Cutaneous model of invasive aspergillosis

Abstract

Cutaneous models have proven useful in studies of the pathogenesis and treatment of Gram-positive bacterial infections. Because cutaneous invasive aspergillosis (IA) occurs in the clinical setting, we sought to develop a nonlethal murine cutaneous model of IA. We induced cutaneous IA in cyclophosphamide-treated nude BALB/c mice by subcutaneous injection of Aspergillus fumigatus conidia. Skin lesion areas correlated well with tissue fungal burdens, allowing dynamic visual monitoring of cutaneous infections. The cutaneous model accurately reflected alterations in A. fumigatus pathogenicity resulting from deletions of recognized virulence genes (pabaA, sidA, and pksP). Moreover, analysis of the roles of conidial and mycelial catalases revealed that the former is required for the initiation of cutaneous aspergillosis, whereas the latter contributes to its propagation. Finally, posaconazole treatment reduced skin lesion areas relative to those of untreated and fluconazole-treated controls. This novel cutaneous model system should be applicable to comparative studies of the pathogenesis, treatment, and tissue specificity of IA.

FIG. 1.

Cutaneous model of IA. (A) A circumscribed skin lesion with peripheral enhancement appeared 48 h after subcutaneous injection of 5 × 10⁶ A. fumigatus conidia in cyclophosphamide-treated nude BALB/c mice. (B, C) Histopathologic examination of thigh tissue sections revealed hyphal proliferation in the dermis and invasion of muscle tissue (hematoxylin and eosin, ×200 magnification [B]; GMS, ×200 magnification [C]).

FIG. 2.

Correlation between skin lesion area and tissue galactomannan content. A significant linear correlation between skin lesion area and thigh tissue galactomannan content is described by a Pearson's coefficient of 0.76 (P < 0.0001).

FIG. 3.

Features of murine thigh infection with hypovirulent A. fumigatus mutants and their parental wild-type strains. (A to F) GMS-stained thigh tissue sections of mice infected with A. fumigatus isolates with deletion mutations in known virulence-associated genes and parental wild-type strains are shown (original magnification, ×200). (A to C) In tissue infected with the ΔpksP, ΔsidA, and ΔpabaA mutants, fungal elements were few, clustered, and limited to subcutaneous tissue. (C) ΔpabaA conidia failed to germinate. (D to F) In contrast, the parental wild types produced widespread infection with deep invasion of muscle tissue. (G to I) The corresponding differences in skin lesion areas (black bars) and tissue galactomannan content (gray bars) are shown. Bars and error bars represent means and standard errors, respectively (15 mice per A. fumigatus strain). *, P < 0.05.

FIG. 4.

Importance of catalase production by A. fumigatus spores and hyphae for cutaneous IA. Nude BALB/c mice were inoculated simultaneously with catalase-deficient mutants and their parental wild-type strains in opposite thighs. (A and C) Deletion of the spore-specific catA gene was associated with significant reductions in the mean skin lesion area and galactomannan content compared with those after infection with the wild-type strain. (C) Left arrow, site of strain G10 inoculation; right arrow, site of ΔcatA mutant inoculation. (E) Failure of ΔcatA spores (arrows) to germinate and produce invasive hyphae can be seen in GMS-stained tissue. (B) Deletion of the mycelium-specific catalase genes cat1 and cat2 was associated with a small reduction in skin lesion area and a nonsignificant reduction in tissue galactomannan content compared with those of mice infected with the parental wild-type strain. (D and F) Histopathologic analysis revealed that Δcat1 Δcat2 conidia germinated but formed shorter hyphal elements (F) than the wild-type strain (D) (original magnification, ×200). Bars and error bars represent means and standard errors, respectively (10 mice per A. fumigatus strain). *, P < 0.05.

FIG. 5.

Posaconazole treatment reduces skin lesion areas. Compared with no drug treatment or fluconazole treatment (40 mg/kg/day), treatment with posaconazole (40 mg/kg/day) resulted in a 47% to 49% reduction in the mean skin lesion area on days 2 through 5 after inoculation with strain Af293 (P < 0.001). Each datum point represents the mean skin lesion area (±standard error); 20 mice were used for each treatment group. *, P < 0.001.