Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen^1,2,3. Its conidia production is prolific, and so human respiratory tract exposure is almost constant⁴. A. fumigatus is isolated from human habitats⁵ and vegetable compost heaps^6,7. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis⁸. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.

In the yeast Saccharomyces cerevisiae, environmental stress conditions that damage the cell wall lead to activation of the so-called “compensatory mechanism,” aimed at preserving cell integrity through a remodeling of this extracellular matrix. Here we used DNA microarrays to investigate the molecular basis of this response to two agents that induce transient cell wall damage; namely Congo Red and Zymolyase. Treatment of the cells with these two agents elicited the up-regulation of 132 and 101 genes respectively, the main functional groups among them being involved in cell wall construction and metabolism. The main response does not occur until hours after exposure to the cell wall-perturbing agent. In some cases, this response was transient, but more sustained in others, especially in the case of the genes involved in cell wall remodeling. Clustering of these data together with those from the response to constitutive cell wall damage, revealed the existence of a cluster of co-regulated genes that was strongly induced under all conditions assayed. Those genes induced by cell wall damage showed an enrichment in DNA binding motifs for Rlm1p, Crz1p, SBF (Swi4p/Swi6p), Msn2p/Msn4p, Ste12p, and Tec1p transcription factors, suggesting a complex regulation of this response together with the possible involvement of several signaling pathways. With the exception of PHO89 and FKS2, none of the genes induced by Congo Red was up-regulated in a slt2 strain. Moreover, characterization of the transcriptional response to Congo Red in a rlm1 mutant strain revealed that only a few genes (i.e. PHO89, FKS2, YLR042C, and CHA1) were induced at least partially independently of the transcription factor Rlm1p, the rest being totally dependent on this transcription factor for their activation. Our findings consistently demonstrate that the cell integrity signaling pathway regulates the cell wall damage compensatory response, mainly through transcriptional activation mediated by Rlm1p.

Yeast mitogen-activated protein kinase (MAPK) signaling pathways transduce external stimuli into cellular responses very precisely. The MAPKs Slt2/Mpk1 and Hog1 regulate transcriptional responses of adaptation to cell wall and osmotic stresses, respectively. Unexpectedly, we observe that the activation of a cell wall integrity (CWI) response to the cell wall damage caused by zymolyase (β-1,3 glucanase) requires both the HOG and SLT2 pathways. Zymolyase activates both MAPKs and Slt2 activation depends on the Sho1 branch of the HOG pathway under these conditions. Moreover, adaptation to zymolyase requires essential components of the CWI pathway, namely the redundant MAPKKs Mkk1/Mkk2, the MAPKKK Bck1, and Pkc1, but it does not require upstream elements, including the sensors and the guanine nucleotide exchange factors of this pathway. In addition, the transcriptional activation of genes involved in adaptation to cell wall stress, like CRH1, depends on the transcriptional factor Rlm1 regulated by Slt2, but not on the transcription factors regulated by Hog1. Consistent with these findings, both MAPK pathways are essential for cell survival under these circumstances because mutant strains deficient in different components of both pathways are hypersensitive to zymolyase. Thus, a sequential activation of two MAPK pathways is required for cellular adaptation to cell wall damage.

Two mitogen‐activated protein kinase (MAPK) pathways, viz. the high‐osmolarity glycerol (HOG) and the cell wall integrity (CWI) pathways, regulate stress responses in the yeast Saccharomyces cerevisiae. Whereas the former is mainly involved in adaptation of yeast cells to hyperosmotic stress, the latter is activated under conditions leading to cell wall instability. Although MAPK signalling specificity can be conceived as requiring insulation of the different pathways, it is also becoming clear that the two pathways do not compete with each other but can be positively coordinated to regulate many stress responses. This review highlights our current knowledge about the collaboration between these two MAPK pathways to counteract different kinds of environmental stress. Copyright © 2010 John Wiley & Sons, Ltd.

The adaptation of Saccharomyces cerevisiae to situations in which cell wall integrity is seriously compromised mainly involves the cell wall integrity (CWI) pathway. However, in a recent work ( Bermejo, C., Rodriguez, E., García, R., Rodríguez-Peña, J. M., Rodríguez de la Concepción, M. L., Rivas, C., Arias, P., Nombela, C., Posas, F., and Arroyo, J. (2008) Mol. Biol. Cell 19, 1113-1124 ) we have demonstrated the co-participation of the high osmotic response (HOG) pathway to ensure yeast survival to cell wall stress mediated by zymolyase, which hydrolyzes the β-1,3 glucan network. Here we have characterized the role of both pathways in the regulation of the overall yeast transcriptional responses to zymolyase treatment using whole genome expression profiling. A main group of yeast genes is dependent on both MAPKs, Slt2 and Hog1, for their induction. The transcriptional activation of these genes depends on the MAPKKK Bck1, the transcription factor Rlm1, and elements of the sho1 branch of the HOG pathway, but not on the sensors of the CWI pathway. A second group of genes is dependent on Slt2 but not Hog1 or Pbs2. However, the induction of these genes is dependent on upstream elements of the HOG pathway such as Sho1, Ste50, and Ste11, in accordance with a sequential activation of the HOG and CWI pathways. Zymolyase also promotes an osmotic-like transcriptional response with the activation of a group of genes dependent on elements of the Sho1 branch of HOG pathway but not on Slt2, with the induction of many of them dependent on Msn2/4. Additionally, in the absence of Hog1, zymolyase induces an alternative response related to mating and filamentation as a consequence of the cross-talk between these pathways and the HOG pathway. Finally, in the absence of Slt2, zymolyase increases the induction of genes associated with osmotic adaptation with respect to the wild type, suggesting an inhibitory effect of the CWI pathway over the HOG pathway. These studies clearly reveal the complexity of the signal transduction machinery responsible for regulating yeast adaptation responses to cell wall stress.

Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of β1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect β1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation.