A possible role for fumagillin in cellular damage during host infection by Aspergillus fumigatus

Figures & data

Figure 1. Survival analysis of immunosuppressed mice intranasally infected with Aspergillus fumigatus. Mice were immunosuppressed with two intraperitoneal doses of cyclophosphamide, 150 mg/Kg and 100 mg/Kg, four days and one day before infection, respectively. Animals were infected with 1 x 10⁶, 1 × 10⁷ and 1 × 10⁸ A. fumigatus resting conidia/animal. Non-infected animals were used as controls.

Figure 2. Histology of infected lungs. To study the fungal progression and the subsequent tissue damage, lung slices were stained with Grocott’s methenamine silver (G.M.S.) and haematoxylin-eosin (H.E.). White arrows show: (A) conidia in the lumen airways, (B and C) hyphae in lung parenchyma, (D) hyphae in lung parenchyma and inside venous vessels. Black arrows show vascular walls: (C) arterial and (D) venous vessels.

Figure 3. Gene ontology enrichment of differential expressed genes (DEGs). Percentage of down-regulated and up-regulated genes belonging to one of GO categories: (A and D) cellular components, (B and E) molecular functions and (C and F) biological process. The graphics only included the most representative groups (> 4%). Raw data are included in the table S4.

Table 1. Results of Differential Expressed Genes (DEGs) detected using microarray technologies versus expression data using specific primers in RT-qPCR.

			Fold Change^a
			Day3 vs Day1		Day4 vs Day1
Product description^b	Systematic Name^c	Standard Name^d	AWAFUGE	RT-qPCR	AWAFUGE	RT-qPCR
Fumagillin/Pseurotin pathway
Polyketide synthase	Afu8g00370	fma-PKS / fmaB		6.28	5.66	7.10
O-methyltransferase	Afu8g00390	fmaD	6.37	7.97	5.71	8.46
Hypothetical protein	Afu8g00400		7.8	15.90	6.84	16.23
Methionine aminopeptidase. type II	Afu8g00410	metAP / fpaII	4.53	5.03	4.09	4.88
C6 finger transcription factor	Afu8g00420	fumR / fapR	4.28	5.48		5.52
Hypothetical protein	Afu8g00430		7.21	9.52	6.97	9.61
Steroid monooxygenase	Afu8g00440	psoF	5.22	8.63	4.89	9.05
Phytanoyl-CoA dioxygenase family protein	Afu8g00480	fmaF	4.63	11.24	4.65	11.56
Acetate-CoA ligase	Afu8g00500		5.03	9.95	4.68	10.36
Cytochrome P450 oxidoreductase OrdA-like	Afu8g00510	fmaG	5.87	11.07	5.02	10.92
α/β hydrolase	Afu8g00530	psoB	5.41	8.30	5.29	8.47
Methyltransferase	Afu8g00550	psoC	7.09	10.12	6.90	10.70
Cytochrome P450 oxidoreductase	Afu8g00560		4.61	ND	4.46	ND
Glutathione S-transferase like	Afu8g00580	elfB / psoE	5.06	9.48	4.96	9.87
Lytic enzymes
Secreted dipeptidyl peptidase DppV	Afu2g09030	dppV	3.47	3.25	3.92	3.83
Vacuolar carboxypeptidase Cps1	Afu3g07040	cps1			2.82	3.43
Extracellular dipeptidyl-peptidase Dpp4	Afu4g09320	dppIV			3.50	0.63
Extracellular lipase	Afu5g02040		3.93	3.20	4.21	3.97
Major allergen and cytotoxin AspF1	Afu5g02330	aspf1			4.64	4.09
Lipase	Afu7g04020		5.64	3.62	5.95	4.90
Class V chitinase ChiB1	Afu8g01410	chiB1			3.50	5.17
Elastinolytic metalloproteinase Mep	Afu8g07080	Mep			3.88	3.59
Others
Acetyltransferase. GNAT family family	Afu1g09260		−3.40	1.43	−3.05	4.12
Hypothetical protein	Afu1g10450		−3.39	−0.41	−3.39	−0.23
Methionine aminopeptidase. type II	Afu2g01750		3.60	2.37	4.10	3.02
4-hydroxyphenylpyruvate dioxygenase	Afu2g04200	hppD			4.21	4.87
Maleylacetoacetate isomerase MaiA	Afu2g04240	maiA			3.72	3.79
Hypothetical protein	Afu2g16440		−3.35	0.58	−3.66	0.73
Short chain dehydrogenase/ oxidoreductase CpoX2	Afu2g18000	fgaDH	−3.43	1.01	−3.31	−0.17
Hypothetical protein	Afu3g00410				−3.47	0.06
MFS sugar transporter	Afu3g03700		3.39	2.31	3.77	3.65
C6 sexual development transcription factor NosA	Afu4g09710	rosA			3.87	6.59
C6 transcription factor	Afu5g00950		−3.71	1.26	−4.09	0.72
Hypothetical protein	Afu5g08800		3.99	4.82
Pectin lyase	Afu5g10170				−3.79	−4.50
Pectin lyase	Afu5g10380				−3.57	−3.31
C6 transcription factor	Afu5g14290		4.36	1.16
Aldehyde dehydrogenase	Afu7g01000				4.11	7.86
Indoleamine 2.3-dioxygenase	Afu7g02010		4.19	5.94	5.72	8.21
Defensin domain protein	Afu7g05180		5.06	6.71	4.81	4.21
Integral membrane protein Pth11-like	Afu7g06620		3.41	4.72	3.38	4.41

^aThis value represents the difference of the fold change in log₂ obtained for each gene between days of infection compared in each case. A negative or positive value indicated down or up-regulation relative to the first day post-infection. respectively. AWAFUGE: data obtained with Agilent Whole A. fumigatus Genome Expression 44K v.1; RT-qPCR: data obtained with RT-qPCR using the A. fumigatus specific primers designed.

^bProduct description of the genes found on the microarray following RefSeq nomenclature.

^cSystematic name of the gene following AspGD nomenclature.

^dGene name following AspGD nomenclature (http://www.aspergillusgenome.org). except fumagillin/pseurotin pathway that also follow the nomenclature published by Wieman P. et al. (2013).

Figure 4. Schematic representation of the intertwined fumagillin/pseurotin gene cluster. Red arrows indicate the up-regulated DEGs relative to the first day post-infection. Graphics represent the log₂ fold change (FC) obtained by AWAFUGE microarray and RT-qPCR assays. FumR/FapR is the transcription factor that regulates fumagillin and pseurotin production. PKS/NRPS regulates pseurotin production. ND: Not determined by RT-qPCR.

Figure 5. Cell damage and fumagillin production during co-incubation of A. fumigatus with A549 cells. (A and B) Extent of A. fumigatus-induced damage to the A549 pulmonary epithelial cell line. The indicated strains of A. fumigatus were incubated with A549 cells for 20h, and the extent of epithelial cell damage was determined using a ⁵¹Cr release assay. Results in (A) are a box-whisker plot of three independent experiments, each performed in duplicate. Results in (B) are from three independent experiments, each performed in triplicate. **p < 0.01; **** p < 0.0001. (C) HPLC chromatograms of indicated sample fractions from A549 cells co-incubated with A. fumigatus conidia after 24 h and respective controls (cells without A. fumigatus conidia, conidia without cells and media only). Samples were prepared as described in Material and Methods. The wavelength of the shown chromatograms was set to 340 nm, which is the absorption maximum for fumagillin detected at 14.5 min. Fumagillin was absent in the ∆fmaA mutant.

Figure 6. Scheme of the development of an Aspergillus fumigatus intranasal infection in a murine model. In the initial stages of the infection (1st and 2nd days), fungal conidia colonize the lung tissue and start to germinate invading the parenchyma. By contrast, during the final phase of the infection (3rd and 4th days), which are associated with angioinvasion and tissue necrosis, the fungus continues growing and forming invasive hyphae and increasing the expression of genes related to virulence such as lytic enzymes and fumagillin.