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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

Role of SrbA in Aspergillus fumigatus virulence.(A) Outbred CD-1 mice (n = 12) were immunosuppressed by i.p. injection of cyclophosphamide (150 mg/kg) 2 days prior to infection and s.c. injection of Kenalog (40 mg/kg) 1 day prior to infection and injection of 150 mg/kg cyclophosphamide 3 days post-inoculation and 40 mg/kg Kenalog 6 days post-inoculation. Mice were inoculated intranasally with 106 conidia in a volume of 40 µl of wild type CEA10, ΔsrbA mutant strain SDW1 and the srbA reconstituted strain SDW2. P value for comparison between SDW1 and wild type CEA10, P = 0.0002. (B) gp91phox−/− mice (n = 6) were challenged intratracheally with 106 conidia in a volume of 40 µl of wild type CEA10, ΔsrbA mutant strain SDW1 and the srbA reconstituted strain SDW2. A log rank test was used for pair wise comparisons of survival levels among the strain groups. P value for comparison between SDW1 and wild type CEA10, P = 0.0054. SDW1 is significantly less virulent than the wild type CEA10 and the reconstituted strain SDW2 in both murine models. All animal experiments were repeated in duplicate with similar results.
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ppat-1000200-g008: Role of SrbA in Aspergillus fumigatus virulence.(A) Outbred CD-1 mice (n = 12) were immunosuppressed by i.p. injection of cyclophosphamide (150 mg/kg) 2 days prior to infection and s.c. injection of Kenalog (40 mg/kg) 1 day prior to infection and injection of 150 mg/kg cyclophosphamide 3 days post-inoculation and 40 mg/kg Kenalog 6 days post-inoculation. Mice were inoculated intranasally with 106 conidia in a volume of 40 µl of wild type CEA10, ΔsrbA mutant strain SDW1 and the srbA reconstituted strain SDW2. P value for comparison between SDW1 and wild type CEA10, P = 0.0002. (B) gp91phox−/− mice (n = 6) were challenged intratracheally with 106 conidia in a volume of 40 µl of wild type CEA10, ΔsrbA mutant strain SDW1 and the srbA reconstituted strain SDW2. A log rank test was used for pair wise comparisons of survival levels among the strain groups. P value for comparison between SDW1 and wild type CEA10, P = 0.0054. SDW1 is significantly less virulent than the wild type CEA10 and the reconstituted strain SDW2 in both murine models. All animal experiments were repeated in duplicate with similar results.

Mentions: Next, we sought to determine whether SrbA was required for A. fumigatus virulence. To answer this important question, we utilized two distinct murine models of IPA. In the first model, outbred CD1 neutropenic mice infected with SDW1 displayed no symptoms associated with IPA (Figure 8A). This was in contrast to mice infected with the wild type CEA10 and reconstituted strain SDW2 that displayed well described symptoms of A. fumigatus infection including hunched posture, ruffled fur, weight loss, and increased respiration. Consequently, a significant difference in mortality was observed between the mice infected with SDW1 and mice infected with either SDW2 or CEA1O (P = 0.0002). Indeed, in this murine model, the SDW1 strain was completely avirulent (Figure 8A). We next asked the question whether mice infected with SDW1 were able to clear the infection. After 28 days, SDW1 infected mice displayed no visible or microscopic signs of infection. In particular, at days 14, 21, and 28 lung homogenates were taken from SDW1 infected mice and with the exception of one mouse, no fungal colonies were recoverable indicating that the mice had cleared the infection. Histopathological analyses of mice on days 14, 21 and 28 in this neutropenic model also confirmed the lack of fungal persistence and inflammation in mice infected with SDW1 (Figure 9).


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)

Role of SrbA in Aspergillus fumigatus virulence.(A) Outbred CD-1 mice (n = 12) were immunosuppressed by i.p. injection of cyclophosphamide (150 mg/kg) 2 days prior to infection and s.c. injection of Kenalog (40 mg/kg) 1 day prior to infection and injection of 150 mg/kg cyclophosphamide 3 days post-inoculation and 40 mg/kg Kenalog 6 days post-inoculation. Mice were inoculated intranasally with 106 conidia in a volume of 40 µl of wild type CEA10, ΔsrbA mutant strain SDW1 and the srbA reconstituted strain SDW2. P value for comparison between SDW1 and wild type CEA10, P = 0.0002. (B) gp91phox−/− mice (n = 6) were challenged intratracheally with 106 conidia in a volume of 40 µl of wild type CEA10, ΔsrbA mutant strain SDW1 and the srbA reconstituted strain SDW2. A log rank test was used for pair wise comparisons of survival levels among the strain groups. P value for comparison between SDW1 and wild type CEA10, P = 0.0054. SDW1 is significantly less virulent than the wild type CEA10 and the reconstituted strain SDW2 in both murine models. All animal experiments were repeated in duplicate with similar results.
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Related In: Results  -  Collection

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ppat-1000200-g008: Role of SrbA in Aspergillus fumigatus virulence.(A) Outbred CD-1 mice (n = 12) were immunosuppressed by i.p. injection of cyclophosphamide (150 mg/kg) 2 days prior to infection and s.c. injection of Kenalog (40 mg/kg) 1 day prior to infection and injection of 150 mg/kg cyclophosphamide 3 days post-inoculation and 40 mg/kg Kenalog 6 days post-inoculation. Mice were inoculated intranasally with 106 conidia in a volume of 40 µl of wild type CEA10, ΔsrbA mutant strain SDW1 and the srbA reconstituted strain SDW2. P value for comparison between SDW1 and wild type CEA10, P = 0.0002. (B) gp91phox−/− mice (n = 6) were challenged intratracheally with 106 conidia in a volume of 40 µl of wild type CEA10, ΔsrbA mutant strain SDW1 and the srbA reconstituted strain SDW2. A log rank test was used for pair wise comparisons of survival levels among the strain groups. P value for comparison between SDW1 and wild type CEA10, P = 0.0054. SDW1 is significantly less virulent than the wild type CEA10 and the reconstituted strain SDW2 in both murine models. All animal experiments were repeated in duplicate with similar results.
Mentions: Next, we sought to determine whether SrbA was required for A. fumigatus virulence. To answer this important question, we utilized two distinct murine models of IPA. In the first model, outbred CD1 neutropenic mice infected with SDW1 displayed no symptoms associated with IPA (Figure 8A). This was in contrast to mice infected with the wild type CEA10 and reconstituted strain SDW2 that displayed well described symptoms of A. fumigatus infection including hunched posture, ruffled fur, weight loss, and increased respiration. Consequently, a significant difference in mortality was observed between the mice infected with SDW1 and mice infected with either SDW2 or CEA1O (P = 0.0002). Indeed, in this murine model, the SDW1 strain was completely avirulent (Figure 8A). We next asked the question whether mice infected with SDW1 were able to clear the infection. After 28 days, SDW1 infected mice displayed no visible or microscopic signs of infection. In particular, at days 14, 21, and 28 lung homogenates were taken from SDW1 infected mice and with the exception of one mouse, no fungal colonies were recoverable indicating that the mice had cleared the infection. Histopathological analyses of mice on days 14, 21 and 28 in this neutropenic model also confirmed the lack of fungal persistence and inflammation in mice infected with SDW1 (Figure 9).

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