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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.

SUBMITTER: Gautam P

PROVIDER: S-EPMC2592866 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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

Gautam Poonam P Shankar Jata J Madan Taruna T Sirdeshmukh Ravi R Sundaram Curam Sreenivasacharlu CS Gade Wasudev Namdeo WN Basir Seemi Farhat SF Sarma Puranam Usha PU

Antimicrobial agents and chemotherapy 20081006 12

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, ...[more]

PMID: 18838595

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Project description:Aspergillus fumigatus is a mold responsible for the majority of cases of aspergillosis in humans. To survive in the human body, A. fumigatus must adapt to microenvironments that are often characterized by low nutrient and oxygen availability. Recent research suggests that the ability of A. fumigatus and other pathogenic fungi to adapt to hypoxia contributes to their virulence. However, molecular mechanisms of A. fumigatus hypoxia adaptation are poorly understood. Thus, to better understand how A. fumigatus adapts to hypoxic microenvironments found in vivo during human fungal pathogenesis, the dynamic changes of the fungal transcriptome and proteome in hypoxia were investigated over a period of 24 hours utilizing an oxygen-controlled fermenter system.Significant increases in transcripts associated with iron and sterol metabolism, the cell wall, the GABA shunt, and transcriptional regulators were observed in response to hypoxia. A concomitant reduction in transcripts was observed with ribosome and terpenoid backbone biosynthesis, TCA cycle, amino acid metabolism and RNA degradation. Analysis of changes in transcription factor mRNA abundance shows that hypoxia induces significant positive and negative changes that may be important for regulating the hypoxia response in this pathogenic mold. Growth in hypoxia resulted in changes in the protein levels of several glycolytic enzymes, but these changes were not always reflected by the corresponding transcriptional profiling data. However, a good correlation overall (R(2) = 0.2, p < 0.05) existed between the transcriptomic and proteomics datasets for all time points. The lack of correlation between some transcript levels and their subsequent protein levels suggests another regulatory layer of the hypoxia response in A. fumigatus.Taken together, our data suggest a robust cellular response that is likely regulated both at the transcriptional and post-transcriptional level in response to hypoxia by the human pathogenic mold A. fumigatus. As with other pathogenic fungi, the induction of glycolysis and transcriptional down-regulation of the TCA cycle and oxidative phosphorylation appear to major components of the hypoxia response in this pathogenic mold. In addition, a significant induction of the transcripts involved in ergosterol biosynthesis is consistent with previous observations in the pathogenic yeasts Candida albicans and Cryptococcus neoformans indicating conservation of this response to hypoxia in pathogenic fungi. Because ergosterol biosynthesis enzymes also require iron as a co-factor, the increase in iron uptake transcripts is consistent with an increased need for iron under hypoxia. However, unlike C. albicans and C. neoformans, the GABA shunt appears to play an important role in reducing NADH levels in response to hypoxia in A. fumigatus and it will be intriguing to determine whether this is critical for fungal virulence. Overall, regulatory mechanisms of the A. fumigatus hypoxia response appear to involve both transcriptional and post-transcriptional control of transcript and protein levels and thus provide candidate genes for future analysis of their role in hypoxia adaptation and fungal virulence.

Project description:Aspergillus fumigatus (A. fumigatus) is a wide-spread fungus that is a potent allergen in hypersensitive individuals but also an opportunistic pathogen in immunocompromised patients. It reproduces asexually by releasing airborne conidiospores (conidia). Upon inhalation, fungal conidia are capable of reaching the airway epithelial cells (AECs) in bronchial and alveolar tissues. Previous studies have predominantly used submerged monolayer cultures for studying this host-pathogen interaction; however, these cultures do not recapitulate the mucocililary differentiation phenotype of the in vivo epithelium in the respiratory tract. Thus, the aim of this study was to use well-differentiated primary human bronchial epithelial cells (HBECs) grown at the air-liquid interface (ALI) to determine their transcriptomic and proteomic responses following interaction with A. fumigatus conidia. We visualized conidial interaction with HBECs using confocal laser scanning microscopy (CLSM), and applied NanoString nCounter and shotgun proteomics to assess gene expression changes in the human cells upon interaction with A. fumigatus conidia. Western blot analysis was used to assess the expression of top three differentially expressed proteins, CALR, SET and NUCB2. CLSM showed that, unlike submerged monolayer cultures, well-differentiated ALI cultures of primary HBECs were estimated to internalize less than 1% of bound conidia. Nevertheless, transcriptomic and proteomic analyses revealed numerous differentially expressed host genes; these were enriched for pathways including apoptosis/autophagy, translation, unfolded protein response and cell cycle (up-regulated); complement and coagulation pathways, iron homeostasis, nonsense mediated decay and rRNA binding (down-regulated). CALR and SET were confirmed to be up-regulated in ALI cultures of primary HBECs upon exposure to A. fumigatus via western blot analysis. Therefore, using transcriptomics and proteomics approaches, ALI models recapitulating the bronchial epithelial barrier in the conductive zone of the respiratory tract can provide novel insights to the molecular response of bronchial epithelial cells upon exposure to A. fumigatus conidia.

Dataset Information

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

Publications

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

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