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A role for the unfolded protein response (UPR) in virulence and antifungal susceptibility in Aspergillus fumigatus.

Richie DL, Hartl L, Aimanianda V, Winters MS, Fuller KK, Miley MD, White S, McCarthy JW, Latgé JP, Feldmesser M, Rhodes JC, Askew DS - PLoS Pathog. (2009)

Bottom Line: Failure to induce the UPR did not affect radial growth on rich medium at 37 degrees C, but cell wall integrity was disrupted at 45 degrees C, resulting in a dramatic loss in viability.The DeltahacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates.These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology & Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America.

ABSTRACT
Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The DeltahacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37 degrees C, but cell wall integrity was disrupted at 45 degrees C, resulting in a dramatic loss in viability. The DeltahacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the DeltahacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy.

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The ΔhacA mutant is hypersensitive to antifungal drugs.(A) Antifungal susceptibility using Etest: conidia were spread evenly onto a 150 mm plate of RPMI agar. Four Etest strips, each impregnated with a concentration gradient of caspofungin (CS), fluconazole (FL), amphotericin B (AmB), and itraconazole (IT) were applied to the surface, with the highest concentration oriented at the plate edge. (B) Microscopic analysis of caspofungin-treated hyphae. Equal numbers of conidia from the indicated strains were spread onto the surface of an IMA plate and allowed to germinate at 30°C for 24 h. A caspofungin Etest strip was then applied to each plate and incubated overnight at 37°C. The morphology of the hyphae surrounding the highest concentration on the strip was observed by DIC microscopy. Scale bar represents 50 µm.
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ppat-1000258-g008: The ΔhacA mutant is hypersensitive to antifungal drugs.(A) Antifungal susceptibility using Etest: conidia were spread evenly onto a 150 mm plate of RPMI agar. Four Etest strips, each impregnated with a concentration gradient of caspofungin (CS), fluconazole (FL), amphotericin B (AmB), and itraconazole (IT) were applied to the surface, with the highest concentration oriented at the plate edge. (B) Microscopic analysis of caspofungin-treated hyphae. Equal numbers of conidia from the indicated strains were spread onto the surface of an IMA plate and allowed to germinate at 30°C for 24 h. A caspofungin Etest strip was then applied to each plate and incubated overnight at 37°C. The morphology of the hyphae surrounding the highest concentration on the strip was observed by DIC microscopy. Scale bar represents 50 µm.

Mentions: All major classes of antifungal drugs that are currently in use against A. fumigatus attack the integrity of the membrane or cell wall. Fungi respond to these agents by upregulating cell wall and membrane repair systems [37],[38],[39], which may increase stress on the secretory system. To determine how loss of UPR function would affect growth in the presence of antifungal stress, susceptibility to amphotericin B, caspofungin, itraconazole and fluconazole was compared using the Etest method. Conidia were spread onto the surface of an agar plate, and 4 Etest strips were placed on top, each impregnated with a concentration gradient of a different antifungal drug, and incubated at 37°C for 48 h. The ΔhacA mutant had larger areas of growth inhibition surrounding each strip, indicating heightened susceptibility to each of these drugs and a decrease in the minimal inhibitory concentration (Figure 8A). The incomplete clearing around the caspofungin strip on plates inoculated with the wt or complemented strains is consistent with the known fungistatic activity of this class of drug for A. fumigatus [40]. It is therefore striking that a complete zone of clearing was evident around the caspofungin strip on the plate inoculated with the ΔhacA mutant. Agar plugs taken from the zone of growth inhibition surrounding the caspofungin strip on wt-inoculated plates were able to grow when transferred to medium lacking any drug. However, no viable organism could be recovered from agar plugs taken from the cleared zone surrounding the caspofungin strip on ΔhacA-inoculated plates, indicating that caspofungin becomes fungicidal in the absence of UPR function. Microscopic analysis of caspofungin-treated hyphae revealed normal morphology in the wt, but abnormal swelling and lysis in the ΔhacA mutant (Figure 8B). These defects were localized to hyphal tips and branch points, similar to what was observed under conditions of thermal stress and CFW treatment. This experiment was performed on RPMI agar in accordance with the manufacturer's specifications, but comparable results were also obtained using IMA as the medium (Figure S4). Remarkably, the corresponding Δhac1 mutant in S. cerevisiae did not show increased sensitivity to either caspofungin, ketoconazole, amphotericin B or fluconazole (Figure S5).


