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Noda Institute for Scientific Research GRANT2013 Young Investigator Research GrantOutline of Research Result

Molecular mechanism of stress tolerance in conidia of Aspergillus speciesDaisuke HagiwaraMycology Research Center (MMRC), Chiba University

Research aimsConidia are stress-resistant spores that are produced bymany fungal species, including the genera Aspergillus andPenicillium. The ATF/CREB-type transcription factor AtfAhas been characterized as a regulator of conidia stress tolerance in A. nidulans and A. oryzae.1, 2) Nonetheless, the roleof ATF-type transcription factors in the pathogenic fungusA. fumigatus remains unclear. The conidium is an infectiousagent that is transmitted to the human lungs from environmental niches. Therefore, a better understanding of the molecular mechanisms that regulate conidia stress tolerancewould be beneficial, both for the prevention of aspergillosisand the development of new antifungals. In this study, weconstructed an A. fumigatus ΔatfA deletion strain to characterize the role of AtfA in conidia stress tolerance.
MethodsA. fumigatus strain AfS35 (FGSC A1159) (akuA::loxP)
was utilized to generate the following deletion strains usedin this study: ΔatfA, ΔsakA, ΔmpkC, and ΔsakA, ΔmpkC.
For heat stress assays, diluted conidia suspensions were incubated at 55°C. For oxidative stress assays, H2O2 (Final100 mM) was added to conidia suspensions. After 15 min,the conidia were appropriately diluted and spread onto normal medium agar. After 24 h, the number of colonies wascounted. The polyol and trehalose contents of the conidiawere measured by HPLC. To determine gene expressionlevels, quantitative real-time PCR (qRT-PCR) analysis wasperformed using the Life Technology 7300 system withSYBR Green detection. To obtain transcriptomes, IlluminaMiseq and CLC Genomics Workbench were used.

Fig. 1. The HOG pathway of A. fumgiatus

significantly less trehalose in the conidia of the ΔatfAstrain, suggesting that AtfA is involved in trehalose accumulation in A. fumigatus conidia. It was suggested that thisdecrease in trehalose levels results in the heat stress-sensitive phenotype of the ΔatfA conidia.

Results

2. Transcriptome analysis of the asexual developmental stage of A. fumigatus

1. The role of AtfA in the stress resistance of A. fumigatus conidiaThe conidia of the atfA deletion mutant (named ΔatfA)exhibited remarkable sensitivity to both heat stress and oxidative stress, indicating that AtfA plays an indispensiblerole in the stress resistance of conidia. Carbohydrate quantification revealed that trehalose was more abundant in A.
fumigatus conidia than other carbohydrates such as mannitol, arabitol, erythritol, and glycerol. In contrast, there wasTo gain a greater insight into the role of AtfA in conidiastress tolerance, we compared the transcriptomes of the WTand ΔatfA strains at the conidiation stage. We found that738 genes were markedly induced at the conidiation stage.
Of these, 54 were AtfA-dependent, including the genes thatencode the conidia-specific catalase CatA, the dehydrinlike protein DprA, the HSP9/12 family protein Scf1, andthe conidiation-specific protein ConJ. Because CatA andDprA were previously shown to be involved in the oxida-

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tive stress resistance of conidia,3, 4) the impaired expressionof these genes during conidiation in the ΔatfA strain mightbe the cause of the observed sensitivity to oxidative stress.
3. The High Osmolarity Glycerol (HOG) pathway isinvolved in the stress tolerance of conidiaWe questioned whether the A. fumigatus SakA MAP kinase is required for the stress tolerance of the conidia. Unexpectedly, conidia of the ΔsakA strain exhibited a level ofstress tolerance comparable to that of the WT strain. Because A. fumigatus also encodes MpkC, a paralog of theSakA MAP kinase, we tested whether this protein confersstress tolerance to conidia in the presence or absence ofSakA. Whereas the conidia of the ΔmpkC strain exhibitedWT levels of tolerance to oxidative and heat stresses, theconidia of the ΔsakA,mpkC strain were sensitive to bothstresses, suggesting that SakA and MpkC played a redundant or cooperative role in the conidial stress response.

ConclusionIn this study, we demonstrated the following pointsconcerning the stress tolerance of A. fumigatus conidia:(1) trehalose is the predominant compatible solute; (2) thetranscription factor AtfA governs the stress-tolerance ofconidia; (3) the HOG pathway and, more specifically,(4) the SakA and MpkC MAPKs are required for conidiastress resistance.
References1)2)3)4)

Hagiwara, D., Asano, Y., Yamashino, T., and Mizuno, T. (2008)Characterization of bZip-type transcription factor AtfA with reference to stress responses of conidia of Aspergillus nidulans. Biosci.
Biotechnol. Biochem. 72: 2756–2760.
Sakamoto, K., et al. (2009) Aspergillus oryzae atfA controls conidialgermination and stress tolerance. Fungal Genet. Biol. 46: 887–897Paris, S., et al. (2003) Catalases of Aspergillus fumigatus. Infect.
Immun. 71: 3551–3562.
Hoi, J.W.S., et al. (2011) A novel family of dehydrin-like proteins isinvolved in stress response in the human fungal pathogen Aspergillus fumigates. Mol. Biol. Cell 22: 1896–1906.