Proteomics of Aspergillus fumigatus conidia-containing phagolysosomes identifies processes governing immune evasion

several pathogenic microorganisms, proteomic studies unraveled the specific protein composition of phagolysosomes for a better understanding of the phagosomal maturation process and its modulation by pathogens (15-23). These studies demonstrated that the protein composition of the phagosome is highly dynamic, specific for the ingested particle, depends on the stage of phagosomal maturation, the type or cell line and the activation status of the phagocyte. Therefore, we set out to analyze the phagolysosomal proteome after phagocytosis of wt and pksP mutant conidia in order to shed light on processes inhibited by wt conidia during the maturation of phagosomes. In this study, a magnetic label– based purification protocol for conidia-containing phagolysosomes and the application of a label-free protein quantification method was developed. Bioinformatics was employed to determine the regulatory modules of differentially abundant proteins of the macrophage phagolysosome. We confirmed a differential regulation of vATPase subunits and apoptosis induction. These processes were already identified by us in previous studies as targets of A. fumigatus for immune evasion. Moreover, the combination of quantitative proteomics and bioinformatics led to the identification of A. fumigatus modulated processes and of macrophage intracellular functions. These processes include vesicle trafficking and endocytosis, degradative and immune functions of the phagolysosome, NADPH oxidase activity to produce ROS, MAPK and mTOR signaling as well as metabolic reprogramming of the macrophage. biotin (Thermo Fisher Scientific) in 50 mM Na 2 CO 3 . Initial experiments demonstrated that for the same amount of pksP mutant conidia 10 μ g/mL NHS-biotin linker yielded a comparable efficiency of magnetic loading. Cells were incubated with the linker for 2 h at 4 °C on a rotator and then washed. 50 μ L of the streptavidin-coupled magnetic beads (Miltenyi, Germany) in the concentration provided by the supplier (Thermo Fisher Scientific) were added in labeling buffer (PBS with 2 mM EDTA) to the conidia suspension and incubated for 15 min at 4 °C on a rotator. Co-incubation was performed in 4-well plates. Each well contained 4×10 6 RAW 264.7 macrophage cells. at a resolution of 70k/140k FWHM (M#1/M#2) using a maximum injection time of 120 ms and an AGC (automatic gain control) target of 10 6 . Up to 10 of the most abundant precursor ions per scan cycle with an assigned charge state of z = 2-6 were selected for data-dependent acquisition using an isolation width of m/z 2.0. HCD fragmentation was conducted at a normalized collision energy of 30 V using N 2 . Dynamic exclusion of precursor ions was 35 s (M#1) or 40 s (M#2). Fragment ions were monitored at a resolution of 17.5k (FWHM) using a maximum injection time of 120 ms and an AGC target of 2x10 5 . The fixed first mass was set to m/z 120. The LC-MS/MS instrument was operated by means of the Thermo/Dionex Chromeleon Xpress 6.8 software and the Thermo QExactive Plus Tune / Xcalibur 3.0.63 software. comparison of the protein abundances in the wt and pksP mutant conidia-containing phagolysosomes complexity of the experimental set-up and high variations of the biological replicates. The results of studies on the phagolysosomal proteome vary depending on different factors: (i) the usage of cell lines or primary cells (ii) the ingested particles e.g. latex beads, apoptotic cells, pathogen-associated structures or pathogens (16-18, 22), (iii) the age of the phagolysosome (15, 17), (iv) the analyzed fraction e.g . entire phagolysosome or membrane domains (detergent-resistant or detergent-soluble membranes) (15) and (v) the activation status of the cells, i.e. stimulation with cytokines prior to the infection Components of the oxidative phosphorylation (OXPHOS) system, i.e., protein complexes of the mitochondrial electron transport chain, are enriched in the pksP conidia-containing phagolysosomal sample. The occurrence of those components, although they are of non-phagolysosomal origin, might represent a specific effect of mitochondrial involvement in phagolysosomal processes. We found subunits of all four complexes of the electron transport chain as well as 3-ketoacyl-CoA thiolase and acyl- (cid:18)(cid:381)(cid:4)(cid:3)(cid:282)(cid:286)(cid:346)(cid:455)(cid:282)(cid:396)(cid:381)(cid:336)(cid:286)(cid:374)(cid:258)(cid:400)(cid:286)(cid:3)(cid:381)(cid:296)(cid:3)(cid:410)(cid:346)(cid:286)(cid:3)(cid:296)(cid:258)(cid:410)(cid:410)(cid:455)(cid:3)(cid:258)(cid:272)(cid:349)(cid:282)(cid:3)(cid:628) -oxidation enriched in the pksP mutant conidia-containing phagolysosome, indicating that wt conidia influence the host cell energy metabolism. In line with this assumption, we found mTOR, a regulator of cellular metabolism (50), enriched in phagolysosomes of macrophages that were challenged with pksP conidia. Furthermore, mitochondrial ROS, which are generated by the electron transport chain, were described to contribute to the killing of intracellular pathogens (42, 51). Altogether, this study provides a detailed map of proteins and a comprehensive overview of macrophage intracellular processes that are regulated upon ingestion of A. fumigatus conidia. It delivers a protocol to obtain conidia-containing phagolysosomes, a bioinformatics method for integrating quantitative LC-MS/MS data into the context of the entire protein-protein interaction network to identify significantly regulated processes and confirms previous findings about the A. fumigatus immune evasion strategy, i.e. , inhibition of vATPase-dependent phagolysosomal acidification and the prevention of apoptosis induction. Intriguingly, our data suggest an interference of wt conidia with endocytic vesicle trafficking, MAPK and mTOR signaling, ROS production via NADPH oxidase, and degradative functions of the phagolysosome. Further, the reprogramming of energy metabolism and activation of macrophage immune responses, has not been reported before. These findings lay the basis for mechanistic studies that will help to unravel the complexity of immune evasion strategies of A. fumigatus .


Introduction
Infections with the opportunistic pathogen Aspergillus fumigatus pose a major threat to human health.
It is estimated that there are worldwide more than 200,000 cases of invasive aspergillosis per year affecting patients with a severe underlying immune suppression due to hematologic diseases, genetic immune deficiencies or solid organ or hematopoietic stem cell transplantation (1,2). A. fumigatus produces asexual spores (conidia) that are distributed via the air. Upon inhalation conidia reach the lower airway tract of human individuals. In immunocompromised hosts, the impaired immune cell function allows fungal colonization of the lung tissue and the establishment of a life-threatening infection (3).
Resident alveolar macrophages belong to the first line of immune defense (4). They are activated by fungal surface structures such as -1,3-glucans of the conidial cell wall which bind to the C-type lectin receptor Dectin-1 and thereby inducing phagocytosis. Activated macrophages engulf conidia in a phagosome, which gradually acquires biocidal properties from fusion with lysosomes to generate a mature phagolysosome with acidic luminal pH due to vacuolar ATPase (vATPase) activity (4,5). Small Rab GTPases govern the dynamic fusion and fission processes required for generation of mature phagolysosomes and thus belong to the targets of intracellular pathogens to interfere with the maturation process (6)(7)(8). For example, Mycobacterium tuberculosis prevents the recruitment of Rab14 to the phagolysosome and thereby arrests its maturation (7).
The grey-green pigment of A. fumigatus conidia consists of 1,8-dihydroxynaphthalene (DHN) melanin that represents an important virulence determinant (9). Due to melanin wt conidia are able to reduce the production of and quench reactive oxygen species (ROS) and thus lower the amounts of ROS in the phagolysosome. Furthermore, wt conidia inhibit apoptosis, survive in host cells, germinate and cause damage to host cells (10)(11)(12)(13). A mutant strain producing white conidia was shown to be defective in the polyketide synthase gene pksP and, hence, unable to produce DHN melanin (12). Inside the phagolysosome pksP conidia are faster degraded than wt conidia by luminal acidification and activity of lytic enzymes (14). DHN melanin is the crucial and sufficient factor to block acidification as demonstrated in experiments with melanin particles, i.e., 'melanin ghosts' (14). However, the molecular mechanisms of the interference of the wt conidium with the phagosomal maturation are still not well understood. Previous work of our group showed that A. fumigatus wt conidia-containing phagolysosomes fused with vesicles of the endocytic compartment to a certain extent, but showed reduced acidification required to establish the fungicidal environment for degradation of conidia (13).
The activity of the vATPase is essential to drive the acidification of conidia-containing phagolysosomes (14).
For several pathogenic microorganisms, proteomic studies unraveled the specific protein composition of phagolysosomes for a better understanding of the phagosomal maturation process and its modulation by pathogens (15)(16)(17)(18)(19)(20)(21)(22)(23). These studies demonstrated that the protein composition of the phagosome is highly dynamic, specific for the ingested particle, depends on the stage of phagosomal maturation, the type or cell line and the activation status of the phagocyte. Therefore, we set out to analyze the phagolysosomal proteome after phagocytosis of wt and pksP mutant conidia in order to shed light on processes inhibited by wt conidia during the maturation of phagosomes. In this study, a magnetic labelbased purification protocol for conidia-containing phagolysosomes and the application of a label-free protein quantification method was developed. Bioinformatics was employed to determine the regulatory modules of differentially abundant proteins of the macrophage phagolysosome. We confirmed a differential regulation of vATPase subunits and apoptosis induction. These processes were already identified by us in previous studies as targets of A. fumigatus for immune evasion. Moreover, the combination of quantitative proteomics and bioinformatics led to the identification of A. fumigatusmodulated processes and of macrophage intracellular functions. These processes include vesicle trafficking and endocytosis, degradative and immune functions of the phagolysosome, NADPH oxidase activity to produce ROS, MAPK and mTOR signaling as well as metabolic reprogramming of the macrophage.

Magnetic labeling of conidia and purification of phagolysosomes
The protocol for isolation of conidia-containing phagolysosomes by magnetic separation was adapted from Steinhäuser et al. (24). 2×10 8  Roche, Switzerland) and benzonase nuclease (Merck Millipore, Germany)). Cell lysis was achieved by pressing the cell suspension 60 times through a needle (27G) and was monitored microscopically. DNase (Epicentre, USA) was added and the lysate was incubated for 5 min at 37 °C. For sampling of the whole cell proteome, proteins of the lysate were precipitated and processed as described below. To purify the phagolysosomal fraction the lysate was loaded onto a QuadroMACS separator (Miltenyi).
Phagolysosomes with magnetically labeled conidia were retained on the stand by magnetic force and proteins were directly eluted from the column with 98 °C heated elution buffer (2 % (w/v) SDS, 100 mM TEAB, 10 % (v/v) glycerol, 1 mM TCEP).

Processing of proteins
The eluted protein solution was reduced by evaporation in a SpeedVac at 60 °C to a volume of 100 μL.
Proteins were precipitated with methanol and chloroform as described elsewhere (25)  Fluorescence and chemiluminescence were detected with the Fusion FX7 system (Vilber Lourmat, Germany) and signal intensities and quantifications were determined with the Bio-1D analysis software (Vilber Lourmat) as described in the manufacturer's instructions. Western blots were conducted for each of the three replicates of conidia-containing phagolysosome purifications.

Experimental design and statistical rational
By using a quantitative label-free proteomics approach, we compared the subproteome of enriched

