Proteomic and transcriptomic analysis of Aspergillus fumigatus on exposure to amphotericin B

Abstract

Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections, including invasive aspergillosis. It has been our aim to understand the molecular targets of AMB in Aspergillus fumigatus by genomic and proteomic approaches. In transcriptomic analysis, a total of 295 genes were found to be differentially expressed (165 upregulated and 130 downregulated), including many involving the ergosterol pathway, cell stress proteins, cell wall proteins, transport proteins, and hypothetical proteins. Proteomic profiles of A. fumigatus alone or A. fumigatus treated with AMB showed differential expression levels for 85 proteins (76 upregulated and 9 downregulated). Forty-eight of them were identified with high confidence and belonged to the above-mentioned categories. Differential expression levels for Rho-GDP dissociation inhibitor (Rho-GDI), secretory-pathway GDI, clathrin, Sec 31 (a subunit of the exocyst complex), and RAB GTPase Ypt51 in response to an antifungal drug are reported here for the first time and may represent a specific response of A. fumigatus to AMB. The expression of some of these genes was validated by real-time reverse transcription-PCR. The AMB responsive genes/proteins observed to be differentially expressed in A. fumigatus may be further explored for novel drug development.

FIG. 1.

(a) Determination of MIC₅₀s of AMB for A. fumigatus. (b, c) Microscopic images of A. fumigatus alone (b) and treated with AMB at 0.125 μg/ml (the MIC₅₀) (c). The values in the graph (a) represent concentrations of AMB and mean absorbance values ± standard deviations. The MIC₅₀ of AMB for A. fumigatus was determined to be 0.125 μg/ml. The microscopic images were taken at ×40 magnification on a Nikon Eclipse TE2000-S phase contrast inverted microscope (Nikon, Melville, New York, NY). Spores (1 × 10⁶ per ml) of A. fumigatus were incubated for 24 h at 37°C in RPMI medium with or without different concentrations of AMB. An MTT assay was performed to study the inhibition of growth of A. fumigatus by AMB (at the MIC₅₀) as described in Materials and Methods.

FIG. 2.

2-D PAGE of proteins extracted from A. fumigatus alone (a) and treated with AMB at 0.125 μg/ml (the MIC₅₀) (b). Proteins (600 μg) were separated by IEF on an IPG strip (17 cm; pI range, 5 to 8) and SDS-PAGE on 12.0% Laemmli gels and stained with Coomassie blue R 250. 2-D gel images were compared to identify differential expression levels of A. fumigatus proteins on exposure to AMB, using PDQuest software. Differentially expressed proteins were then subjected to MALDI-TOF and MALDI-TOF-TOF analysis, which resulted in identification of a total of 48 proteins (marked with arrows). Forty-four proteins were observed to be upregulated, and four proteins were downregulated (b). The functions and functional categories of the differentially expressed proteins identified are given in Table 2 and Table S2 in the supplemental material. MW, molecular mass.

FIG. 3.

Pie chart grouping responsive proteins (n = 48) (a) and genes (n = 295) (b) of A. fumigatus with 2.0-fold changes or more on exposure to AMB. According to the proteomic study, 22% of the identified proteins are associated with cell stress, 4% are cell wall maintenance proteins, 4% of the responsive proteins belong to transport proteins, 2% are associated with the ergosterol biosynthesis pathway, and 6% of the responsive proteins are hypothetical proteins (a). According to the microarray study, 48% of the responsive genes encode hypothetical proteins, 4% of the genes are associated with cell stress, 1% are associated with cell wall maintenance or ergosterol biosynthesis, and 9% of the responsive genes are involved in transport (b). Differentially expressed proteins and genes belonging to various functional categories were annotated from the ExPASy database (http://expasy.org/uniprot). TCA, tricarboxylic acid.