PrtT-regulated proteins secreted by Aspergillus fumigatus activate MAPK signaling in exposed A549 lung cells leading to necrotic cell death

Abstract

Aspergillus fumigatus is the most commonly encountered mold pathogen of humans, predominantly infecting the respiratory system. Colonization and penetration of the lung alveolar epithelium is a key but poorly understood step in the infection process. This study focused on identifying the transcriptional and cell-signaling responses activated in A549 alveolar carcinoma cells incubated in the presence of A. fumigatus wild-type and ΔPrtT protease-deficient germinating conidia and culture filtrates (CF). Microarray analysis of exposed A549 cells identified distinct classes of genes whose expression is altered in the presence of germinating conidia and CF and suggested the involvement of both NFkB and MAPK signaling pathways in mediating the cellular response. Phosphoprotein analysis of A549 cells confirmed that JNK and ERK1/2 are phosphorylated in response to CF from wild-type A. fumigatus and not phosphorylated in response to CF from the ΔPrtT protease-deficient strain. Inhibition of JNK or ERK1/2 kinase activity substantially decreased CF-induced cell damage, including cell peeling, actin-cytoskeleton damage, and reduction in metabolic activity and necrotic death. These results suggest that inhibition of MAPK-mediated host responses to treatment with A. fumigatus CF decreases cellular damage, a finding with possible clinical implications.

Figure 1. Validation of changes in cytokine gene expression levels.

A549 cells were grown with either WT conidia or WT-CF for 8 h. Control cells had no added fungi or CF. RT-PCR was used for semi-quantitative analysis of IL-8 and MCP1 transcripts. IL8 levels showed a 16-fold rise in response to conidial infection (black) and a 6-fold increase in response to WT CF (grey). Conidial infection and WT-CF treatment activated MCP1 transcription to similar levels (approximately 3-fold) as measured by the microarrays. Data are presented as mean ± SD.

Figure 2. Phosphorylation of ERK1/2, JNK, and p38 is activated by WT CF.

A549 cells were treated for 30 min, 1 h, 2 h or 3 h with (A–B) WT or ΔPrtT conidia, or inert polystyrene beads, (C–D) WT CF or ΔPrtT CF (lacking protease activity) or for 3 h with (E-F) WT-CF and heat-inactivated (HI) CF lacking protease activity. We used hydrogen peroxide (H₂O₂)-treated cells as a positive control (+), and carrier-treated cells as a negative control (−). Phosphorylation of ERK1/2, JNK, and p38 was monitored by western blotting of whole-cell extracts with specific anti-p-ERK (Thr183/Tyr185), anti-p-SAPK/JNK (Thr183/Tyr185), and anti-p-p38 (Thr180/Tyr182) MAPK antibodies (A, C, E). Total MAPK levels were monitored by ERK1/2, JNK and p38-specific antibodies (B, D, F).

Figure 3. MAPK inhibitors protect A549 lung epithelial cells from loss of viability induced by CF treatment.

(A) ERK1/2 and JNK inhibitors block CF-induced cell peeling. A549 cells pretreated for 2 h with 50 µM ERK-I (FR180204), 25 µM JNK-I (SP600125) or 25 µM p38-I (SB203580) washed, incubated for 12 h in the presence of WT CF, stained with hemacolor and analyzed by microscopy (bar  = 100 µm for all images). Cells pretreated with ERK-I or JNK-I remained attached to the plate and retained their elongated shape. Cells treated with heat-inactivated WT CF lacking protease activity retained their normal shape (B) Verification of ERK1/2 and JNK inhibitor specificity. A549 cells pretreated with ERK-I, JNK-I or p38-I were washed, incubated with WT CF for 3 h, lysed and analyzed by western blot with ERK1/2 and c-Jun-specific antibodies. (C) ERK1/2 and JNK inhibitors block CF-induced cell death. A549 cells pretreated with ERK-I (grey), JNK-I (unshaded) or a carrier control (black) were washed and incubated with WT CF for 12, 16, 24 or 36 h. Cell viability was determined by the XTT colorimetric assay which measures mitochondrial metabolic activity. Shown are the means standard deviations (error bars) for three independent experiments. *, P value of <0.05 for cell viability following inhibitor treatment relative to untreated cells at the same time-point.

Figure 4. ERK1/2 and JNK inhibitors block CF-induced actin-fiber depolymerization and necrotic death.

A549 cells pretreated with ERK-I, JNK-I or p38-I were washed and incubated with WT CF for 8 h. Cells treated with ΔPrtT CF (lacking protease activity) or carrier (untreated) served as negative controls. Cells were (A) stained for actin with phalloidin-FITC and analyzed by confocal microscopy. Cells treated with ERK-I or JNK-I retained their flattened shape and network of actin fibers. (bar  = 20 µm for all images) whereas cells treated with p38-I did not (B) assayed for apoptosis/necrosis by flow cytometry following annexin V (x-axis)/PI staining (y-axis) of the cells. WT CF induces protease-dependent cellular necrosis which is blocked by ERK1/2 and JNK inhibitors. Upper panel: necrosis is induced in A549 cells after 12 h of incubation in the presence of WT CF but not ΔPrtT CF. Lower panel: ERK-I and JNK-I inhibit the induction of necrosis in A549 cells under WT CF treatment. Experiments were repeated three times with similar results. A representative experiment is shown.