The Aspergillus fumigatus pkcA G579R Mutant Is Defective in the Activation of the Cell Wall Integrity Pathway but Is Dispensable for Virulence in a Neutropenic Mouse Infection Model

Abstract

Aspergillus fumigatus is an opportunistic human pathogen, which causes the life-threatening disease, invasive pulmonary aspergillosis. In fungi, cell wall homeostasis is controlled by the conserved Cell Wall Integrity (CWI) pathway. In A. fumigatus this signaling cascade is partially characterized, but the mechanisms by which it is activated are not fully elucidated. In this study we investigated the role of protein kinase C (PkcA) in this signaling cascade. Our results suggest that pkcA is an essential gene and is activated in response to cell wall stress. Subsequently, we constructed and analyzed a non-essential A. fumigatus pkcAG579R mutant, carrying a Gly579Arg substitution in the PkcA C1B regulatory domain. The pkcAG579R mutation has a reduced activation of the downstream Mitogen-Activated Protein Kinase, MpkA, resulting in the altered expression of genes encoding cell wall-related proteins, markers of endoplasmic reticulum stress and the unfolded protein response. Furthermore, PkcAG579R is involved in the formation of proper conidial architecture and protection to oxidative damage. The pkcAG579R mutant elicits increased production of TNF-α and phagocytosis but it has no impact on virulence in a murine model of invasive pulmonary aspergillosis. These results highlight the importance of PkcA to the CWI pathway but also indicated that additional regulatory circuits may be involved in the biosynthesis and/or reinforcement of the A. fumigatus cell wall during infection.

Fig 1. Chelerythrine treatment leads to a fungicidal effect of A. fumigatus pkcA ^G579R mutant.

(A) 2x10³ conidia from each strain were grown for 24 hours in liquid MM at 30°C in the presence of the indicated concentration of chelerythrine. After growth, cells were centrifuged, washed in pre-warmed medium and allow to recover in fresh MM medium for an additional 24 hours. After this time, survival was determined via the MTT assay comparing the formanzan salt formation at each time point in comparison to the untreated control. * p ≤ 0.01. (B) Western blot for MpkA phosphorylation. Wild-type and pkcA ^G579R strains were grown for 16 h in YG at 37°C and then chelerythrine (25 μM) was added, or not (control), to the medium for 120 minutes. Anti-phospho-p44/42 MAPK antibody was used to detect the phosphorylation of MpkA. The γ-tubulin antibody and Coomassie Brilliant Blue stained gels were used as loading sample controls. Signal intensity was quantified using the ImageJ software by dividing the intensity of phosphorylatred MpkA/γ-tubulin and expressed as arbitrary units.

Fig 2. PkcA contributes to thermotolerant growth and germination in A. fumigatus.

1x10⁴ conidia of each strain were inoculated onto the center of a YAG medium and radial growth was measured after 3 days at the indicated temperatures (A-B). 1x10⁶ conidia of each strain were inoculated in 2 ml liquid YG culture and incubated at 37°C and 45°C during 2, 4, 6 and 8 hours before the percentage of germination was evaluated (C). Experiments were performed in triplicate and the results are the mean ± standard deviation. * Statistically significant by Student’s T-test (p ≤ 0.01).

Fig 3. Growth phenotypes of the pkcA ^G579R mutant.

(A) 1x10⁵ conidia of each strain were inoculated onto the center of a solid YG plates supplemented with different cell wall perturbing agents: Congo Red (CR); Calcofluor White (CFW); caffeine (CAF); anidulafungin (AF); Sodium Dodecyl Sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). (B) The indicated number of conidia was spotted onto solid YG plates supplemented with nikkomycin Z (NKZ) and fluconazole (FLUC). The plates were incubated for 3 days (A) or 2 days (B) at 37°C.

Fig 4. pkcA ^G579R mutant strain presents increased protoplasts production and β-1,3-glucan content, lower biofilm formation and reduced hydrophobin content.

(A) The digestion mixture included the Lallzyme MMX and 100 mg of mycelium into 50 ml of osmotic stabilizer solution at the indicated incubation times. Protoplasts were quantified using a Neubauer chamber. Detection of the exposed chitin (B) and β-1,3−glucan (C) contents on the cell surface. Conidia were cultured in liquid MM to the hyphal stage, UV-fixed, and stained with CFW or soluble dectin-1to detect the content of exposed chitin and β-glucan, respectively. The intensity of staining was calculated by averaging the amount of staining to the total area of each fungal cell using ImageJ software. These experiments were performed in triplicate, and the results are displayed as mean values ± standard deviation. * Student’s T-test (p ≤ 0.05). (D) Biofilm formation was evaluated by crystal violet absorbance at 570 nm and expressed as the percentage of adhesion considering 100% for the wild-type strain. Experiments were performed in quintuplicate and the results are the mean ± standard deviation. (*p ≤ 0.01). (E) Silver stained SDS-PAGE profiles of HF acid extracts from resting conidia showing the hydrophobin concentration of the wild-type, pkcA ^G579R and complementing strains. The number of conidia subjected to the hydrophobin extraction was validated by CFU counting on solid YG medium. RodAp corresponds to the native RodA and RodAp* to partially degraded or processed RodA.

Fig 5. The pkcA ^G579R mutant is sensitive to antifungal drugs.

Antifungal susceptibility using E-test gradient strips for voriconazole (A) and caspofungin (B).

Fig 6. Growth phenotypes of the pkcA ^G579R mutant strain in the presence of oxidative damage.

The strains were grown in liquid MM in 24-well plates supplemented with in the indicated concentrations of paraquat (A) or menadione (B) during 48 hours at 37°C.

Fig 7. The pkcA ^G579R mutant strain is defective in activating the CWI pathway.

Conidia of the wild-type and mutant strains were grown overnight in liquid YG medium and cell wall stress was induced by exposure to CR for 0, 15, 30 and 60 minutes. Samples were collected at the indicated time points for western blot preparation. Phosphorylated and the total fraction of MpkA were detected using anti-phospho p44/42 and anti-p44/42 MAPK antibodies, respectively. γ-tubulin antibody and Coomassie Brilliant Blue stained gel were used as loading sample controls (A). Densitometry analysis of western blots using the ImageJ software presenting the difference in the phosphorylated MpkA/non-phosphorylated MpkA ratio in the wild-type and mutant strain expressed as percentage in the histogram (B).

Fig 8. Cell wall stress caused by loss of pkcA is connected to UPR signaling in A. FUMIGATUS.

(A) The indicated number of conidia of each strain was spotted on solid YG medium supplemented with BFA (brefeldin A) or in a 24 well plate (1x10⁴ conidia/well) containing the indicated concentration of TM (tunicamycin) (B); or (C) DTT. (D) Analysis of hacA mRNA splicing in the wild-type and pkcA ^G579R mutant. Overnight liquid cultures of each strain were subjected to cell wall stress by the addition of CR during the indicated amount of time before total RNA was extracted. RT-PCR was used to amplify the hacA transcript using primers that span de border of the 20 nucleotide unconventional intron to distinguish induced (spliced hacA form = hacA ⁱ) from the uninduced (non-spliced hacA form = hacA ^u) yielding fragments of 100 and 120 nucleotides, respectively. PCR products were separated in an acrylamide gel and stained. As a control, both wild-type and mutant strain were treated with 1 mM of DTT to induce UPR. Genomic DNA was also used as template in the same reactions yielding only the 120 bp band (upper panel). Images were subjected to densitometric analysis of pixel intensity and normalized by the expression of tubA run as loading control in each RT-PCR.

Fig 9. Transcriptional analysis of cell wall-related genes.

The wild-type, and pkcA ^G579R strains were grown for 24 hours in YG medium and transferred to fresh pre-warmed YG with either 0 or 300 μg/ml of CR and grown for a further 15, 30 and 60 minutes. mRNA abundance for each gene was assessed by real time RT-PCR and normalized to β-tubulin. Fold increase in each strain represents the normalized mRNA abundance relative to the wild-type strain at time point 0 (i. e. prior to CR exposure). Data represent the average value of at least three biological replicates, each repeated in duplicate in the same run and standard deviation. * p ≤ 0.05.

Fig 10. Transcriptional analysis of chitin synthase genes.

The wild-type, and pkcA ^G579R strains were grown for 24 hours in YG medium and transferred to fresh pre-warmed YG with either 0 or 300 μg/ml of CR and grown for a further 15, 30 and 60 minutes. mRNA abundance for each gene was assessed by real time RT-PCR and normalized to β-tubulin. Fold increase in each strain represents the normalized mRNA abundance relative to the wild-type strain at time point 0 (i. e. prior to CR exposure). Data represent the average value of at least three biological replicates, each repeated in duplicate in the same run and standard deviation. * p ≤ 0.05.

Fig 11. pkcA ^G579R strain presents no virulence attenuation in a mouse model but activates innate immunity against A. fumigatus.

(A) Secretion of TNF-α from bone marrow derived macrophages (BMDM) after co-incubation with A. fumigatus hyphae of wild-type, pkcA ^G579R and complementing strains. TNF-α levels were quantified by ELISA from the culture supernatant after 18 hours of BMDMs infection. Data show average ± standard deviation (* p ≤ 0.005). NI: Non−infected; LPS: lipopolysaccharide (positive control). (B) Phagocytosis index is increased in the pkcA ^G579R mutant strain (average ± standard deviation, *p ≤ 0.01 compared to the wild−type and the complemented strain). (C) Comparative analysis of wild type, pkcA ^G579R and complemented strains in a neutropenic murine model of invasive pulmonary aspergillosis.