Limits...
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.

Show MeSH

Related in: MedlinePlus

Histopathology of X-CGD mouse model day 4 after infection.Mock = 0.01% Tween inoculated, WT = CEA10, SDW1 = ΔsrbA, SDW2 = ΔsrbA+srbA. Mice were inoculated with 1×106 conidia intratracheally, euthanized on day +4 after inoculation, lungs removed, fixed in formaldehyde, and stained with hematoxylin and eosin (H&E) or Gommori's methenamine silver (GMS) stain. Significant inflammation, necrosis, and an influx of immune effector cells (primarily neutrophils) is observed on day +4 in all infected animals but not the mock control. However, lesions are more localized and not as extensive in mice infected with SDW1. Open alveoli and more localized inflammation are clearly observed in mice infected with SDW1. Interestingly, GMS staining revealed that fungal growth is less extensive in SDW1 as well. This result indicates that as the infection progresses, SDW1 is incapable of continued hyphal growth despite the absence of NADPH oxidase in this murine model. Bar = 500 µm for 40×; Bar = 100 µm for 200×.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2572145&req=5

ppat-1000200-g011: Histopathology of X-CGD mouse model day 4 after infection.Mock = 0.01% Tween inoculated, WT = CEA10, SDW1 = ΔsrbA, SDW2 = ΔsrbA+srbA. Mice were inoculated with 1×106 conidia intratracheally, euthanized on day +4 after inoculation, lungs removed, fixed in formaldehyde, and stained with hematoxylin and eosin (H&E) or Gommori's methenamine silver (GMS) stain. Significant inflammation, necrosis, and an influx of immune effector cells (primarily neutrophils) is observed on day +4 in all infected animals but not the mock control. However, lesions are more localized and not as extensive in mice infected with SDW1. Open alveoli and more localized inflammation are clearly observed in mice infected with SDW1. Interestingly, GMS staining revealed that fungal growth is less extensive in SDW1 as well. This result indicates that as the infection progresses, SDW1 is incapable of continued hyphal growth despite the absence of NADPH oxidase in this murine model. Bar = 500 µm for 40×; Bar = 100 µm for 200×.

Mentions: Next, we examined the virulence of SDW1 in a murine model of X-linked chronic granulomatous disease (X-CGD) utilizing gp91phox−/− mice. These mice are deficient in NADPH oxidase activity and display hyper-susceptibility to Aspergillus species without the need for immunosuppression with chemotherapeautic agents [45],[46]. Similar to the neutropenic mouse model, X-CGD mice infected with strain SDW1 had significant differences in survival compared with mice infected with wild type and reconstituted strains (Figure 8B) (P = 0.005). Unlike the neutropenic mouse model, these mice all displayed symptoms of IPA during the preliminary stages of infection. These symptoms, likely due to the large inflammatory response characteristic of these mice when exposed to fungal antigens, included ruffled fur, hunched posture, and lethargic movement as early as 24 hours post-infection. However, only one mouse infected with SDW1 succumbed to the infection. In a repeat experiment, 3 additional X-CGD mice infected with SDW1 also succumbed on day 4 to the infection. Most likely, this was due to the hyper-inflammatory response that occurs in X-CGD mice and not death due to invasive fungal growth. Regardless, the majority of X-CGD mice infected with SDW1 survived the infection and displayed no symptoms of IPA by day 14. Histopathological analyses of these mice displayed standard pathological findings associated with Aspergillus infections in X-CGD mice including the development of granulomatous like lesions, massive influx of inflammatory cells (primarily neutrophils) to sites of infection, subsequent peribronchiolar and alveolar inflammation, and substantial fungal growth in silver stained tissue (Figures 10 and 11). On day 1 of the infection, fungal germination and growth is observed in mice infected respectively with all 3 strains of the fungus. This observation confirms the viability of SDW1 conidia in vivo (Figure 10). Semi-quantitative assessment of the percent of the lung affected by the infection, measured by inflammation and necrosis, of mice infected with the 3 strains respectively revealed no difference at this early time point (CEA10 = 1.3±0.5, SDW1 = 1.3±0.5, SDW2 = 1±0.0). Histopathology on day 4 of the infection, however, revealed extensive growth and proliferation of the wild type and reconstituted SDW2 strain, but minimal fungal growth and proliferation in mice infected with the SrbA mutant SDW1 (Figure 11). Semi-quantitative assessment of the inflammation and necrosis observed in the lungs of mice infected with the 3 strains respectively at this time point revealed significant differences in the percent of the lung affected by the infection (CEA10 = 3.3±0.5, SDW1 = 2.3±0.5, SDW2 = 3.8±0.5). Lung homogenates from these mice also revealed that viable SDW1 fungus was recoverable from these mice at this time point. This data is consistent with the observed in vitro phenotype of the SDW1 strain in hypoxia. Histopathological analysis of SDW1 infected survivors in this model revealed persistence of granuloma like structures and fungal tissue (Figure 12). Lung homogenates from these animals revealed that the observed fungal tissue was still viable. These results indicate that despite normal growth rates in vitro in normoxic conditions, the SDW1 strain is severely attenuated in its ability to cause lethal disease in two distinct murine models of IPA.


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)

Histopathology of X-CGD mouse model day 4 after infection.Mock = 0.01% Tween inoculated, WT = CEA10, SDW1 = ΔsrbA, SDW2 = ΔsrbA+srbA. Mice were inoculated with 1×106 conidia intratracheally, euthanized on day +4 after inoculation, lungs removed, fixed in formaldehyde, and stained with hematoxylin and eosin (H&E) or Gommori's methenamine silver (GMS) stain. Significant inflammation, necrosis, and an influx of immune effector cells (primarily neutrophils) is observed on day +4 in all infected animals but not the mock control. However, lesions are more localized and not as extensive in mice infected with SDW1. Open alveoli and more localized inflammation are clearly observed in mice infected with SDW1. Interestingly, GMS staining revealed that fungal growth is less extensive in SDW1 as well. This result indicates that as the infection progresses, SDW1 is incapable of continued hyphal growth despite the absence of NADPH oxidase in this murine model. Bar = 500 µm for 40×; Bar = 100 µm for 200×.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000200-g011: Histopathology of X-CGD mouse model day 4 after infection.Mock = 0.01% Tween inoculated, WT = CEA10, SDW1 = ΔsrbA, SDW2 = ΔsrbA+srbA. Mice were inoculated with 1×106 conidia intratracheally, euthanized on day +4 after inoculation, lungs removed, fixed in formaldehyde, and stained with hematoxylin and eosin (H&E) or Gommori's methenamine silver (GMS) stain. Significant inflammation, necrosis, and an influx of immune effector cells (primarily neutrophils) is observed on day +4 in all infected animals but not the mock control. However, lesions are more localized and not as extensive in mice infected with SDW1. Open alveoli and more localized inflammation are clearly observed in mice infected with SDW1. Interestingly, GMS staining revealed that fungal growth is less extensive in SDW1 as well. This result indicates that as the infection progresses, SDW1 is incapable of continued hyphal growth despite the absence of NADPH oxidase in this murine model. Bar = 500 µm for 40×; Bar = 100 µm for 200×.
Mentions: Next, we examined the virulence of SDW1 in a murine model of X-linked chronic granulomatous disease (X-CGD) utilizing gp91phox−/− mice. These mice are deficient in NADPH oxidase activity and display hyper-susceptibility to Aspergillus species without the need for immunosuppression with chemotherapeautic agents [45],[46]. Similar to the neutropenic mouse model, X-CGD mice infected with strain SDW1 had significant differences in survival compared with mice infected with wild type and reconstituted strains (Figure 8B) (P = 0.005). Unlike the neutropenic mouse model, these mice all displayed symptoms of IPA during the preliminary stages of infection. These symptoms, likely due to the large inflammatory response characteristic of these mice when exposed to fungal antigens, included ruffled fur, hunched posture, and lethargic movement as early as 24 hours post-infection. However, only one mouse infected with SDW1 succumbed to the infection. In a repeat experiment, 3 additional X-CGD mice infected with SDW1 also succumbed on day 4 to the infection. Most likely, this was due to the hyper-inflammatory response that occurs in X-CGD mice and not death due to invasive fungal growth. Regardless, the majority of X-CGD mice infected with SDW1 survived the infection and displayed no symptoms of IPA by day 14. Histopathological analyses of these mice displayed standard pathological findings associated with Aspergillus infections in X-CGD mice including the development of granulomatous like lesions, massive influx of inflammatory cells (primarily neutrophils) to sites of infection, subsequent peribronchiolar and alveolar inflammation, and substantial fungal growth in silver stained tissue (Figures 10 and 11). On day 1 of the infection, fungal germination and growth is observed in mice infected respectively with all 3 strains of the fungus. This observation confirms the viability of SDW1 conidia in vivo (Figure 10). Semi-quantitative assessment of the percent of the lung affected by the infection, measured by inflammation and necrosis, of mice infected with the 3 strains respectively revealed no difference at this early time point (CEA10 = 1.3±0.5, SDW1 = 1.3±0.5, SDW2 = 1±0.0). Histopathology on day 4 of the infection, however, revealed extensive growth and proliferation of the wild type and reconstituted SDW2 strain, but minimal fungal growth and proliferation in mice infected with the SrbA mutant SDW1 (Figure 11). Semi-quantitative assessment of the inflammation and necrosis observed in the lungs of mice infected with the 3 strains respectively at this time point revealed significant differences in the percent of the lung affected by the infection (CEA10 = 3.3±0.5, SDW1 = 2.3±0.5, SDW2 = 3.8±0.5). Lung homogenates from these mice also revealed that viable SDW1 fungus was recoverable from these mice at this time point. This data is consistent with the observed in vitro phenotype of the SDW1 strain in hypoxia. Histopathological analysis of SDW1 infected survivors in this model revealed persistence of granuloma like structures and fungal tissue (Figure 12). Lung homogenates from these animals revealed that the observed fungal tissue was still viable. These results indicate that despite normal growth rates in vitro in normoxic conditions, the SDW1 strain is severely attenuated in its ability to cause lethal disease in two distinct murine models of IPA.

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