Nanoparticle decoration impacts airborne fungal pathobiology

Abstract

Airborne fungal pathogens, predominantly Aspergillus fumigatus, can cause severe respiratory tract diseases. Here we show that in environments, fungal spores can already be decorated with nanoparticles. Using representative controlled nanoparticle models, we demonstrate that various nanoparticles, but not microparticles, rapidly and stably associate with spores, without specific functionalization. Nanoparticle-spore complex formation was enhanced by small nanoparticle size rather than by material, charge, or "stealth" modifications and was concentration-dependently reduced by the formation of environmental or physiological biomolecule coronas. Assembly of nanoparticle-spore surface hybrid structures affected their pathobiology, including reduced sensitivity against defensins, uptake into phagocytes, lung cell toxicity, and TLR/cytokine-mediated inflammatory responses. Following infection of mice, nanoparticle-spore complexes were detectable in the lung and less efficiently eliminated by the pulmonary immune defense, thereby enhancing A. fumigatus infections in immunocompromised animals. Collectively, self-assembly of nanoparticle-fungal complexes affects their (patho)biological identity, which may impact human health and ecology.

Keywords: fungal infection; nanomedicine; nanoparticles.

Fig. 1.

NP physicochemical properties affect their assembly on spores. (A) SEM images of NP-covered conidia harvested from a construction site. Arrows indicate NPs. (Scale bar: 2 µm.) (B) A. fumigatus^GFP conidia incubated with fluorescent silica (SiO_R) or polymer NPs (OSi_RN or OSi_RPEG). Negatively charged SiO_R or PEGylated OSi_RPEG efficiently adsorbed to conidia in situ, whereas positively charged OSi_RN bound less efficiently. (Scale bar: 2 µm.) (C) TEM indicates better fitting of small (∅ ∼30 nm; Left) compared with larger (∅ ∼140 nm; Right) SiO NPs into groove structures on the conidia surface. (Scale bars: 50 nm.) (D) SEM showing assembly of SiO (∅ ∼30/140 nm) and ZnO. (Scale bars: 200 nm.) (E) Kinetic analysis of complex formation demonstrating rapid NP binding (within <30 s). (Scale bars: 2 µm.) (F and G) NP size, but not charge, is critical for binding. (F) Fluorescence microscopy of vital conidia demonstrating that increasing NP size reduced conidia binding. (Scale bar: 2 µm.) (G) Quantification of NP-conidia interaction by automated microscopy. Compared with small SiO_R NPs (∅ ∼30 nm), larger silica SiO_140R NPs (∅ ∼140 nm) exhibited reduced binding (Left). Reduced binding was also observed for positively charged (OSi_RN; ζ = +24 mV) vs. negatively charged (OSi_RC; ζ = −32 mV) polymer NPs. Surface modification with steric molecules (OSi_RPEG/OSi_RPEtO) did not affect binding. *P = 0.05; **P = 0.01.

Fig. 2.

Concentration-dependent reduction of NP-conidia assembly by biomolecule coronas. (A and B) Environmental coronas reduce SiO_R/G-conidia complex formation, as visualized by fluorescence microscopy. (A) Tannic acid. (B) Humic acids or sweet water NOM. (C–F) Inhibition by Curosurf corona. (C) Quantification by automated microscopy; the ratio of SiO_R-conidia complex formation is displayed. (D) SEM visualizing SiO_R assembled on conidia in the presence of undiluted Curosurf. (E) Curosurf corona inhibits complex formation, visualized by fluorescence microscopy. (F) Conidia preincubated in Curosurf still bind NPs; undiluted Curosurf does not trigger dissociation of NP-conidia complexes. (Scale bars: 2 µm.)

Fig. 3.

Pathobiological impact of NP-conidia complex formation. (A) NP coating increases resistance against defensins, HNP-1, or hbD3. Conidia were exposed to defensins, and their minimal inhibitory concentration (MIC) was determined by the AlamarBlue assay. (B) Binding of ZnO NP to conidia reduced NP toxicity. HPECs were cultivated at the ALI and exposed to aerosol containing either ZnO or ZnO-conidia complexes. Cell vitality was assessed after 6 h. (C) NP coating increases IL-1β secretion. THP-1M cells were exposed to SiO_R, conidia, or SiO_R-conidia complexes for 90 min. IL-1β was analyzed by ELISA. (D–F) NP coating inhibits TLR2-dependent uptake by phagocytes. (D) Fluorescence microscopy showing internalization of NP-conidia complexes into primary human monocytes and neutrophils. (Scale bar: 10 µm.) (E) Automated microscopy demonstrating reduced internalization of SiO_R-conidia complexes into THP-1M macrophages. (F) Reduced internalization of NP-coated conidia in murine neutrophils from wild-type, but not TLR2^−/−-deficient, animals. (G) Reduced internalization of NP-coated conidia in THP-1M cells increases fungal burden (CFU). *P = 0.05; **P = 0.01.

Fig. 4.

NP coating increases fungal lung pathobiology. (A) SiO_G-conidia complexes visualized in alveoli and internalized into macrophages by two-photon microscopy of lung slices. (Scale bars: 10 µm.) (B) NP coating reduces the uptake of conidia into lung neutrophils and macrophages, as quantified by flow cytometry. (C) NP coating enhances IL-1β and TNF-α secretion. Cytokine response in bronchoalveolar lavage fluid from mice analyzed by ELISA at 24 h after infection. (D) NP coating increases the severity of IPA in immunocompromised mice. Mice were pretreated with an anti–Gr-1 mAb to induce neutropenia. Subsequent infection with conidia or SiO_G-conidia complexes resulted in increased mortality for NP-conidia complexes. (E) Model showing how NP-spore assembly may impact fungal pathobiology. *P = 0.05; ***P = 0.005.