Isolate-Dependent Growth, Virulence, and Cell Wall Composition in the Human Pathogen <i>Aspergillus fumigatus</i>

<div><p>The ubiquitous fungal pathogen <i>Aspergillus fumigatus</i> is a mediator of allergic sensitization and invasive disease in susceptible individuals. The significant genetic and phenotypic variability between and among clinical and environmental isolates are important considerations in host-pathogen studies of <i>A. fumigatus</i>-mediated disease. We observed decreased radial growth, rate of germination, and ability to establish colony growth in a single environmental isolate of <i>A. fumigatus</i>, Af5517, when compared to other clinical and environmental isolates. Af5517 also exhibited increased hyphal diameter and cell wall β-glucan and chitin content, with chitin most significantly increased. Morbidity, mortality, lung fungal burden, and tissue pathology were decreased in neutropenic Af5517-infected mice when compared to the clinical isolate Af293. Our results support previous findings that suggest a correlation between <i>in vitro</i> growth rates and <i>in viv</i>o virulence, and we propose that changes in cell wall composition may contribute to this phenotype.</p></div

Increased cell wall chitin and β-glucan in <i>A. fumigatus</i> isolate Af5517.

<p>(<b>A,B</b>) Representative dot-blot images of processed <i>A. fumigatus</i> mycelial extracts and chitin (shrimp shell chitin) (<b>A</b>) or β-glucan (curdlan) (<b>B</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. (<b>C,D</b>) Chitin (<b>C</b>) or β-glucan (<b>D</b>) vs. total fungal protein in mycelial extracts as determined by dot blot assay. (<b>C</b>) Chitin was significantly increased compared to other isolates at 25, 50, or 75 µg of total protein, while β-glucan (<b>D</b>) was significant at 50 and 100 µg total protein. ****p<0.0001. (<b>A–D</b>) Panels are representative of two experiments. (<b>E,F</b>) 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 (<b>F</b>). Negative controls are unstained conidia (<b>E</b>) or goat anti-mouse alexa-fluor 488 only (<b>F</b>) and depicted as dotted histograms. (<b>G,H</b>) Median fluorescence intensities of WGA (<b>G</b>) or anti-β-glucan (<b>H</b>) stained conidia after 0 or 5 hours incubation at 37°C. (<b>G</b>) 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.</p

<i>A. fumigatus</i> isolate Af5517 is less virulent than Af293 in an animal model of pulmonary IA.

<p>Neutropenic BALB/c mice were infected with 2×10⁶ (circles) or 5×10⁶ (squares) conidia of isolates Af293 (closed symbols) or Af5517 (open symbols). (<b>A</b>) Survival curves depict death or moribund status for experimental animals over the course of the experiment. (<b>B</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. (<b>C,D</b>) Disease scores of mice infected with 2×10⁶ (<b>C</b>) or 5×10⁶ (<b>D</b>) 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.</p

Decreased radial growth of <i>A. fumigatus</i> isolate Af5517.

<p>(<b>A</b>) Representative <i>Aspergillus</i> minimal media (AMM) plates 10 days after inoculation with 100 conidia of the indicated <i>A. fumigatus</i> isolates in triplicate. Plates were incubated at either 22°C or 37°C, as indicated. (<b>B</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. (<b>C</b>) 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. (<b>D</b>) 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). (<b>E</b>) 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.</p

Decreased rate of germination and ability to establish colony growth by <i>A. fumigatus</i> isolate Af5517.

<p>(<b>A,B</b>) Flow cytometric analysis of conidial swelling in <i>A. fumigatus</i> isolates during 5 hours of incubation at 37°C. (<b>A</b>) Representative histograms from three experiments of forward scatter (FSC) from each isolate at 0 and 5 hours. (<b>B</b>) Increase in size (Conidial swelling) = FSC 5 h/FSC 0 h. Data are a summary of three experiments (n = 5). (<b>C</b>) Temporal quantification of germling formation by microscopic analysis. Summary of two experiments (n = 6). (<b>D</b>) 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.</p

List of <i>A. fumigatus</i> isolates used in this study.

<p>List of <i>A. fumigatus</i> isolates used in this study.</p

Histopathology of Af293 and Af5517 infection.

<p>Neutropenic BALB/c mice were infected with Af293 or Af5517 and sacrificed after 3 days for lung histological analysis. (<b>A</b>) 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>B</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.</p

A Murine Inhalation Model to Characterize Pulmonary Exposure to Dry <i>Aspergillus fumigatus</i> Conidia

<div><p>Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from <i>Aspergillus fumigatus</i> wild-type (WT) or a melanin-deficient (<i>Δalb1</i>) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1×10⁵ twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8⁺ IL17⁺ (Tc17) cells were significantly higher in BALF and positively correlated with germination of <i>A. fumigatus</i> WT spores. Germination was associated with specific IgG to intracellular proteins while <i>Δalb1</i> spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants.</p></div

Histopathology of sections derived from <i>Aspergillus fumigatus</i> WT exposed mice.

<p>A) Representative histopathology sections from WT exposed mice sacrificed at the indicated time points. Top panel-H&E stained sections at 100× magnification, Bottom panel-PAS stained sections at 100× magnification. B) GMS stained sections at 400× magnification. Black arrow heads indicate swollen conidia (24 hr), while red arrow heads indicate conidia germination and emergence of hyphal tubes (48 hr). C) Quantification of conidia and germination (swollen conidia+germ tube formation) over time. Values were obtained by quantifying the number of conidia visualized by counting 100 random fields of view covering both lung fields at a magnification of 400×. Conidia were considered swollen when the size was >2× that of resting conidia. Data are presented as the average ± standard error of measure. ****P≤0.0001, ***P≤0.001, **P≤0.01.</p

Total cell counts in the BALF.

<p>Following 8 dry conidial exposures, mice were sacrificed at the indicated time points to determine the kinetics of the cellular influx to the lung. Total cell numbers were obtained through acridine orange staining and quantified using an automated cell counter. Each cell population was quantified by multiplying the frequency of each by the total cell counts. Data are presented as the average ± standard error of measure. (Control n = 30 mice/time point, Exposed n = 7–10 mice/group/time point). ****P≤0.0001, ***P≤0.001, **P≤0.01, *P≤0.05.</p