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A sterol-regulatory element binding protein is required for cell polarity, hypoxia adaptation, azole drug resistance, and virulence in Aspergillus fumigatus.

Willger SD, Puttikamonkul S, Kim KH, Burritt JB, Grahl N, Metzler LJ, Barbuch R, Bard M, Lawrence CB, Cramer RA - PLoS Pathog. (2008)

Bottom Line: At the site of microbial infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments in which both the pathogen and host cells must survive.Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment and consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA).Significantly, the SrbA mutant was highly susceptible to fluconazole and voriconazole.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT, USA.

ABSTRACT
At the site of microbial infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments in which both the pathogen and host cells must survive. Currently, whether hypoxia adaptation is an important virulence attribute of opportunistic pathogenic molds is unknown. Here we report the characterization of a sterol-regulatory element binding protein, SrbA, in the opportunistic pathogenic mold, Aspergillus fumigatus. Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment and consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA). Transcriptional profiling revealed 87 genes that are affected by loss of SrbA function. Annotation of these genes implicated SrbA in maintaining sterol biosynthesis and hyphal morphology. Further examination of the SrbA mutant consequently revealed that SrbA plays a critical role in ergosterol biosynthesis, resistance to the azole class of antifungal drugs, and in maintenance of cell polarity in A. fumigatus. Significantly, the SrbA mutant was highly susceptible to fluconazole and voriconazole. Thus, these findings present a new function of SREBP proteins in filamentous fungi, and demonstrate for the first time that hypoxia adaptation is likely an important virulence attribute of pathogenic molds.

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C4-demethylation is altered in the absence of SrbA.Representative GC-MS chromatograms of sterol extracts from wild type (A) and SDW1 (B). Key: A- ergosta-5,8,22-trien-3β-ol, B- ergosterol, C- ergosta-5,7,22,24(28)-tetraen-3β-ol, D- ergosta-5,7,24(28)-trien-3β-ol, E- 24-ethylcholesta-5,7,22-trien-3β-ol, F- 4-methylfecosterol, G- 4methylergosta-5,8,24(28)-trien-3β-ol, H- 4,4-demethylergosta-8,24(28)-dien-3β-ol. An accumulation of 4-methyl sterols is observed in the absence of SrbA, suggesting a blockage in enzymes involved in sterol C-4 demethylation. The ratio of C-4 methylated sterols to ergosterol in the absence of SrbA was 1.94 whereas no C-4 methylated sterols accumulated in the wild type.
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ppat-1000200-g007: C4-demethylation is altered in the absence of SrbA.Representative GC-MS chromatograms of sterol extracts from wild type (A) and SDW1 (B). Key: A- ergosta-5,8,22-trien-3β-ol, B- ergosterol, C- ergosta-5,7,22,24(28)-tetraen-3β-ol, D- ergosta-5,7,24(28)-trien-3β-ol, E- 24-ethylcholesta-5,7,22-trien-3β-ol, F- 4-methylfecosterol, G- 4methylergosta-5,8,24(28)-trien-3β-ol, H- 4,4-demethylergosta-8,24(28)-dien-3β-ol. An accumulation of 4-methyl sterols is observed in the absence of SrbA, suggesting a blockage in enzymes involved in sterol C-4 demethylation. The ratio of C-4 methylated sterols to ergosterol in the absence of SrbA was 1.94 whereas no C-4 methylated sterols accumulated in the wild type.

Mentions: Transcriptional profiling of SDW1 under hypoxia suggested that SrbA was involved in both early and late steps of the sterol biosynthesis pathway. In addition, the abnormal conidial and hyphal morphology observed via light microscopy and TEM micrographs in SDW1 also suggested possible alterations in sterol content in the absence of SrbA. Thus, we examined the sterol profile of the SrbA mutant SDW1 by GC-MS and compared it with the wild type strain CEA10. The GC-MS profiles demonstrated a significant accumulation of 4-methyl sterols in the SrbA mutant, SDW1, that was not observed in the wild type strain CEA10 (Figure 7). Interestingly, both strains possessed significant amounts of ergosterol (Figure 7). The ratio of C-4 methylated sterols to ergosterol in the absence of SrbA is 1.94 whereas no C-4 methylated sterols accumulated in the wild type. Specifically, the accumulation of 4-methylfecosterol and 4,4-dimethylergosta-8,24(28)-dien-3β-ol in the absence of SrbA suggests a blockage at ERG25 in the sterol biosynthesis pathway in SDW1. These alterations are supported by the transcriptional profiling data, which suggests transcriptional regulation of ERG25 by SrbA in A. fumigatus (Table 1). Consequently, these results suggest a blockage of C4 demethylation in the absence of SrbA in A. fumigatus. In addition, these results suggest that ergosterol can still be synthesized in the absence of SrbA in A. fumigatus.


