First report of coexistence of blaKPC-2 and blaNDM-1 in carbapenem-resistant clinical isolates of Klebsiella aerogenes in Brazil.

Klebsiella aerogenes is an important opportunistic pathogen with the potential to develop resistance against last-line antibiotics, such as carbapenems, limiting the treatment options. Here, we investigated the antibiotic resistance profiles of 10 K. aerogenes strains isolated from patient samples in the intensive-care unit of a Brazilian tertiary hospital using conventional PCR and a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the blaKPC-2 and blaNDM-1 genes simultaneously. All isolates were completely resistant to ß-lactam antibiotics, including ertapenem, imipenem, and meropenem with differencing levels of resistance to aminoglycosides, quinolones, and tigecycline also observed. Half of the strains studied were classified as multidrug-resistant. The carbapenemase-producing isolates carried many genes of interest including: ß-lactams (blaNDM-1, blaKPC-2, blaTEM-1, blaCTX-M-1 group, blaOXA-1 group and blaSHVvariants in 20-80% of the strains), aminoglycoside resistance genes [aac(6')-Ib and aph(3')-VI, 70 and 80%], a fluoroquinolone resistance gene (qnrS, 80%), a sulfonamide resistance gene (sul-2, 80%) and a multidrug efflux system transporter (mdtK, 70%) while all strains carried the efflux pumps Acr (subunit A) and tolC. Moreover, we performed a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the blaKPC-2 and blaNDM-1 genes simultaneously. The draft genome assembly of the CRK317 had a total length of 5,462,831 bp and a GC content of 54.8%. The chromosome was found to contain many essential genes. In silico analysis identified many genes associated with resistance phenotypes, including ß-lactamases (blaOXA-9, blaTEM-1, blaNDM-1, blaCTX-M-15, blaAmpC-1, blaAmpC-2), the bleomycin resistance gene (bleMBL), an erythromycin resistance methylase (ermC), aminoglycoside-modifying enzymes [aac(6')-Ib, aadA/ant(3")-Ia, aph(3')-VI], a sulfonamide resistance enzyme (sul-2), a chloramphenicol acetyltransferase (catA-like), a plasmid-mediated quinolone resistance protein (qnrS1), a glutathione transferase (fosA), PEtN transferases (eptA, eptB) and a glycosyltransferase (arnT). We also detected 22 genomic islands, eight families of insertion sequences, two putative integrative and conjugative elements with a type IV secretion system, and eight prophage regions. This suggests the significant involvement of these genetic structures in the dissemination of antibiotic resistance. The results of our study show that the emergence of carbapenemase-producing K. aerogenes, co-harboring blaKPC-2 and blaNDM-1, is a worrying phenomenon which highlights the importance of developing strategies to detect, prevent, and control the spread of these microorganisms.

Aspergillus fumigatus mitogen-activated protein kinase MpkA is involved in gliotoxin production and self-protection.

Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. The Mitogen-Activated Protein kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. The sensor histidine kinase SlnASln1 is important for modulation of MpkA phosphorylation. Our work emphasizes the importance of MpkA and compartmentalization of cellular events for GT production and self-defense.

Vacuoles and peroxisomes are involved in Aspergillus fumigatus gliotoxin production and self-protection.

Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. Peroxisomes are also required for proper GT production and self-defense. The Mitogen-Activated Protein (MAP) kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. Our work emphasizes the importance of dynamic compartmentalization of cellular events for GT production and self-defense.

The Heat Shock Transcription Factor HsfA Plays a Role in Membrane Lipids Biosynthesis Connecting Thermotolerance and Unsaturated Fatty Acid Metabolism in Aspergillus fumigatus.

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.

Brevundimonas brasiliensis sp. nov.: a New Multidrug-Resistant Species Isolated from a Patient in Brazil.

To increase knowledge on Brevundimonas pathogens, we conducted in-depth genomic and phenotypic characterization of a Brevundimonas strain isolated from the cerebrospinal fluid of a patient admitted in a neonatal intensive care unit. The strain was identified as a member of the genus Brevundimonas based on Vitek 2 system results and 16S rRNA gene sequencing and presented a multidrug resistance profile (MDR). Several molecular and biochemical tests were used to characterize and identify the species for in-depth results. The draft genome assembly of the isolate has a total length of 3,261,074 bp and a G+C of 66.86%, similar to other species of the genus. Multilocus sequence analysis, Type (Strain) Genome Server, digital DNA-DNA hybridization, and average nucleotide identity confirmed that the Brevundimonas sp. studied represents a distinct species, for which we propose the name Brevundimonas brasiliensis sp. nov. In silico analysis detected antimicrobial resistance genes (AMRGs) mediating resistance to ß-lactams (penP, blaTEM-16, and blaBKC-1) and aminoglycosides [strA, strB, aac(6')-Ib, and aac(6')-Il]. We also found AMRGs encoding the AcrAB efflux pump that confers resistance to a broad spectrum of antibiotics. Colistin and quinolone resistance can be attributed to mutation in qseC and/or phoP and GyrA/GyrB, respectively. The Brevundimonas brasiliensis sp. nov. genome contained copies of type IV secretion system (T4SS)-type integrative and conjugative elements (ICEs); integrative mobilizable elements (IME); and Tn3-type and IS3, IS6, IS5, and IS1380 families, suggesting an important role in the development and dissemination of antibiotic resistance. The isolate presented a range of virulence-associated genes related to biofilm formation, adhesion, and invasion that can be relevant for its pathogenicity. Our findings provide a wealth of data to hinder the transmission of MDR Brevundimonas and highlight the need for monitoring and identifying new bacterial species in hospital environments. IMPORTANCE Brevundimonas species is considered an opportunistic human pathogen that can cause multiple types of invasive and severe infections in patients with underlying pathologies. Treatment of these pathogens has become a major challenge because many isolates are resistant to most antibiotics used in clinical practice. Furthermore, there are no consistent therapeutic results demonstrating the efficacy of antibacterial agents. Although considered a rare pathogen, recent studies have provided evidence of the emergence of Brevundimonas in clinical settings. Hence, we identified a novel pathogenic bacterium, Brevundimonas brasiliensis sp. nov., that presented a multidrug resistance (MDR) profile and carried diverse genes related to drug resistance, virulence, and mobile genetic elements. Such data can serve as a baseline for understanding the genomic diversity, adaptation, evolution, and pathogenicity of MDR Brevundimonas.

Assessment of cecal microbiota modulation from piglet dietary supplementation with copper.

BACKGROUND: Swine production expanded in the last decades. Efforts have been made to improve meat production and to understand its relationship to pig gut microbiota. Copper (Cu) is a usual supplement to growth performance in animal production. Here, two performance studies were conducted to investigate the effects of three different sources of Cu on the microbiota of piglets. A total of 256 weaned piglets were randomly allocated into 4 treatments (10 replicates per treatment of 4 piglets per pen in Trial 1 and 8 replicates of 3 piglets per pen in Trial 2). Treatments included a control group (fed 10 mg/kg of Cu from CuSO4), a group fed at 160 mg/kg of Copper (II) sulfate (CuSO4) or tri-basic copper chloride (TBCC), and a group fed with Cu methionine hydroxy analogue chelated (Cu-MHAC) at 150, 80, and 50 mg/kg in Phases 1 (24-35 d), 2 (36-49 d), and 3 (50-70 d), respectively. At 70 d, the cecum luminal contents from one pig per pen were collected and polled for 16 S rRNA sequencing (V3/V4 regions). Parameters were analyzed in a completely randomized block design, in which each experiment was considered as a block. RESULTS: A total of 1337 Operational Taxonomic Units (OTUs) were identified. Dominance and Simpson ecological metrics were statistically different between control and treated groups (P < 0.10) showing that different Cu sources altered the gut microbiota composition with the proliferation of some bacteria that improve gut health. A high abundance of Prevotella was observed in all treatments while other genera were enriched and differentially modulated, according to the Cu source and dosage. The supplementation with Cu-MHAC can modify a group of bacteria involved in feed efficiency (FE) and short chain fatty acids (SCFA) production (Clostridium XIVa, Desulfovibrio, and Megasphera). These bacteria are also important players in the activation of ghrelin and growth hormones that were previously reported to correlate with Cu-MHAC supplementation. CONCLUSIONS: These results indicated that some genera seem to be directly affected by the Cu source offered to the animals. TBCC and Cu-MHAC (even in low doses) can promote healthy modifications in the gut bacterial composition, being a promising source of supplementation for piglets.

Identification, cloning, and characterization of a novel chitinase from leaf-cutting ant Atta sexdens: An enzyme with antifungal and insecticidal activity.

Chitinases are enzymes that degrade chitin, a polysaccharide found in the exoskeleton of insects, fungi, yeast, and internal structures of other vertebrates. Although chitinases isolated from bacteria, fungi and plants have been reported to have antifungal or insecticide activities, chitinases from insects with these activities have been seldomly reported. In this study, a leaf-cutting ant Atta sexdens DNA fragment containing 1623 base pairs was amplified and cloned into a vector to express the protein (AsChtII-C4B1) in Pichia pastoris. AsChtII-C4B1, which contains one catalytic domain and one carbohydrate-binding module (CBM), was secreted to the extracellular medium and purified by ammonium sulfate precipitation followed by nickel column chromatography. AsChtII-C4B1 showed maximum activity at pH 5.0 and 55 °C when tested against colloidal chitin substrate and maintained >60% of its maximal activity in different temperatures during 48 h. AsChtII-C4B1 decreased the survival of Spodoptera frugiperda larvae fed with an artificial diet that contained AsChtII-C4B1. Our results have indicated that AsChtII-C4B1 has a higher effect on larva-pupa than larva-larva molts. AsChtII-C4B1 activity targets more specifically the growth of filamentous fungus than yeast. This work describes, for the first time, the obtaining a recombinant chitinase from ants and the characterization of its insecticidal and antifungal activities.

Vaccination with Live or Heat-Killed Aspergillus fumigatus ΔsglA Conidia Fully Protects Immunocompromised Mice from Invasive Aspergillosis.

Aspergillus fumigatus causes invasive aspergillosis (IA) in immunocompromised patients, resulting in high mortality rates. Currently, no vaccine formulations to promote immune protection in at-risk individuals have been developed. In this work, we deleted the sterylglucosidase-encoding gene, sglA, in Aspergillus fumigatus and investigated its role in fungal virulence and host vaccine protection. The ΔsglA mutant accumulated sterylglucosides (SGs), newly studied immunomodulatory glycolipids, and exhibited reduced hyphal growth and altered compositions of cell wall polysaccharides. Interestingly, the ΔsglA mutant was avirulent in two murine models of IA and was fully eliminated from the lungs. Both corticosteroid-induced immunosuppressed and cyclophosphamide-induced leukopenic mice vaccinated with live or heat-killed ΔsglA conidia were fully protected against a lethal wild-type A. fumigatus challenge. These results highlight the potential of SG-accumulating strains as safe and promising vaccine formulations against invasive fungal infections. IMPORTANCE Infections by Aspergillus fumigatus occur by the inhalation of environmental fungal spores called conidia. We found that live mutant conidia accumulating glycolipids named sterylglucosides are not able to cause disease when injected into the lung. Interestingly, these animals are now protected against a secondary challenge with live wild-type conidia. Remarkably, protection against a secondary challenge persists even with vaccination with heat-killed mutant conidia. These results will significantly advance the field of the research and development of a safe fungal vaccine for protection against the environmental fungus A. fumigatus.

Enhancing the biological properties of zinc complexes with bis(indolyl)methane groups: Synthesis, characterization, DNA interaction, and biocide activity.

The unprecedented mononucleated ligand (6,6-di(1H-indol-3-yl)-N,N-bis(pyridin-2-ylmethyl)hexan-1-amine (LC5) with an N3-donor set and its complexes [Zn(LC5)Cl2] • 2CH3OH (1) and [Zn(LC5)2](ClO4)2 (2), were successfully prepared. All compounds were fully characterized by a suite of physicochemical methods. Fluid 1H and 13C NMR spectroscopy, as well as DFT and TD-DFT calculations, were carried out to propose a viable structural arrangement for both complexes. The interaction between these compounds and DNA was monitored in the UV region where binding constants (Kb) were estimated (2 > 1 > LC5). These data were corroborated by DNA cleavage assays using groove binders, circular dichroism, and docking studies. Both complexes confirmed their biocide activity against selected microorganisms: Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria, the filamentous fungi A. fumigatus and S. cerevisiae. Finally, the cytotoxic activities of 1 and 2 were tested against the erythroleukemia K562 cell line. For all biological studies, it was probed that the presence of the indole moieties and the zinc atoms in the chemical composition of the complexes studied could increase the magnitude of the activity following the order: 2 > 1 > LC5, where a linear relationship between the biological activity upon K562 cells (IC50) and DNA binding studies (Kb) was found.

The Penicillium brasilianum Histone Deacetylase Clr3 Regulates Secondary Metabolite Production and Tolerance to Oxidative Stress.

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.

Identification of a New Endo-ß-1,4-xylanase Prospected from the Microbiota of the Termite Heterotermes tenuis.

Xylanases are hemicellulases that break down xylan to soluble pentoses. They are used for industrial purposes, such as paper whitening, beverage clarification, and biofuel production. The second-generation bioethanol production is hindered by the enzymatic hydrolysis step of the lignocellulosic biomass, due to the complex arrangement established among its constituents. Xylanases can potentially increase the production yield by improving the action of the cellulolytic enzyme complex. We prospected endo-ß-1,4-xylanases from meta-transcriptomes of the termite Heterotermes tenuis. In silico structural characterization and functional analysis of an endo-ß-1,4-xylanase from a symbiotic protist of H. tenuis indicate two active sites and a substrate-binding groove needed for the catalytic activity. No N-glycosylation sites were found. This endo-ß-1,4-xylanase was recombinantly expressed in Pichia pastoris and Escherichia coli cells, presenting a molecular mass of approximately 20 kDa. Enzymatic activity assay using recombinant endo-ß-1,4-xylanase was also performed on 1% xylan agar stained with Congo red at 30 °C and 40 °C. The enzyme expressed in both systems was able to hydrolyze the substrate xylan, becoming a promising candidate for further analysis aiming to determine its potential for application in industrial xylan degradation processes.

Identification and selection of a new Saccharomyces cerevisiae strain isolated from Brazilian ethanol fermentation process for application in beer production.

The fermented beverage industry is always pursuing alternatives to make products that delight consumers with special or unique characteristics. The identification and improvement of new yeast strains emerge as an opportunity; however, wild strains usually have a limitation in maltose fermentation and/or off-flavors production. Here we report the production of a Blond-style ale beer using a bioethanol isolated strain (LBGA-287) with flavor complexity approved in sensorial panels. LBGA-287 also showed an increase in maltose consumption, growth and fermentation rates when compared to the commercial yeast. Using qPCR analysis, genes related to the (i) efficiency of fermentation (ii) production of aromas/off-flavors, and (iii) metabolization of carbohydrates were found as differentially expressed in the isolated strains when compared to industrial yeast. This suggests that LBGA-287 could have an important impact on beer production, improving brewing efficiency, quality and diversity of this beverage, and most importantly satisfying the final consumer.

Heterogeneity in the transcriptional response of the human pathogen Aspergillus fumigatus to the antifungal agent caspofungin.

Aspergillus fumigatus is the main causative agent of invasive pulmonary aspergillosis (IPA), a severe disease that affects immunosuppressed patients worldwide. The fungistatic drug caspofungin (CSP) is the second line of therapy against IPA but has increasingly been used against clinical strains that are resistant to azoles, the first line antifungal therapy. In high concentrations, CSP induces a tolerance phenotype with partial reestablishment of fungal growth called CSP paradoxical effect (CPE), resulting from a change in the composition of the cell wall. An increasing number of studies has shown that different isolates of A. fumigatus exhibit phenotypic heterogeneity, including heterogeneity in their CPE response. To gain insights into the underlying molecular mechanisms of CPE response heterogeneity, we analyzed the transcriptomes of two A. fumigatus reference strains, Af293 and CEA17, exposed to low and high CSP concentrations. We found that there is a core transcriptional response that involves genes related to cell wall remodeling processes, mitochondrial function, transmembrane transport, and amino acid and ergosterol metabolism, and a variable response related to secondary metabolite (SM) biosynthesis and iron homeostasis. Specifically, we show here that the overexpression of a SM pathway that works as an iron chelator extinguishes the CPE in both backgrounds, whereas iron depletion is detrimental for the CPE in Af293 but not in CEA17. We next investigated the function of the transcription factor CrzA, whose deletion was previously shown to result in heterogeneity in the CPE response of the Af293 and CEA17 strains. We found that CrzA constitutively binds to and modulates the expression of several genes related to processes involved in CSP tolerance and that crzA deletion differentially impacts the SM production and growth of Af293 and CEA17. As opposed to the ΔcrzACEA17 mutant, the ΔcrzAAf293 mutant fails to activate cell wall remodeling genes upon CSP exposure, which most likely severely affects its macrostructure and extinguishes its CPE. This study describes how heterogeneity in the response to an antifungal agent between A. fumigatus strains stems from heterogeneity in the function of a transcription factor and its downstream target genes.

Dietary Intervention, When Not Associated With Exercise, Upregulates Irisin/FNDC5 While Reducing Visceral Adiposity Markers in Obese Rats.

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.

The Heat Shock Transcription Factor HsfA Is Essential for Thermotolerance and Regulates Cell Wall Integrity in Aspergillus fumigatus.

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.

High-Intensity Interval Training Does Not Change Vaspin and Omentin and Does Not Reduce Visceral Adipose Tissue in Obese Rats.

This study aimed to determine the expression of omentin and vaspin, inflammatory markers, body composition, and lipid profile in diet-induced obese rats and high-intensity interval training (HIIT). Forty Wistar rats were divided into four groups: untrained normal diet, trained normal diet (T-ND), untrained high-fat diet (Unt-HFD), and trained high-fat diet (T-HFD). For the animals of the Unt-HFD and T-HFD groups, a high-fat diet was offered for 4 weeks. After that, all the animals in the T-ND and T-HFD groups were submitted to HITT, three times per week, for 10 weeks (2 weeks of adaptation and 8 weeks of HIIT). Muscle (gastrocnemius), liver, epididymal adipose tissue, retroperitoneal adipose tissue, visceral adipose tissue (VAT), and serum were collected to analyze TNF-α, IL-6, PCR, IL-8, IL-10, IL-4, vaspin, and omentin. A body composition analysis was performed before adaptation to HIIT protocol and after the last exercise session using dual-energy X-ray absorptiometry. Omentin and vaspin in the VAT were quantified using Western blotting. The results showed that, when fed a high-fat diet, the animals obtained significant gains in body fat and elevated serum concentrations of vaspin and blood triglycerides. The HIIT was able to minimize body fat gain but did not reduce visceral fat despite the increase in maximum exercise capacity. Moreover, there was a reduction in the serum levels of adiponectin, IL-6, and IL-10. Finally, we concluded that, although the training protocol was able to slow down the weight gain of the animals, there was no reduction in visceral fat or an improvement in the inflammatory profile, including no changes in omentin and vaspin.

Transcriptional Control of the Production of Aspergillus fumigatus Conidia-Borne Secondary Metabolite Fumiquinazoline C Important for Phagocytosis Protection.

Aspergillus fumigatus produces diverse secondary metabolites whose biological functions and regulation remain to be understood. Despite the importance of the conidia for this fungus, the role of the conidia-born metabolite fumiquinazoline C (FqC) is unclear. Here, we describe a dual function of the cell-wall integrity pathway in regulating FqC biosynthesis dictated by the MAPK kinase MpkA, which phosphorylates one of the nonribosomal peptide synthetases enzymes of the cluster (FmqC), and the transcription factor RlmA, which directly regulates the expression of fmq genes. Another level of crosstalk between the FqC regulation and the cell physiology is described since the deletion of the stress-responsive transcription factor sebA provokes derepression of the fmq cluster and overproduction of FqC. Thus, we describe a mechanism by which A. fumigatus controls FqC biosynthesis orchestrated by MpkA-RlmA and SebA and hence enabling survival and adaptation to the environmental niche, given that FqC is a deterrent of ameba predation.

Novel Biological Functions of the NsdC Transcription Factor in Aspergillus fumigatus.

The fungal zinc finger transcription factor NsdC is named after, and is best known for, its essential role in sexual reproduction (never in sexual development). In previous studies with Aspergillus nidulans, it was also shown to have roles in promotion of vegetative growth and suppression of asexual conidiation. In this study, the function of the nsdC homologue in the opportunistic human pathogen A. fumigatus was investigated. NsdC was again found to be essential for sexual development, with deletion of the nsdC gene in both MAT1-1 and MAT1-2 mating partners of a cross leading to complete loss of fertility. However, a functional copy of nsdC in one mating partner was sufficient to allow sexual reproduction. Deletion of nsdC also led to decreased vegetative growth and allowed conidiation in liquid cultures, again consistent with previous findings. However, NsdC in A. fumigatus was shown to have additional biological functions including response to calcium stress, correct organization of cell wall structure, and response to the cell wall stressors. Furthermore, virulence and host immune recognition were affected. Gene expression studies involving chromatin immunoprecipitation (ChIP) of RNA polymerase II (PolII) coupled to next-generation sequencing (Seq) revealed that deletion of nsdC resulted in changes in expression of over 620 genes under basal growth conditions. This demonstrated that this transcription factor mediates the activity of a wide variety of signaling and metabolic pathways and indicates that despite the naming of the gene, the promotion of sexual reproduction is just one among multiple roles of NsdC.IMPORTANCEAspergillus fumigatus is an opportunistic human fungal pathogen and the main causal agent of invasive aspergillosis, a life-threatening infection especially in immunocompromised patients. A. fumigatus can undergo both asexual and sexual reproductive cycles, and the regulation of both cycles involves several genes and pathways. Here, we have characterized one of these genetic determinants, the NsdC transcription factor, which was initially identified in a screen of transcription factor null mutants showing sensitivity when exposed to high concentrations of calcium. In addition to its known essential roles in sexual reproduction and control of growth rate and asexual reproduction, we have shown in the present study that A. fumigatus NsdC transcription factor has additional previously unrecognized biological functions including calcium tolerance, cell wall stress response, and correct cell wall organization and functions in virulence and host immune recognition. Our results indicate that NsdC can play novel additional biological functions not directly related to its role played during sexual and asexual processes.

Aspergillus Fumigatus ZnfA, a Novel Zinc Finger Transcription Factor Involved in Calcium Metabolism and Caspofungin Tolerance.

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.

Aspergillus fumigatus Hsp90 interacts with the main components of the cell wall integrity pathway and cooperates in heat shock and cell wall stress adaptation.

The initiation of Aspergillus fumigatus infection occurs via dormant conidia deposition into the airways. Therefore, conidial germination and subsequent hyphal extension and growth occur in a sustained heat shock (HS) environment promoted by the host. The cell wall integrity pathway (CWIP) and the essential eukaryotic chaperone Hsp90 are critical for fungi to survive HS. Although A. fumigatus is a thermophilic fungus, the mechanisms underpinning the HS response are not thoroughly described and important to define its role in pathogenesis, virulence and antifungal drug responses. Here, we investigate the contribution of the CWIP in A. fumigatus thermotolerance. We observed that the CWIP components PkcA, MpkA and RlmA are Hsp90 clients and that a PkcAG579R mutation abolishes this interaction. PkcAG579R also abolishes MpkA activation in the short-term response to HS. Biochemical and biophysical analyses indicated that Hsp90 is a dimeric functional ATPase, which has a higher affinity for ADP than ATP and prevents MpkA aggregation in vitro. Our data suggest that the CWIP is constitutively required for A. fumigatus to cope with the temperature increase found in the mammalian lung environment, emphasising the importance of this pathway in supporting thermotolerance and cell wall integrity.