Isoeugenol affects expression pattern of conidial hydrophobin gene RodA and transcriptional regulators MedA and SomA responsible for adherence and biofilm formation in Aspergillus fumigatus

Abstract

Aspergillus fumigatus is one of the major pathogenic fungal species, causing life-threatening infections. Due to a limited spectrum of available antifungals, exploration of new drug targets as well as potential antifungal molecules has become pertinent. Rodlet layer plays an important role in adherence of fungal conidia to hydrophobic cell surfaces in host, which also leads to A. fumigatus biofilm formation, contributing factor to fungal pathogenicity. From decades, natural sources have been known for the development of new active molecules. The present study investigates effect of isoeugenol on genes responsible for hydrophobins (RodA), adhesion as well as biofilm formation (MedA and SomA) of A. fumigatus. Minimum inhibitory concentrations (MIC and IC₅₀) of isoeugenol against A. fumigatus were determined using broth microdilution assay. The IC₅₀ results showed reduced hydrophobicity and biofilm formation as well as eradication after treatment with the compound and electron micrograph data corroborated these findings. The qRT-PCR showed a significant downregulation of genes RodA, MedA, SomA and pksP involved in hydrophobicity and biofilm formation. SwissADME studies potentiated drug-like propensity for isoeugenol which formed four hydrogen bonds with low binding energy (- 4.54 kcal/mol) at the catalytic site of RodA protein studied via AutoDock4. Hence, the findings conclude that isoeugenol inhibits conidial hydrophobicity and biofilm formation of A. fumigatus and further investigations are warranted in this direction.

Keywords: Aspergillus fumigatus; Hydrophobicity; Isoeugenol; Transcription regulators; Virulence.

Fig. 1

A) Reduction in CSH percentage of A. fumigatus conidia in the presence of isoeugenol. B) 10 µL of sterile water was dropped onto the surface of isoeugenol treated and untreated A. fumigatus culture. Loss of hydrophobicity is indicated by absorption of the water droplet into the culture

Fig. 2

Evaluation of A. fumigatus conidia formation in presence of isoeugenol and graph depicted statistical increase in number of A. fumigatus conidia after treating with isoeugenol

Fig. 3

Visualisation of scanning electron micrographs of A. fumigatus conidial surface with (A) presence of protrusions on wild-type conidia, whereas (B) surface morphology with absence of protrusions in isoeugenol treated conidia at magnification of 40 K × (C) closely packed hydrophobic wild-type conidia (D) isoeugenol treated A. fumigatus conidia at 5 K × magnification (E) and F loss of protrusions and electron dense melanin layer on A. fumigatus conidial cell wall in comparison to control (untreated) under transmission electron microscope

Fig. 4

Electron micrographs of A. fumigatus biofilm morphology (A) wild type control (without treatment); B isoeugenol treatment with lack of ECM and reduced hyphae; C pre-formed A. fumigatus biofilm and D distorted hyphae with absence of ECM at IC₅₀ at 2 K × magnification

Fig. 5

Confocal laser scanning microscope image of A. fumigatus biofilm stained with calcofluor white dye depicted (A–C) control; D–F disintegrated hyphae without ECM in isoeugenol treated sample at 20 × magnification. The chitin component shown in three images with and without fluorochrome and overlapping image. Scale Bar: 100 µm

Fig. 6

Relative quantification of RodA, MedA SomA and pksP gene expression (normalised to house-keeping gene β-tubulin) in A. fumigatus treated with IC₅₀ of isoeugenol. Data reported as mean of fold changes with standard deviation from three independent experiments amplified in triplicates. p ≤ 0.05 was considered statistically significant

Fig. 7

The oral bioavailability radar of isoeugenol using SwissADME predictor. The six physiochemical properties taken into account to consider a molecule as drug-like are FLEX, LIPO, INSATU, SIZE, POLAR and INSOLU. The pink area of radar plot showed that compound satisfies all the above-mentioned properties except saturation

Fig. 8

Binding interactions of isoeugenol with the active site of RodA hydrophobin protein target site (PDB ID: 6GCJ). Green dotted lines depicted hydrogen bonds; pink dotted line showed pi-pi bond