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Termites are well recognized by their complex development trajectories, involving dynamic differentiation process between non‐reproductive castes, workers and soldiers. These insects are associated with endosymbiotic microorganisms, which help in lignocellulose digestion and nitrogen metabolism. Aiming to identify genes harbouring biotechnological potential, we analyzed workers and soldiers RNA‐Seq data of three neotropical termites: Heterotermes tenuis (Isoptera: Rhinotermitidae), Velocitermes heteropterus (Isoptera: Termitidae) and Cornitermes cumulans (Isoptera: Termitidae). We observed differences in the microbiota associated with each termite family, and found protists' genes in both Termitidae species. We found an opposite pattern of caste‐biased gene expression between H. tenuis and the termitids studied. Moreover, the two termitids are considerably different concerning the number of differentially expressed genes (DEGs). Functional annotation indicated considerable differences in caste‐biased gene content between V. heteropterus and C. cumulans, even though they share similar diet and biological niche. Among the most DEGs, we highlighted those involved in caste differentiation and cellulose digestion, which are attractive targets for studying more efficient technologies for termite control, biomass digestion and other biotechnological applications. [ABSTRACT FROM AUTHOR]

O objetivo deste estudo foi investigar a prevalência do papilomavírus humano 6/11 e 16/18 em pacientes, com lesões orais clínicamente diagnosticadas como leucoplasias, atendidas na Faculdade de Odontologia de Araraquara, UNESP, Brasil. Após a inclusão em parafina, os cortes corados com H&E, foram selecionadas 30 biópsias e separadas em 3 grupos: lesões sem displasia (n=10), lesões com diferentes graus de displasia (n=10) e carcinoma espinocelular invasivo(n=10). As lesões que apresentaram displasia epitelial foram classificadas de acordo com os critérios histopatológicos propostos por Van Der Waal. As lesões foram investigadas para a presença de HPV por hibridização in situ com sondas biotiniladas de amplo espectro, 6/11 e 16/18. HPV 16/18 foi detectado em 20% (n=2) das biópsias com displasia severa. A presença de HPV 16/18 em lesões malignas sugere sua importância como fator de risco na carcinogênese oral.

Abstract 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.

Abstract 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 characterize Aspergillus fumigatus CRZ1 homologue, AfCrzA. Here, we investigate which pathways are influenced by A. fumigatus AfCrzA during a short pulse of calcium by comparatively determining the transcriptional profile of A. fumigatus wild type and ΔAfcrzA mutant 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 the ΔcrzA was 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 in A. fumigatus increases 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 MnCl2 25 mM. We constructed deletion, overexpression, and GFP fusion protein for the closely related A. nidulans AnRcnA. 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 the A. fumigatus ΔAfcrzA mutant 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 with A. fumigatus AfrcnA molecular analysis, we decided to exploit the conserved features of A. nidulans calcineurin system and investigated the A. nidulans AnRcnA homologue. A. nidulans AnRcnA mutation is suppressing CnaA mutation and it is responsible for modulating the calcineurin activity and mRNA accumulation of genes encoding calcium transporters.

Abstract Background Microarray techniques have become an important tool to the investigation of genetic relationships and the assignment of different phenotypes. Since microarrays are still very expensive, most of the experiments are performed with small samples. This paper introduces a method to quantify dependency between data series composed of few sample points. The method is used to construct gene co-expression subnetworks of highly significant edges. Results The results shown here are for an adapted subset of a Saccharomyces cerevisiae gene expression data set with low temporal resolution and poor statistics. The method reveals common transcription factors with a high confidence level and allows the construction of subnetworks with high biological relevance that reveals characteristic features of the processes driving the organism adaptations to specific environmental conditions. Conclusion Our method allows a reliable and sophisticated analysis of microarray data even under severe constraints. The utilization of systems biology improves the biologists ability to elucidate the mechanisms underlying celular processes and to formulate new hypotheses.

Cullin-1-RING ubiquitin ligases (CRL1) or SCF1 (SKP1-CUL1-RBX1) E3 ubiquitin ligases are the largest and most extensively investigated class of E3 ligases in mammals that regulate fundamental processes, such as the cell cycle and proliferation. These enzymes are multiprotein complexes comprising SKP1, CUL1, RBX1, and an F-box protein that acts as a specificity factor by interacting with SKP1 through its F-box domain and recruiting substrates via other domains. E3 ligases are important players in the ubiquitination process, recognizing and transferring ubiquitin to substrates destined for degradation by proteasomes or processing by deubiquitinating enzymes. The ubiquitin-proteasome system (UPS) is the main regulator of intracellular proteolysis in eukaryotes and is required for parasites to alternate hosts in their life cycles, resulting in successful parasitism. Leishmania UPS is poorly investigated, and CRL1 in L. infantum, the causative agent of visceral leishmaniasis in Latin America, is yet to be described. Here, we show that the L. infantum genes LINF_110018100 (SKP1-like protein), LINF_240029100 (cullin-like protein-like protein), and LINF_210005300 (ring-box protein 1 -putative) form a LinfCRL1 complex structurally similar to the H. sapiens CRL1. Mass spectrometry analysis of the LinfSkp1 and LinfCul1 interactomes revealed proteins involved in several intracellular processes, including six F-box proteins known as F-box-like proteins (Flp) (data are available via ProteomeXchange with identifier PXD051961). The interaction of LinfFlp 1-6 with LinfSkp1 was confirmed, and using in vitro ubiquitination assays, we demonstrated the function of the LinfCRL1(Flp1) complex to transfer ubiquitin. We also found that LinfSKP1 and LinfRBX1 knockouts resulted in nonviable L. infantum lineages, whereas LinfCUL1 was involved in parasite growth and rosette formation. Finally, our results suggest that LinfCul1 regulates the S phase progression and possibly the transition between the late S to G2 phase in L. infantum. Thus, a new class of E3 ubiquitin ligases has been described in L. infantum with functions related to various parasitic processes that may serve as prospective targets for leishmaniasis treatment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rolemberg Santana Travaglini Berti de Correia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 bla KPC-2 and bla NDM-1 genes simultaneously. All isolates were completely resistant to β-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: β-lactams ( bla NDM-1 , bla KPC-2 , bla TEM-1 , bla CTX-M-1 group, bla OXA-1 group and bla SHVvariants 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 bla KPC-2 and bla NDM-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 β-lactamases ( bla OXA-9 , bla TEM-1 , bla NDM-1 , bla CTX-M-15 , bla AmpC-1 , bla AmpC-2 ), the bleomycin resistance gene ( ble MBL ), 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 bla KPC-2 and bla NDM-1 , is a worrying phenomenon which highlights the importance of developing strategies to detect, prevent, and control the spread of these microorganisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rodrigues, Nunes, Soares, Ferreira, Damas, Laprega, Shilling, Campos, Costa, Malavazi, Cunha and Pranchevicius.)

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 SlnA Sln1 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., (© 2024. The Author(s).)

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., Competing Interests: Maurizio Del Poeta is a Co-Founder and Chief Scientific Officer of MicroRid Technologies Inc. The other authors declare no conflict of interest.

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 β-lactams ( penP , bla TEM-16 , and bla BKC-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 Tn 3 -type and IS 3 , IS 6 , IS 5 , and IS 1380 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., Competing Interests: The authors declare no conflict of interest.

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 CuSO 4 ), a group fed at 160 mg/kg of Copper (II) sulfate (CuSO 4 ) 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., (© 2023. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare there is no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The unprecedented mononucleated ligand (6,6-di(1H-indol-3-yl)-N,N-bis(pyridin-2-ylmethyl)hexan-1-amine (L C5 ) with an N 3 -donor set and its complexes [Zn(L C5 )Cl 2 ] • 2CH 3 OH (1) and [Zn(L C5 ) 2 ](ClO 4 ) 2 (2), were successfully prepared. All compounds were fully characterized by a suite of physicochemical methods. Fluid 1 H and 13 C 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 (K b ) were estimated (2 > 1 > L C5 ). 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 > L C5 , where a linear relationship between the biological activity upon K562 cells (IC 50 ) and DNA binding studies (K b ) was found., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 Δ sglA mutant accumulated sterylglucosides (SGs), newly studied immunomodulatory glycolipids, and exhibited reduced hyphal growth and altered compositions of cell wall polysaccharides. Interestingly, the Δ sglA 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 Δ sglA 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.

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.

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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-β-1,4-xylanases from meta-transcriptomes of the termite Heterotermes tenuis . In silico structural characterization and functional analysis of an endo-β-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-β-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-β-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.

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Furino, Alves, Marine, Sene-Fiorese, Rodrigues, Arrais-Lima, Mattiello, Castro, Borra, Rocha, Malavazi and Duarte.)

