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ABSTRACT Thermotolerance is a remarkable virulence attribute of Aspergillus fumigatus, but the consequences of heat shock (HS) to the cell membrane of this fungus are unknown, although this structure is one of the first to detect changes in ambient temperature that imposes on the cell a prompt adaptative response. Under high-temperature stress, fungi trigger the HS response controlled by heat shock transcription factors, such as HsfA, which regulates the expression of heat shock proteins. In yeast, smaller amounts of phospholipids with unsaturated fatty acid (FA) chains are synthesized in response to HS, directly affecting plasma membrane composition. The addition of double bonds in saturated FA is catalyzed by Δ9-fatty acid desaturases, whose expression is temperature-modulated. However, the relationship between HS and saturated/unsaturated FA balance in membrane lipids of A. fumigatus in response to HS has not been investigated. Here, we found that HsfA responds to plasma membrane stress and has a role in sphingolipid and phospholipid unsaturated biosynthesis. In addition, we studied the A. fumigatus Δ9-fatty acid desaturase sdeA and discovered that this gene is essential and required for unsaturated FA biosynthesis, although it did not directly affect the total levels of phospholipids and sphingolipids. sdeA depletion significantly sensitizes mature A. fumigatus biofilms to caspofungin. Also, we demonstrate that hsfA controls sdeA expression, while SdeA and Hsp90 physically interact. Our results suggest that HsfA is required for the adaptation of the fungal plasma membrane to HS and point out a sharp relationship between thermotolerance and FA metabolism in A. fumigatus. IMPORTANCE Aspergillus fumigatus causes invasive pulmonary aspergillosis, a life-threatening infection accounting for high mortality rates in immunocompromised patients. The ability of this organism to grow at elevated temperatures is long recognized as an essential attribute for this mold to cause disease. A. fumigatus responds to heat stress by activating heat shock transcription factors and chaperones to orchestrate cellular responses that protect the fungus against damage caused by heat. Concomitantly, the cell membrane must adapt to heat and maintain physical and chemical properties such as the balance between saturated/unsaturated fatty acids. However, how A. fumigatus connects these two physiological responses is unclear. Here, we explain that HsfA affects the synthesis of complex membrane lipids such as phospholipids and sphingolipids and controls the enzyme SdeA, which produces monounsaturated fatty acids, raw material for membrane lipids. These findings suggest that forced dysregulation of saturated/unsaturated fatty acid balance might represent novel strategies for antifungal therapy.

Invasive pulmonary aspergillosis is a life-threatening fungal infection especially in the immunocompromised patients. The low diversity of available antifungal drugs coupled with the emergence of antifungal resistance has become a worldwide clinical concern. The echinocandin Caspofungin (CSP) is recommended as a second-line therapy but resistance and tolerance mechanisms have been reported. However, how the fungal cell articulates the response to CSP is not completely understood. This work provides a detailed characterization of ZnfA, a transcription factor (TF) identified in previous screening studies that is involved in the A. fumigatus responses to calcium and CSP. This TF plays an important role in the regulation of iron homeostasis and cell wall organization in response to high CSP concentrations as revealed by Chromatin Immunoprecipitation coupled to DNA sequencing (ChIP-seq) analysis. Furthermore, ZnfA acts collaboratively with the key TF CrzA in modulating the response to calcium as well as cell wall and osmotic stresses. This study therefore describes the existence of an additional, previously unknown TF that bridges calcium signaling and the CSP cellular response and further exposes the complex connections that exist among different pathways which govern stress sensing and signaling in A. fumigatus.

Obesity is an epidemic disease and the expansion of adipose tissue, especially visceral fat, promotes the secretion of factors that lead to comorbidities such as diabetes and cardiovascular diseases. Thus, diet and exercise have been proposed as an intervention to reverse these complications. An adipocytokine, known as irisin, mediates the beneficial effects of exercise. It has been proposed as a therapeutic potential in controlling obesity. In view of the above, this paper attempts to determine the modulation of irisin, visceral adiposity and biochemical markers in response to dietary intervention and aerobic exercise. To do this, 52 diet-induced obese male Wistar rats were divided into the following four groups: high-fat diet and exercise (HFD-Ex); HFD-Sedentary (HFD-Sed); chow-diet and exercise (CD-Exercise); and CD-Sed. The exercise-trained group performed a treadmill protocol for 60 min/day, 3 days/week for 8 weeks. Body mass (BM), body fat (BF), fat mass (FM), and fat-free mass (FFM) were analyzed. Mesenteric (MES), epididymal (EPI), and retroperitoneal (RET) adipose tissue was collected and histological analysis was performed. Biochemical irisin, triglycerides, glucose, insulin and inflammatory markers were determined and, FNDC5 protein expression was analyzed. In this study, the diet was the most important factor in reducing visceral adiposity in the short and long term. Exercise was an important factor in preserving muscle mass and reducing visceral depots after a long term. Moreover, the combination of diet and exercise can enhance these effects. Diet and exercise exclusively were the factors capable of increasing the values of irisin/FNDC5, however it did not bring cumulative effects of both interventions. Prescriptions to enhance the obesity treatments should involve reducing visceral adiposity by reducing the fat content in the diet associated with aerobic exercise.

Abstract Background 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. Results 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. Conclusions This work points to important insights into a better understanding of the cellular mechanisms underlying the regulation of cellulolytic enzyme secretion in this fungus.

Abstract Background Trichophyton rubrum is the main etiological agent of skin and nail infections worldwide. Because of its keratinolytic activity and anthropophilic nature, infection models based on the addition of protein substrates have been employed to assess transcriptional profiles and to elucidate aspects related to host-pathogen interactions. Chalcones are widespread compounds with pronounced activity against dermatophytes. The toxicity of trans-chalcone towards T. rubrum is not fully understood but seems to rely on diverse cellular targets. Within this context, a better understanding of the mode of action of trans-chalcone may help identify new strategies of antifungal therapy and reveal new chemotherapeutic targets. This work aimed to assess the transcriptional profile of T. rubrum grown on different protein sources (keratin or elastin) to mimic natural infection sites and exposed to trans-chalcone in order to elucidate the mechanisms underlying the antifungal activity of trans-chalcone. Results Overall, the use of different protein sources caused only slight differences in the transcriptional profile of T. rubrum. The main differences were the modulation of proteases and lipases in gene categories when T. rubrum was grown on keratin and elastin, respectively. In addition, some genes encoding heat shock proteins were up-regulated during the growth of T. rubrum on keratin. The transcriptional profile of T. rubrum exposed to trans-chalcone included four main categories: fatty acid and lipid metabolism, overall stress response, cell wall integrity pathway, and alternative energy metabolism. Consistently, T. rubrum Mapk was strongly activated during the first hours of trans-chalcone exposure. Noteworthy, trans-chalcone inhibited genes involved in keratin degradation. The results also showed effects of trans-chalcone on fatty acid synthesis and metabolic pathways involved in acetyl-CoA supply. Conclusion Our results suggest that the mode of action of trans-chalcone is related to pronounced changes in fungal metabolism, including an imbalance between fatty acid synthesis and degradation that interferes with cell membrane and cell wall integrity. In addition, this compound exerts activity against important virulence factors. Taken together, trans-chalcone acts on targets related to dermatophyte physiology and the infection process.

The deleterious effects of human-induced climate change have long been predicted. However, the imminent emergence and spread of new diseases, including fungal infections through the rise of thermotolerant strains, is still neglected, despite being a potential consequence of global warming. Thermotolerance is a remarkable virulence attribute of the mold Aspergillus fumigatus. Under high-temperature stress, opportunistic fungal pathogens deploy an adaptive mechanism known as heat shock (HS) response controlled by heat shock transcription factors (HSFs). In eukaryotes, HSFs regulate the expression of several heat shock proteins (HSPs), such as the chaperone Hsp90, which is part of the cellular program for heat adaptation and a direct target of HSFs. We recently observed that the perturbation in cell wall integrity (CWI) causes concomitant susceptibility to elevated temperatures in A. fumigatus, although the mechanisms underpinning the HS response and CWI cross talking are not elucidated. Here, we aim at further deciphering the interplay between HS and CWI. Our results show that cell wall ultrastructure is severely modified when A. fumigatus is exposed to HS. We identify the transcription factor HsfA as essential for A. fumigatus viability, thermotolerance, and CWI. Indeed, HS and cell wall stress trigger the coordinated expression of both hsfA and hsp90. Furthermore, the CWI signaling pathway components PkcA and MpkA were shown to be important for HsfA and Hsp90 expression in the A. fumigatus biofilms. Lastly, RNA-sequencing confirmed that hsfA regulates the expression of genes related to the HS response, cell wall biosynthesis and remodeling, and lipid homeostasis. Our studies collectively demonstrate the connection between the HS and the CWI pathway, with HsfA playing a crucial role in this cross-pathway regulation, reinforcing the importance of the cell wall in A. fumigatus thermophily.

Aspergillus fumigatusA. fumigatus

Most of the biosynthetic gene clusters (BGCs) found in microbes are silent under standard laboratory cultivation conditions due to the lack of expression triggering stimuli, representing a considerable drawback in drug discovery. To access the full biosynthetic potential, studies towards the activation of cryptic BGCs are essential. Histone acetylation status is an important regulator of chromatin structure, which impacts cell physiology and the expression of BGCs. In this study, clr3, a gene encoding a histone deacetylase in Penicillium brasilianum LaBioMMi 136, is deleted and associated phenotypic and metabolic changes are evaluated. The results indicate reduced growth under oxidative stress conditions in the ∆clr3 strain, higher intracellular reactive oxygen species (ROS) levels, and a different transcriptional profile of 13 ROS-related genes of both strains under basal and ROS-induced conditions. Moreover, the production of 14 secondary metabolites, including austin-related meroterpenoids, brasiliamides, verruculogen, penicillic acid, and cyclodepsipeptides was evaluated in the ∆clr3 strain, most of them being reduced. Accordingly, the addition of epigenetic modulators responsible for HDAC inhibition into P. brasilianum’s growth media also culminated in the reduction in secondary metabolite production. The results suggest that Clr3 plays an essential role in secondary metabolite biosynthesis in P. brasilianum, thus offering new strategies for the regulation of natural product synthesis by assessing chromatin modification.

ABSTRACT G-protein coupled receptors (GPCRs) are extracellular signaling receptors that sense environmental cues. Fungi sense their environment primarily through GPCR-mediated signaling pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. Aspergillus fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that present a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate compared to the wild-type (WT) strain and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the ΔgprM and ΔgprJ strains and decreased in the overexpression mutants compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. Transcriptome sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the ΔgprM and ΔgprJ strains, at least partially through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signaling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis. IMPORTANCE A. fumigatus is the main etiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immunocompromised humans. Withstanding the host environment is essential for A. fumigatus virulence, and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin, and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella. This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.

ABSTRACT Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, in the last 10 years there have been several reports of azole resistance in A. fumigatus and new strategies are needed to combat invasive aspergillosis. Caspofungin is effective against other human-pathogenic fungal species, but it is fungistatic only against A. fumigatus. Resistance to caspofungin in A. fumigatus has been linked to mutations in the fksA gene that encodes the target enzyme of the drug β-1,3-glucan synthase. However, tolerance of high caspofungin concentrations, a phenomenon known as the caspofungin paradoxical effect (CPE), is also important for subsequent adaptation and drug resistance evolution. Here, we identified and characterized the transcription factors involved in the response to CPE by screening an A. fumigatus library of 484 null transcription factors (TFs) in CPE drug concentrations. We identified 11 TFs that had reduced CPE and that encoded proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism, and cell wall remodeling. One of these TFs, FhdA, was important for mitochondrial respiratory function and iron metabolism. The ΔfhdA mutant showed decreased growth when exposed to Congo red or to high temperature. Transcriptome sequencing (RNA-seq) analysis and further experimental validation indicated that the ΔfhdA mutant showed diminished respiratory capacity, probably affecting several pathways related to the caspofungin tolerance and resistance. Our results provide the foundation to understand signaling pathways that are important for caspofungin tolerance and resistance. IMPORTANCE Aspergillus fumigatus, one of the most important human-pathogenic fungal species, is able to cause aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Invasive pulmonary aspergillosis is the most serious pathology in terms of patient outcome and treatment, with a high mortality rate ranging from 50% to 95% primarily affecting immunocompromised patients. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, there were several reports of evolution of clinical azole resistance in the last decade. Caspofungin, a noncompetitive β-1,3-glucan synthase inhibitor, has been used against A. fumigatus, but it is fungistatic and is recommended as second-line therapy for invasive aspergillosis. More information about caspofungin tolerance and resistance is necessary in order to refine antifungal strategies that target the fungal cell wall. Here, we screened a transcription factor (TF) deletion library for TFs that can mediate caspofungin tolerance and resistance. We have identified 11 TFs that are important for caspofungin sensitivity and/or for the caspofungin paradoxical effect (CPE). These TFs encode proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism or cell wall remodeling, and mitochondrial respiratory function. The study of those genes regulated by TFs identified in this work will provide a better understanding of the signaling pathways that are important for caspofungin tolerance and resistance.

