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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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Related in: MedlinePlus

Disruption of hacA.Deletion strategy: The hacA gene was deleted by replacing the coding region with the hygromycin resistance cassette (HYG). Southern blot analysis of BamHI–digested genomic DNA using Probe B (flanking region) identified the predicted 11.5 kb wt band which was truncated to 4.0 kb in the ΔhacA mutant. A second probe (Probe A) derived from the deleted region of the hacA gene was used to confirm that no duplication had occurred.
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ppat-1000258-g002: Disruption of hacA.Deletion strategy: The hacA gene was deleted by replacing the coding region with the hygromycin resistance cassette (HYG). Southern blot analysis of BamHI–digested genomic DNA using Probe B (flanking region) identified the predicted 11.5 kb wt band which was truncated to 4.0 kb in the ΔhacA mutant. A second probe (Probe A) derived from the deleted region of the hacA gene was used to confirm that no duplication had occurred.

Mentions: Deletion of hacA was accomplished by replacing the hacAi open reading frame with the hygromycin resistance cassette (Figure 2). To determine whether loss of hacA was sufficient to disrupt UPR signaling in A. fumigatus, the expression of four known UPR target genes was examined by northern blot analysis, including bipA (ER chaperone), pdiA and tigA (protein disulfide isomerases) and hacA itself. Each of these genes contains an unfolded protein response element (UPRE) in its promoter [31], and the abundance of each mRNA increases in response to UPR activation [30]. As expected, treatment of wt A. fumigatus with DTT increased hacA abundance and induced the conversion of hacAu into hacAi, indicating activation of the UPR under these conditions (Figure 3A). The smaller size of the hacAi mRNA is consistent with a 5′ mRNA truncation that has been reported following UPR induction in other filamentous fungi [29],[30]. In contrast to wt A. fumigatus, the ΔhacA mutant was unable to increase the level of three other UPR target genes when treated with DTT, indicating a defect in UPR-regulated gene expression. Complementation of the ΔhacA mutant (C') restored UPR signaling to the ΔhacA mutant (Figure 3B).


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)

Disruption of hacA.Deletion strategy: The hacA gene was deleted by replacing the coding region with the hygromycin resistance cassette (HYG). Southern blot analysis of BamHI–digested genomic DNA using Probe B (flanking region) identified the predicted 11.5 kb wt band which was truncated to 4.0 kb in the ΔhacA mutant. A second probe (Probe A) derived from the deleted region of the hacA gene was used to confirm that no duplication had occurred.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000258-g002: Disruption of hacA.Deletion strategy: The hacA gene was deleted by replacing the coding region with the hygromycin resistance cassette (HYG). Southern blot analysis of BamHI–digested genomic DNA using Probe B (flanking region) identified the predicted 11.5 kb wt band which was truncated to 4.0 kb in the ΔhacA mutant. A second probe (Probe A) derived from the deleted region of the hacA gene was used to confirm that no duplication had occurred.
Mentions: Deletion of hacA was accomplished by replacing the hacAi open reading frame with the hygromycin resistance cassette (Figure 2). To determine whether loss of hacA was sufficient to disrupt UPR signaling in A. fumigatus, the expression of four known UPR target genes was examined by northern blot analysis, including bipA (ER chaperone), pdiA and tigA (protein disulfide isomerases) and hacA itself. Each of these genes contains an unfolded protein response element (UPRE) in its promoter [31], and the abundance of each mRNA increases in response to UPR activation [30]. As expected, treatment of wt A. fumigatus with DTT increased hacA abundance and induced the conversion of hacAu into hacAi, indicating activation of the UPR under these conditions (Figure 3A). The smaller size of the hacAi mRNA is consistent with a 5′ mRNA truncation that has been reported following UPR induction in other filamentous fungi [29],[30]. In contrast to wt A. fumigatus, the ΔhacA mutant was unable to increase the level of three other UPR target genes when treated with DTT, indicating a defect in UPR-regulated gene expression. Complementation of the ΔhacA mutant (C') restored UPR signaling to the ΔhacA mutant (Figure 3B).

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