LC-MS/MS analysis of protein samples
Two different methods were applied. For the sub-proteome analysis of the conidia-containing phagolysosomal fraction an analytical method designated as method M#1 was used. For comparison of the phagolysosomal sub-proteome with the whole cell proteome method M#2 was applied.
LC-MS/MS analysis was carried out on an Ultimate 3000 RSLC nano system coupled to a QExactive Plus mass spectrometer (both Thermo Fisher Scientific). Peptides were enriched on a nano-trap column (Acclaim PepMap 100, length 20 mm, diameter 75 μm, particle size 3 μm) at a flow rate of 5 μL/min. cleavages were allowed for tryptic peptides. The precursor mass tolerance was 10 ppm and the fragment mass tolerance was 0.02 Da. Dynamic modification was oxidation of methionine. Static modification was cysteine carbamidomethylation. Percolator node and a reverse decoy database was used for q-value validation of the peptide spectral matches (PSMs) using a strict target false discovery (FDR) rate of < 1%. At least 2 peptides per protein were required for positive protein hits. Label-free quantification was performed with the precursor ions area -Top3 method included in PD. It compares the 3 most abundant peptides of each protein by using the peak area of the respective precursor ion (27). The mass tolerance was set to 2 ppm and the signal-to-noise ratio should be > 3. The abundance values were normalized based on the total peptide amount. Only unique peptides were considered for quantification. The significance threshol 2.0 (upor down-regulation). The mass spectrometry proteomics data were deposited at the ProteomeXchange Consortium via the PRIDE (28) partner repository with the dataset identifiers PXD005724, 10.6019/PXD005724.

Identification of the regulatory module from LC-MS/MS data
Identification of the murine regulatory module was performed using ModuleDiscoverer (29) similar to the approach described in (30). In brief, differentially abundant proteins were mapped onto the murine protein-protein interaction network obtained from the STRING database. Identification of the regulatory module was then performed by the extraction of network regions that are significantly enriched with differentially abundant proteins. These sub-networks were analyzed regarding their biological function using the GOstats package for R (31). Mapping of different protein identifiers was achieved by applying the org.Mm.eg.db annotation package for R (32) as well as the Ensembl BioMarts resource (33) using the biomaRt package for R (34). A detailed description is provided in the supplement.

Prediction of host-pathogen interactions
In order to predict the protein interactions of the M. musculus macrophages and A. fumigatus conidia within the phagolysosome, the regulatory module and the detected fungal proteins were mapped to an existing database of predicted host-pathogen interactions (HPI). The dataset HPI was calculated by using experimental verified interactions from model organisms. These sources for interactions deliver the backbone to predict interfacial interaction of host and pathogen (35). By using traits that characterize the source interacting proteins, such as amino acid sequence, GO annotation, pathway membership,

Development of a protocol for isolation of conidia-containing phagolysosomes
To address the question, whether the manipulation of the phagosome maturation by wt conidia is reflected in the phagolysosomal proteome, proteins were extracted from phagolysosomes containing melanized wt and non-melanized pksP mutant conidia based on a protocol for the isolation of Mycobacteria-containing phagolysosomes (24). Magnetic labeling of conidia enabled the isolation of the phagolysosomal fraction after lysis of infected macrophages (Fig. 1). The concentration of NHS-biotin linker was adjusted to the different surface properties (11) (Fig. 1).

Identification of differentially abundant proteins from LC-MS/MS data
LC-MS/MS measurement and protein identification resulted in a dual proteome set consisting of 2431 murine phagolysosomal and 65 A. fumigatus proteins (Table S1). 95 % of the proteins were detected in both wt und pksP mutant conidia-containing phagolysosomes, suggesting that quantitative differences predominate. To identify the differences, a label-free quantification was performed using the 'Top Three' method (27)

