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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

Generation and confirmation of a SrbA  mutant in Aspergillus fumigatus.(A) Schematic of wild type (CEA10) and SDW1 (SrbA  mutant) genomic loci. (B) Southern blot analysis of wild type, SDW1, and SDW2 strains. Genomic DNA from the respective strains was isolated and digested overnight with NcoI. An approximate 1 kb genomic region of the SrbA locus was utilized as a probe. The expected hybridization patterns and sizes were observed for the wild type CEA10 (5721 bp) and SrbA mutant (SDW1) (3622 bp) strains. In addition, confirmation of ectopic reconstitution of the SrbA  mutant was confirmed by the presence of the wild type srbA locus hybridization signal and persistence of the SrbA  mutant locus (strain SDW2).
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ppat-1000200-g001: Generation and confirmation of a SrbA mutant in Aspergillus fumigatus.(A) Schematic of wild type (CEA10) and SDW1 (SrbA mutant) genomic loci. (B) Southern blot analysis of wild type, SDW1, and SDW2 strains. Genomic DNA from the respective strains was isolated and digested overnight with NcoI. An approximate 1 kb genomic region of the SrbA locus was utilized as a probe. The expected hybridization patterns and sizes were observed for the wild type CEA10 (5721 bp) and SrbA mutant (SDW1) (3622 bp) strains. In addition, confirmation of ectopic reconstitution of the SrbA mutant was confirmed by the presence of the wild type srbA locus hybridization signal and persistence of the SrbA mutant locus (strain SDW2).

Mentions: To determine whether SrbA is involved in hypoxia adaptation and fungal virulence in filamentous fungi, we generated a mutant of the gene encoding SrbA by replacement of the srbA coding sequence in A. fumigatus strain CEA17 with the auxotrophic marker pyrG from A. parasiticus as previously described [41],[42] (Figure 1). The resulting ΔsrbA strain was named SDW1. Ectopic re-introduction of the wild type srbA allele into SDW1 (resulting in strain SDW2) allowed us to attribute all resulting phenotypes specifically to the absence of srbA in SDW1. All strains were rigorously confirmed with Southern blot (Figure 1) and PCR analyses (data not shown). The re-introduced srbA allele in SDW2 displayed similar mRNA abundance in response to hypoxia as the srbA allele in the wild type strain (data not shown). SDW1 and SDW2 both displayed normal hyphal growth rates compared to the wild type strain CEA10 in normoxic conditions on glucose minimal medium (GMM) (Figure 2A) (P>0.01). However, no hyphal growth of SDW1 was observed in hypoxic (1% O2, 5% CO2, 94% N2) conditions whereas wild type strain CEA10 and reconstituted strain SDW2 grew at a normal rate with visual phenotypic differences in colony color and conidiation compared to growth in normoxia (Figure 2A and 2B). In hypoxia, the wild type strains displayed increased aerial hyphae, decreased conidia production, and consequently exhibited a fluffy colony morphology (Figure 2B). After 96 hours of incubation in hypoxia, SDW1 continued to display undetectable growth. However, upon transfer back to normoxic conditions, wild type growth rate was restored (data not shown). Addition of exogenous ergosterol or lanosterol did not rescue the SDW1 growth defect or alter wild type growth morphology in hypoxia (data not shown). These results indicate that A. fumigatus can rapidly adapt to hypoxic microenvironments, and that SrbA in A. fumigatus is involved in mediating this response 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)

Generation and confirmation of a SrbA  mutant in Aspergillus fumigatus.(A) Schematic of wild type (CEA10) and SDW1 (SrbA  mutant) genomic loci. (B) Southern blot analysis of wild type, SDW1, and SDW2 strains. Genomic DNA from the respective strains was isolated and digested overnight with NcoI. An approximate 1 kb genomic region of the SrbA locus was utilized as a probe. The expected hybridization patterns and sizes were observed for the wild type CEA10 (5721 bp) and SrbA mutant (SDW1) (3622 bp) strains. In addition, confirmation of ectopic reconstitution of the SrbA  mutant was confirmed by the presence of the wild type srbA locus hybridization signal and persistence of the SrbA  mutant locus (strain SDW2).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000200-g001: Generation and confirmation of a SrbA mutant in Aspergillus fumigatus.(A) Schematic of wild type (CEA10) and SDW1 (SrbA mutant) genomic loci. (B) Southern blot analysis of wild type, SDW1, and SDW2 strains. Genomic DNA from the respective strains was isolated and digested overnight with NcoI. An approximate 1 kb genomic region of the SrbA locus was utilized as a probe. The expected hybridization patterns and sizes were observed for the wild type CEA10 (5721 bp) and SrbA mutant (SDW1) (3622 bp) strains. In addition, confirmation of ectopic reconstitution of the SrbA mutant was confirmed by the presence of the wild type srbA locus hybridization signal and persistence of the SrbA mutant locus (strain SDW2).
Mentions: To determine whether SrbA is involved in hypoxia adaptation and fungal virulence in filamentous fungi, we generated a mutant of the gene encoding SrbA by replacement of the srbA coding sequence in A. fumigatus strain CEA17 with the auxotrophic marker pyrG from A. parasiticus as previously described [41],[42] (Figure 1). The resulting ΔsrbA strain was named SDW1. Ectopic re-introduction of the wild type srbA allele into SDW1 (resulting in strain SDW2) allowed us to attribute all resulting phenotypes specifically to the absence of srbA in SDW1. All strains were rigorously confirmed with Southern blot (Figure 1) and PCR analyses (data not shown). The re-introduced srbA allele in SDW2 displayed similar mRNA abundance in response to hypoxia as the srbA allele in the wild type strain (data not shown). SDW1 and SDW2 both displayed normal hyphal growth rates compared to the wild type strain CEA10 in normoxic conditions on glucose minimal medium (GMM) (Figure 2A) (P>0.01). However, no hyphal growth of SDW1 was observed in hypoxic (1% O2, 5% CO2, 94% N2) conditions whereas wild type strain CEA10 and reconstituted strain SDW2 grew at a normal rate with visual phenotypic differences in colony color and conidiation compared to growth in normoxia (Figure 2A and 2B). In hypoxia, the wild type strains displayed increased aerial hyphae, decreased conidia production, and consequently exhibited a fluffy colony morphology (Figure 2B). After 96 hours of incubation in hypoxia, SDW1 continued to display undetectable growth. However, upon transfer back to normoxic conditions, wild type growth rate was restored (data not shown). Addition of exogenous ergosterol or lanosterol did not rescue the SDW1 growth defect or alter wild type growth morphology in hypoxia (data not shown). These results indicate that A. fumigatus can rapidly adapt to hypoxic microenvironments, and that SrbA in A. fumigatus is involved in mediating this response 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