Figure 1.
Decreased radial growth of A. fumigatus isolate Af5517.
(A) Representative Aspergillus minimal media (AMM) plates 10 days after inoculation with 100 conidia of the indicated A. fumigatus isolates in triplicate. Plates were incubated at either 22°C or 37°C, as indicated. (B) Colony diameter during this incubation, measured daily. Clinical isolates are depicted with filled symbols connected by solid lines, while environmental isolates are open symbols connected by dotted lines. Growth of Af5517 was significantly different from all isolates after 4 days of growth. (C) Top panels, hyphal morphology of isolates after 24 hours growth in liquid culture at 37°C without shaking. Bottom panels, hyphal width was measured using SPOT Basic Software. (D) Summary of hyphal diameter measurements (n = 20–22/group). The diameters of Af5517 and Af164 hyphae were significantly increased when compared to Af293 and Af13073, and Af5517 diameter was increased compared to Af164 (p<0.01). (E) Hyphal mass accumulation of isolates after 24 hours growth in liquid culture with shaking. Data depict a summary of two experiments (n = 6/group). Each panel displayed is representative of two experiments. ***p<0.001, ****p<0.0001.
Table 1.
List of A. fumigatus isolates used in this study.
Figure 2.
Decreased rate of germination and ability to establish colony growth by A. fumigatus isolate Af5517.
(A,B) Flow cytometric analysis of conidial swelling in A. fumigatus isolates during 5 hours of incubation at 37°C. (A) Representative histograms from three experiments of forward scatter (FSC) from each isolate at 0 and 5 hours. (B) Increase in size (Conidial swelling) = FSC 5 h/FSC 0 h. Data are a summary of three experiments (n = 5). (C) Temporal quantification of germling formation by microscopic analysis. Summary of two experiments (n = 6). (D) Percent colony growth (CFU = colony forming unit), averaged from inoculations of 1000, 100, and 10 conidia, each in triplicate. Graphed data are a summary of two experiments. Decreased ability of Af5517 to establish colony growth was significantly different from all other isolates. ***p<0.001.
Figure 3.
Increased cell wall chitin and β-glucan in A. fumigatus isolate Af5517.
(A,B) Representative dot-blot images of processed A. fumigatus mycelial extracts and chitin (shrimp shell chitin) (A) or β-glucan (curdlan) (B) standards, probed with chitin binding probe or anti-(1,3)-β-glucan, respectively. Blots of mycelial extracts depict 75 µg (A) and 100 µg (B) of total protein of each isolates. (C,D) Chitin (C) or β-glucan (D) vs. total fungal protein in mycelial extracts as determined by dot blot assay. (C) Chitin was significantly increased compared to other isolates at 25, 50, or 75 µg of total protein, while β-glucan (D) was significant at 50 and 100 µg total protein. ****p<0.0001. (A–D) Panels are representative of two experiments. (E,F) Representative flow cytometric histograms of dormant (0 h) or swollen (5 h at 37°C) conidia of each isolate stained with the chitin binding wheat germ agglutinin (WGA, panel E) or anti-(1,3)-β-glucan (F). Negative controls are unstained conidia (E) or goat anti-mouse alexa-fluor 488 only (F) and depicted as dotted histograms. (G,H) Median fluorescence intensities of WGA (G) or anti-β-glucan (H) stained conidia after 0 or 5 hours incubation at 37°C. (G) Chitin exposure in Af5517 conidia was significantly increased in comparison will all other isolates after 0 and 5 hours incubation. *p<0.05. **p<0.01. Panels are a summary of three experiments.
Figure 4.
A. fumigatus isolate Af5517 is less virulent than Af293 in an animal model of pulmonary IA.
Neutropenic BALB/c mice were infected with 2×106 (circles) or 5×106 (squares) conidia of isolates Af293 (closed symbols) or Af5517 (open symbols). (A) Survival curves depict death or moribund status for experimental animals over the course of the experiment. (B) Fungal burden of mice infected with 5×106 conidia of Af293 (closed symbols) or Af5517 (open symbols) was determined by quantification of fungal 18S rDNA. (C,D) Disease scores of mice infected with 2×106 (C) or 5×106 (D) conidia. Mice were scored daily for progression of disease as described in Materials and Methods. Graphed data depicts the summary of two experiments with 5–8 mice per group in panels A, C, and D. *p<0.05. ****p<0.0001.
Figure 5.
Histopathology of Af293 and Af5517 infection.
Neutropenic BALB/c mice were infected with Af293 or Af5517 and sacrificed after 3 days for lung histological analysis. (A) Hematoxylin and Eosin (H&E)-stained sections (left panels) depict lung inflammation in mice infected with 5×106 conidia. Adjacent Gomori’s Methanamine Silver (GMS)-stained sections (right panels) show areas of fungal growth. The black bar (bottom right panel) is equivalent to 100 µm. (B) GMS staining representing fungal growth was quantified in sections from mice infected with 2×106 or 5×106 conidia, with the mean of four representative fields displayed for each sample. *p<0.05.