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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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Related in: MedlinePlus

SrbA mediates resistance to Fluconazole (FL) and Voriconazole (VO) in Aspergillus fumigatus.A clear ellipse indicates the susceptibility to the respective drug. As expected, Fluconazole has no effect on CEA10 and SDW2, but in the absence of SrbA, SDW1 is highly susceptible to Fluconazole (MIC = 1.0 µg/ml). CEA10 and SDW2 are susceptible to Voriconazole (MIC for both = 0.125 µg/ml); however, SDW1 also displays increased susceptibility (MIC = 0.012 µg/ml) to this important antifungal agent. The numbers on the scale correspond to the Fluconazole and Voriconazole concentrations on the E-test strip (in micrograms per milliliter).
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ppat-1000200-g003: SrbA mediates resistance to Fluconazole (FL) and Voriconazole (VO) in Aspergillus fumigatus.A clear ellipse indicates the susceptibility to the respective drug. As expected, Fluconazole has no effect on CEA10 and SDW2, but in the absence of SrbA, SDW1 is highly susceptible to Fluconazole (MIC = 1.0 µg/ml). CEA10 and SDW2 are susceptible to Voriconazole (MIC for both = 0.125 µg/ml); however, SDW1 also displays increased susceptibility (MIC = 0.012 µg/ml) to this important antifungal agent. The numbers on the scale correspond to the Fluconazole and Voriconazole concentrations on the E-test strip (in micrograms per milliliter).

Mentions: Given the number of ergosterol biosynthesis genes apparently regulated by SrbA, we next asked the question whether SrbA mediated resistance to the azole class of antifungal drugs that target ergosterol biosynthesis. In a screen for susceptibility to antifungal drugs using E-Test strips (AB Biodisk, kindly provided by Dr. Theodore White, Seattle Biomedical Research Institute) we found that SrbA is required for resistance to Fluconazole and Voriconazole, but not Amphotericin B or Caspofungin. All 3 strains showed equivalent minimal inhibitory concentrations (MIC) to Amphotericin B (0.25 µg/ml) and Caspofungin (0.125 µg/ml). The lack of effect of Caspofungin provides support for our hypothesis that the mutant is likely not directly affected in cell wall biosynthesis as possibly suggested by the transcriptional profiling data. However, while CEA10 and SDW2 showed resistance to Fluconazole as expected, SDW1 growth was inhibited at the surprisingly low MIC of 1 µg/ml (Figure 3). On the plates with Voriconazole we could observe that CEA10 and SDW2 were susceptible as expected (MIC of 0.125 µg/ml respectively). Similar to the results with Fluconazole, SDW1 was significantly more susceptible to Voriconazole and showed a MIC of only 0.012 µg/ml (Figure 3). These clinically significant results suggest that SrbA mediates resistance to the azole class of antifungal drugs by an undefined mechanism.


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)

SrbA mediates resistance to Fluconazole (FL) and Voriconazole (VO) in Aspergillus fumigatus.A clear ellipse indicates the susceptibility to the respective drug. As expected, Fluconazole has no effect on CEA10 and SDW2, but in the absence of SrbA, SDW1 is highly susceptible to Fluconazole (MIC = 1.0 µg/ml). CEA10 and SDW2 are susceptible to Voriconazole (MIC for both = 0.125 µg/ml); however, SDW1 also displays increased susceptibility (MIC = 0.012 µg/ml) to this important antifungal agent. The numbers on the scale correspond to the Fluconazole and Voriconazole concentrations on the E-test strip (in micrograms per milliliter).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000200-g003: SrbA mediates resistance to Fluconazole (FL) and Voriconazole (VO) in Aspergillus fumigatus.A clear ellipse indicates the susceptibility to the respective drug. As expected, Fluconazole has no effect on CEA10 and SDW2, but in the absence of SrbA, SDW1 is highly susceptible to Fluconazole (MIC = 1.0 µg/ml). CEA10 and SDW2 are susceptible to Voriconazole (MIC for both = 0.125 µg/ml); however, SDW1 also displays increased susceptibility (MIC = 0.012 µg/ml) to this important antifungal agent. The numbers on the scale correspond to the Fluconazole and Voriconazole concentrations on the E-test strip (in micrograms per milliliter).
Mentions: Given the number of ergosterol biosynthesis genes apparently regulated by SrbA, we next asked the question whether SrbA mediated resistance to the azole class of antifungal drugs that target ergosterol biosynthesis. In a screen for susceptibility to antifungal drugs using E-Test strips (AB Biodisk, kindly provided by Dr. Theodore White, Seattle Biomedical Research Institute) we found that SrbA is required for resistance to Fluconazole and Voriconazole, but not Amphotericin B or Caspofungin. All 3 strains showed equivalent minimal inhibitory concentrations (MIC) to Amphotericin B (0.25 µg/ml) and Caspofungin (0.125 µg/ml). The lack of effect of Caspofungin provides support for our hypothesis that the mutant is likely not directly affected in cell wall biosynthesis as possibly suggested by the transcriptional profiling data. However, while CEA10 and SDW2 showed resistance to Fluconazole as expected, SDW1 growth was inhibited at the surprisingly low MIC of 1 µg/ml (Figure 3). On the plates with Voriconazole we could observe that CEA10 and SDW2 were susceptible as expected (MIC of 0.125 µg/ml respectively). Similar to the results with Fluconazole, SDW1 was significantly more susceptible to Voriconazole and showed a MIC of only 0.012 µg/ml (Figure 3). These clinically significant results suggest that SrbA mediates resistance to the azole class of antifungal drugs by an undefined mechanism.

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