The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Background: Considering that the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, new studies aimed at understanding and improving cellulase efficiency and productivity are of paramount importance. Aspergillus niger has been shown to produce a wide spectrum of polysaccharide hydrolytic enzymes. To understand how to improve enzymatic cocktails that can hydrolyze pretreated sugarcane bagasse, we used a genomics approach to investigate which genes and pathways are transcriptionally modulated during growth of A. niger on steam-exploded sugarcane bagasse (SEB). Results: Herein we report the main cellulase- and hemicellulase-encoding genes with increased expression during growth on SEB. We also sought to determine whether the mRNA accumulation of several SEB-induced genes encoding putative transporters is induced by xylose and dependent on glucose. We identified 18 (58% of A. niger predicted cellulases) and 21 (58% of A. niger predicted hemicellulases) cellulase- and hemicellulase-encoding genes, respectively, that were highly expressed during growth on SEB. Conclusions: Degradation of sugarcane bagasse requires production of many different enzymes which are regulated by the type and complexity of the available substrate. Our presently reported work opens new possibilities for understanding sugarcane biomass saccharification by A. niger hydrolases and for the construction of more efficient enzymatic cocktails for second-generation bioethanol.

The Cell Wall Integrity (CWI) pathway is the primary signaling cascade that controls the de novo synthesis of the fungal cell wall and in Saccharomyces cerevisiae, this event is highly dependent on the RLM1 transcription factor. Here, we investigated the function of RlmA in the fungal pathogen Aspergillus fumigatus. We show that the ΔrlmA strain exhibits an altered cell wall organization in addition to defects related to vegetative growth and tolerance to cell wall-perturbing agents. A genetic analysis indicated that rlmA is positioned downstream of the pkcA and mpkA genes in the CWI pathway. As a consequence, rlmA loss-of-function leads to the altered expression of genes encoding cell wall-related proteins. RlmA positively regulates the phosphorylation of MpkA and is induced at both protein and transcriptional levels during cell wall stress. The rlmA was also involved in tolerance to oxidative damage and transcriptional regulation of genes related to oxidative stress adaptation. Moreover, the ΔrlmA strain had attenuated virulence in a neutropenic murine model of invasive pulmonary aspergillosis. Our results suggest that RlmA functions as a transcription factor in the A. fumigatus CWI pathway acting downstream of PkcA-MpkA signaling and contributing to the virulence of this fungus.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes invasive aspergillosis (IA), a life-threatening disease in immunocompromised humans. The echinocandin caspofungin, adopted as a second-line therapy in combating IA, is a β-1,3-glucan synthase inhibitor, which, when used in high concentrations, reverts the anticipated A. fumigatus growth inhibition, a phenomenon called the “caspofungin paradoxical effect” (CPE). The CPE has been widely associated with increased chitin content in the cell wall due to a compensatory upregulation of chitin synthase-encoding genes. Here, we demonstrate that the CPE is dependent on the cell wall integrity (CWI) mitogen-activated protein kinase MpkA ^MPK1 and its associated transcription factor (TF) RlmA ^RLM1 , which regulate chitin synthase gene expression in response to different concentrations of caspofungin. Furthermore, the calcium- and calcineurin-dependent TF CrzA binds to and regulates the expression of specific chitin synthase genes during the CPE. These results suggest that the regulation of cell wall biosynthetic genes occurs by several cellular signaling pathways. In addition, CrzA is also involved in cell wall organization in the absence of caspofungin. Differences in the CPE were also observed between two A. fumigatus clinical isolates, which led to the identification of a novel basic leucine zipper TF, termed ZipD. This TF functions in the calcium-calcineurin pathway and is involved in the regulation of cell wall biosynthesis genes. This study therefore unraveled additional mechanisms and novel factors governing the CPE response, which ultimately could aid in developing more effective antifungal therapies. IMPORTANCE Systemic Aspergillus fumigatus infections are often accompanied by high mortality rates. The fungal cell wall is important for infection as it has immunomodulatory and immunoevasive properties. Paradoxical growth of A. fumigatus in the presence of high concentrations of the cell wall-disturbing agent caspofungin has been observed for more than a decade, although the mechanistic nature of this phenomenon remains largely uncharacterized. Here, we show that the CWI pathway components MpkA and RlmA as well as the calcium/calcineurin-responsive transcription factor CrzA regulate the expression of cell wall biosynthetic genes during the caspofungin paradoxical effect (CPE). Furthermore, an additional, novel calcium/calcineurin-responsive transcription factor was identified to play a role in cell wall biosynthesis gene expression during the CPE. This work paints a crucial role for calcium metabolism in the CPE and provides further insight into the complex regulation of cell wall biosynthesis, which could ultimately lead to the development of more efficient antifungal therapies.

Trichoderma reesei is the most important industrial producer of lignocellulolytic enzymes. These enzymes play an important role in biomass degradation leading to novel applications of this fungus in the biotechnology industry, specifically biofuel production. The secretory pathway of fungi is responsible for transporting proteins addressed to different cellular locations involving some cellular endomembrane systems. Although protein secretion is an extremely efficient process in T. reesei, the mechanisms underlying protein secretion have remained largely uncharacterized in this organism. Here, we report for the first time the isolation and characterization of T. reesei extracellular vesicles (EVs). Using proteomic analysis under cellulose culture condition, we have confidently identified 188 vesicular proteins belonging to different functional categories. Also, we characterized EVs production using transmission electron microscopy in combination with light scattering analysis. Biochemical assays revealed that T. reesei extracellular vesicles have an enrichment of filter paper (FPase) and β-glucosidase activities in purified vesicles from 24, 72 and 96, and 72 and 96 h, respectively. Furthermore, our results showed that there is a slight enrichment of small RNAs inside the vesicles after 96 h and 120 h, and presence of hsp proteins inside the vesicles purified from T. reesei grown in the presence of cellulose. This work points to important insights into a better understanding of the cellular mechanisms underlying the regulation of cellulolytic enzyme secretion in this fungus.

Background: Calcineurin, a serine/threonine-specific protein phosphatase, plays an important role in the control of cell morphology and virulence in fungi. Calcineurin regulates localization and activity of a transcription factor called CRZ1. Recently, we characterizeAspergillus fumigatus CRZ1homologue, AfCrzA. Here, we investigate which pathways are influenced byA. fumigatusAfCrzA during a short pulse of calcium by comparatively determining the transcriptional profile ofA. fumigatuswild type andΔAfcrzAmutant strains.Results: We were able to observe 3,622 genes modulated in at least one timepoint in the mutant when compared to the wild type strain (3,211 and 411 at 10 and 30 minutes, respectively). Decreased mRNA abundance in theΔcrzAwas seen for genes encoding calcium transporters, transcription factors and genes that could be directly or indirectly involved in calcium metabolism. Increased mRNA accumulation was observed for some genes encoding proteins involved in stress response. AfCrzA overexpression inA. fumigatusincreases the expression of several of these genes. The deleted strain of one of these genes, AfRcnA, belonging to a class of endogenous calcineurin regulators, calcipressins, had more calcineurin activity after exposure to calcium and was less sensitive to menadione 30 μM, hydrogen peroxide 2.5 mM, EGTA 25 mM, and MnCl₂25 mM. We constructed deletion, overexpression, and GFP fusion protein for the closely relatedA. nidulansAnRcnA. GFP::RcnA was mostly detected along the germling, did not accumulate in the nuclei and its location is not affected by the cellular response to calcium chloride.Conclusion: We have performed a transcriptional profiling analysis of theA. fumigatus ΔAfcrzAmutant strain exposed to calcium stress. This provided an excellent opportunity to identify genes and pathways that are under the influence of AfCrzA. AfRcnA, one of these selected genes, encodes a modulator of calcineurin activity. Concomitantly withA. fumigatus AfrcnAmolecular analysis, we decided to exploit the conserved features ofA. nidulanscalcineurin system and investigated theA. nidulansAnRcnA homologue.A. nidulansAnRcnA mutation is suppressing CnaA mutation and it is responsible for modulating the calcineurin activity and mRNA accumulation of genes encoding calcium transporters.