Abstract Standing among the front defense strategies against pathogens, host phagocytic cells release various oxidants. Therefore, pathogens have to cope with stressful conditions at the site of infection. Peroxiredoxins (Prx) are highly reactive and abundant peroxidases that can support virulence and persistence of pathogens in distinct hosts. Here, we revealed that the opportunistic human pathogen A. fumigatus presents three 1-Cys Prx (Prx6 subfamily), which is unprecedented. We showed that PrxB and PrxC were in mitochondria, while Prx1 was in cytosol. As observed for other Prxs, recombinant Prx1 and PrxC decomposed H2O2 at elevated velocities (rate constants in the 107 M−1s−1 range). Deletion mutants for each Prx displayed higher sensitivity to oxidative challenge in comparison with the wild-type strain. Additionally, cytosolic Prx1 was important for A. fumigatus survival upon electron transport dysfunction. Expression of Prxs was dependent on the SakAHOG1 MAP kinase and the Yap1YAP1 transcription factor, a global regulator of the oxidative stress response in fungi. Finally, cytosolic Prx1 played a major role in pathogenicity, since it is required for full virulence, using a neutropenic mouse infection model. Our data indicate that the three 1-Cys Prxs act together to maintain the redox balance of A. fumigatus.

The pyruvate dehydrogenase complex (PDH), that converts pyruvate to acetyl-coA, is regulated by pyruvate dehydrogenase kinases (PDHK) and phosphatases (PDHP) that have been shown to be important for morphology, pathogenicity and carbon source utilization in different fungal species. The aim of this study was to investigate the role played by the three PDHKs PkpA, PkpB and PkpC in carbon source utilization in the reference filamentous fungus Aspergillus nidulans, in order to unravel regulatory mechanisms which could prove useful for fungal biotechnological and biomedical applications. PkpA and PkpB were shown to be mitochondrial whereas PkpC localized to the mitochondria in a carbon source-dependent manner. Only PkpA was shown to regulate PDH activity. In the presence of glucose, deletion of pkpA and pkpC resulted in reduced glucose utilization, which affected carbon catabolite repression (CCR) and hydrolytic enzyme secretion, due to de-regulated glycolysis and TCA cycle enzyme activities. Furthermore, PkpC was shown to be required for the correct metabolic utilization of cellulose and acetate. PkpC negatively regulated the activity of the glyoxylate cycle enzyme isocitrate lyase (ICL), required for acetate metabolism. In summary, this study identified PDHKs important for the regulation of central carbon metabolism in the presence of different carbon sources, with effects on the secretion of biotechnologically important enzymes and carbon source-related growth. This work demonstrates how central carbon metabolism can affect a variety of fungal traits and lays a basis for further investigation into these characteristics with potential interest for different applications.

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.

Extracellular vesicles (EVs) has been considered an alternative process for intercellular communication. EVs release by filamentous fungi and the role of vesicular secretion during fungus-host cells interaction remain unknown. Here, we identified the secretion of EVs from the pathogenic filamentous fungus, Aspergillus fumigatus. Analysis of the structure of EVs demonstrated that A. fumigatus produces round shaped bilayer structures ranging from 100 to 200 nm size, containing ergosterol and a myriad of proteins involved in REDOX, cell wall remodeling and metabolic functions of the fungus. We demonstrated that macrophages can phagocytose A. fumigatus EVs. Phagocytic cells, stimulated with EVs, increased fungal clearance after A. fumigatus conidia challenge. EVs were also able to induce the production of TNF-α and CCL2 by macrophages and a synergistic effect was observed in the production of these mediators when the cells were challenged with the conidia. In bone marrow-derived neutrophils (BMDN) treated with EVs, there was enhancement of the production of TNF-α and IL-1β in response to conidia. Together, our results demonstrate, for the first time, that A. fumigatus produces EVs containing a diverse set of proteins involved in fungal physiology and virulence. Moreover, EVs are biologically active and stimulate production of inflammatory mediators and fungal clearance.

