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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

Abnormal cell wall-plasma membrane interface and hyphal morphology is evident in the absence of SrbA.(A–C) Transmission electron micrographs showing sections of conidia of wild type CEA10 (A) and SDW1 (B,C). Compared with the round wild type conidia having clear boundaries between plasma membrane and cell wall layers, most of the SDW1 conidia were distorted in shape and possessed faint, somewhat shriveled boundaries. Note that frequent “tearing” took place mainly at the cell wall – plasma membrane interface during microscopic examination of the SDW1 conidia (arrows). This phenotype was observed in over 80% of SDW1 conidia examined. Inset panels depict a 3× magnified view of the conidial cell wall region. Bars = 500 nm. (D–H). Transmission electron micrographs showing longitudinal and transverse hyphal sections of wild type CEA10 (D,F) and SDW1 (E,G,H). Close observation of the hyphal tips show phenotypic differences between wild type and SDW1. Abnormal cell wall – plasma membrane interfaces and apical swellings in SDW1 hyphae were frequently observed, while the wild type showed normal round-shaped apexes. With respect to cell wall morphology around the hyphal apex, SDW1 had an abnormally expanded cell wall (arrows) containing numerous electron dense objects (arrowheads), which likely resulted in hyphal tip bending (H). Inset panels depict a magnified view of the boxed region. Bars = 1 µm, except for the inset panels of E and H where they denote 500 nm.
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ppat-1000200-g005: Abnormal cell wall-plasma membrane interface and hyphal morphology is evident in the absence of SrbA.(A–C) Transmission electron micrographs showing sections of conidia of wild type CEA10 (A) and SDW1 (B,C). Compared with the round wild type conidia having clear boundaries between plasma membrane and cell wall layers, most of the SDW1 conidia were distorted in shape and possessed faint, somewhat shriveled boundaries. Note that frequent “tearing” took place mainly at the cell wall – plasma membrane interface during microscopic examination of the SDW1 conidia (arrows). This phenotype was observed in over 80% of SDW1 conidia examined. Inset panels depict a 3× magnified view of the conidial cell wall region. Bars = 500 nm. (D–H). Transmission electron micrographs showing longitudinal and transverse hyphal sections of wild type CEA10 (D,F) and SDW1 (E,G,H). Close observation of the hyphal tips show phenotypic differences between wild type and SDW1. Abnormal cell wall – plasma membrane interfaces and apical swellings in SDW1 hyphae were frequently observed, while the wild type showed normal round-shaped apexes. With respect to cell wall morphology around the hyphal apex, SDW1 had an abnormally expanded cell wall (arrows) containing numerous electron dense objects (arrowheads), which likely resulted in hyphal tip bending (H). Inset panels depict a magnified view of the boxed region. Bars = 1 µm, except for the inset panels of E and H where they denote 500 nm.

Mentions: Visual inspection of SDW1 colony morphology in standard laboratory conditions did not reveal any apparent morphological phenotypes (Figure 2B). However, our transcriptional profiling experiments suggested possible alterations in cell wall biosynthesis, a critical component of hyphal morphology and growth, in the absence of SrbA. Consequently, we performed a more in depth analysis of SDW1 morphology. First, we utilized light microscopy to examine the growing edges of SDW1 colonies in normoxia. We observed a significant defect in hyphal tip branching in SDW1 that is not apparent in strains CEA10 and SDW2 (Figure 4). SDW1 hyphal tips display hyper-branching and a “blunted” abnormal morphological phenotype (Figure 4). This phenotype suggests that SrbA is involved in maintaining cell polarity that directs hyphal growth. Interestingly, this phenotype does not appear to alter the growth rate of the colony, which was comparable to the wild type under normoxic conditions (Figure 2A). Next, we utilized transmission electron microscopy (TEM) to further examine the cell wall and morphology of conidia and hyphae of SDW1. Confirming our suspicions that the mutant was not directly affected in cell wall biosynthesis we observed no clear cell wall defects. However, a general thickening of the intracellular space between the cell wall and plasma membrane is observed in SDW1 conidia and hyphae compared with the wild type (Figure 5A and 5B and 5D and 5E). A striking phenotype was consequently observed in conidia from SDW1 that suggested a significant defect in the cell wall-plasma membrane interface occurs in the absence of SrbA (Figure 5A and 5B). This defect is apparently exacerbated by the electron beam, which causes a separation between the cell wall and plasma membrane in SDW1 conidia (Figure 5C). This phenotype was observed in over 80% of the SDW1 conidia examined. However, the size and density of the mutant conidia were comparable to the wild type strain as measured by flow cytometry (data not shown). Since a defect in the cell wall plasma membrane interface was suggested, we examined viability of the SDW1 conidia by monitoring germination. These experiments revealed that viability, as measured by conidia germination, was not significantly different between the wild type, SDW1 and SDW2 strains (Figure 6) (P>0.01). Similar cell wall-plasma membrane defects were observed in the SDW1 hyphae compared with the wild type hyphae (Figure 5D and 5E). Importantly, an accumulation of electron dense objects was observed in the SDW1 hyphae. We hypothesize that these objects may be vesicles of the Spitzenkörper, and their abnormal location in the SDW1 hyphae may cause the observed altered cell polarity (Figure 5E and 5H). This phenotype was observed in over 50% of the SDW1 hyphae examined and never observed in the wild type strain. These results suggest that SrbA is critical for maintaining the cell wall – plasma membrane interface, and that SrbA is critical for normal hyphal branching and cell polarity in filamentous fungi 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)