Regulatory module implies interaction of regulated proteins
To deduce regulated processes from the dataset of differentially abundant proteins, we used a bioinformatics approach that identified regulatory modules within the protein-protein interaction (PPI) network provided by the STRING database (37). This analysis was based on the projection of experimentally identified differentially abundant proteins. As a result, a host regulatory module was defined that contained the regulated proteins but also non-regulated and non-detected proteins depicted as knots and their connections visualized as edges. Within the network, submodules represent groups of proteins with a higher connectivity based on STRING that are functionally or structurally related.
The assembled host regulatory module is composed of 302 proteins connected by 3448 edges. 178 of the 302 proteins were detected by LC-MS/MS. 109 of these 178 proteins were identified as differentially abundant. 79 proteins were enriched in the pksP mutant and 30 in the wt conidia-containing phagolysosome. In the host regulatory module 17 submodules were classified. 14 of these submodules comprising five or more proteins were considered for further discussion and used in enrichment analysis of GO and KEGG pathway terms (Table S3). The submodules representing vATPase-dependent vacuolar acidification, signaling, endocytosis and vesicle transport, immune response, generation of ROS via NADPH oxidase and electron transport chain were the focus for further validation experiments and discussion (Fig. 3). In detail, these submodules include proteins with annotated functions in MAPK signaling pathway, actin polymerization, SNARE-mediated membrane trafficking and Rab GTPaseregulated vesicle fusion, hydrolytic activities and NADPH oxidase-dependent ROS production.
To verify the data obtained by LC-MS/MS analysis, label-free quantification and the regulatory module analysis, the abundances of several representative proteins were quantified by western blot analyses or immunofluorescence (Fig. 4). First, to exclude that magnetic labeling interferes with recruitment of phagolysosomal proteins the localization of cathepsin D was monitored by immunofluorescence. Both, labeled and unlabeled wt conidia reduced recruitment of cathepsin D to the phagolysosomal membrane, whereas pksP conidia-containing phagolysosomes showed a typical pattern of cathepsin D recruitment, independent of magnetic labeling (Fig. 4A). Representative proteins included proteins enriched in pksP conidia-containing phagolysosomes: Cathepsin Z, a lysosomal protease, which was 2fold higher abundant; Lamp1, a phagolysosomal marker protein represented in the 'endocytosis' module with a 3.8-fold higher abundance; Nduf9, a component of the NADH dehydrogenase complex represented in the module 'electron transport chain' with 5.9-fold higher abundance. The higher abundance of selected proteins in pksP conidia-containing phagolysosomes was confirmed by western blot analysis (Fig. 4B). Because we found LAMTOR, a regulator of the mTOR signaling pathway, enriched in pksP conidia-containing phagolysosomes, and, secondly, the regulatory module indicated a regulation of carboxylic acid metabolism and cellular respiration, we analyzed the abundance of the kinase mTOR.
Both aforementioned processes are potentially controlled by mTOR. Although not detected by LC-MS/MS, we could confirm a differential regulation of mTOR in conidia-containing phagolysosomes by western blot (Fig. 4B). The vesicle trafficking protein 8 (Vamp8), which was assigned to the 'vesicle trafficking' module and which was 2.8-fold enriched in pksP conidia-containing phagolysosomes, was analyzed for its localization by immunofluorescence. A differential recruitment was clearly confirmed (Fig. 4C).

Fungal proteins in the phagolysosome
The host regulatory module provided information about processes that are regulated in the macrophage upon phagocytosis of A. fumigatus conidia. The dual proteome analysis allowed us to investigate differentially abundant fungal proteins as potential effectors interfering with the host endocytic pathway. It is likely that fungal proteins in the phagolysosome are either actively secreted or released from the fungal cell surface. However, it needs to be considered that the two strains face different conditions: While pksP mutant conidia are exposed to an acidic environment and tackled by phagolysosomal degrading enzymes 2 h after phagocytosis, the wt conidia reside in more favorable conditions in a phagolysosome with neutral pH. Accordingly, the fungal proteome enriched from pksP mutant conidia-containing phagolysosomes was composed of proteins induced upon oxidative stress or upon encounter with immune cells, e.g., a GTPase regulating vesicular transport, an RNA helicase, alcohol dehydrogenases and a transaldolase of the pentose phosphate pathway (Table S4).
Proteins enriched in wt conidia-containing phagolysosomes included a catalase, drug response and mitochondrial unfolded protein response elements and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Further proteins were histone H2A1, a component of ribosome biogenesis, transcription and mRNA processing, the signaling protein 14-3-3 and a high abundant conidial protein with unknown function that was found by Asif et al. (38) to be present on conidia (Table S5).

