Proteome analysis of bronchoalveolar lavage fluids reveals host and fungal proteins highly expressed during invasive pulmonary aspergillosis in mice and humans

Figures & data

Table 1. Demographic and clinical characteristics of IPA and non-IPA patients.

	IPA patients (n = 27)	non- IPA patients (n = 27)
Demographic variables
Age	58 (±16)	53 (±19)
Sex ratio (male/female)	14/13	13/14
Underlying disease
Acute Leukemia	2	0
Allo-HSCT	6	0
Influenza	2	0
Lung disease	4	3
Solid tumor	1	1
SOT	11	20
Other disease	1	3

SOT: solid organ transplants, Allo HSCT: allogeneic hematopoietic stem cell transplantation.

Figure 1. Detection of proteins in human BAL samples by LC-MS/MS analysis. (a) Total numbers of identified proteins in each patient sample. (b) Volcano plot of proteins with different abundance in infected versus non-infected samples. The adjusted p-value is plotted against the expression fold change of all detected proteins within the different groups. Data points in the lower center area of the plots (empty circles) display unchanged or proteins with no significant fold change. Data points in the upper quadrants indicate proteins (filled circles) with significant negative (left) and positive (right) changes in protein abundances, respectively. (c) Graphical overview of the numbers of proteins with increased abundance (Up) and decreased abundance (Down) in IPA patients compared to the control group.

Figure 2. Proteins with most significant differential abundance determined by LC-MS/MS analysis in human BAL samples. (a) Heat map of the 30 most abundant proteins during IPA. Levels of proteins with significantly increased abundance in each patient sample are visualized by LFQ intensities with a significant fold change >2 of the IPA group vs non-IPA group. FC values are fold changes. (b) The most significantly overrepresented GO terms of highly abundant proteins for biological process, molecular function and cellular component were determined with an adjusted p-value < 0.05. A full list of overrepresented proteins and GO terms are provided in Supplementary Tables 2 and 3.

Table 2. Common host proteins that were significantly upregulated during IPA in both human and mice.

Name	Gene	FC in human	FC in mice
Apolipoprotein B	APOB	12	82
CD177 molecule	CD177	11	121
C-Reactive Protein	CRP	27	782
Fibrinogen Beta Chain	FGB	5	3
Hexose-6-Phosphate Dehydrogenase/Glucose 1-Dehydrogenase	H6PD	4	10
Histone H3.1	HIST1H3A	14	12
Inter-Alpha-Trypsin Inhibitor Heavy Chain 3	ITIH3	14	3
Lamin B1	LMNB1	5	14
Lipopolysaccharide Binding Protein	LBP	10	5
Protein Z-dependent protease inhibitor	SERPINA10	3	3
Serum amyloid protein-A1	SAA1	15	390

FC values are fold changes calculated from measured LFQ values.

Figure 3. Detection of proteins in murine BAL samples after pulmonary infection with A. fumigatus by LC-MS/MS analysis. (a) Fungal infection of murine lungs visualized by in vivo imaging in five exemplary mice of the infection group versus five mice of the exposition group. (b) Total numbers of identified proteins in each mouse sample. (c) Volcano plot of differentially expressed proteins from infected versus non-infected mice. The adjusted p-value is plotted against the abundance fold change of all detected proteins within the different groups. Data points in the lower center area of the plots (gray) display unchanged or proteins with no significant fold change. Data points in the upper quadrants indicate proteins (filled circles) with significant negative (left) and positive (right) changes in protein abundances, respectively. (d) Graphical overview of the numbers of proteins with increased (Up) and decreased (Down) abundance in samples from infected mice compared to non-infected mice. (e) Volcano plot of differentially abundant proteins from infected versus exposed mice. The adjusted p-value is plotted against the abundance fold change of all detected proteins within the different groups. Data points in the lower center area of the plots (gray) display unchanged or proteins with no significant fold change. Data points in the upper quadrants indicate proteins (filled circles) with significant negative (left) and positive (right) changes in protein abundances, respectively. (f) Graphical overview of the numbers of proteins with increased (Up) and decreased (Down) abundance in samples from infected mice compared to exposed mice.

Figure 4. Proteins with most significant differential abundance in infected versus non-infected mice determined by LC-MS/MS analysis. (a) Heat map of the Top 30 abundant proteins during IPA. Abundance of significantly upregulated proteins in each animal sample are visualized by LFQ intensities with a significant fold change (FC) >2 between infected and non-infected group. The asterisk indicates proteins that were also significantly increased in comparison to the exposed group in comparison to the exposed group. (b) The most significantly overrepresented GO terms of highly abundant proteins for biological process, molecular function and cellular component were determined with an adjusted p-value. A full list of proteins with significantly differential abundance is provided in Supplementary Table 4.

Table 3. Fungal Proteins identified in BAL of mice.

Protein Number	Peptide counts (all)	Razor + unique peptides	Protein ID	Fasta header	Gene	Mice EX INF NI (n = 9) (n = 19) (n = 10)			Signal peptide	Description in other studies
Unknown function
M1	5	5	B0Y9E0	SH3b domain-containing protein	AFUB_080700	0	12	0	+	32
M2	3	3	B0YAY0	NlpC/P60-like cell-wall peptidase, putative	AFUB_086210	0	9	0	+	32, 33
M3	8	8	B0YEL6	Uncharacterized protein	AFUB_099610	0	5	0	+
M4	4	2	B0Y269	NlpC/P60-like cell-wall peptidase, putative	AFUB_061470	0	4	0	+	31,32, 33
M5	2	2	B0Y402	Uncharacterized protein	AFUB_056040	0	2	0
Allergen
M6	2	2	B0Y6E9	Major allergen Asp F2	AFUB_066690	0	4	0	+	31,32,33,36,37,59,66
M7	4	4	B0XNL0	Extracellular cell wall glucanase Crf1/allergen Asp F9	AFUB_015530	0	3	0	+	31,34,36,37,8, 35
M8	7	7	B0XUQ5	Allergen Asp F4	AFUB_020900	2	10	1	+	36, 37
Defense mechanisms
M9	2	2	B0Y5K4	Glutamate dehydrogenase	AFUB_063700	0	4	0		66, 7
M10	3	3	B0Y818	Thioredoxin reductase GliT	AFUB_075790	0	3	0		31, 34
M11	5	5	B0Y1Q1	Nonribosomal peptide synthase, putative	AFUB_060400	0	4	3
M12	5	5	B0YAW2	Hybrid NRPS/PKS enzyme, putative	AFUB_086030	5	4	5		33
Nutrient aquisition
M13	3	3	B0XT32	Probable pectate lyase A	plyA	0	3	0	+	8, 31
M14	3	3	B0Y9M8	Probable pectate lyase E	plyE	0	5	0	+	32
M15	2	2	B0Y3V5	Extracellular cellulase CelA/allergen Asp F7-like, putative	AFUB_055560	0	5	0	+	31
M16	2	2	B0Y9F1	Glycosyl hydrolase, putative	AFUB_080810	0	3	0	+
M17	3	3	B0XQF0	RNase L inhibitor of the ABC superfamily, putative	AFUB_009720	4	5	3
M18	2	2	B0XSS1	Beta-alanine synthase, putative	AFUB_017390	4	12	4
Signaling
M19	3	3	B0Y182	FacC-like extracellular signaling protein, putative	AFUB_049010	0	5	0	+	32, 33
Cell wall maintenance
M20	5	5	B0YCQ5	SUN domain protein (Uth1), putative	AFUB_091030	0	7	0	+	38
M21	4	4	B0XXF8	Probable glucan endo-1,3-beta-glucosidase	eglC	0	4	0	+	8, 31, 35

[32]: Kale et al. 2017 SciRep, [33]: Mac Donagh et al. 2018 PlosPath: Transcriptome studies: upregulation in vivo

[31]: Sriranganadane et al. 2009 JProt, [59]: Fekkar et al. 2012, JProt; [8]: Wartenberg et al. 2011 IJMM, [35]: Champer et al. 2016 JFungi; Proteome studies: detection of secreted proteins

[7]: Virginio et al. 2014 IJMS, [34]: Bacher et al. 2014 JImmun, [36]: Crameri 1998 Int Arch Allergy Immunol, [37]: Singh et al. 2010 JProtRes, [66]: Teutschbein et al. 2016 JProtRes: Immune Proteome Studies: Antigenic protein detected by patient sera

[38]: Gastebois et al. 2013 JBC; study on SUN proteins: Transcriptome study: upregulation during mycelial growth.

Table 4. Fungal proteins detected in human BAL fluid by LC-MS/MS.

Protein number	Peptide counts (all)	Razor + unique peptides	Protein IDs	Fasta header	Gene	Patients IP (n = 27) non IPA (n = 27)
H1	3	3	P79017	Major allergen Asp f 2	AFUA_4G09580	2	0
H2	5	2	Q4WND4	14-3-3 family protein	AFUA_6G06750	3	0
H3	2	2	Q4WQP9	Gamma-glutamyltranspeptidase	AFUA_4G13580	6	2
H4	5	5	Q4WE46	MYB DNA-binding domain protein	AFUA_5G01730	5	2
H5	2	2	Q4X1H5	Mitochondrial Hsp70 chaperone (Ssc70), putative	AFUA_2G09960	4	2
H6	2	2	Q4WLD0	Polyprenyl transferase pyr6	pyr6	1	3
H7	3	2	Q4WHW6	Serine/threonine-protein phosphatase	AFUA_2G03950	9	4
H8	5	5	Q4X0G8	Fermentation associated protein (Csf1), putative	AFUA_2G1352	2	6
H9	2	2	Q4X1P9	60S ribosomal protein L30, putative	AFUA_2G09200	9	6
H10	4	4	Q4WQU7	Glycosyl transferase, putative	AFUA_4G14070	5	8
H11	3	2	Q4WN57	T-complex protein 1 subunit epsilon	AFUA_6G07540	9	9
H12	2	2	Q4WR07	C6 transcription factor, putative	AFUA_4G14680	11	19

List of all fungal proteins that were identified in individual patient samples with at least two unique peptides.

Sriranganadane D, Waridel P, Salamin K, et al. Aspergillus protein degradation pathways with different secreted protease sets at neutral and acidic pH. J Proteome Res. 2010;9:3511–3519.

 PubMed Web of Science ®Google Scholar

Kale SD, Ayubi T, Chung D, et al. Modulation of immune signaling and metabolism highlights host and fungal transcriptional responses in mouse models of invasive pulmonary aspergillosis. Sci Rep. 2017;7:17096.

 PubMed Web of Science ®Google Scholar

De Pauw B, Walsh TJ, Donnelly JP, et al. Revised definitions of invasive fungal disease from the European organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (EORTC/MSG) consensus group. Clin Infect Dis. 2008;46:1813–1821.

 PubMed Web of Science ®Google Scholar

Fekkar A, Balloy V, Pionneau C, et al. Secretome of human bronchial epithelial cells in response to the fungal pathogen Aspergillus fumigatus analyzed by differential in-gel electrophoresis. J Infect Dis. 2012;205:1163–1172.

 PubMed Web of Science ®Google Scholar

Wartenberg D, Lapp K, Jacobsen ID, et al. Secretome analysis of Aspergillus fumigatus reveals Asp-hemolysin as a major secreted protein. Int J Med Microbiol. 2011;301:602–611.

 PubMed Web of Science ®Google Scholar

Champer J, Diaz-Arevalo D, Champer M, et al. Protein targets for broad-spectrum mycosis vaccines: quantitative proteomic analysis of Aspergillus and Coccidioides and comparisons with other fungal pathogens. Ann N Y Acad Sci. 2012;1273:44–51.

 PubMed Web of Science ®Google Scholar

Virginio ED, Kubitschek-Barreira PH, Batista MV, et al. Immunoproteome of Aspergillus fumigatus using sera of patients with invasive aspergillosis. Int J Mol Sci. 2014;15:14505–14530.

 PubMed Web of Science ®Google Scholar

Bacher P, Kniemeyer O, Teutschbein J, et al. Identification of immunogenic antigens from Aspergillus fumigatus by direct multiparameter characterization of specific conventional and regulatory CD4+ T cells. J Immunol. 2014;193:3332–3343.

 PubMed Web of Science ®Google Scholar

Crameri R. Recombinant Aspergillus fumigatus allergens: from the nucleotide sequences to clinical applications. Int Arch Allergy Immunol. 1998;115:99–114.

 PubMed Web of Science ®Google Scholar

Singh B, Oellerich M, Kumar R, et al. Immuno-reactive molecules identified from the secreted proteome of Aspergillus fumigatus. J Proteome Res. 2010;9:5517–5529.

 PubMed Web of Science ®Google Scholar

Lass-Florl C. How to make a fast diagnosis in invasive aspergillosis. Med Mycol. 2019;57:S155–S60.

 PubMed Web of Science ®Google Scholar

Gastebois A, Aimanianda V, Bachellier-Bassi S, et al. SUN proteins belong to a novel family of beta-(1,3)-glucan-modifying enzymes involved in fungal morphogenesis. J Biol Chem. 2013;288:13387–13396.

 PubMed Web of Science ®Google Scholar