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

Barker BM, Kroll K, Vödisch M, Mazurie A, Kniemeyer O, Cramer RA - BMC Genomics (2012)

Bottom Line: To survive in the human body, A. fumigatus must adapt to microenvironments that are often characterized by low nutrient and oxygen availability.A concomitant reduction in transcripts was observed with ribosome and terpenoid backbone biosynthesis, TCA cycle, amino acid metabolism and RNA degradation.However, a good correlation overall (R(2) = 0.2, p < 0.05) existed between the transcriptomic and proteomics datasets for all time points.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology and Infectious Disease, Montana State University, Bozeman, MT, USA.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

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Hypoxia increases transcript levels of enzymes involved in heme biosynthesis, iron-associated and SreA-associated processes. Microarray datasets include three biological and two technical replicates to create heat maps using a median values to compare wild type Aspergillus fumigatus strain CBS144.89 at the indicated times after exposure to hypoxia to time point immediately prior to hypoxia exposure (0 hours). Yellow indicates transcript level is higher under exposure to hypoxia. Data are sorted by the late time point (24 hours post exposure to hypoxia).
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Figure 9: Hypoxia increases transcript levels of enzymes involved in heme biosynthesis, iron-associated and SreA-associated processes. Microarray datasets include three biological and two technical replicates to create heat maps using a median values to compare wild type Aspergillus fumigatus strain CBS144.89 at the indicated times after exposure to hypoxia to time point immediately prior to hypoxia exposure (0 hours). Yellow indicates transcript level is higher under exposure to hypoxia. Data are sorted by the late time point (24 hours post exposure to hypoxia).

Mentions: Taking only transcripts that were most significantly changed in abundance using SAM (Additional file 1), analysis with FungiFun https://sbi.hki-jena.de/FungiFun/FungiFun.cgi showed additional significant categories. In addition to the previous pathways identified by GSEA, oxidation-reduction and iron related transcripts are significantly increased in response to hypoxia (Additional file 6). Importantly, iron is a required cofactor for many of the transcripts coding for enzymes associated with ergosterol biosynthesis steps that were increased in response to hypoxia (erg25 and erg11). A literature search provides a list of iron-related transcripts to investigate the microarray results [47-49]. The majority of transcripts associated with iron acquisition and biosynthesis are somewhat increased in response to hypoxia (Figure 9). However, transcripts related to siderophore biosynthesis (i.e. sidA, Afu2g07680) and iron uptake (i.e. sit1, Afu7g06060) were slightly transcriptionally reduced. SreA (Afu5g11260), a previously identified negative regulator of iron homeostasis, showed increased transcript levels at 2, 6, and 12 hours, so that the iron-limitation response was apparently not induced under the tested conditions. Importantly, we must distinguish A. fumigatus SreA from SrbA the sterol regulatory element binding protein in this mold (SREBPs in yeast have been named Sre1). There is also an apparent decrease in terpenoid intermediates in response to hypoxia which may negatively affect siderophore biosynthesis [45]. A further discussion on the implications in changes in iron homeostasis mechanisms is below in the transcription factor analysis. In general, these results are consistent with a requirement for iron in the hypoxia response that has been observed in mammalian systems and was shown for A. fumigatus when it was cultivated in a glucose-limited chemostat at low oxygen levels [21,25]. Moreover, in C. albicans an increase in iron uptake transcripts was also observed upon exposure to hypoxia [11,17]. Recently, we observed that SrbA also regulates iron uptake and homeostasis in iron replete conditions under hypoxia and in low iron conditions, further suggesting an important link between iron uptake and the A. fumigatus hypoxia response that remains to be further explored [27].


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

Barker BM, Kroll K, Vödisch M, Mazurie A, Kniemeyer O, Cramer RA - BMC Genomics (2012)

Hypoxia increases transcript levels of enzymes involved in heme biosynthesis, iron-associated and SreA-associated processes. Microarray datasets include three biological and two technical replicates to create heat maps using a median values to compare wild type Aspergillus fumigatus strain CBS144.89 at the indicated times after exposure to hypoxia to time point immediately prior to hypoxia exposure (0 hours). Yellow indicates transcript level is higher under exposure to hypoxia. Data are sorted by the late time point (24 hours post exposure to hypoxia).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293747&req=5

Figure 9: Hypoxia increases transcript levels of enzymes involved in heme biosynthesis, iron-associated and SreA-associated processes. Microarray datasets include three biological and two technical replicates to create heat maps using a median values to compare wild type Aspergillus fumigatus strain CBS144.89 at the indicated times after exposure to hypoxia to time point immediately prior to hypoxia exposure (0 hours). Yellow indicates transcript level is higher under exposure to hypoxia. Data are sorted by the late time point (24 hours post exposure to hypoxia).
Mentions: Taking only transcripts that were most significantly changed in abundance using SAM (Additional file 1), analysis with FungiFun https://sbi.hki-jena.de/FungiFun/FungiFun.cgi showed additional significant categories. In addition to the previous pathways identified by GSEA, oxidation-reduction and iron related transcripts are significantly increased in response to hypoxia (Additional file 6). Importantly, iron is a required cofactor for many of the transcripts coding for enzymes associated with ergosterol biosynthesis steps that were increased in response to hypoxia (erg25 and erg11). A literature search provides a list of iron-related transcripts to investigate the microarray results [47-49]. The majority of transcripts associated with iron acquisition and biosynthesis are somewhat increased in response to hypoxia (Figure 9). However, transcripts related to siderophore biosynthesis (i.e. sidA, Afu2g07680) and iron uptake (i.e. sit1, Afu7g06060) were slightly transcriptionally reduced. SreA (Afu5g11260), a previously identified negative regulator of iron homeostasis, showed increased transcript levels at 2, 6, and 12 hours, so that the iron-limitation response was apparently not induced under the tested conditions. Importantly, we must distinguish A. fumigatus SreA from SrbA the sterol regulatory element binding protein in this mold (SREBPs in yeast have been named Sre1). There is also an apparent decrease in terpenoid intermediates in response to hypoxia which may negatively affect siderophore biosynthesis [45]. A further discussion on the implications in changes in iron homeostasis mechanisms is below in the transcription factor analysis. In general, these results are consistent with a requirement for iron in the hypoxia response that has been observed in mammalian systems and was shown for A. fumigatus when it was cultivated in a glucose-limited chemostat at low oxygen levels [21,25]. Moreover, in C. albicans an increase in iron uptake transcripts was also observed upon exposure to hypoxia [11,17]. Recently, we observed that SrbA also regulates iron uptake and homeostasis in iron replete conditions under hypoxia and in low iron conditions, further suggesting an important link between iron uptake and the A. fumigatus hypoxia response that remains to be further explored [27].

Bottom Line: To survive in the human body, A. fumigatus must adapt to microenvironments that are often characterized by low nutrient and oxygen availability.A concomitant reduction in transcripts was observed with ribosome and terpenoid backbone biosynthesis, TCA cycle, amino acid metabolism and RNA degradation.However, a good correlation overall (R(2) = 0.2, p < 0.05) existed between the transcriptomic and proteomics datasets for all time points.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology and Infectious Disease, Montana State University, Bozeman, MT, USA.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus