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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 UPR contributes to cell wall integrity at 45°C.(A) The temperature-sensitivity of ΔhacA is remediated by sorbitol: Conidia were inoculated onto the center of an IMA plate containing 1.2 M sorbitol and incubated at 45°C for 3 days. The experiment was performed in triplicate and colony diameter was measured daily. Values represent the mean±SD. Colony morphology after 3 days of incubation at 45°C is shown in the photograph below. (B) Loss of cell wall integrity at 45°C: wild type and ΔhacA conidia were inoculated onto coverslips in liquid AMM–glucose and incubated at 37°C for 24 h. The plates were then shifted to 45°C and the hyphae were photographed after 4 and 8 h by DIC microscopy. Experiments in Figure 6 were performed twice with similar results. Scale bar represents 30 µm.
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ppat-1000258-g006: The UPR contributes to cell wall integrity at 45°C.(A) The temperature-sensitivity of ΔhacA is remediated by sorbitol: Conidia were inoculated onto the center of an IMA plate containing 1.2 M sorbitol and incubated at 45°C for 3 days. The experiment was performed in triplicate and colony diameter was measured daily. Values represent the mean±SD. Colony morphology after 3 days of incubation at 45°C is shown in the photograph below. (B) Loss of cell wall integrity at 45°C: wild type and ΔhacA conidia were inoculated onto coverslips in liquid AMM–glucose and incubated at 37°C for 24 h. The plates were then shifted to 45°C and the hyphae were photographed after 4 and 8 h by DIC microscopy. Experiments in Figure 6 were performed twice with similar results. Scale bar represents 30 µm.

Mentions: The inability of the ΔhacA mutant to grow at 45°C could be reversed by osmotic stabilization of the medium with sorbitol (Figure 6A) or KCl (data not shown), suggesting that the impaired growth of ΔhacA at elevated temperature is due, in part, to loss of cell wall integrity. To test this more directly, conidia were inoculated onto cover-slips in liquid medium and germinated overnight at 37°C. After shifting to 45°C, the hyphae were examined microscopically. Within 4 h of incubation at the elevated temperature, the hyphal tips began to swell, and tip lysis became apparent within 8 h (Figure 6B). Occasional areas of cytoplasmic leakage were also observed in subapical hyphae, possibly representing emerging branch points (data not shown). The ΔhacA mutant was severely growth impaired following the temperature shift, suggesting an important role for HacA in the maintenance of cell wall integrity at the hyphal tips during thermal stress.


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 UPR contributes to cell wall integrity at 45°C.(A) The temperature-sensitivity of ΔhacA is remediated by sorbitol: Conidia were inoculated onto the center of an IMA plate containing 1.2 M sorbitol and incubated at 45°C for 3 days. The experiment was performed in triplicate and colony diameter was measured daily. Values represent the mean±SD. Colony morphology after 3 days of incubation at 45°C is shown in the photograph below. (B) Loss of cell wall integrity at 45°C: wild type and ΔhacA conidia were inoculated onto coverslips in liquid AMM–glucose and incubated at 37°C for 24 h. The plates were then shifted to 45°C and the hyphae were photographed after 4 and 8 h by DIC microscopy. Experiments in Figure 6 were performed twice with similar results. Scale bar represents 30 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2606855&req=5

ppat-1000258-g006: The UPR contributes to cell wall integrity at 45°C.(A) The temperature-sensitivity of ΔhacA is remediated by sorbitol: Conidia were inoculated onto the center of an IMA plate containing 1.2 M sorbitol and incubated at 45°C for 3 days. The experiment was performed in triplicate and colony diameter was measured daily. Values represent the mean±SD. Colony morphology after 3 days of incubation at 45°C is shown in the photograph below. (B) Loss of cell wall integrity at 45°C: wild type and ΔhacA conidia were inoculated onto coverslips in liquid AMM–glucose and incubated at 37°C for 24 h. The plates were then shifted to 45°C and the hyphae were photographed after 4 and 8 h by DIC microscopy. Experiments in Figure 6 were performed twice with similar results. Scale bar represents 30 µm.
Mentions: The inability of the ΔhacA mutant to grow at 45°C could be reversed by osmotic stabilization of the medium with sorbitol (Figure 6A) or KCl (data not shown), suggesting that the impaired growth of ΔhacA at elevated temperature is due, in part, to loss of cell wall integrity. To test this more directly, conidia were inoculated onto cover-slips in liquid medium and germinated overnight at 37°C. After shifting to 45°C, the hyphae were examined microscopically. Within 4 h of incubation at the elevated temperature, the hyphal tips began to swell, and tip lysis became apparent within 8 h (Figure 6B). Occasional areas of cytoplasmic leakage were also observed in subapical hyphae, possibly representing emerging branch points (data not shown). The ΔhacA mutant was severely growth impaired following the temperature shift, suggesting an important role for HacA in the maintenance of cell wall integrity at the hyphal tips during thermal stress.

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