Summary: It is estimated that fungal infections, caused most commonly by Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, result in more deaths annually than malaria or tuberculosis. It has long been hypothesized the fungal metabolism plays a critical role in virulence though specific nutrient sources utilized by human pathogenic fungi in vivo has remained enigmatic. However, the metabolic utilisation of preferred carbon and nitrogen sources, encountered in a host niche‐dependent manner, is known as carbon catabolite and nitrogen catabolite repression (CCR, NCR), and has been shown to be important for virulence. Several sensory and uptake systems exist, including carbon and nitrogen source‐specific sensors and transporters, that allow scavenging of preferred nutrient sources. Subsequent metabolic utilisation is governed by transcription factors, whose functions and essentiality differ between fungal species. Furthermore, additional factors exist that contribute to the implementation of CCR and NCR. The role of the CCR and NCR‐related factors in virulence varies greatly between fungal species and a substantial gap in knowledge exists regarding specific pathways. Further elucidation of carbon and nitrogen metabolism mechanisms is therefore required in a fungal species‐ and animal model‐specific manner in order to screen for targets that are potential candidates for anti‐fungal drug development.

Aspergillus fumigatus is responsible for a disproportionate number of invasive mycosis cases relative to other common filamentous fungi. While many fungal factors critical for infection establishment are known, genes essential for disease persistence and progression are ill defined. We propose that fungal factors that promote navigation of the rapidly changing nutrient and structural landscape characteristic of disease progression represent untapped clinically relevant therapeutic targets. To this end, we find that A. fumigatus requires a carbon catabolite repression (CCR) mediated genetic network to support in vivo fungal fitness and disease progression. While CCR as mediated by the transcriptional repressor CreA is not required for pulmonary infection establishment, loss of CCR inhibits fungal metabolic plasticity and the ability to thrive in the dynamic infection microenvironment. Our results suggest a model whereby CCR in an environmental filamentous fungus is dispensable for initiation of pulmonary infection but essential for infection maintenance and disease progression. Conceptually, we argue these data provide a foundation for additional studies on fungal factors required to support fungal fitness and disease progression and term such genes and factors, DPFs (disease progression factors). Medical treatment advances such as organ transplants and chemotherapies that suppress the immune system have increased the number of patients susceptible to invasive fungal diseases. The most common filamentous fungus isolated from these infections is the environmental mold, Aspergillus fumigatus, the causative agent of invasive aspergillosis (IA). Despite medical intervention, mortality from IA remains high, underscoring the need to understand A. fumigatus pathogenesis mechanisms to uncover new therapeutic targets and strategies. Here, we show that regulation of central metabolism in A. fumigatus is critical for infection maintenance and progression of disease. The dysregulation of carbon catabolite repression results in reduced virulence in an animal model at later stages of the infection because of an inability to navigate infection microenvironment dynamics driven in part by oxygen depletion and alterations in nutrient availability. These results likely not only apply to IA, but are broadly applicable to other infection models stressing the need to understand spatiotemporal dynamics of individual microbial infections particularly at the level of metabolism. We propose that microbial genes which support disease progression, in contrast but not mutually exclusive to disease initiation, be termed DPFs (disease progression factors) and as such represent a novel class of antimicrobial drug targets.

The production of bioethanol from lignocellulosic feedstocks is dependent on lignocellulosic biomass degradation by hydrolytic enzymes. The main component of lignocellulose is cellulose and different types of organisms are able to secrete cellulases. The filamentous fungus Aspergillus nidulans serves as a model organism to study cellulase production and the available tools allow exploring more in depth the mechanisms governing cellulase production and carbon catabolite repression. In A. nidulans, microarray data identified the cAMP-dependent protein kinase A (PkaA) as being involved in the transcriptional modulation and the production of lignocellulolytic enzymes in the presence of cellulose. Deletion of pkaA resulted in increased hydrolytic enzyme secretion, but reduced growth in the presence of lignocellulosic components and various other carbon sources. Furthermore, genes involved in fungal development were increased in the ΔpkaA strain, probably leading to the increased hyphal branching as was observed in this strain. This would allow the secretion of higher amounts of proteins. In addition, the expression of SynA, encoding a V-SNARE synaptobrevin protein involved in secretion, was increased in the ΔpkaA mutant. Deletion of pkaA also resulted in the reduced nuclear localization of the carbon catabolite repressor CreA in the presence of glucose and in partial de-repression when grown on cellulose. PkaA is involved in the glucose signaling pathway as the absence of this protein resulted in reduced glucose uptake and lower hexokinase/glucokinase activity, directing the cell to starvation conditions. Genome-wide transcriptomics showed that the expression of genes encoding proteins involved in fatty acid metabolism, mitochondrial function and in the use of cell storages was increased. This study shows that PkaA is involved in hydrolytic enzyme production in A. nidulans. It appears that this protein kinase blocks the glucose pathway, hence forcing the cell to change to starvation conditions, increasing hydrolytic enzyme secretion and inducing the usage of cellular storages. This work uncovered new regulatory avenues governing the tight interplay between the metabolic states of the cell, which are important for the production of hydrolytic enzymes targeting lignocellulosic biomass. Deletion of pkaA resulted in a strain with increased hydrolytic enzyme secretion and reduced biomass formation.

