Objective:
Aspergillus fumigatus is a pathogenic filamentous fungus capable of causing serious disease in immunosuppressed patients through inhalation of its conidia. These conidia are small and highly hydrophobic, two characteristics that allow them to remain airborne and reach the pulmonary alveoli. Once the organisms reaches the site of infection, it can express a wide range of virulence factors to cause disease. In this work, we studied the fungal transcriptomic changes over the first four days of an intranasal infection in immunosuppressed mice.

Methods:
Ten female BALB/c mice between 16-20 g were used in each experiment. All the mice were immunosuppressed with cyclophosphamide, administered at 150 mg/kg and 100 mg/kg on days -4 and -1, respectively relative to infection. Each mouse was infected with 20 µl of saline solution 0.02% tween 20 (SS-Tw20) containing 1 x 107 conidia of A. fumigatus. Two mice were treated only with 20 µl of SS-Tw20 as control. During the next 4 days, 2 mice were euthanized each day and their lungs were harvested for histological analysis and RNA extraction. For transcriptomic analysis, total RNA was hybridized with the Agilent Whole A. fumigatus Genome Expression 44K v.1 (AWAFUGE) microarray. In all cases, we compared the fungal expression data from day 1 of infection to days 3 and 4. The experiments were carried out in triplicate and key transcriptomic data were verified by RT-qPCR. Finally, we demonstrated the in vitro synthesis of fumagillin by organisms in contact with A549 epithelial cells and its effect on damage to these cells. To do that, we used HPLC to detect the toxin after fungal co-incubation with the A549 pneumocytes and used a 51Cr released assay to assess the extent of host cell damage caused by the ∆fmaA deletion mutant, which did not make fumagillin.

Results:
The histopathological study revealed fungal progression through the lung tissue during the 4 days of infection accompanied by tissue necrosis. Angioinvasion was visible by day 3 and it progressed during the 4th day of infection. Transcriptional analysis showed that only 103 genes were detected as differentially expressed genes (DEGs), 85 of which were up-regulated during pulmonary infection. Among these up-regulated DEGs, 60% were either members of the fumagillin biosynthesis cluster or specified classical virulence factors such as DppIV, DppV, Aspf1, Aspf5 and ChiB1. Because the transcriptomic data suggest that fumagillin was produced during invasive infection, we performed in vitro studies.  We verified that fumagillin was present in the conditioned media above pulmonary epithelial cells that had been infected with wild-type A. fumigatus for 24 h.  Furthermore, we found that the fumagillin-deficient ∆fmaA mutant had reduced capacity to damage pulmonary epithelial cells after 16 h of incubation.

Conclusion:
This work suggests a correlation between the histopathological data found during the progression of the infection and the genes differentially expressed detected by transcriptomics. The production of fumagillin during the infection process likely contributes to virulence by promoting the destruction of the lung parenchyma.

Financial support:
This work was partly funded by the UPV/EHU grants (GIU15/36 and PPG17/41). Xabier Guruceaga has been supported Pre-doctoral Research Grants of the Government of the Basque Country.


8 / Aspergillus fumigatus fumagillin as possible virulence factor