetent C57BL/6 mice intranasally with WT conidia or ΔfleA conidia. H&E staining of infected lung tissue showed that mice infected with WT conidia had well-contained pneumonia (Fig 7A) whereas ΔfleA conidia treated animals had a poorly contained pneumonia (Fig 7B). GMS staining showed limited numbers of conidia with little evidence of germination in sections from WT treated animals (Fig 7C and 7E). In contrast, a large number of conidia from ΔfleA treated animals showed hyphae generation (germlings) evident on the GMS stain, typical of invasive aspergillosis (Fig 7D and 7F). Both the total number and percentage of germinating conidia are significantly higher in mice infected with ΔfleA conidia than WT conidia (Fig 7G and 7H). Lung injury is more severe in ΔfleA-infected mice, as evidenced by a higher concentration of primary human alveolar macrophages. 100mM fucose (dark grey) inhibits binding almost down to the background level (light grey). Z-stack confocal images show binding and phagocytosis of GFP expressing WT conidia by (C) RAW264.7 cells, (E) primary human alveolar macrophages and that ΔfleA conidia are not bound well or phagocytosed effectively. Cells were dyed with CellMask Deep red (red); internalized conidia express GFP (green); calcofluor white (noninternalized conidia) stain blue. Quantitative data (expressed as an index of total number of conidia internalized/total cell number) demonstrate how loss of FleA significantly inhibits binding and phagocytosis of A. fumigatus conidia by (D) RAW264.7 cells or (F) primary human alveolar macrophages. Calcofluor white stained conidia were excluded from these counts to reflect only internalized conidia. The data shown in D (6 replicates) reflects the mean ± SD of three independent experiments whereas the data in panel F (6 replicates) reflects the mean ± SD of three independent donors. (G) FACS plots showing that binding of Dectin-1 is not significantly different between WT (dark grey) and ΔfleA (black) conidia compared to control (light grey). (H) Binding and internalization of FleA coated microspheres by RAW264.7 cells is significantly higher than control and inhibited by 500mM fucose. *Denotes significantly different from control, p = <0.05. Scale bar is 10μM. hemoglobin in bronchoalveolar lavage (BAL) (Fig 7I). Aspergillus 18S gene expression in lung homogenates from ΔfleA-infected mice is higher than in lung homogenates from WT infected mice, indicative of higher fungal burden (Fig 7J).
BAL cells from mice infected with WT and ΔfleA conidia were analyzed by flow cytometry to quantify multiple immune cells and by multiplex immunoassay to quantify multiple cytokines, chemokines and growth factors. Mouse BAL cells were labeled with a panel of antibodies including CD11c, F4/80, CD11b, MHCII, Ly6G, Ly6C, NK1.1, TCRβ, B220, CD4 and CD8. Compared to mice infected with WT conidia, we found that several cell types were decreased in BAL from mice infected with ΔfleA conidia, including alveolar macrophages, neutrophils, NK and NKT cells and CD4 and γΔ T cells (Fig 8). No significant differences were observed for eosinophils, dendritic cells, B cells, monocytes and inflammatory monocytes and CD8 T cells. Compared to mice infected with WT conidia, we found that the concentrations of multiple cytokines, chemokines and growth factors were similar in BAL from mice infected with ΔfleA conidia, including IL-6, IFNγ, KC and VEGF in BAL fluid (S1 Table).

Discussion
We have discovered that FleA produced by A. fumigatus conidia allows airway mucins to bind conidia and macrophages to effectively phagocytose them. Notably, when we engineer conidia