A sterol-regulatory element binding protein is required for cell polarity, hypoxia adaptation, azole drug resistance, and virulence in Aspergillus fumigatus.

Willger SD, Puttikamonkul S, Kim KH, Burritt JB, Grahl N, Metzler LJ, Barbuch R, Bard M, Lawrence CB, Cramer RA - PLoS Pathog. (2008)

C4-demethylation is altered in the absence of SrbA.Representative GC-MS chromatograms of sterol extracts from wild type (A) and SDW1 (B). Key: A- ergosta-5,8,22-trien-3β-ol, B- ergosterol, C- ergosta-5,7,22,24(28)-tetraen-3β-ol, D- ergosta-5,7,24(28)-trien-3β-ol, E- 24-ethylcholesta-5,7,22-trien-3β-ol, F- 4-methylfecosterol, G- 4methylergosta-5,8,24(28)-trien-3β-ol, H- 4,4-demethylergosta-8,24(28)-dien-3β-ol. An accumulation of 4-methyl sterols is observed in the absence of SrbA, suggesting a blockage in enzymes involved in sterol C-4 demethylation. The ratio of C-4 methylated sterols to ergosterol in the absence of SrbA was 1.94 whereas no C-4 methylated sterols accumulated in the wild type.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2572145&req=5

ppat-1000200-g007: C4-demethylation is altered in the absence of SrbA.Representative GC-MS chromatograms of sterol extracts from wild type (A) and SDW1 (B). Key: A- ergosta-5,8,22-trien-3β-ol, B- ergosterol, C- ergosta-5,7,22,24(28)-tetraen-3β-ol, D- ergosta-5,7,24(28)-trien-3β-ol, E- 24-ethylcholesta-5,7,22-trien-3β-ol, F- 4-methylfecosterol, G- 4methylergosta-5,8,24(28)-trien-3β-ol, H- 4,4-demethylergosta-8,24(28)-dien-3β-ol. An accumulation of 4-methyl sterols is observed in the absence of SrbA, suggesting a blockage in enzymes involved in sterol C-4 demethylation. The ratio of C-4 methylated sterols to ergosterol in the absence of SrbA was 1.94 whereas no C-4 methylated sterols accumulated in the wild type.
Mentions: Transcriptional profiling of SDW1 under hypoxia suggested that SrbA was involved in both early and late steps of the sterol biosynthesis pathway. In addition, the abnormal conidial and hyphal morphology observed via light microscopy and TEM micrographs in SDW1 also suggested possible alterations in sterol content in the absence of SrbA. Thus, we examined the sterol profile of the SrbA mutant SDW1 by GC-MS and compared it with the wild type strain CEA10. The GC-MS profiles demonstrated a significant accumulation of 4-methyl sterols in the SrbA mutant, SDW1, that was not observed in the wild type strain CEA10 (Figure 7). Interestingly, both strains possessed significant amounts of ergosterol (Figure 7). The ratio of C-4 methylated sterols to ergosterol in the absence of SrbA is 1.94 whereas no C-4 methylated sterols accumulated in the wild type. Specifically, the accumulation of 4-methylfecosterol and 4,4-dimethylergosta-8,24(28)-dien-3β-ol in the absence of SrbA suggests a blockage at ERG25 in the sterol biosynthesis pathway in SDW1. These alterations are supported by the transcriptional profiling data, which suggests transcriptional regulation of ERG25 by SrbA in A. fumigatus (Table 1). Consequently, these results suggest a blockage of C4 demethylation in the absence of SrbA in A. fumigatus. In addition, these results suggest that ergosterol can still be synthesized in the absence of SrbA in A. fumigatus.

Bottom Line: At the site of microbial infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments in which both the pathogen and host cells must survive.Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment and consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA).Significantly, the SrbA mutant was highly susceptible to fluconazole and voriconazole.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT, USA.

ABSTRACT
At the site of microbial infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments in which both the pathogen and host cells must survive. Currently, whether hypoxia adaptation is an important virulence attribute of opportunistic pathogenic molds is unknown. Here we report the characterization of a sterol-regulatory element binding protein, SrbA, in the opportunistic pathogenic mold, Aspergillus fumigatus. Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment and consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA). Transcriptional profiling revealed 87 genes that are affected by loss of SrbA function. Annotation of these genes implicated SrbA in maintaining sterol biosynthesis and hyphal morphology. Further examination of the SrbA mutant consequently revealed that SrbA plays a critical role in ergosterol biosynthesis, resistance to the azole class of antifungal drugs, and in maintenance of cell polarity in A. fumigatus. Significantly, the SrbA mutant was highly susceptible to fluconazole and voriconazole. Thus, these findings present a new function of SREBP proteins in filamentous fungi, and demonstrate for the first time that hypoxia adaptation is likely an important virulence attribute of pathogenic molds.

Show MeSH
Related in: MedlinePlus