Functional convergence of gliP and aspf1 in Aspergillus fumigatus pathogenicity

Figures & data

Table 1. A. fumigatus genes that were differentially expressed in the ΔgliP mutant relative to the wild-type strain at 5 days post-infection in the lungs of immunosuppressed mice. These results are based on nanoString probe counts (Table S1). Data are the mean of 3 biological replicates.

Gene	Gene Name	Putative Function	ΔgliP/WT
Afu5g02330	aspf1	Cell surface – allergen, cytotoxin, upregulated in biofilm and in vivo	6.8
Afu3g12890	hasA	Transcription factor – regulates biosynthesis of hexadehydroastechrome	5.1
Afu1g01550	zrfA	Zinc transporter – acidic environments	3.8
Afu4g14070		Cell surface – secreted protein, glycosyl transferase	2.3
Afu5g14800		Metabolism – lactate dehydrogenase, downregulated in biofilm	2.2
Afu4g09560	zrfC	Zinc transporter – neutral or alkaline environments	2.1
Afu4g09580	aspf2	Cell surface – fibrinogen binding protein, required for growth in zinc-limited conditions	2.1
Afu1g14560	msdS	Putative 1,2-α-mannosidase; secreted protein; fibrinogen-binding	0.43
Afu6g03540	acuE	Metabolism – malate synthase, glyoxylate cycle	0.39
Afu2g12850	gel3	Putative GPI anchored β(1–3)glucanosyltransferase	0.34
Afu8g01310		Iron acquisition – metalloreductase	0.15
Afu4g11800	alp1	Cell surface – secreted protein alkaline serine protease	0.13
Afu6g09660	gliP	Gliotoxin – non-ribosomal peptide synthetase	0.03

Figure 1. Up-regulation of aspf1 in the ΔgliP mutant during pulmonary infection. Corticosteroid-treated mice were infected via an aerosol with the A. fumigatus wild-type (Af293) strain and ΔgliP and Δaspf1 mutants. After 5 days of infection, the lungs were harvested and total RNA was extracted for real-time PCR analysis. The transcript levels of aspf1 and gliP were normalized to GAPDH. Result are from 4 mice per strain, each tested in triplicate. Horizontal line indicates the median value. *p < 0.02 compared to wild type strain by the Mann-Whitney test.

Figure 2. Effects of deletion of aspf1 and/or gliP on the virulence of A. fumigatus. Mice were immunosuppressed with cortisone acetate, infected with an aerosol of the indicated strains of A. fumigatus, and monitored for survival. Data are the combined results of two independent experiments for a total 16 mice per strain. *P < 0.001 compared to Af293 and the Δaspf1+ aspf1 complemented strain; †p < 0.01 compared to the Δglip and Δaspf1 mutants by the log-rank test.

Figure 3. Deletion of gliP reduces leukocyte death. Photomicrographs of periodic acid-Schiff (PAS) stained sections of the lungs of mice after 5 days infection with the indicated strains of A. fumigatus. Insets show magnified images of the regions indicated by the arrows. Filled arrows indicate leukocyte fragmentation and hollow arrows indicate intact leukocytes. Results are representative of 3 mice per strain. Scale bar, 20 µm.

Figure 4. gliP is required for A. fumigatus to induce leukocyte apoptosis at foci of infection. Photomicrographs of the lungs of mice after 5 days of infection with the indicated A. fumigatus strains. Apoptotic cells (brown) were detected by TUNEL staining. Results are representative of 3 mice per strain. Scale bar, 30 µm.

Figure 5. Effects of deletion of aspf1 and/or gliP on the capacity of A. fumigatus to damage a mouse macrophage cell line in vitro. The mouse RAW 264.7 macrophage cell line was infected with the indicated strains of A. fumigatus for 16–24 h and the extent of host cell damage after 16, 20, and 24 h of infection was measured using a ⁵¹Cr release assay. Results are mean ± SD of 3 experiments, each performed in triplicate. *P < 0.01 vs Af293 and the Δaspf1+ aspf1 complemented strain; ^†p < 0.01 vs. the Δaspf1 and gliP mutants. Statistical significance was analyzed by the student’s t-test with the Holm-Sidak correction for multiple comparisons.

Table 2. Expression ratios of host genes that were differentially expressed in mice infected for 5 days with the indicated A. fumigatus mutants. These results are based on nanoString probe counts (Table S2). Data are the mean of 4–5 biological replicates.

Host Gene	Gene Product	Δaspf1/WT	ΔgliP/WT	Δaspf1 Δglip/WT
CXCL2	Macrophage inflammatory protein 2α	0.8	5.8	2.2
CCL4	Macrophage inflammatory protein 1β	0.9	5.7	3.5
CCL3	Macrophage inflammatory protein 1α	0.9	4.9	3.0
TNF	Tumor necrosis factor-α	0.9	3.0	1.4

Figure 6. Effects of deletion of aspf1 and/or gliP on the pulmonary inflammatory response. Corticosteroid-treated mice were infected with the indicated strains for 5 days, after which the mice were sacrificed and the lungs were harvested for RNA extraction. The transcript levels of the indicated chemokine and cytokine genes were determined by real-time PCR. Each symbol indicates the result from an individual mouse and the horizontal lines indicate the median values. *P < 0.03 vs Af293; ^†p < 0.03 vs. the gliP mutant by the Mann-Whitney test.

Figure 7. Deletion of gliP results in increased pulmonary fungal burden. Mice were infected with the indicated strains of A. fumigatus and their pulmonary fungal burden was determined at day 5 by quantitative PCR. Results are from a single experiment using 8–10 mice per strain. Each symbol represents the fungal burden of an individual mouse. Horizontal lines indicate the median value. *P < 0.02 vs Af293 by the Mann-Whitney test.

Table 3. Strains of A. fumigatus used in the experiments.

Strain	Genotype	Reference
Af293	Wild-type	[45]
Δaspf1	Aspf1::hph	This study
Δaspf1+ aspf1	Aspf1::hph paspf1-ble	This study
ΔgliP	gliP::hph
Δaspf1+ ΔgliP	Aspf1::ble gliP::hph	This study

Nierman WC, Pain A, Anderson MJ, et al. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature. 2005;438:1151–1156.

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