A Sterol-Regulatory Element Binding Protein Is Required for Cell Polarity, Hypoxia Adaptation, Azole Drug Resistance, and Virulence in Aspergillus fumigatus

Besides srbA itself, the gene with the highest fold difference in expression in SDW1 is a non-ribosomal peptide synthetase, AFUA_1g10380 (NRPS1 or pes1).

“Interestingly, besides srbA itself, the gene with the highest fold difference in expression in SDW1 is a non-ribosomal peptide synthetase, AFUA_1g10380 (NRPS1 or pes1) [43],[44].”

• cites [43] RA Cramer Jr, 2006, Phylogenomic analysis of non-ribosomal peptide synthetases in the genus <italic>Aspergillus</italic>., Gene sourceNot checked yet

  Not checked in the free run.

• cites [44] EP Reeves, 2006, A nonribosomal peptide synthetase (Pes1) confers protection against oxidative stress in <italic>Aspergillus fumigatus</italic>., Febs J sourceNot checked yet

  Not checked in the free run.

No obvious defects in cell wall biosynthesis were observed in the mutant strain.

“However, no obvious defects in cell wall biosynthesis were observed in the mutant strain”

Several genes previously shown to be involved in ergosterol biosynthesis in fungi were transcriptionally repressed in the absence of SrbA, including ERG25, ERG24, and ERG3.

“Several genes previously shown to be involved in ergosterol biosynthesis in fungi were found to be transcriptionally repressed in the absence of SrbA including ERG25, ERG24, and ERG3 (Table 1).”

After 96 hours of incubation in hypoxia, SDW1 continued to display undetectable growth.

“After 96 hours of incubation in hypoxia, SDW1 continued to display undetectable growth.”

A significant number of genes involved in cell wall biosynthesis or homeostasis were repressed in SDW1 compared to wild type.

“In addition, a significant number of genes involved in cell wall biosynthesis or homeostasis were observed to be repressed in SDW1 compared to wild type.”

SDW1 and SDW2 both displayed normal hyphal growth rates compared to the wild type strain CEA10 in normoxic conditions on glucose minimal medium (GMM).

“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).”

The SrbA null mutant is incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA).

“consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA)”

AFUA_2g01260 (srbA) is induced 5.02 fold in response to hypoxia.

“only one, AFUA_2g01260 (induced 5.02 fold in response to hypoxia), had similarity with a functionally characterized protein, Sre1 from S. pombe [25]”

• cites [25] AL Hughes, 2005, SREBP pathway responds to sterols and functions as an oxygen sensor in fission yeast., Cell sourceNot checked yet

  Not checked in the free run.

The SrbA null mutant is highly susceptible to fluconazole and voriconazole.

“the SrbA null mutant was highly susceptible to fluconazole and voriconazole”

• Also stated in: § SrbA mediates resistance to the azole class of antifungal drugs · plain

  “SDW1 growth was inhibited at the surprisingly low MIC of 1 µg/ml”

• Also stated in: § SrbA mediates resistance to the azole class of antifungal drugs · plain

  “SDW1 was significantly more susceptible to Voriconazole and showed a MIC of only 0.012 µg/ml”

The overall set of genes putatively regulated by SrbA in A. fumigatus is significantly different from data obtained from Sre1 mutants in S. pombe and C. neoformans.

“However, the overall set of genes putatively regulated by SrbA in A. fumigatus is significantly different from data obtained from Sre1 mutants in the yeast S. pombe and C. neoformans.”

Loss of SrbA results in a mutant strain of Aspergillus fumigatus that is incapable of growth in a hypoxic environment.

“Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment”

• Also stated in: § SrbA is required for hypoxia adaptation in Aspergillus fumigatus · plain

  “no hyphal growth of SDW1 was observed in hypoxic (1% O2, 5% CO2, 94% N2) conditions”

Transcriptional profiling revealed 87 genes that are affected by loss of SrbA function.

“Transcriptional profiling revealed 87 genes that are affected by loss of SrbA function.”

• Also stated in: § Transcriptional profiling of SDW1 in response to hypoxia · hedged

  “Microarray experiments comparing the transcriptional profiles of wild type strain CEA10 and SDW1 exposed to hypoxia for 24 hours revealed 87 significant genes possibly regulated by SrbA (Table 1).”

We did not observe any increased susceptibility to oxidative stress in the SrbA null mutant.

“Interestingly, we did not observe any increased susceptibility to oxidative stress in the SrbA null mutant.”

Histopathological analyses confirmed the lack of fungal persistence and inflammation in mice infected with SDW1.

“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).”

• Also stated in: § SrbA is required for fungal virulence in two distinct murine models · plain

  “No sign of inflammation or fungal burden was observed in any surviving animal on day +14, +21 and +28 of the infection.”

Histopathological analysis of SDW1 infected survivors in the X-CGD model revealed persistence of granuloma like structures and fungal tissue.

“Histopathological analysis of SDW1 infected survivors in this model revealed persistence of granuloma like structures and fungal tissue (Figure 12).”

After 28 days, SDW1 infected mice displayed no visible or microscopic signs of infection.

“After 28 days, SDW1 infected mice displayed no visible or microscopic signs of infection.”

Lung homogenates from these animals revealed that the observed fungal tissue was still viable.

“Lung homogenates from these animals revealed that the observed fungal tissue was still viable.”

NRPS1 (or pes1) had the highest change in expression between wild type and the SrbA null mutant.

“For example, a non-ribosomal peptide synthetase, NRPS1 (or pes1), had the highest change in expression between wild type and the SrbA null mutant [43],[44].”

• cites [43] RA Cramer Jr, 2006, Phylogenomic analysis of non-ribosomal peptide synthetases in the genus <italic>Aspergillus</italic>., Gene sourceNot checked yet

  Not checked in the free run.

• cites [44] EP Reeves, 2006, A nonribosomal peptide synthetase (Pes1) confers protection against oxidative stress in <italic>Aspergillus fumigatus</italic>., Febs J sourceNot checked yet

  Not checked in the free run.

After 48 hours, no detectable difference in growth morphology or colony diameter was observed among CEA10, SDW1, and SDW2 in the presence of 1 mM and 2.5 mM hydrogen peroxide on glucose minimal media.

“After 48 hours, we observed no detectable difference in growth morphology or colony diameter.”

Semi-quantitative assessment of the percent of lung affected by infection at the early time point revealed no difference among strains (CEA10 = 1.3±0.5, SDW1 = 1.3±0.5, SDW2 = 1±0.0).

“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).”

At days 14, 21, and 28, with the exception of one mouse, no fungal colonies were recoverable from lung homogenates of SDW1 infected mice.

“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.”

Similar to C. neoformans and S. pombe, we observed SrbA dependent genes involved in ergosterol biosynthesis including ERG25, ERG24, and ERG3.

“Similar to C. neoformans and S. pombe, we observed SrbA dependent genes involved in ergosterol biosynthesis including ERG25, ERG24, and ERG3 [31]–[33].”

• cites [31]–[33]Not checked yet

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Semi-quantitative assessment at day 4 revealed significant differences in the percent of lung affected by infection among strains (CEA10 = 3.3±0.5, SDW1 = 2.3±0.5, SDW2 = 3.8±0.5).

“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).”

In the X-CGD murine model, mice infected with SDW1 had significant differences in survival compared with mice infected with wild type and reconstituted strains (P = 0.005).

“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).”

No clear cell wall defects were observed in SDW1.

“Confirming our suspicions that the mutant was not directly affected in cell wall biosynthesis we observed no clear cell wall defects.”

This phenotype was observed in over 80% of the SDW1 conidia examined.

“This phenotype was observed in over 80% of the SDW1 conidia examined.”

Similar cell wall-plasma membrane defects were observed in the SDW1 hyphae compared with the wild type hyphae.

“Similar cell wall-plasma membrane defects were observed in the SDW1 hyphae compared with the wild type hyphae (Figure 5D and 5E).”

An accumulation of electron dense objects was observed in the SDW1 hyphae.

“Importantly, an accumulation of electron dense objects was observed in the SDW1 hyphae.”

CEA10 and SDW2 were susceptible to Voriconazole with MIC of 0.125 μg/ml.

“On the plates with Voriconazole we could observe that CEA10 and SDW2 were susceptible as expected (MIC of 0.125 µg/ml respectively).”

A significant difference in mortality was observed between mice infected with SDW1 and mice infected with either SDW2 or CEA10 (P = 0.0002).

“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).”

A significant defect in the cell wall-plasma membrane interface occurs in the absence of SrbA.

“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).”

The size and density of the mutant conidia were comparable to the wild type strain as measured by flow cytometry.

“However, the size and density of the mutant conidia were comparable to the wild type strain as measured by flow cytometry (data not shown).”

In the first murine model, outbred CD1 neutropenic mice infected with SDW1 displayed no symptoms associated with IPA.

“In the first model, outbred CD1 neutropenic mice infected with SDW1 displayed no symptoms associated with IPA (Figure 8A).”

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.

“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).”

Only one mouse infected with SDW1 succumbed to the infection in the X-CGD model.

“However, only one mouse infected with SDW1 succumbed to the infection.”

Unlike C. neoformans, we did not observe SrbA dependent genes involved in iron or copper uptake in A. fumigatus.

“Unlike C. neoformans, we did not observe SrbA dependent genes involved in iron or copper uptake in A. fumigatus [32].”

• cites [32] YC Chang, 2007, Sre1p, a regulator of oxygen sensing and sterol homeostasis, is required for virulence in <italic>Cryptococcus neoformans</italic>., Mol Microbiol sourceNot checked yet

  Not checked in the free run.

No significant difference in conidia killing was observed between CEA10, SDW1, and SDW2 (P>0.01).

“As presented in figure 13, no significant difference in conidia killing was observed between CEA10, SDW1, and SDW2 (P>0.01).”

The cell wall-plasma membrane defect is apparently exacerbated by the electron beam, which causes separation between the cell wall and plasma membrane in SDW1 conidia.

“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).”

Unlike the neutropenic mouse model, X-CGD mice all displayed symptoms of IPA during the preliminary stages of infection.

“Unlike the neutropenic mouse model, these mice all displayed symptoms of IPA during the preliminary stages of infection.”

In the first murine model, the SDW1 strain was completely avirulent.

“Indeed, in this murine model, the SDW1 strain was completely avirulent (Figure 8A).”

Mice infected with wild type CEA10 and reconstituted strain SDW2 displayed symptoms of A. fumigatus infection including hunched posture, ruffled fur, weight loss, and increased respiration.

“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.”

On day 1 of infection, fungal germination and growth is observed in mice infected with all 3 strains of the fungus.

“On day 1 of the infection, fungal germination and growth is observed in mice infected respectively with all 3 strains of the fungus.”

• Also stated in: § SrbA is required for fungal virulence in two distinct murine models · plain

  “GMS staining revealed that fungal colonization and germination is observed in all infected animals but not the mock control.”

GC-MS profiles demonstrated a significant accumulation of 4-methyl sterols in the SrbA null mutant SDW1 that was not observed in the wild type strain CEA10.

“The GC-MS profiles demonstrated a significant accumulation of 4-methyl sterols in the SrbA null mutant, SDW1, that was not observed in the wild type strain CEA10 (Figure 7).”

In a repeat experiment, 3 additional X-CGD mice infected with SDW1 succumbed on day 4 to the infection.

“In a repeat experiment, 3 additional X-CGD mice infected with SDW1 also succumbed on day 4 to the infection.”

SDW1 has a significant defect in hyphal tip branching that is not apparent in strains CEA10 and SDW2.

“We observed a significant defect in hyphal tip branching in SDW1 that is not apparent in strains CEA10 and SDW2 (Figure 4).”

Viability, as measured by conidia germination, was not significantly different between the wild type, SDW1 and SDW2 strains.

“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).”

Visual inspection of SDW1 colony morphology in standard laboratory conditions did not reveal any apparent morphological phenotypes.

“Visual inspection of SDW1 colony morphology in standard laboratory conditions did not reveal any apparent morphological phenotypes (Figure 2B).”

Unlike C. neoformans and S. pombe, we did not observe any genes with an annotation that would clearly point to a role in allowing Aspergillus to adapt to hypoxia.

“Overall, however, unlike C. neoformans and S. pombe, we did not observe any genes with an annotation that would clearly point to a role in allowing Aspergillus to adapt to hypoxia.”

The SrbA mutant did not display increased sensitivity to hydrogen peroxide.

“In addition, the SrbA mutant did not display increased sensitivity to hydrogen peroxide.”

By day 14 of the infection, little evidence of inflammation or fungal burden was evident in mice infected with the SrbA null mutant.

“Indeed, by day 14 of the infection, little evidence of inflammation or fungal burden was evident in mice infected with the SrbA null mutant.”

Loss of SrbA resulted in a strain of A. fumigatus highly susceptible to fluconazole, an azole that normally has minimal activity against A. fumigatus.

“Interestingly, loss of SrbA resulted in a strain of A. fumigatus highly susceptible to fluconazole, an azole that normally has minimal activity against A. fumigatus [58],[59].”

• cites [58] F Sabatelli, 2006, In vitro activities of posaconazole, fluconazole, itraconazole, voriconazole, and amphotericin B against a large collection of clinically important molds and yeasts., Antimicrob Agents Chemother sourceNot checked yet

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• cites [59] TC White, 1998, Clinical, cellular, and molecular factors that contribute to antifungal drug resistance., Clin Microbiol Rev sourceNot checked yet

  Not checked in the free run.

The SrbA null mutant conidia showed no difference in susceptibility to macrophage (RAW264.7 cells) killing compared with the wild type strain.

“Furthermore, we also have examined the susceptibility of the SrbA null mutant conidia to macrophage (RAW264.7 cells) killing and found no difference with the wild type strain.”

Histopathological findings with the SrbA mutant strain revealed fungal growth in the neutropenic model early in the infection.

“Our histopathological findings with the SrbA mutant strain revealed fungal growth in this model early in the infection.”

We identified SrbA in a transcriptional profiling screen of A. fumigatus in response to hypoxia (induced >5 fold).

“First, we identified SrbA in a transcriptional profiling screen of A. fumigatus in response to hypoxia (induced >5 fold).”

In vitro experiments revealed that the growth defect of the SrbA mutant in hypoxia was not fungicidal but fungistatic.

“Furthermore, our in vitro experiments revealed that the growth defect of the SrbA mutant in hypoxia was not fungicidal but fungistatic.”

Unlike the neutropenic mice, we could detect persistence of viable SDW1 in the lungs of surviving X-CGD mice out to day 14.

“Furthermore, unlike the neutropenic mice, we could detect the persistence of viable SDW1 in the lungs of these surviving mice out to day 14.”

Despite extensive bioinformatic analyses, we were unable to identify a clear homolog of the sterol cleavage activating protein (SCAP).

“First, despite extensive bioinformatic analyses, we were unable to identify a clear homolog of the sterol cleavage activating protein (SCAP).”

By day 14, we were unable to recover viable colonies from mice infected with the SrbA null mutant strain.

“However, by day 14, we were unable to recover viable colonies from mice infected with the SrbA null mutant strain.”

The A. fumigatus SrbA null mutant produced substantial levels of ergosterol even in the absence of SrbA.

“We observed that the A. fumigatus SrbA null mutant produced substantial levels of ergosterol even in the absence of SrbA.”

Generation of null mutants in putative site-1 (S1P) and site-2 (S2P) protease homologs in A. fumigatus did not demonstrate expected defects in hypoxic growth.

“Third, though sequence identity was extremely low, generation of null mutants in putative site-1 (S1P) and site-2 (S2P) protease homologs in A. fumigatus did not demonstrate expected defects in hypoxic growth (Willger and Cramer, unpublished data).”

We could identify a clear Insig1 homolog, which we have named InsA.

“We could, however, identify a clear Insig1 homolog, which we have named InsA.”

Transcriptional profiling of the SrbA mutant revealed numerous transporters possibly regulated by SrbA.

“Transcriptional profiling of the SrbA mutant revealed numerous transporters possibly regulated by SrbA.”

Exogenous addition of ergosterol or lanosterol did not alter growth of the wild type strain in hypoxia.

“Nor did exogenous addition of these sterols alter growth of the wild type strain in hypoxia as is the case for S. cerevisiae, which requires exogenous sterols for anaerobic growth.”

Unlike the neutropenic mouse model, extensive signs of chronic inflammation remained evident in the X-CGD mice post-day 14.

“Unlike the neutropenic mouse model, extensive signs of chronic inflammation remained evident in the X-CGD mice post-day 14, consistent with previously reported results in these animals [45].”

• cites [45] DE Morgenstern, 1997, Absence of respiratory burst in X-linked chronic granulomatous disease mice leads to abnormalities in both host defense and inflammatory response to <italic>Aspergillus fumigatus</italic>., J Exp Med sourceNot checked yet

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In the neutropenic mouse model, significant inflammation and tissue necrosis is observed in histopathological examinations.

“In this model, significant inflammation and tissue necrosis is observed in histopathological examinations.”

We could not rescue the SrbA phenotype in hypoxia with addition of ergosterol or lanosterol.

“We could not rescue the SrbA phenotype in hypoxia with addition of ergosterol or lanosterol (data not shown).”

Hypoxia adaptation is likely an important virulence attribute of pathogenic molds.

“demonstrate for the first time that hypoxia adaptation is likely an important virulence attribute of pathogenic molds”

Ectopic re-introduction of the wild type srbA allele into SDW1 (resulting in strain SDW2) allowed attribution of all resulting phenotypes specifically to the absence of srbA in 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.”

SrbA in A. fumigatus is involved in mediating the hypoxic response by an undefined mechanism.

“and that SrbA in A. fumigatus is involved in mediating this response by an undefined mechanism”

Invasive fungal pathogens like A. fumigatus are likely subjected to hypoxia during fungal pathogenesis.

“it follows that invasive fungal pathogens like A. fumigatus are likely subjected to hypoxia during fungal pathogenesis”

The transcriptional profiling results are likely indirect effects of the altered cell polarity of the mutant.

“and thus the transcriptional profiling results are likely indirect effects of the altered cell polarity of the mutant (discussed below)”

SrbA plays a critical role in ergosterol biosynthesis in A. fumigatus.

“SrbA plays a critical role in ergosterol biosynthesis”

• Also stated in: § Transcriptional profiling of SDW1 in response to hypoxia · plain

  “Several genes previously shown to be involved in ergosterol biosynthesis in fungi were found to be transcriptionally repressed in the absence of SrbA including ERG25, ERG24, and ERG3 (Table 1).”

The lack of effect of Caspofungin provides support for the hypothesis that the mutant is likely not directly affected in cell wall biosynthesis.

“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.”

Pathogenic molds such as A. fumigatus must possess mechanisms to adapt to hypoxic microenvironments found in vivo during infection.

“Consequently, it seems probable that pathogenic molds such as A. fumigatus must possess mechanisms to adapt to hypoxic microenvironments found in vivo during infection.”

SrbA plays a distinct role in filamentous fungal biology.

“Consequently, these results strongly suggest that SrbA plays a distinct role in filamentous fungal biology.”

There is an important link between sterol biosynthesis, hypoxia adaptation, azole drug resistance, and the virulence of pathogenic yeasts.

“Taken together, these observations demonstrate an important link between sterol biosynthesis, hypoxia adaptation, azole drug resistance, and the virulence of pathogenic yeasts.”

• cites [32] YC Chang, 2007, Sre1p, a regulator of oxygen sensing and sterol homeostasis, is required for virulence in <italic>Cryptococcus neoformans</italic>., Mol Microbiol sourceNot checked yet

  Not checked in the free run.

• cites [33] CD Chun, 2007, A Link between virulence and homeostatic responses to hypoxia during infection by the human fungal pathogen <italic>Cryptococcus neoformans</italic>., PLoS Pathog sourceNot checked yet

  Not checked in the free run.

A. fumigatus can rapidly adapt to hypoxic microenvironments.

“These results indicate that A. fumigatus can rapidly adapt to hypoxic microenvironments”

These findings present a new function of SREBP proteins in filamentous fungi.

“Thus, these findings present a new function of SREBP proteins in filamentous fungi”

SrbA plays a critical role in resistance to the azole class of antifungal drugs.

“resistance to the azole class of antifungal drugs”

• Also stated in: § SrbA mediates resistance to the azole class of antifungal drugs · plain

  “we found that SrbA is required for resistance to Fluconazole and Voriconazole, but not Amphotericin B or Caspofungin”

AFUA_2g01260 is likely the SREBP homolog in A. fumigatus, named srbA.

“Consequently, these results suggest that AFUA_2g01260 is likely the SREBP homolog in A. fumigatus, and we consequently named this gene srbA”

Sites of inflammation contain significantly low levels of oxygen (hypoxia).

“it is well established that sites of inflammation contain significantly low levels of oxygen (hypoxia) [5]–[7]”

• cites [5]–[7]Not checked yet

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SrbA plays a critical role in maintenance of cell polarity in A. fumigatus.

“maintenance of cell polarity in A. fumigatus”

Mechanisms of hypoxia adaptation are almost certainly different in molds than yeast.

“This result further illustrates that mechanisms of hypoxia adaptation are almost certainly different in molds than yeast.”

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.

“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.”

• Also stated in: § SrbA is required for fungal virulence in two distinct murine models · plain

  “SDW1 is significantly less virulent than the wild type CEA10 and the reconstituted strain SDW2 in both murine models.”

Increased susceptibility to oxidative stress and macrophage killing is not responsible for the virulence defect observed in the absence of SrbA.

“We conclude that increased susceptibility to oxidative stress and macrophage killing is not responsible for the virulence defect observed in the absence of SrbA.”

• Also stated in: § SrbA is not required for oxidative stress resistance and resistance to macrophage killing · plain

  “Loss of SrbA does not affect susceptibility to conidia killing by RAW264.7 cells.”

Mice infected with SDW1 had cleared the 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.”

• Also stated in: § SrbA is required for fungal virulence in two distinct murine models · hedged

  “This result indicates that in this murine model, the immune system is capable of clearing the fungal infection in the absence of SrbA.”

SrbA is involved in maintaining cell polarity that directs hyphal growth.

“This phenotype suggests that SrbA is involved in maintaining cell polarity that directs hyphal growth.”

SDW1 conidia are viable in vivo during the early stages of infection.

“This observation confirms the viability of SDW1 conidia in vivo (Figure 10).”

• Also stated in: § SrbA is required for fungal virulence in two distinct murine models · hedged

  “This result indicates that SDW1 conidia are viable in vivo during the early stages of infection.”

Abnormal conidial and hyphal morphology in SDW1 suggested possible alterations in sterol content in the absence of SrbA.

“In addition, the abnormal conidial and hyphal morphology observed via light microscopy and TEM micrographs in SDW1 also suggested possible alterations in sterol content in the absence of SrbA.”

Regulation of ergosterol biosynthesis is a conserved function of SREBPs in fungi.

“This result suggests that regulation of ergosterol biosynthesis is a conserved function of SREBPs in fungi.”

These results suggest that ergosterol can still be synthesized in the absence of SrbA in A. fumigatus.

“In addition, these results suggest that ergosterol can still be synthesized in the absence of SrbA in A. fumigatus.”

This data is consistent with the observed in vitro phenotype of the SDW1 strain in hypoxia.

“This data is consistent with the observed in vitro phenotype of the SDW1 strain in hypoxia.”

In the yeasts S. pombe and C. neoformans, SREBP homologs are crucial for sterol biosynthesis, survival under hypoxic conditions, resistance to azole antifungal agents, and fungal virulence.

“In the yeasts S. pombe and C. neoformans, SREBP homologs are crucial for sterol biosynthesis, survival under hypoxic conditions, resistance to azole antifungal agents, and fungal virulence [25],[32],[33].”

• cites [25] AL Hughes, 2005, SREBP pathway responds to sterols and functions as an oxygen sensor in fission yeast., Cell sourceNot checked yet

  Not checked in the free run.

• cites [32] YC Chang, 2007, Sre1p, a regulator of oxygen sensing and sterol homeostasis, is required for virulence in <italic>Cryptococcus neoformans</italic>., Mol Microbiol sourceNot checked yet

  Not checked in the free run.

• cites [33] CD Chun, 2007, A Link between virulence and homeostatic responses to hypoxia during infection by the human fungal pathogen <italic>Cryptococcus neoformans</italic>., PLoS Pathog sourceNot checked yet

  Not checked in the free run.

Transcriptional profiling data suggests transcriptional regulation of ERG25 by SrbA in A. fumigatus.

“These alterations are supported by the transcriptional profiling data, which suggests transcriptional regulation of ERG25 by SrbA in A. fumigatus (Table 1).”

SrbA is critical for maintaining the cell wall - plasma membrane interface.

“These results suggest that SrbA is critical for maintaining the cell wall – plasma membrane interface”

SrbA mediates resistance to the azole class of antifungal drugs by an undefined mechanism.

“These clinically significant results suggest that SrbA mediates resistance to the azole class of antifungal drugs by an undefined mechanism.”

Our results suggest additional functions of SREBPs in filamentous fungi, most importantly a role in maintenance of cell polarity.

“However, our results suggest additional functions of SREBPs in filamentous fungi, most importantly a role in maintenance of cell polarity.”

These results suggest a blockage of C4 demethylation in the absence of SrbA in A. fumigatus.

“Consequently, these results suggest a blockage of C4 demethylation in the absence of SrbA in A. fumigatus.”

Accumulation of 4-methylfecosterol and 4,4-dimethylergosta-8,24(28)-dien-3β-ol in the absence of SrbA suggests a blockage at ERG25 in the sterol biosynthesis pathway in SDW1.

“Specifically, the accumulation of 4-methylfecosterol and 4,4-dimethylergosta-8,24(28)-dien-3β-ol in the absence of SrbA suggests a blockage at ERG25 in the sterol biosynthesis pathway in SDW1.”

SrbA is critical for normal hyphal branching and cell polarity in filamentous fungi by an undefined mechanism.

“and that SrbA is critical for normal hyphal branching and cell polarity in filamentous fungi by an undefined mechanism”

TEM examination of the mutant suggested altered vesicle translocation or formation in the hyphae.

“Further examination of the mutant with TEM suggested altered vesicle translocation or formation in the hyphae.”

Transcriptional profiling of SDW1 under hypoxia suggested that SrbA is involved in both early and late steps of the sterol biosynthesis pathway.

“Transcriptional profiling of SDW1 under hypoxia suggested that SrbA was involved in both early and late steps of the sterol biosynthesis pathway.”

A. fumigatus contains complex regulatory mechanisms, of which SrbA is a part, for the production of ergosterol that remain to be elucidated.

“Thus, it appears that A. fumigatus contains complex regulatory mechanisms, of which SrbA is a part, for the production of ergosterol that remain to be elucidated.”

The altered cell polarity of the SrbA mutant did not significantly affect fungal growth.

“Though the altered cell polarity of the SrbA mutant may contribute to the virulence defect, the fact that SrbA null mutant displayed normal growth rates in vitro in standard laboratory growth conditions suggests to us that the altered cell polarity did not significantly affect fungal growth.”

The observation that sterol biosynthesis was altered in normoxia, likely resulting in altered cell polarity, suggests that in molds, SrbA plays a significant role in the biology of filamentous fungi in normoxic conditions.

“Second, the observation that sterol biosynthesis was altered in normoxia, likely resulting in altered cell polarity, suggests that in molds, SrbA plays a significant role in the biology of filamentous fungi in normoxic conditions.”

The inability of the SrbA null mutant to grow in hypoxic microenvironments is primarily responsible for the avirulent phenotype of the mutant.

“Consequently, we conclude that these observations strongly suggest that the inability of the SrbA null mutant to grow in hypoxic microenvironments is primarily responsible for the avirulent phenotype of the mutant.”

• AssumptionThe authors state this as an assumption: Sites of infection and inflammation in the neutropenic mouse model are hypoxic.
• AssumptionThe authors state this as an assumption: The hyper-inflammatory response in X-CGD mice generates significant hypoxia in the lung.
• AssumptionRests on an assumption: A. fumigatus must overcome significant hypoxia during pulmonary infections.

SREBPs play critical roles in the biology of fungi that have important implications for fungal virulence and how we manage and treat invasive fungal infections.

“What is clear, however, is that SREBPs play critical roles in the biology of fungi that have important implications for fungal virulence and how we manage and treat invasive fungal infections.”

The inability of the SrbA null mutant to grow in hypoxia prevents invasive disease from being established.

“First, and we believe most likely, the inability of the SrbA null mutant to grow in hypoxia prevents invasive disease from being established.”

• AssumptionThe authors state this as an assumption: Sites of infection and inflammation in the neutropenic mouse model are hypoxic.
• AssumptionRests on an assumption: A. fumigatus must overcome significant hypoxia during pulmonary infections.

The virulence defect in the SrbA null mutant is due to its inability to grow in hypoxia.

“Taken together, we feel these observations strongly suggest that the virulence defect in the SrbA null mutant is due to its inability to grow in hypoxia.”

• AssumptionThe authors state this as an assumption: Sites of infection and inflammation in the neutropenic mouse model are hypoxic.
• AssumptionRests on an assumption: A. fumigatus must overcome significant hypoxia during pulmonary infections.

The inability of the mutant to grow in hypoxia is related to the perturbation in sterol biosynthesis, a highly oxygen dependent pathway reported to require at least 22 molecules of oxygen.

“It is also likely then that the inability of the mutant to grow in hypoxia is related to the perturbation in sterol biosynthesis, a highly oxygen dependent pathway reported to require at least 22 molecules of oxygen.”

SrbA mediates resistance to the azole class of antifungal drugs.

“An additional observation of clinical significance was the finding that SrbA mediates resistance to the azole class of antifungal drugs.”

X-CGD gp91phox−/− mice exhibit a hyper-inflammatory response when exposed to A. fumigatus and other Aspergillus species.

“These mice exhibit a hyper-inflammatory response when exposed to A. fumigatus and other Aspergillus species.”

• cites [45] DE Morgenstern, 1997, Absence of respiratory burst in X-linked chronic granulomatous disease mice leads to abnormalities in both host defense and inflammatory response to <italic>Aspergillus fumigatus</italic>., J Exp Med sourceNot checked yet

  Not checked in the free run.

• cites [46] JD Pollock, 1995, Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production., Nat Genet sourceNot checked yet

  Not checked in the free run.

Hughes et al. (2007) identified 4-methyl sterols as the primary activating agent of Sre1 in S. pombe.

“Indeed, Hughes et al. (2007) have identified 4-methyl sterols as the primary activating agent of Sre1 in S. pombe [64].”

• cites [64] AL Hughes, 2007, 4-Methyl sterols regulate fission yeast SREBP-Scap under low oxygen and cell stress., J Biol Chem sourceNot checked yet

  Not checked in the free run.

Based on current knowledge of the pathophysiology of IPA, the in vitro phenotypes observed in the SrbA mutant would not predict a role for this protein in A. fumigatus virulence.

“Based on our current knowledge of the pathophysiology of IPA, the in vitro phenotypes observed in the SrbA mutant would not predict a role for this protein in A. fumigatus virulence.”

Limited mortality in X-CGD mice inoculated with the SrbA null mutant strongly suggests that the mutant cannot grow effectively in vivo to cause invasive disease.

“However, in contrast, we observed limited mortality in these mice when inoculated with the SrbA null mutant, strongly suggesting that the mutant simply cannot grow effectively in vivo to cause invasive disease.”

As the infection progresses, SDW1 is incapable of continued hyphal growth despite the absence of NADPH oxidase in the X-CGD murine model.

“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.”

Increased hypoxia prevents proliferation of fungal tissue in the absence of SrbA.

“This result confirms the importance of a functional NADPH oxidase in resistance to Aspergillus infections, and suggests that increased hypoxia prevents proliferation of fungal tissue in the absence of SrbA.”

• AssumptionThe authors state this as an assumption: The hyper-inflammatory response in X-CGD mice generates significant hypoxia in the lung.