Aspergillus terreus accessory conidia are multinucleated, hyperpolarizing structures that display differential dectin staining and can induce heightened inflammatory responses in a pulmonary model of aspergillosis

Abstract

In addition to phialidic conidia (PC), A. terreus produces accessory conidia (AC) both in vitro and in vivo. AC are distinct from PC in cell surface architecture, with the AC surfaces displaying more β-glucan, a molecule that can be a trigger for the induction of inflammatory responses. The present study follows β-glucan cell surface presentation throughout the course of germination of both types of conidia, and analyzes the differential capacity of AC and PC to elicit immune responses. Results show that AC display early, increased dectin-1 labeling on their cell surfaces compared to PC, and this differential dectin-1 labeling is sustained on the cell surface from the time of breaking dormancy through early germ tube emergence. Mouse alveolar macrophages showed a stronger inflammatory cytokine/chemokine response when challenged with AC than with PC in both ex vivo and in vivo experiments, correlating with the greater exposure of β-glucan exhibited by AC. Further, histopathologic staining of the lungs from mice challenged with AC demonstrated heightened cell recruitment and increased inflammatory response compared to the lungs of mice challenged with PC. Our study also demonstrates that AC are multinucleate structures with the ability to germinate rapidly, polarizing in multiple directions and producing several hyphal extensions. We present evidence that A. terreus AC are phenotypically distinct from PC and can be potent activators of the innate immune mechanism thus possibly playing a role in this organism's pathogenesis.

Disclaimer

The findings and conclusions in this article are those of the author(s) and do not necessarily represent the views of the CDC.

Figures and Tables

Figure 1 Differential dectin-1 binding on Aspergillus fumigatus and Aspergillus terreus phialidic conidia. Soluble dectin-1 binding A. fumigatus and A. terreus phialidic conidia at various stages of germination (A) dormant conidia (B) swollen condia (C) early germ tube formation and (D) late germination. Differential interference microscopy (DIC) and fluorescence images were captured by microscopy at 40x (A) and 100x (B–D), and are representative of 3 experiments. Scale bars denote 5 µm and 10 µm for 40x and 100x magnification, respectively.

Figure 2 Dectin-1 binding on phialidic conidia and accessory conidia at different germination stages. Soluble dectin-1 binding on A. terreus phialidic and accessory conidia at (A) dormant conidia (B) swollen conidia, (C) early germ tube formation and (D) late germination. DIC and fluorescence images were captured by microscopy at 40x (A) and 100x (B–D), and are representative of 3 experiments. Within windows, arrows depict the ring-like staining pattern on AC at 100x (A). Scale bars denote 5 µm and 10 µm for 40x and 100x magnification, respectively.

Figure 3 Accessory conidia are multinucleated prior to germination. Hoechst staining was performed on A. terreus accessory conidia, both attached (A) and detached (B) from the hyphae, and A. terreus PC (C) and A. fumigatus PC (D). DIC and UV images were captured by microscopy at 100x, and are representative of 3 experiments. Scale bars denote 10 µm.

Figure 4 Accessory conidia undergo hyperpolarization during germination. Early germ tube formation and Hoechst nuclei staining was assessed for A. terreus accessory conidia. DIC and UV images were captured by microscopy at 100x, and are representative of 3 experiments. Scale bars denote 10 µm.

Figure 5 Aspergillus terreus accessory conidia elicit a heightened inflammatory response by alveolar macrophages. Alveolar macrophages were co-cultured with A. terreus AC or PC. Supernatants were collected at (A) 6 or (B) 20 h and assayed for chemokine/cytokine levels by Bio-Plex or ELISA. Experiments were performed three times.

Figure 6 Mice intratracheally challenged with Aspergillus terreus accessory conidia exhibit enhanced chemokine/cytokine production and cell recruitment in lungs. C57BL/6 mice were intratracheally challenged with AC or PC. (A) Chemokine/cytokine levels in the supernatants of homogenized lungs collected at 18 h post-challenge with AC or PC (need to indicate from figure). (B) Hematoxylin and eosin staining of sections of lungs post-challenge with AC or PC (also denote figure labels). All experiments were performed 3 times.