Abnormal cell wall-plasma membrane interface and hyphal morphology is evident in the absence of SrbA.(A–C) Transmission electron micrographs showing sections of conidia of wild type CEA10 (A) and SDW1 (B,C). Compared with the round wild type conidia having clear boundaries between plasma membrane and cell wall layers, most of the SDW1 conidia were distorted in shape and possessed faint, somewhat shriveled boundaries. Note that frequent “tearing” took place mainly at the cell wall – plasma membrane interface during microscopic examination of the SDW1 conidia (arrows). This phenotype was observed in over 80% of SDW1 conidia examined. Inset panels depict a 3× magnified view of the conidial cell wall region. Bars = 500 nm. (D–H). Transmission electron micrographs showing longitudinal and transverse hyphal sections of wild type CEA10 (D,F) and SDW1 (E,G,H). Close observation of the hyphal tips show phenotypic differences between wild type and SDW1. Abnormal cell wall – plasma membrane interfaces and apical swellings in SDW1 hyphae were frequently observed, while the wild type showed normal round-shaped apexes. With respect to cell wall morphology around the hyphal apex, SDW1 had an abnormally expanded cell wall (arrows) containing numerous electron dense objects (arrowheads), which likely resulted in hyphal tip bending (H). Inset panels depict a magnified view of the boxed region. Bars = 1 µm, except for the inset panels of E and H where they denote 500 nm.
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Related In: Results  -  Collection

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ppat-1000200-g005: Abnormal cell wall-plasma membrane interface and hyphal morphology is evident in the absence of SrbA.(A–C) Transmission electron micrographs showing sections of conidia of wild type CEA10 (A) and SDW1 (B,C). Compared with the round wild type conidia having clear boundaries between plasma membrane and cell wall layers, most of the SDW1 conidia were distorted in shape and possessed faint, somewhat shriveled boundaries. Note that frequent “tearing” took place mainly at the cell wall – plasma membrane interface during microscopic examination of the SDW1 conidia (arrows). This phenotype was observed in over 80% of SDW1 conidia examined. Inset panels depict a 3× magnified view of the conidial cell wall region. Bars = 500 nm. (D–H). Transmission electron micrographs showing longitudinal and transverse hyphal sections of wild type CEA10 (D,F) and SDW1 (E,G,H). Close observation of the hyphal tips show phenotypic differences between wild type and SDW1. Abnormal cell wall – plasma membrane interfaces and apical swellings in SDW1 hyphae were frequently observed, while the wild type showed normal round-shaped apexes. With respect to cell wall morphology around the hyphal apex, SDW1 had an abnormally expanded cell wall (arrows) containing numerous electron dense objects (arrowheads), which likely resulted in hyphal tip bending (H). Inset panels depict a magnified view of the boxed region. Bars = 1 µm, except for the inset panels of E and H where they denote 500 nm.
Mentions: Visual inspection of SDW1 colony morphology in standard laboratory conditions did not reveal any apparent morphological phenotypes (Figure 2B). However, our transcriptional profiling experiments suggested possible alterations in cell wall biosynthesis, a critical component of hyphal morphology and growth, in the absence of SrbA. Consequently, we performed a more in depth analysis of SDW1 morphology. First, we utilized light microscopy to examine the growing edges of SDW1 colonies in normoxia. We observed a significant defect in hyphal tip branching in SDW1 that is not apparent in strains CEA10 and SDW2 (Figure 4). SDW1 hyphal tips display hyper-branching and a “blunted” abnormal morphological phenotype (Figure 4). This phenotype suggests that SrbA is involved in maintaining cell polarity that directs hyphal growth. Interestingly, this phenotype does not appear to alter the growth rate of the colony, which was comparable to the wild type under normoxic conditions (Figure 2A). Next, we utilized transmission electron microscopy (TEM) to further examine the cell wall and morphology of conidia and hyphae of SDW1. Confirming our suspicions that the mutant was not directly affected in cell wall biosynthesis we observed no clear cell wall defects. However, a general thickening of the intracellular space between the cell wall and plasma membrane is observed in SDW1 conidia and hyphae compared with the wild type (Figure 5A and 5B and 5D and 5E). A striking phenotype was consequently observed in conidia from SDW1 that suggested a significant defect in the cell wall-plasma membrane interface occurs in the absence of SrbA (Figure 5A and 5B). This defect is apparently exacerbated by the electron beam, which causes a separation between the cell wall and plasma membrane in SDW1 conidia (Figure 5C). This phenotype was observed in over 80% of the SDW1 conidia examined. However, the size and density of the mutant conidia were comparable to the wild type strain as measured by flow cytometry (data not shown). Since a defect in the cell wall plasma membrane interface was suggested, we examined viability of the SDW1 conidia by monitoring germination. These experiments revealed that viability, as measured by conidia germination, was not significantly different between the wild type, SDW1 and SDW2 strains (Figure 6) (P>0.01). Similar cell wall-plasma membrane defects were observed in the SDW1 hyphae compared with the wild type hyphae (Figure 5D and 5E). Importantly, an accumulation of electron dense objects was observed in the SDW1 hyphae. We hypothesize that these objects may be vesicles of the Spitzenkörper, and their abnormal location in the SDW1 hyphae may cause the observed altered cell polarity (Figure 5E and 5H). This phenotype was observed in over 50% of the SDW1 hyphae examined and never observed in the wild type strain. These results suggest that SrbA is critical for maintaining the cell wall – plasma membrane interface, and that SrbA is critical for normal hyphal branching and cell polarity in filamentous fungi 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