(I) Hemoglobin concentration is significantly higher in lung lavage from mice infected with
ΔfleA conidia than WT conidia (n = 10 mice per group) (J). Gene expression for Aspergillus 18S is significantly higher in lung homogenates from mice infected with ΔfleA conidia than from mice infected with WT conidia (n = 10 per group). All mouse samples were harvested 3 days after infection. Scale bar is 10μM. Data are mean +/-SD. *Denotes significantly different from control, p = <0.05. that lack FleA, the resultant ΔfleA conidia show increased virulence in a mouse model of A. fumigatus pneumonia. Together these data uncover a novel mechanism of host defense against A. fumigatus infection in which fucosylated receptors in the airway mucus gel and on the surface of macrophages bind FleA to hasten the elimination of A. fumigatus conidia.
To date, research on binding of A. fumigatus conidia to lung proteins has focused on binding to basement membrane proteins in the airway epithelium [29][30][31][32]. But mucins provide the first line of defense against inhaled pathogens in the bronchi and bronchioles [25], and studies of binding of A. fumigatus conidia to human airway mucins are highly relevant to mechanisms of infection and invasion. Mucin glycans include multiple fucosylated structures [22], and we report avid binding of FleA to human airway mucins that is inhibited by a wide range of fucose structures, including fucose in α1,2, α1,3, α1,4, or α1,6 linkages. We also report that deletion of FleA in Aspergillus fumigatus conidia markedly decreases mucin binding and that this role for FleA is conserved in the pathogenic A. flavus. We conclude that a range of sterically available fucosylated glycans in mucins can act as FleA ligands and the airway mucus gel is a powerful "sticky" barrier that can capture and remove conidia to guard against invasive infection.
Phagocytosis of inhaled conidia by alveolar macrophages represents an important innate immune defense against A. fumigatus infection, especially in the alveolar lung compartment where mucocilliary clearance does not operate. We explored the role of FleA in phagocytosis of A. fumigatus conidia by murine and human macrophages using two approaches. First, we compared the phagocytosis of wild type (WT) conidia and fleA-deficient (ΔfleA) conidia and showed that macrophage phagocytosis of the ΔfleA conidia is decreased by 40-50%. Second, we screened a library of fucopyranosides to reveal that (2E)-hexenyl α-L-fucopyranoside (2EHex) inhibits FleA with nanomolar efficacy and also inhibits the binding and phagocytosis of WT A. fumigatus conidia by lung macrophages. Together, these two lines of evidence leads us to conclude that A. fumigatus conidia interact with macrophages in a mechanism that requires the fucose binding activity of FleA. Multiple glycoproteins on the surface of macrophages could act as the receptors for FleA, including membrane-tethered mucins [24]. Neutrophils are also important in cellular immunity against Aspergillus [33,34], but we did not study whether neutrophils have defective uptake of fleA-deficient conidia, and it remains unknown whether fucosylated receptors on neutrophils have a role in host defense against Aspergillus. The identity of all of the cell types participating in FleA-dependent host defense in the lung has not been elucidated in this study. While we have described an alveolar macrophage dependent mechanism, it is also likely that other non-alveolar macrophage dependent cellular mechanisms, including neutrophils or monocytes, can contribute to FleA mediated host defense.
The FleA-dependent binding of A. fumigatus conidia to mucin and the poor uptake of ΔfleA conidia by lung macrophages indicate that FleA is not a virulence factor. Instead, we propose it is a fungal protein recognized by the host to promote defense against invasive aspergillosis. We provide evidence for this in our experiments in which we infected immunocompetent mice with A. fumigatus WT or ΔfleA conidia. The mice infected with the ΔfleA conidia developed invasive aspergillosis whereas those infected with WT conidia did not. Notably, the ΔfleA infected mice showed blunted recruitment of immune cells integral to the fungal immune response. Specifically, FACS analysis of BAL cells showed a significant decrease in the number of alveolar macrophages, neutrophils, NK and NKT cells and CD4 and γΔ T cells. Thus, it appears that ΔfleA conidia fail to elicit the same inflammatory response as WT conidia resulting in reduced recruitment of key effector cells in the fungal immune response in the lung. We conclude that the absence of FleA in A. fumigatus conidia results in a hypo-inflammatory response and promotes invasive infection.
Our experiments prove that FleA is a pathogen-associated molecular pattern that can be recognized by lung mucins and macrophages to protect the host from infection. The fact that we show that A. flavus also uses FleA to bind lung mucin and that phylogenetic analysis reveals FleA present in several pathogenic species may suggest a conserved role for FleA in fucosemediated host pathogen interactions. It is unclear why A. fumigatus and other pathogenic fungi have evolved to express fleA. Since fungi commonly grow on carbon-rich carbohydrate substrates, it is possible that FleA is involved in helping these organisms to establish a niche on the surface of carbohydrate-rich substrates.
Our finding for a role for FleA-fucose mediated mechanism of host defense against A. fumigatus does not cast any doubt on the validity of the well established role for β-glucan/Dectin-1 interactions [35]. β-glucan is abundant on swollen conidia and hyphae, and Dectin-1 binds βglucan on swollen conidia to trigger a robust host defense response [36]. But Dectin-1 does not bind to resting conidia, because these conidia have masked exposure of β-glucans due to the action of hydrophobin proteins [37,38] and the mechanism of clearance of resting conidia from the lung is not completely understood. We propose a model whereby fucosylated receptors on mucins and macrophages interact with FleA on resting conidia to facilitate clearance of resting conidia via the mucociliary escalator and/or macrophage ingestion. Conidia that escape this initial response and mature (swell and germinate), possibly through the shedding of FleA, would be cleared by Dectin-1 mediated phagocytosis [9]. Our data thus reveal contrasting roles for distinct protein carbohydrate interactions in host immune responses to A. fumigatus. On one hand, a macrophage expressed lectin-Dectin-1-recognizes and binds a carbohydrate structure (β-glucan) on A. fumigatus conidia to promote phagocytosis and killing. On the other hand, we reveal here that fucosylated carbohydrates on macrophages engage a conidial lectin (FleA) to promote phagocytosis and killing. We conclude that protein-and carbohydrate-based defenses on macrophages provide complimentary mechanisms to prevent potentially fatal Aspergillus lung infections.

