Antifungal Effect of All- trans Retinoic Acid against Aspergillus fumigatus In Vitro and in a Pulmonary Aspergillosis In Vivo Model

Abstract

Aspergillus fumigatus is the most common opportunistic fungal pathogen and causes invasive pulmonary aspergillosis (IPA), with high mortality among immunosuppressed patients. The fungistatic activity of all-trans retinoic acid (ATRA) has been recently described in vitro We evaluated the efficacy of ATRA in vivo and its potential synergistic interaction with other antifungal drugs. A rat model of IPA and in vitro experiments were performed to assess the efficacy of ATRA against Aspergillus in association with classical antifungal drugs and in silico studies used to clarify its mechanism of action. ATRA (0.5 and 1 mM) displayed a strong fungistatic activity in Aspergillus cultures, while at lower concentrations, synergistically potentiated fungistatic efficacy of subinhibitory concentration of amphotericin B (AmB) and posaconazole (POS). ATRA also enhanced macrophagic phagocytosis of conidia. In a rat model of IPA, ATRA reduced mortality similarly to posaconazole. Fungistatic efficacy of ATRA alone and synergistically with other antifungal drugs was documented in vitro, likely by inhibiting fungal heat shock protein 90 (Hsp90) expression and Hsp90-related genes. ATRA treatment reduced mortality in a model of IPA in vivo Those findings suggest ATRA as a suitable fungistatic agent that can also reduce dosage and adverse reactions of classical antifungal drugs and add to the development of new therapeutic strategies against IPA and systemic fungal infections.

Keywords: ATRA; Aspergillus; aspergillosis; invasive pulmonary aspergillosis; pneumonia; trans-retinoic acid.

FIG 1

ATRA inhibits germination of Aspergillus fumigatus conidia. Representative images of the effect of different ATRA concentrations on Aspergillus fumigatus conidia germination. The conidia germination was followed using an optical microscope with a ×40 magnification objective lens. Microscopic images were recorded 18 and 30 h after the treatments. One of three representative experiments is shown. Bars indicate 50 µm.

FIG 2

Synergic effect of ATRA and amphotericin B on Aspergillus fumigatus conidia germination. (A) Representative images of the effect of AmB and ATRA alone and in combination on Aspergillus fumigatus conidia germination, followed using an optical microscope with a ×40 magnification objectives lens. Microscopic images were recorded 30 h after the treatments. One of three representative experiments is shown. Bars indicate 50 µm.

FIG 3

ATRA and posaconazole show a similar efficacy in a rat model of invasive pulmonary aspergillosis. Kaplan-Meier analysis of overall survival of 10 rats/group treated with ATRA (2 mg/kg i.p.), posaconazole (4 mg/kg per os), or vehicle (control group). Statistical significance of ATRA and posaconazole treatments versus control group was evaluated by log-rank test analysis; *, P = 0.05.

FIG 4

Effect of ATRA on macrophage phagocytosis of Aspergillus fumigatus conidia in vitro. (A) Representative image of bar graph showing an increased phagocytosis of Aspergillus fumigatus conidia by human macrophage cell line U-937 incubated overnight with DMSO (control) or different concentrations of ATRA and exposed to unopsonized Aspergillus conidia., Data collected from three independent experiments and expressed as percentage of control. Student's t test was used to determine the significance of values in experimental groups and defined as P < 0.05. *, P < 0.05; **, P < 0.01. (B) Representative image of macrophage phagocytosis also performed in 24-well plates. All images of the cell culture plates were recorded at the end of the incubation period using an optical microscope with a ×40 magnification objective lens. One of three representative experiments was shown. Bars indicate 50 µm.

FIG 5

ATRA inhibits the heat shock Hsp90 protein and mRNA expression of Aspergillus. Representative blots (A) and bar graphs (B) of densitometric evaluation of Hsp90 protein expression on Aspergillus conidia treated with ATRA. Aspergillus conidia were cultured in L15 with 2% FCS at 37°C for 24 h in the presence of 1 mM ATRA. The average and SEM of triplicate experiments are shown. Significant changes are reported as P < 0.001 (***) (Student's t test). (C) Hsp90 mRNA expression level assessed by qRT-PCR and normalized to untreated Aspergillus conidia. (D). CrzA, WetA, and AbaA mRNA levels assessed by qRT-PCR and normalized to untreated Aspergillus conidia. The expression ratios were normalized to elongation factor 1α expression and were calculated according to the threshold cycle (ΔΔC_T) method. All experiments were performed in triplicate, and data were presented as the mean ± SEM. **, P < 0.01; ***, P < 0.001; Student's t test.

FIG 6

Heat shock protein 90 is a pharmacological target of ATRA. (A) Structural model of Aspergillus fumigatus Hsp90 dimeric structure. Magnification of Aspergillus fumigatus ATP-binding site, including the ATP molecule from the template, colored by atom type (right). (B) Schematic view of the best molecular docking complexes between Hsp90 Aspergillus fumigatus. ATP (left) and ATRA molecule (right). The hydrogen bonds have been indicated with green dashed lines between the interaction partners. The residues composing the active sites that are in proximity of the molecules are shown. Images have been produced using the LigPlot+ software. (C) Best molecular docking complexes between Aspergillus fumigatus Hsp90 with ATP (left) and ATRA molecules (right). The α-helices and loops are shown as light blue spirals and wires, respectively. The molecular surface is shown, and the ligand molecules, hosted in the Hsp90 ATP-binding site, are depicted by sphere representations. Panels A and C have been produced using the Chimera program.