ABSTRACT Aspergillus fumigatus is a filamentous fungus which causes invasive pulmonary aspergillosis in immunocompromised individuals. In fungi, cell signaling and cell wall plasticity are crucial for maintaining physiologic processes. In this context, Msb2 is an important signaling mucin responsible for activation of a variety of mitogen-activated protein kinase (MAPK)-dependent signaling pathways that regulate cell growth in several organisms, such as the cell wall integrity (CWI) pathway. Here, we aimed to characterize the MSB2 homologue in A. fumigatus. Our results showed that MsbA plays a role in the vegetative and reproductive development of the fungus, in stress adaptation, and in resistance to antifungal drugs by modulating the CWI pathway gene expression. Importantly, cell wall composition is also responsible for activation of diverse receptors of the host immune system, thus leading to a proper immune response. In a model of acute Aspergillus pulmonary infection, results demonstrate that the ΔmsbA mutant strain induced less inflammation with diminished cell influx into the lungs and lower cytokine production, culminating in increased lethality rate. These results characterize for the first time the role of the signaling mucin MsbA in the pathogen A. fumigatus, as a core sensor for cell wall morphogenesis and an important regulator of virulence. IMPORTANCE Aspergillus fumigatus is an opportunistic fungus with great medical importance. During infection, Aspergillus grows, forming hyphae that colonize the lung tissue and invade and spread over the mammal host, resulting in high mortality rates. The knowledge of the mechanisms responsible for regulation of fungal growth and virulence comprises an important point to better understand fungal physiology and host-pathogen interactions. Msb2 is a mucin that acts as a sensor and an upstream regulator of the MAPK pathway responsible for fungal development in Candida albicans and Aspergillus nidulans. Here, we show the role of the signaling mucin MsbA in the pathogen A. fumigatus, as a core sensor for cell wall morphogenesis, fungal growth, and virulence. Moreover, we show that cell wall composition, controlled by MsbA, is detrimental for fungal recognition and clearance by immune cells. Our findings are important for the understanding of how fungal sensors modulate cell physiology.

ABSTRACT The pathogenic fungus Aspergillus fumigatus is able to adapt to extremely variable environmental conditions. The A. fumigatus genome contains four genes coding for mitogen-activated protein kinases (MAPKs), which are important regulatory knots involved in diverse cellular responses. From a clinical perspective, MAPK activity has been connected to salvage pathways, which can determine the failure of effective treatment of invasive mycoses using antifungal drugs. Here, we report the characterization of the Saccharomyces cerevisiae Fus3 ortholog in A. fumigatus, designated MpkB. We demonstrate that MpkB is important for conidiation and that its deletion induces a copious increase of dihydroxynaphthalene (DHN)-melanin production. Simultaneous deletion of mpkB and mpkA, the latter related to maintenance of the cell wall integrity, normalized DHN-melanin production. Localization studies revealed that MpkB translocates into the nuclei when A. fumigatus germlings are exposed to caspofungin stress, and this is dependent on the cross-talk interaction with MpkA. Additionally, DHN-melanin formation was also increased after deletion of genes coding for the Gα protein GpaA and for the G protein-coupled receptor GprM. Yeast two-hybrid and coimmunoprecipitation assays confirmed that GpaA and GprM interact, suggesting their role in the MpkB signaling cascade. IMPORTANCE Aspergillus fumigatus is the most important airborne human pathogenic fungus, causing thousands of deaths per year. Its lethality is due to late and often inaccurate diagnosis and the lack of efficient therapeutics. The failure of efficient prophylaxis and therapy is based on the ability of this pathogen to activate numerous salvage pathways that are capable of overcoming the different drug-derived stresses. A major role in the protection of A. fumigatus is played by melanins. Melanins are cell wall-associated macromolecules classified as virulence determinants. The understanding of the various signaling pathways acting in this organism can be used to elucidate the mechanism beyond melanin production and help to identify ideal drug targets.

Sphingolipids (SL) are complex lipids and components of the plasma membrane which are involved in numerous cellular processes, as well as important for virulence of different fungal pathogens. In yeast, SL biosynthesis is regulated by the “AGC kinases” Ypk1 and Ypk2, which also seem to connect the SL biosynthesis with the cell wall integrity (CWI) and the High Osmolarity Glycerol (HOG) pathways. Here, we investigate the role of ypkAY PK1 in SL biosynthesis and its relationship with the CWI and the HOG pathways in the opportunistic human pathogen Aspergillus fumigatus. We found that ypkA is important for fungal viability, since the ΔypkA strain presented a drastically sick phenotype and complete absence of conidiation. We observed that under repressive condition, the conditional mutant niiA::ypkA exhibited vegetative growth defects, impaired germination and thermosensitivity. In addition, the ypkA loss of function caused a decrease in glycosphingolipid (GSL) levels, especially the metabolic intermediates belonging to the neutral GSL branch including dihydroceramide (DHC), ceramide (Cer), and glucosylceramide (GlcCer), but interestingly a small increase in ergosterol content. Genetic analyzes showed that ypkA genetically interacts with the MAP kinases of CWI and HOG pathways, mpkA and sakA, respectively, while only SakA physically interacts with YpkA. Our results suggest that YpkA is important for fungal survival through the regulation of GSL biosynthesis and cross talks with A. fumigatus MAP kinase pathways.

This study aims to analyze the effects of resisted, aerobic, and combined exercises on omentin levels in visceral adipose tissue and muscle of rats with experimental diabetes to verify whether these adipokines are related to the glucose pathway and inflammation process in this model. Male Wistar rats received a high-fat diet for 4 weeks and a low dose of streptozotocin (35 mg/kg) to induce experimental diabetes. After inducing diabetes, the animals were divided into 4 experimental groups (n = 10): diabetic control (C); resistance training (RT); aerobic training (AT); and combined training (CT). The groups were exercised for 12 weeks, 3 times/week, where: RT means the stair climbing protocol until exhaustion; AT is the 30 min/day reaching 20 m/min protocol, and CT is the combination of RT and AT. The AT group showed reduced retroperitoneal and mesenteric adipose tissue and abdominal fat deposits. Our study also showed a possible control of blood glucose, as well as decreased Interleukin 6 (IL-6) and C-reactive protein, increased circulating adiponectin and increased omentin in visceral adipose tissue. In addition, the AT group affected the glucose pathway by stimulating phosphorylation of Akt in muscle tissue. Omentin also showed a strong positive correlation with adiponectin and a moderate negative correlation with IL-6. Thus, our findings indicated that omentin in type 2 diabetes is changed by AT. Furthermore, increased omentin levels had a close association with the glucose pathway by stimulating phosphorylation of Akt in muscle tissue and with IL-6 in serum, suggesting that omentin is likely to have anti-inflammatory and protective action in experimental diabetes.

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.

ABSTRACT 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 MpkAMPK1 and its associated transcription factor (TF) RlmARLM1, 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.

Aspergillus fumigatus is an opportunistic human pathogen, which causes the life-threatening disease, invasive pulmonary aspergillosis. In fungi, cell wall homeostasis is controlled by the conserved Cell Wall Integrity (CWI) pathway. In A. fumigatus this signaling cascade is partially characterized, but the mechanisms by which it is activated are not fully elucidated. In this study we investigated the role of protein kinase C (PkcA) in this signaling cascade. Our results suggest that pkcA is an essential gene and is activated in response to cell wall stress. Subsequently, we constructed and analyzed a non-essential A. fumigatus pkcAG579R mutant, carrying a Gly579Arg substitution in the PkcA C1B regulatory domain. The pkcAG579R mutation has a reduced activation of the downstream Mitogen-Activated Protein Kinase, MpkA, resulting in the altered expression of genes encoding cell wall-related proteins, markers of endoplasmic reticulum stress and the unfolded protein response. Furthermore, PkcAG579R is involved in the formation of proper conidial architecture and protection to oxidative damage. The pkcAG579R mutant elicits increased production of TNF-α and phagocytosis but it has no impact on virulence in a murine model of invasive pulmonary aspergillosis. These results highlight the importance of PkcA to the CWI pathway but also indicated that additional regulatory circuits may be involved in the biosynthesis and/or reinforcement of the A. fumigatus cell wall during infection.