Subjects and clinical samples
Induced sputum was collected from 5 healthy nonsmoking, non-allergic subjects aged between 24-55 years (4 male) as described [39]. Bronchoalveolar lavage (BAL) was collected from 3 healthy non-smoking, non-allergic subjects aged 30-45 years (2 male, 1 female) by instilling 4 aliquots of 50mLs of warmed (37°C) normal saline into a segmental bronchus in the right middle lobe or lingula. After the first two 50mL aliquots were instilled and aspirated from one segmental bronchus, the bronchoscope was moved to an adjacent bronchus in the same segment for collection of two additional 50 mL aliquots.
Purification of high molecular weight mucin from sputum 8M guanidine hydrochloride was added in 1:1 volume to sputum samples and the samples rotated at 4°C until homogenized. Mucins were then purified from the sputum as described previously [40,41] with additional details in S1 Text.

Synthesis of carbohydrates
The disaccharides were synthesized by glycosylating appropriately protected glucose acceptors with a fucosyl bromide donor, 2,3,4-tri-O-benzyl-L-fucopyranosyl bromide, using Lemieux's halide assisted conditions [42], followed by deprotection of the α-linked disaccharides using catalytic hydrogenolysis to give the target structures. Detailed synthesis methods are in S1 Text.
Aspergillus strains and culture (S2 Table) Unless noted, all A. fumigatus strains were propagated on solid glucose minimal media (GMM) at 37°C [43]. A. fumigatus asexual spore suspensions were fixed, where appropriate, in 4% formaldehyde in PBS.

Immunofluorescence of Aspergillus conidia
A. fumigatus strains were cultured on GMM at 37°C for 3 days, spores were harvested, placed on a pre-cleaned glass slide and coverslipped. Images were taken of GFP and RFP fluorescence using a Nikon Ti inverted microscope equipped with a Nikon Plan Apo VC 60x/1.40 Oil DIC/ 1/0.17 WD. Time-course microscopy was carried out over 27 hours at 37°C. The average fluorescent intensity at each developmental state (resting conidia, swollen conidia, and hyphae) of untagged FleA (TJMP131.5 or wild type) was subtracted from the mean fluorescent intensity value of two different transformants (TGJF7.11 and TGJF7.15) expressing RFP-tagged versions of FleA. The adjusted mean fluorescence was then standardized to area.

