Transcriptomic and proteomic analyses of the Aspergillus fumigatus hypoxia response using an oxygen-controlled fermenter

Abstract

BackgroundAspergillus 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.ResultsSignificant 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 (R2 = 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.ConclusionsTaken 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-...

Identification of Hypoxia-Inducible Target Genes of Aspergillus fumigatus by Transcriptome Analysis Reveals Cellular Respiration as an Important Contributor to Hypoxic Survival

“…A comparison with the transcriptome data from the hypoxic response of A. fumigatus cultivated in a batch fermenter at low oxygen levels for 24 h by Barker et al (12) revealed that only 27% of the 867 significantly altered genes were also differentially expressed in our study (see Fig. S2 in the supplemental material).…”

“…The role of fermentation during hypoxic growth remains ambiguous, and the electron transport chain (ETC) is still active at low oxygen levels for energy production (11). This was also shown in a previous report on hypoxia-induced changes of the transcriptome and proteome in A. fumigatus over a period of 24 h (here defined as a short-term response) (12). In this work, the authors used a batch cultivation and monitored the A. fumigatus hypoxic response at the time points 2, 6, 12, and 24 h. In a recent proteomic study from our group on the long-term response (5 to 6 days) to hypoxia, A. fumigatus was cultivated in an oxygen-controlled chemostat.…”

“…Fermentation was carried out as described previously (12). For expression analysis of the flavohemoprotein fhpA gene, either 70 mM NaNO 3 or 20 mM L-glutamine was used as the nitrogen source in batch cultivations of A. fumigatus.…”

“…However, for A. fumigatus no growth phenotype was observed under oxygen-limiting conditions for strains devoid of ethanol fermentation (19). The ␥-aminobutyric acid (GABA) shunt pathway has also been proposed to bypass the accumulation of NADH in A. fumigatus and A. nidulans during hypoxic growth (12,20,21). Furthermore, in A. nidulans and Fusarium oxysporum, growth under oxygen-limiting conditions is supported by using nitrate as an alternative electron acceptor, a process also referred to as ammonia fermentation (22,23).…”

“…A follow-up proteomic study on the short-term response of A. fumigatus to hypoxia (2 to 24 h) revealed a surprisingly low correlation between the proteomic data of the two studies (12). This may be explained by the different incubation times (6 days versus 2 to 24 h) or culture conditions (glucose-limited chemostat versus batch culture without glucose limitation).…”