Deciphering cell wall integrity signalling in Aspergillus fumigatus: Identification and functional characterization of cell wall stress sensors and relevant Rho GTPases

Max von Pettenkofer-Institut, Ludwig-Maximilians-Universität, Pettenkoferstraße 9a, Munich, Germany.
Molecular Microbiology (Impact Factor: 4.42). 02/2012; 83(3):506-19. DOI: 10.1111/j.1365-2958.2011.07946.x
Source: PubMed


The fungal cell wall, a conserved and highly dynamic structure, is essential for virulence and viability of fungal pathogens and is an important antifungal drug target. The cell wall integrity (CWI) signalling pathway regulates shape and biosynthesis of the cell wall. In this work we identified, localized and functionally characterized four putative CWI stress sensors of Aspergillus fumigatus, an airborne opportunistic human pathogen and the cause of invasive aspergillosis. We show that Wsc1 is specifically required for resistance to echinocandin antifungals. MidA is specifically required for elevated temperature tolerance and resistance to the cell wall perturbing agents congo red and calcofluor white. Wsc1, Wsc3 and MidA additionally have overlapping functions and are redundantly required for radial growth and conidiation. We have also analysed the roles of three Rho GTPases that have been implicated in CWI signalling in other fungi. We show that Rho1 is essential and that conditional downregulation of rho1 or deletion of rho2 or rho4 results in severely impaired CWI. Our data indicate significant functional differences between the CWI stress sensors of yeasts and moulds.

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    • "Alternatively, replacing the native promoter of a gene with the Tet-on cassette can be used for gene functional analysis. For example, the Rho GTPase rho1 in the pathogenic mould A. fumigatus was demonstrated to be an essential gene by replacement of the native rho1 promoter with the Tet-on cassette, which resulted in a mutant isolate which could not grow in the absence of Dox (Dichtl et al., 2012). An alternative strategy is to place genes of interest under control of the Tet-on system and subsequently delete the wild-type allele, enabling characterisation of gene function by quantitative transcript downregulation by reducing Dox in growth media, an approach validated using the A. niger γ-actin encoding gene (Meyer et al., 2011). "
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    ABSTRACT: In Aspergillus, controlled gene expression is often achieved using the reverse tetracycline-controlled transactivator (rtTA) dependent Tet-on system, whereby transcription is titratably activated by addition of the tetracycline derivative doxycycline. The complementary Tet-off system utilises the tetracycline-controlled transactivator (tTA) component to quantitatively reduce gene expression. In this study, we utilized a synthetic biological approach to engineer highly optimized Tet-off conditional expression systems in Aspergillus niger and Aspergillus fumigatus. Steps for delivery of these tools include utilizing codon optimized cassette components, testing several promoters for improved genetic stability and validating two modified luciferase reporters for highly accurate measurements of gene expression. The Tet-off cassettes developed in this study enable facile and quantitative functional analysis, as validated by Tet-off analysis of genes involved in chitin synthesis and cell wall polarity in A. niger, and para-aminobenzoic acid synthesis in A. fumigatus. We also used a racA(G18V) dominant allele to demonstrate that Tet-off in A. niger enables gene over-expression and downregulation in a single isolate. Additionally, we used the improved luciferase reporters to show that the Tet-off cassette in A. niger enables quantification of gene oscillations.In order to demonstrate that synthetic biological approaches developed here are broadly applicable to engineering transcriptional circuits in filamentous fungi, we used our strategy for improving cassette stability by promoter replacement in the A. niger Tet-on system, which resulted in a modified Tet-on cassette with higher stability in recipient genomes.
    Fungal Genetics and Biology 11/2015; DOI:10.1016/j.fgb.2015.11.003 · 2.59 Impact Factor
    • "essentially as described before (Dichtl et al., 2012). The complemented strains dnm1 and pcp1 were constructed by PCR amplifying and cloning the coding region of the respective genes into the PmeI sites of pSK379 (dnm1) or pSK379-phleo (pcp1) (Dirr et al., 2010) and transforming the resulting vectors (pCH004 and pNM002, respectively) in the respective deletion mutants. "
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    ABSTRACT: Mitochondria within eukaryotic cells continuously fuse and divide. This phenomenon is called mitochondrial dynamics and crucial for mitochondrial function and integrity. We performed a comprehensive analysis of mitochondrial dynamics in the pathogenic mould Aspergillus fumigatus. Phenotypic characterization of respective mutants revealed the general essentiality of mitochondrial fusion for mitochondrial genome maintenance and the mould's viability. Surprisingly, it turned out that the mitochondrial rhomboid protease Pcp1 and its processing product s-Mgm1 which are crucial for fusion in yeast are dispensable for fusion, mtDNA maintenance and viability in A. fumigatus. In contrast, mitochondrial fission mutants show drastically reduced growth and sporulation rates and increased heat susceptibility. However, reliable inheritance of mitochondria to newly formed conidia is ensured. Strikingly, mitochondrial fission mutants show a significant and growth condition-dependent increase in azole resistance. Parallel disruption of fusion in a fission mutant partially rescues growth and sporulation defects and further increases the azole resistance phenotype. Taken together, our results indicate an emerging dispensability of the mitochondrial rhomboid protease function in mitochondrial fusion, the suitability of mitochondrial fusion machinery as antifungal target and the involvement of mitochondrial fission in azole susceptibility. This article is protected by copyright. All rights reserved.
    Molecular Microbiology 08/2015; DOI:10.1111/mmi.13167 · 4.42 Impact Factor
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    • "It is here worth highlighting that in our experimental set-up the relevant increase in rhoA expression suggests a role for the CWI signaling pathway in the response of the fungus to DCB. A. nidulans rhoA is indeed the ortholog of Rho1 in A. fumigatus (94% identity and E-value of 2e-136 with BLASTP analysis), which was shown to be essential for CWI in this pathogenic fungus [87] and of S. cerevisae Rho1 [88] (78% identity and E-value of 4e-106 with BLASTP analysis), which acts upstream of the CWI signaling pathway. Although further studies are necessary to confirm the actual role of the CWI signaling pathway in the response to DCB, our results open up interesting avenues for future studies. "
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    ABSTRACT: The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely α-, β-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes' expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage β-1,3;1,4 glucan synthase celA together with the β-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis.
    PLoS ONE 11/2013; DOI:10.1371/journal.pone.0080038 · 3.23 Impact Factor
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