Western blot of A. fumigatus extracts and supernatant
Resting and swollen A. fumigatus conidia and hyphae were isolated from WT and ΔfleA cultures as described in S1 Text and extracted in 50mM Tris/HCl pH 7.4, 50 mM EDTA, 2% SDS, and 40 mM β-Mercaptoethanol. Protein concentrations were determined by BCA assay and 15 μg of protein was loaded into each well of a 4-12% BOLT SDS PAGE gel (Life Technologies, Grand Island, NY) and electrophoresed. Gel was then blotted onto nitrocellulose, blocked with non fat milk and stained with an anti-FleA rabbit polyclonal antibody [18] and donkey antirabbit HRP (Jackson immunoresearch, West Grove, PA) prior to chemiluminescent detection. Culture supernatants were filtered through a 0.2 μM filter and concentrated 10x in a 0.5 ml Amicon Ultra (EMD Millipore, Billirica, MA) before being run as described above.
Mucin conidia binding assay 8 well glass chamber slides (Labtek, Scotts Valley,CA) were coated with 20 μg/ml purified human mucin in dH 2 O overnight at 37°C and then blocked in PBS + 1% BSA for 1 hour. Fixed A. fumigatus conidia suspensions were centrifuged at 6000 x g for 5 minutes to pellet and resuspended in PBS + 1% BSA in the presence or absence of 10mM (2E)-hexenyl α-L-fucopyranoside (2EHex) or 100mM fucose. 2x10 7 conidia were added per well and incubated for 4 hours at room temperature. Unbound conidia were removed by washing in PBS+ BSA, the slides were mounted in Prolong Gold anti-fade reagent (Life Technologies, Grand Island, NY) and allowed to cure for 24 hours prior to sealing. Images were acquired using an FV10i confocal microscope (Olympus, Center Valley, PA) using the multipoint Z-stack mode to acquire 9 fields per well with 3 wells imaged per condition per experiment. Each Z-stack image was compressed into a single plane of focus and conidia were counted using NIH Image J with the ITCN plugin. Each experiment was repeated at least 3 times. A. flavus-mucin interactions were investigated as described above with one exception. These conidia lack GFP so were stained with Calcofluor white for 5 minutes to allow imaging prior to adding to mucin-coated slides.
Cell culture RAW 264.7 cells (UCSF cell culture facility) were maintained in DMEM + 10% fetal bovine serum + 1% penicillin/streptomycin until seeded and grown on 8 well chamber slides (Labtek, Scotts Valley, CA) overnight. Human alveolar macrophages from BAL were centrifuged at 450 x g for 10 minutes and washed with PBS prior to plating on poly-L-lysine coated 8 well chamber slides in RPMI 1640+ 10% fetal bovine serum + 1% penicillin/streptomycin + 0.5μg/ ml amphotericin B. Cells were washed after 2 hours of adherence and cultured overnight prior to experiments. Phagocytosis assay RAW 264.7 cells were plated at 5x10 4 /well on 8 well chamber slides (Labtek, Scotts Valley, CA) and allowed to grow overnight in culture media. 5x10 6 conidia from either the PFA-fixed WT or ΔfleA strain were added per well in the presence or absence of 10mM 2EHex or 500mM fucose and incubated at 37°C for 1 hour. Wells were washed and incubated for a further 2 hours at 37°C for complete uptake. Cells were stained with 7.5μg/ml CellMask Deep Red plasma membrane stain (Life Technologies, Grand Island, NY) and calcofluor white (Sigma, St Louis, MO), washed with PBS and mounted with Fluoromount-G (Southern Biotech, Birmingham, AL). Z-stack images were acquired using an FV10i confocal microscope (Olympus, Center Valley, PA). Each Z-stack image was compressed into a single plane of focus and both internalized conidia and cell number were counted using NIH Image J with the cell counter plugin. Phagocytic index was calculated as the number of conidia internalized per cell. Internalized conidia were counted as conidia within the boundary of the cell that were not stained with calcofluor white. Calcofluor white stained conidia were excluded from the count as they were not internalized. For human macrophages, cells were plated at 5x10 5 per well and grown overnight. 1.5x10 6 conidia from the PFA fixed WT or ΔfleA strains were added per well in the presence or absence of 10mM 2EHEX, incubated at 37°C for 30 minutes then washed, stained and mounted.

Flow cytometry analysis of A. fumigatus conidia
Recombinant Dectin-1 was purchased from R&D Systems (Minneapolis, MN) and biotinylated using the EZ-link sulfo NHS biotin kit (Thermo Fisher Waltham, MA) according to manufacturers recommendations. 1x10 7 WT or ΔfleA conidia were labeled with biotinylated Dectin-1 and Streptavidin-PE (Biolegend, San Diego, CA) and fixed in 4% paraformaldehyde prior to analysis on a Becton Dickenson FACSCalibur and FlowJo software (TreeStar, Ashland, OR).

Phagocytosis of FleA-coated particles
Yellow-green 1μM sulfate treated FluoSpheres (Life Technologies, Grand Island, NY) were coated with recombinant FleA, added to RAW264.7 cells in the presence or absence of 500mM fucose and incubated with agitation for 2 hours at 37°C prior to extensive washing with DMEM to remove unattached FluoSpheres from the cell surface. Cells were fixed in 4% paraformaldehyde prior to analysis on a Becton Dickenson FACSCalibur and FlowJo software (TreeStar, Ashland, OR).

Mice
Male C57BL/6 mice from Charles River Laboratories (Wilmington, Massachusetts, MA) were housed in a pathogen free facility at UCSF. Animal experiments followed protocols approved by the UCSF Institutional Animal Care and Use Committee.

Mouse model of Aspergillus infection
Eight to ten week old C57BL/6 mice were infected intranasally with 5x10 7 of WT or ΔfleA conidia. Mice were sacrificed 3 days post infection. Measures of lung inflammation, infection and lung injury were evaluated using methods described in S1 Text.

Statistical methods
Data analyses were performed GraphPad Prism version 6 (GraphPad, San Diego, CA). ANOVA was used for three-group comparisons followed by pairwise analyses with the Tukey multiple comparisons test when appropriate. Two group comparisons were analyzed using the Students t-test or for non-parametric analyses, a ranked Mann-Whitney test.

Ethics statement
Human samples were obtained from the UCSF Airway Tissue Bank (ATB). All participants signed two informed consent forms-one for the original study protocol and the other for the ATB protocol. All study and ATB procedures were reviewed and approved by the UCSF Committee on Human Research, protocol number 11-05176. All animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. All protocols involving animals were approved by the animal care and use committee at UCSF and are in compliance with Public Health Service Policy (PHS), (IACUC protocol number AN090458-03).