DISCUSSION
Here, we set out to characterize the phagolysosomal proteome and to identify phagolysosomal proteins and processes that are altered after phagocytosis of melanized A. fumigatus conidia to allow their survival inside the macrophage. The phagolysosome is the place of the direct interaction between conidia and the host, hence, it can be expected that modifications in its protein composition influence its fungicidal activity. On the level of the whole cell, changes in abundance of proteins in specific organelles like the phagolysosome might be masked e.g. by highly abundant cytosolic proteins.
Therefore, we analyzed the proteomes of both whole macrophage cells and phagolysosomes. Because The outer phagolysosomal membrane is surrounded by a mesh of sticky actin fibers that trap organelles in its proximity during the isolation procedure (40). The occurrence of proteins typically present in nonphagosomal cell compartments such as the nucleus, ribosomes or mitochondria has been reported earlier (15). Their frequency was especially high in cells with a high autophagocytic activity. This can be explained by the dynamic interaction and exchange between those two organelles (21,41). Additionally, mitochondria have been implicated in a broad number of cellular functions, including TLR-dependent ROS production in response to intracellular pathogens (42) and induction of apoptosis via intrinsic and extrinsic pathways (43). stimulation with cytokines prior to the infection (19).
With the help of a label-free quantification, around 30 % of the identified proteins were classified as significantly regulated. The differential regulation of cathepsin Z, Lamp1 and NADH dehydrogenase was verified by western blotting and the differential recruitment of Vamp8 was confirmed by immunofluorescence. Interestingly, the regulation of mTOR, which was not detected by the LC-MS measurement, but was predicted from the interaction network, was found to be differentially regulated by western blot analysis and thus confirmed the bioinformatics approach. The putative low abundance of mTOR might explain why this protein was only detected by sensitive western blotting but not by LC-MS/MS. The comprehensive LC-MS/MS analysis was based on a bottom-up data-dependent shotgun approach, i.e., not only the general detection sensitivity of the tryptic peptides of a certain protein is decisive. Much more important for highly complex peptide samples is the dynamic range of the analysis.
i.e., relative abundance of the tryptic peptides of mTOR in relation to all other peptides, since the 10 most abundant precursor ions per data-dependent scan cycle are selected for fragmentation. Since murine mTOR is a large protein of about 289 kDa, which implicates numerous potentially detectable peptides, it can be assumed that mTOR is of relative low abundance. At least the abundance of mTOR is lower than for all proteins identified in our study.
Taken together, the bioinformatics compilation of the differentially abundant proteins into the regulatory module allowed for an identification of processes that are modified in the phagolysosome after ingestion of either wt or pksP conidia.

vATPase-dependent acidification
Acidification of the phagolysosome is prerequisite for the degradation of phagocytosed material and is driven by the vATPase, the vacuolar ATP-dependent proton pump (44). Activity of the enzyme complex is regulated via assembly and disassembly of the membrane bound V 0 and cytosolic V 1 domain (45). In a previous study, we showed that activity of the vATPase mainly drives the acidification of phagolysosomes containing pksP conidia (14). Here, the proteomic data confirmed the increased abundance of V 1 subunits in pksP conidia-containing phagolysosomes.

Lysosome-endosomal trafficking
The endocytic trafficking and transport system is also modulated in the wt conidia-containing phagolysosome. We detected several proteins of the SNARE, SNAP and syntaxin family, which mediate intracellular trafficking, docking and fusion processes (46). They were mainly down-regulated in macrophages infected with wt conidia. Also, the small GTPase Rab5 and early endosomal antigen 1 (EEA1) were regulated. Rab5 and its effector EEA1 mediate homotypic fusion of the early phagosome with early endosomes and are regarded as markers for an early stage of phagolysosomal maturation (47,48). Rab5 had a lower abundance in wt conidia-containing phagolysosomes, whereas EEA1 was more abundant. Likewise, EH domain-containing proteins, which control endocytic fusion and co-localize with  (49)).

Generation of energy
Components of the oxidative phosphorylation (OXPHOS) system, i.e., protein complexes of the mitochondrial electron transport chain, are enriched in the pksP conidia-containing phagolysosomal sample. The occurrence of those components, although they are of non-phagolysosomal origin, might represent a specific effect of mitochondrial involvement in phagolysosomal processes. We found subunits of all four complexes of the electron transport chain as well as 3-ketoacyl-CoA thiolase and acyl--oxidation enriched in the pksP mutant conidia-containing phagolysosome, indicating that wt conidia influence the host cell energy metabolism. In line with this assumption, we found mTOR, a regulator of cellular metabolism (50), enriched in phagolysosomes of macrophages that were challenged with pksP conidia. Furthermore, mitochondrial ROS, which are generated by the electron transport chain, were described to contribute to the killing of intracellular pathogens (42, 51).