The production of bioethanol from lignocellulosic feedstocks will only become economically feasible when the majority of cellulosic and hemicellulosic biopolymers can be efficiently converted into bioethanol. The main component of cellulose is glucose, whereas hemicelluloses mainly consist of pentose sugars such as D-xylose and L-arabinose. The genomes of filamentous fungi such as A. nidulans encode a multiplicity of sugar transporters with broad affinities for hexose and pentose sugars. Saccharomyces cerevisiae, which has a long history of use in industrial fermentation processes, is not able to efficiently transport or metabolize pentose sugars (e.g. xylose). Subsequently, the aim of this study was to identify xylose-transporters from A. nidulans, as potential candidates for introduction into S. cerevisiae in order to improve xylose utilization. In this study, we identified the A. nidulans xtrD (x ylose tr ansporter) gene, which encodes a Major Facilitator Superfamily (MFS) transporter, and which was specifically induced at the transcriptional level by xylose in a XlnR-dependent manner, while being partially repressed by glucose in a CreA-dependent manner. We evaluated the ability of xtrD to functionally complement the S. cerevisiae EBY.VW4000 strain which is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae, XtrD was targeted to the plasma membrane and its expression was able to restore growth on xylose, glucose, galactose, and mannose as single carbon sources, indicating that this transporter accepts multiple sugars as a substrate. XtrD has a high affinity for xylose, and may be a high affinity xylose transporter. We were able to select a S. cerevisiae mutant strain that had increased xylose transport when expressing the xtrD gene. This study characterized the regulation and substrate specificity of an A. nidulans transporter that represents a good candidate for further directed mutagenesis. Investigation into the area of sugar transport in fungi presents a crucial step for improving the S. cerevisiae xylose metabolism. Moreover, we have demonstrated that the introduction of adaptive mutations beyond the introduced xylose utilization genes is able to improve S. cerevisiae xylose metabolism.

Acquisition and subsequent metabolism of different carbon and nitrogen sources have been shown to play an important role in virulence attributes of the fungal pathogen Aspergillus fumigatus, such as the secretion of host tissue-damaging proteases and fungal cell wall integrity. We examined the relationship between the metabolic processes of carbon catabolite repression (CCR), nitrogen catabolite repression (NCR) and virulence in a variety of A. fumigatus clinical isolates. A considerable amount of heterogeneity with respect to the degree of CCR and NCR was observed and a positive correlation between NCR and virulence in a neutropenic mouse model of pulmonary aspergillosis (PA) was found. Isolate Afs35 was selected for further analysis and compared to the reference strain A1163, with both strains presenting the same degree of virulence in a neutropenic mouse model of PA. Afs35 metabolome analysis in physiological-relevant carbon sources indicated an accumulation of intracellular sugars that also serve as cell wall polysaccharide precursors. Genome analysis showed an accumulation of missense substitutions in the regulator of protease secretion and in genes encoding enzymes required for cell wall sugar metabolism. Based on these results, the virulence of strains Afs35 and A1163 was assessed in a triamcinolone murine model of PA and found to be significantly different, confirming the known importance of using different mouse models to assess strain-specific pathogenicity. These results highlight the importance of nitrogen metabolism for virulence and provide a detailed example of the heterogeneity that exists between A. fumigatus isolates with consequences for virulence in a strain-specific and host-dependent manner.

Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options. A. fumigatus is the main ethiological agent of one of the most important life-threatening fungal diseases in immuno-compromised humans, invasive aspergillosis. Treatment commonly involves echinocandin antifungal drugs that inhibit cell wall β-1,3-glucan polysaccharide biosynthesis. Calcium is an important messenger for many signaling pathways regulating the fungal response to stress and antifungal drugs. The calcium responsive calcineurin phosphatase influences the localization and activity of the CrzA transcription factor (TF), regulating the activation of several stress responses and cell wall modifications. For many years, CrzA has been recognized as the sole calcium/calcineurin-dependent TF. Here, we identify nine TFs involved in the calcium/calcineurin metabolism, including a novel A. fumigatus calcium/calcineurin dependent TF named ZipD. Transcriptional profiling of the response of A. fumigatus wild-type, plus the ΔcrzA and ΔzipD mutant, strains shows CrzA and ZipD to have shared and unique functions. ZipD was found to be important for not only calcium metabolism, but also for the cell wall organization, osmotic stress and echinocandin tolerance. During host infection, ZipD plays an important role in modulating fungal cell walls, promoting evasion of the host pro-inflammatory immune responses and virulence. Our work emphasizes the complexity and importance of novel additional mechanisms for calcium signaling, including ZipD, to regulate fungal stress responses, cell wall structure, promoting pathogenesis and antifungal resistance, complicating disease control.