A role for the unfolded protein response (UPR) in virulence and antifungal susceptibility in Aspergillus fumigatus.

Richie DL, Hartl L, Aimanianda V, Winters MS, Fuller KK, Miley MD, White S, McCarthy JW, Latgé JP, Feldmesser M, Rhodes JC, Askew DS - PLoS Pathog. (2009)

The ΔhacA mutant is hypersensitive to antifungal drugs.(A) Antifungal susceptibility using Etest: conidia were spread evenly onto a 150 mm plate of RPMI agar. Four Etest strips, each impregnated with a concentration gradient of caspofungin (CS), fluconazole (FL), amphotericin B (AmB), and itraconazole (IT) were applied to the surface, with the highest concentration oriented at the plate edge. (B) Microscopic analysis of caspofungin-treated hyphae. Equal numbers of conidia from the indicated strains were spread onto the surface of an IMA plate and allowed to germinate at 30°C for 24 h. A caspofungin Etest strip was then applied to each plate and incubated overnight at 37°C. The morphology of the hyphae surrounding the highest concentration on the strip was observed by DIC microscopy. Scale bar represents 50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2606855&req=5

ppat-1000258-g008: The ΔhacA mutant is hypersensitive to antifungal drugs.(A) Antifungal susceptibility using Etest: conidia were spread evenly onto a 150 mm plate of RPMI agar. Four Etest strips, each impregnated with a concentration gradient of caspofungin (CS), fluconazole (FL), amphotericin B (AmB), and itraconazole (IT) were applied to the surface, with the highest concentration oriented at the plate edge. (B) Microscopic analysis of caspofungin-treated hyphae. Equal numbers of conidia from the indicated strains were spread onto the surface of an IMA plate and allowed to germinate at 30°C for 24 h. A caspofungin Etest strip was then applied to each plate and incubated overnight at 37°C. The morphology of the hyphae surrounding the highest concentration on the strip was observed by DIC microscopy. Scale bar represents 50 µm.
Mentions: All major classes of antifungal drugs that are currently in use against A. fumigatus attack the integrity of the membrane or cell wall. Fungi respond to these agents by upregulating cell wall and membrane repair systems [37],[38],[39], which may increase stress on the secretory system. To determine how loss of UPR function would affect growth in the presence of antifungal stress, susceptibility to amphotericin B, caspofungin, itraconazole and fluconazole was compared using the Etest method. Conidia were spread onto the surface of an agar plate, and 4 Etest strips were placed on top, each impregnated with a concentration gradient of a different antifungal drug, and incubated at 37°C for 48 h. The ΔhacA mutant had larger areas of growth inhibition surrounding each strip, indicating heightened susceptibility to each of these drugs and a decrease in the minimal inhibitory concentration (Figure 8A). The incomplete clearing around the caspofungin strip on plates inoculated with the wt or complemented strains is consistent with the known fungistatic activity of this class of drug for A. fumigatus [40]. It is therefore striking that a complete zone of clearing was evident around the caspofungin strip on the plate inoculated with the ΔhacA mutant. Agar plugs taken from the zone of growth inhibition surrounding the caspofungin strip on wt-inoculated plates were able to grow when transferred to medium lacking any drug. However, no viable organism could be recovered from agar plugs taken from the cleared zone surrounding the caspofungin strip on ΔhacA-inoculated plates, indicating that caspofungin becomes fungicidal in the absence of UPR function. Microscopic analysis of caspofungin-treated hyphae revealed normal morphology in the wt, but abnormal swelling and lysis in the ΔhacA mutant (Figure 8B). These defects were localized to hyphal tips and branch points, similar to what was observed under conditions of thermal stress and CFW treatment. This experiment was performed on RPMI agar in accordance with the manufacturer's specifications, but comparable results were also obtained using IMA as the medium (Figure S4). Remarkably, the corresponding Δhac1 mutant in S. cerevisiae did not show increased sensitivity to either caspofungin, ketoconazole, amphotericin B or fluconazole (Figure S5).

Bottom Line: Failure to induce the UPR did not affect radial growth on rich medium at 37 degrees C, but cell wall integrity was disrupted at 45 degrees C, resulting in a dramatic loss in viability.The DeltahacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates.These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology & Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America.

ABSTRACT
Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The DeltahacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37 degrees C, but cell wall integrity was disrupted at 45 degrees C, resulting in a dramatic loss in viability. The DeltahacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the DeltahacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy.

Show MeSH
Related in: MedlinePlus