Intracellular signaling
The LC3BII protein that binds to phagolysosomes (55,56). Rubicon recruits the NADPH oxidase (NOX) and activates its ROS producing activity, which in turn is required for LC3B lipidation (54). In line, Chamilos et al. (52,57) demonstrated that DHN melanin of A. fumigatus conidia blocks LAP activation in macrophages.
Previous work of our lab showed that wt conidia of A. fumigatus block apoptosis of macrophages by hijacking the PI3K/Akt signaling pathway (58). The sustained activation of Akt promotes cell survival and inhibits induction of the apoptosis process and likely depicts a strategy of the pathogen to hide inside the macrophage and evade further immune responses (58,59). Among the differential abundant proteins were elements of the apoptosis or cell survival regulation pathways: MAPK, AKT, mTOR and Rac signaling pathway, the ubiquitin-proteasome system, members of the 14-3-3 family proteins, prostaglandin E synthase among others. Pro-apoptotic proteins such as Bak1 (60), Aifm2 and Praf2 were higher abundant in pksP mutant conidia-containing phagolysosomes, whereas the anti-apoptotic regulator Bcl2-like 13 protein was enriched in wt conidia-containing phagolysosomes.

Degradation and antigen processing capacity
Different studies demonstrated the requirement for ROS for an efficient fungal clearance (61,62 Cathepsins, lysosomal peptidases and enzymes to degrade glycans and glycolipids were enriched in the proteome of pksP mutant conidia-containing phagolysosomes indicating a reduced degradation capacity of wt conidia-containing phagolysosomes. In line with this finding is the reduced antigen processing and presentation machinery of those phagolysosomes. For example, phagolysosomes containing wt conidia had lower levels of the macrophage activation marker CD68 or macrophage colony-stimulating factor receptor 1 (CSFR1).
The interference of A. fumigatus with the described intracellular processes of macrophages is conceivably due to an interaction of fungal proteins with host proteins. We performed an initial bioinformatics analysis to predict potential interaction candidates on the basis of already described host-pathogen protein interactions. This analysis suggests fungal heat shock proteins Hsp70 and 90, the translation elongation factor Tef1 and a 14-3-3 protein ArtA to interact with a range of host proteins involved in processes such as vATPase activity, endocytic trafficking and signaling (Fig. S1). Experiments evaluating the prediction of these interactions, e.g. on a biochemical level or using immune cells with a knock down of the genes of interest, are required to identify the role of protein-protein interactions for the immune modulation by A. fumigatus.
Altogether, this study provides a detailed map of proteins and a comprehensive overview of macrophage intracellular processes that are regulated upon ingestion of A. fumigatus conidia. It delivers a protocol to obtain conidia-containing phagolysosomes, a bioinformatics method for integrating quantitative LC-MS/MS data into the context of the entire protein-protein interaction network to identify significantly regulated processes and confirms previous findings about the A. fumigatus immune evasion strategy, i.e., inhibition of vATPase-dependent phagolysosomal acidification and the prevention of apoptosis induction. Intriguingly, our data suggest an interference of wt conidia with endocytic vesicle trafficking, MAPK and mTOR signaling, ROS production via NADPH oxidase, and degradative functions of the phagolysosome. Further, the reprogramming of energy metabolism and activation of macrophage immune responses, has not been reported before. These findings lay the basis for mechanistic studies that will help to unravel the complexity of immune evasion strategies of A. fumigatus.
gratefully acknowledged for initial experiments and Silke Steinbach for excellent technical assistance.

Data Availability
Data sets are available at the PRIDE database under the Project ID: PXD006134 URL: https://www.ebi.ac.uk/pride/archive/projects/PXD006134