LaeA, a regulator of morphogenetic fungal virulence factors

Abstract

Opportunistic animal and plant pathogens, well represented by the genus Aspergillus, have evolved unique mechanisms to adapt to and avoid host defenses. Aspergillus fumigatus, an increasingly serious pathogen owing to expanding numbers of immunocompromised patients, causes the majority of human infections; however, an inability to identify bona fide virulence factors has impeded therapeutic advances. We show that an A. fumigatus mutation in a developmentally expressed transcriptional regulator (deltalaeA) coordinating morphological and chemical differentiation reduces virulence in a murine model; impaired virulence is associated with decreased levels of pulmonary gliotoxin and multiple changes in conidial and hyphal susceptibility to host phagocytes ex vivo. LaeA, a conserved protein in filamentous fungi, is a developmental regulator of virulence genes and, possibly, the first antimicrobial target specific to filamentous fungi that are pathogenic to plants and animals.

FIG. 1.

Filamentous growth and conidiophore development of A. fumigatus WT, ΔlaeA mutant, and ΔlaeA mutant-complemented strains in liquid medium. Images obtained by microscopy of colonies developed after 48 h of incubation in shaking (300 rpm) culture at 25°C.

FIG. 2.

Animal model of invasive aspergillosis. Outbred Swiss ICR mice immunosuppressed by intraperitoneal injection of cyclophosphamide (150 mg/kg) and cortisone acetate (200 mg/kg) were inoculated intranasally with WT, ΔlaeA, and pyrG conidia (50 μl of 5 × 10⁶ conidia/ml). (A) Survival of mice challenged with Aspergillus strains. The log rank test was used to perform pair-wise comparisons of survival levels among the strain groups. P value for comparison of the survival levels of WT- and ΔlaeA mutant-infected mice, 0.002. (B) Fungal burden, measured as number of CFU recovered from homogenized lungs. The Kruskal-Wallis one-way analysis of variance on ranks test was used to compare mean numbers of CFU among strain groups. P value for comparison of WT and pyrG strain CFU counts, >0.05. P value for comparison of ΔlaeA mutant and WT or pyrG strain CFU counts, <0.001. (C) Aspergillus metabolites present in lungs after infection. Chloroform extracts from mouse lungs were cleaned by reverse-phase solid-phase extraction and analyzed by liquid chromatography with diode array detection. The 200- to 600-nm chromatograms are inserted along with the UV spectrum of gliotoxin in the sample and the matching reference standard (upper dotted line) of gliotoxin. The deviation in retention time of gliotoxin (retention time, 6.65 min) was less than 0.01 min. The presence of gliotoxin was confirmed by liquid chromatography-phote diode array detection-high-resolution mass spectrometry (data not shown). mAU, milliabsorbance units.

FIG. 3.

Conidial and hyphal interactions with human phagocytic cells. (A and B) Human CD14⁺ monocyte-derived macrophages ingested higher numbers of ΔlaeA conidia than of WT conidia (internal conidia are stained green). The phagocytosis index was calculated as the percentage of phagocytic macrophages multiplied by the mean number of organisms internalized per cell. Experiments were performed with triplicate wells and repeated at least three times.

FIG. 4.

Conidial properties. (A) Spore metabolites analyzed by thin-layer chromatography. Spore diffusate was obtained by incubation of 10⁸ cells/ml in Hanks balanced salt solution for 1 h; products were filter sterilized, chloroform extracted, and dried under vacuum suction overnight. Products were redissolved with 100 μl chloroform for thin-layer chromatography as previously described (26). Metabolite produced by the WT and not the ΔlaeA mutant is indicated with an arrow. (B) ΔlaeA conidia (right panel) have decreased rodlet protrusions compared to WT conidia. (C) ΔlaeA conidia are less adhesive, as measured by adhesion of latex microspheres (9). Conidia (1 × 10⁸/μl) were suspended in 0.1 mol/liter KNO₃ solution (pH 6.5) and incubated at a 20:1 ratio with latex particles (0.6 μm); the percentage of conidia with adhered particles was calculated from triplicate measurements in two experiments. The two-tailed P value from Student's t test comparing results for the ΔlaeA mutant with those of the WT and the pyrG strain was <0.0001. The P value comparing results for the WT and the pyrG strain was >0.05. (D) Northern blots demonstrating laeA, alb1, rodA, and rodB gene expression in A. fumigatus ΔlaeA mutant and WT strains after growth in GMM from 24 to 72 h. Ethidium bromide-stained rRNA is indicated for loading controls.

FIG. 5.

PMN killing and fungal cytotoxicity. (A) Damage to PMN (calcein release) after 3 h of incubation with live WT and ΔlaeA mutant germ tubes was measured by the release of calcein AM (25). Shown are mean fluorescent units (MFU) of triplicate measurements for six healthy donors (±standard deviations), representative of results obtained for nine different experiments. The two-tailed P value from Student's t test comparing the results for the WT and the pyrG strain was >0.05. The P value for comparisons of the results for the ΔlaeA mutant with the WT and the pyrG strain was <0.001. (B) PMN death after exposure to concentrated culture supernatant from the WT and the ΔlaeA mutant. The percentage of total cells that were PI positive is shown. Controls (not shown) included Triton X-treated PMNs and untreated PMNs, which were >90% and <10% dead, respectively, with the assay. Results are means of triplicate measurements ± standard deviations from two experiments with different donors.

FIG. 6.

Proposed model of LaeA regulation of A. fumigatus phenotypes that confer virulence. LaeA regulation by the protein kinase A pathway was previously reported (13). Virulence factors purported but not yet identified by genetic analyses (spore diffusible metabolite) are shown with dashed lines.