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.

Perturbations of the yeast cell wall trigger a repair mechanism that reconfigures its molecular structure to preserve cell integrity. To investigate this mechanism, we compared the global gene expression in five mutant strains, each bearing a mutation (i.e. fks1, kre6, mnn9, gas1, and knr4 mutants) that affects in a different manner the cell wall construction. Altogether, 300 responsive genes were kept based on high stringency criteria during data processing. Functional classification of these differentially expressed genes showed a substantial subset of induced genes involved in cell wall construction and an enrichment of metabolic, energy generation, and cell defense categories, whereas families of genes belonging to transcription, protein synthesis, and cellular growth were underrepresented. Clustering methods isolated a single group of ∼80 up-regulated genes that could be considered as the stereotypical transcriptional response of the cell wall compensatory mechanism. The in silico analysis of the DNA upstream region of these co-regulated genes revealed pairwise combinations of DNA-binding sites for transcriptional factors implicated in stress and heat shock responses (Msn2/4p and Hsf1p) with Rlm1p and Swi4p, two PKC1-regulated transcription factors involved in the activation genes related to cell wall biogenesis and G₁/S transition. Moreover, this computational analysis also uncovered the 6-bp 5′-AGCCTC-3′ CDRE (calcineurin-dependent response element) motif in 40% of the co-regulated genes. This motif was recently shown to be the DNA binding site for Crz1p, the major effector of calcineurin-regulated gene expression in yeast. Taken altogether, the data presented here lead to the conclusion that the cell wall compensatory mechanism, as triggered by cell wall mutations, integrates three major regulatory systems: namely the PKC1-SLT2 mitogen-activated protein kinase-signaling module, the “global stress” response mediated by Msn2/4p, and the Ca²⁺/calcineurin-dependent pathway. The relative importance of these regulatory systems in the cell wall compensatory mechanism is discussed.

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.

SUMMARY: Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory.