Figure 1.
Immunocompetent mice infected with resting conidia of agsΔ_5T and parental (ku80) strains.
Observations and analysis on mice were done four days post-infection. (A) Fungal load was expressed as log10 CFU/lung. (B) Lung histology (periodic acid-Schiff-staining). Note the polymorphonuclear cells and mononuclear infiltrates surrounding the bronchi in ku80 infected lung. (C) After infection, percentages of monocytes and polymorphonuclear cells found in the lungs alveolar lavage (BAL). (D) Relative expression of TNFα and IL10 assessed by real time RT-PCR on lung total RNA from naïve and infected mice. (E) Conidiocidal activity by purified macrophages from uninfected mice expressed in percentage of CFU inhibition after 2 h incubation of the conidia with macrophages. Data are representative of at least three independent experiments. Ctl, naïve mice; *, P<0.05.
Figure 2.
Cyclophosphamide immunosuppressed mice and anti-Ly6G treated neutropenic mice infected with resting conidia of agsΔ_5T and parental (ku80) strains.
(A–C) Cyclophosphamide immunosuppressed mice; (D–E) anti-Ly6G treated neutropenic mice; (A) Survival (%) and (B) fungal growth estimated as CFUs in lung. (C and E) lung histology (periodic acid-Schiff-staining). Note the polymorphonuclear cells and mononuclear infiltrates surrounding the bronchi in ku80 infected lung. (D) Histological appearance of lungs of anti-Ly6G neutropenic mice infected with conidia of agsΔ_5T and ku80 (Gomori's methanamine silver-staining). Note the absence of mycelial development of agsΔ_5T conidia in neutropenic mice. Data are representative of at least three independent experiments. *: p<0.05.
Figure 3.
Conidiocidal activity of macrophages isolated from uninfected p47phox−/− mice against resting conidia of agsΔ_5T and parental (ku80) strains.
Conidiocidal activity is expressed in percentage of CFU inhibition after 2 and 6 h incubation of the conidia with macrophages. Data are representative from at least three independent experiments. *, P<0.05.
Figure 4.
Phagocytosis activity by isolated macrophages from uninfected mice against resting conidia of agsΔ_5T and parental (ku80) strains.
Index of phagocytosis is expressed in number of conidia per alveolar macrophage after 1*, P<0.05.
Figure 5.
Surface analysis of resting conidia of agsΔ_5T mutant and parental (ku80) strains.
(A): height images (z-range = 1 µm; recorded in water with silicon nitride tips). Atomic Force Microscopy (AFM) images showing the amorphous surface without the rodlet layer on the triple agsΔ_5T mutant conidia whereas the rodlet are observed on the parental strain conidial surface. (B): TEM observations. Note the presence of an extracellular material on the surface of the agsΔ_5T conidia (arrow); CW: cell wall. (C): SDS-PAGE (15% gel) of Hydrofluoric acid (HF) extracts of rodlets from resting conidia showing the two bands, 16 kDa and 14.5 kDa of RodAp classically seen from HF treatment of the conidia [10]. Data are representative of at least three independent experiments.
Figure 6.
Imaging and adhesive properties of A. fumigatus resting conidia of the parental strain and agsΔ_5T mutant.
Structural changes of agsΔ_5T correlate with a loss of cell surface adhesive properties. (A–C) parental strain; (D–F) agsΔ_5T mutant; (A, D) height images (z-range = 1 µm; recorded in water with silicon nitride tips); (B, E) adhesion force maps (z-range: 5 nN) corresponding to the height image; (C, F) Representative force-distance curves and adhesion force histograms (n = 1024) recorded on the surface of parental strain (C) and agsΔ_5T (F).
Figure 7.
ConA-FITC labeling of agsΔ_5T mutant and parental strain (ku80) resting conidia.
Note the increase in the ConA labeling on the agsΔ_5T mutant conidial surface. Histograms represent the calculated fluorescence intensity of the corresponding images, expressed in Einstein per seconds.
Figure 8.
NaCl extracted proteins from the surface of agsΔ_5T resting conidia.
SDS-PAGE (10% gel) of proteins extracted after 2 h incubation of agsΔ_5T and ku80 resting conidia in 0.5 M NaCl.
Table 1.
Proteins identified in the NaCl extract of agsΔ_5T and agsΔ_n8 conidia.
Figure 9.
TNFα production or expression by macrophages (isolated from uninfected immunocompetent mice) upon interaction with resting conidia of parental (ku80) and agsΔ_5T strains or agsΔ_5T conidial NaCl extract (3.2 µg proteins) respectively.
(A) TNFα was quantified after 5 h incubation of the conidia with macrophages; (B) Relative expression of TNFα assessed by real time RT-PCR in total RNA from macrophages after 5 h incubation of the agsΔ_5T conidial NaCl extract with macrophages. NaCl supernatant from ku80 resting conidia incubated for 2 h in 0.5M NaCl was used as a control. NS: Non-stimulated. *, P<0.05.
Figure 10.
Labeling of the surfaces of agsΔ_5T and parental strain swollen conidia by WGA and the β(1,3)-glucan receptor GNBP3.
The surfaces of the swollen conidia were labeled by WGA-FITC (A) and GNBP3 (B) as described in material and methods. (C, D) Histograms represented the calculated fluorescence intensity of the corresponding images (A, B respectively), expressed in Einstein per seconds.
Figure 11.
Working model explaining sequential and differential immune events upon inhalation of the agsΔ mutant and the parental (ku80) strain conidia.
The presence of the glycoprotein layer on the triple agsΔ mutant conidial surface hides the rodlet layer. Increased exposure of PAMPs (WGA and ConA positive molecules and β-(1,3)-glucans) during vegetative growth in the triple agsΔ mutant modifies the host immunological response. This facilitates phagocytosis and killing of the triple agsΔ mutant and stimulates pro-inflammatory immune responses.