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 ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Valero, Colabardini, de Castro, Silva, Ries, Pardeshi, Wang, Rocha, Malavazi, Silva, Martins, Domingos, Pereira-Silva, Bromley, Wong and Goldman.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., Competing Interests: MD is a Co-Founder and Chief Scientific Officer (CSO) of MicroRid Technologies Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fabri, Rocha, Fernandes, Persinoti, Ries, Cunha, Goldman, Del Poeta and Malavazi.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Costa, Castro, Marine, Fabrizzi, Furino, Malavazi, Anibal and Duarte.)

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 PkcA G579R mutation abolishes this interaction. PkcA G579R 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., (© 2020 John Wiley & Sons Ltd.)

The fungal zinc finger transcription factor NsdC is named after, and is best known for, its essential role in sexual reproduction ( n ever in s exual d evelopment). 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. IMPORTANCE Aspergillus 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., (Copyright © 2021 Alves de Castro et al.)

All living beings have an optimal temperature for growth and survival. With the advancement of global warming, the search for understanding adaptive processes to climate changes has gained prominence. In this context, all living beings monitor the external temperature and develop adaptive responses to thermal variations. These responses ultimately change the functioning of the cell and affect the most diverse structures and processes. One of the first structures to detect thermal variations is the plasma membrane, whose constitution allows triggering of intracellular signals that assist in the response to temperature stress. Although studies on this topic have been conducted, the underlying mechanisms of recognizing thermal changes and modifying cellular functioning to adapt to this condition are not fully understood. Recently, many reports have indicated the participation of sphingolipids (SLs), major components of the plasma membrane, in the regulation of the thermal stress response. SLs can structurally reinforce the membrane or/and send signals intracellularly to control numerous cellular processes, such as apoptosis, cytoskeleton polarization, cell cycle arresting and fungal virulence. In this review, we discuss how SLs synthesis changes during both heat and cold stresses, focusing on fungi, plants, animals and human cells. The role of lysophospholipids is also discussed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The use of thermotolerant yeast strains can improve the efficiency of ethanol fermentation, allowing fermentation to occur at temperatures higher than 40 °C. This characteristic could benefit traditional bio-ethanol production and allow simultaneous saccharification and fermentation (SSF) of starch or lignocellulosic biomass., Results: We identified and characterized the physiology of a new thermotolerant strain (LBGA-01) able to ferment at 40 °C, which is more resistant to stressors as sucrose, furfural and ethanol than CAT-1 industrial strain. Furthermore, this strain showed similar CAT-1 resistance to acetic acid and lactic acid, and it was also able to change the pattern of genes involved in sucrose assimilation ( SUC2 and AGT1 ). Genes related to the production of proteins involved in secondary products of fermentation were also differentially regulated at 40 °C, with reduced expression of genes involved in the formation of glycerol ( GPD2 ), acetate ( ALD6 and ALD4 ), and acetyl-coenzyme A synthetase 2 ( ACS2 ). Fermentation tests using chemostats showed that LBGA-01 had an excellent performance in ethanol production in high temperature., Conclusion: The thermotolerant LBGA-01 strain modulates the production of key genes, changing metabolic pathways during high-temperature fermentation, and increasing its resistance to high concentration of ethanol, sugar, lactic acid, acetic acid, and furfural. Results indicate that this strain can be used to improve first- and second-generation ethanol production in Brazil., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

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 Δ gprM and Δ gprJ 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 Δ gprM and Δ gprJ 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 Δ gprM and Δ gprJ 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., (Copyright © 2020 Filho et al.)

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 c aspofungin p aradoxical e ffect (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 Δ fhdA 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 Δ fhdA 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 c aspofungin p aradoxical e ffect (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., (Copyright © 2020 Valero et al.)

Serratia marcescens has emerged as an important opportunistic pathogen responsible for nosocomial and severe infections. Here, we determined phenotypic and molecular characteristics of 54 S. marcescens isolates obtained from patient samples from intensive-care-unit (ICU) and neonatal intensive-care-unit (NIUC) of a Brazilian tertiary hospital. All isolates were resistant to beta-lactam group antibiotics, and 92.6% (50/54) were not susceptible to tigecycline. Furthermore, 96.3% showed intrinsic resistance to polymyxin E (colistin), a last-resort antibiotic for the treatment of infections caused by MDR (multidrug-resistant) Gram-negative bacteria. In contrast, high susceptibility to other antibiotics such as fluoroquinolones (81.5%), and to aminoglycosides (as gentamicin 81.5%, and amikacin 85.2%) was found. Of all isolates, 24.1% were classified as MDR. The presence of resistance and virulence genes were examined by PCR and sequencing. All isolates carried KPC-carbapenemase ( bla KPC ) and extended spectrum beta-lactamase bla TEM genes, 14.8% carried bla OXA- 1 , and 16.7% carried bla F that are associated with efflux pump mediated drug extrusion to fluoroquinolones and tigecycline, respectively, were found in 88.9%. The aac(6')-Ib-cr variant gene that can simultaneously induce resistance to aminoglycoside and fluoroquinolone was present in 24.1% of the isolates. Notably, the virulence genes to (i) pore-forming toxin ( CTX-M- 1 group genes, suggesting that bacterial resistance to β-lactam antibiotics found may be associated with these genes. The genes Sde B/ Has F and Sde Y/ Has F that are associated with efflux pump mediated drug extrusion to fluoroquinolones and tigecycline, respectively, were found in 88.9%. The aac(6')-Ib-cr variant gene that can simultaneously induce resistance to aminoglycoside and fluoroquinolone was present in 24.1% of the isolates. Notably, the virulence genes to (i) pore-forming toxin ( Shl A); (ii) phospholipase with hemolytic and cytolytic activities ( Phl A); (iii) flagellar transcriptional regulator ( Flh D); and (iv) positive regulator of prodigiosin and serratamolide production ( Pig P) were present in 98.2%. The genetic relationship among the isolates determined by ERIC-PCR demonstrated that the vast majority of isolates were grouped in a single cluster with 86.4% genetic similarity. In addition, many isolates showed 100% genetic similarity to each other, suggesting that the S. marcescens that circulate in this ICU are closely related. Our results suggest that the antimicrobial resistance to many drugs currently used to treat ICU and NIUC patients, associated with the high frequency of resistance and virulence genes is a worrisome phenomenon. Our findings emphasize the importance of active surveillance plans for infection control and to prevent dissemination of these strains., (Copyright © 2020 Ferreira, Rezende, Damas, Oliveira-Silva, Pitondo-Silva, Brito, Leonardecz, Góes, Campanini, Malavazi, da Cunha and Pranchevicius.)

Celiac disease (CD) is a chronic illness characterized by an inflammatory process triggered by gluten protein intake. Recent evidence has suggested that the lower relative abundance of bifidobacteria in the intestinal lumen may be associated with CD. Herein, we assessed the effect of the Bifidobacterium species Bifidobacterium bifidum , Bifidobacterium longum , Bembidion breve , Bifidobacterium animalis alone, and also a Bifidobacterium consortium on the digestion of intact gluten proteins (gliadins and glutenins) and the associated immunomodulatory responses elicited by the resulting peptides. The cytotoxicity and proinflammatory responses were evaluated through the activation of NF-kB p65 and the expression of cytokines TNF-α and IL-1β in Caco-2 cell cultures exposed to gluten-derived peptides. The peptides induced a clear reduction in cytotoxic responses and proinflammatory marker levels compared to the gluten fragments generated during noninoculated gastrointestinal digestion. These results highlight the possible use of probiotics based on bifidobacteria as a prospective treatment for CD.

Aspergillus fumigatus is a major cause of human disease. The survival of this fungus is dependent on the cell wall organization and function of its components. The cell wall integrity pathway (CWIP) is the primary signaling cascade that controls de novo synthesis of the cell wall in fungi. Abundant conidiation is a hallmark in A. fumigatus , and uptake of conidia by a susceptible host is usually the initial event in infection. The formation of conidia is mediated by the development of fungus-specific specialized structures, conidiophores, which are accompanied by cell wall remodeling. The molecular regulation of these changes in cell wall composition required for the rise of conidiophore from the solid surface and to disperse the conidia into the air is currently unknown. Here, we investigated the role of CWIP in conidiation. We show that CWIP pkcA G579R , Δ mpkA , and Δ rlmA mutants displayed reduced conidiation during synchronized asexual differentiation. The transcription factor RlmA directly regulated the expression of regulators of conidiation, including flbB , flbC , brlA , abaA , and rasB , as well as genes involved in cell wall synthesis and remodeling, and this affected the chitin content in aerial hyphae. Phosphorylation of RlmA and MpkA was increased during asexual differentiation. We also observed that MpkA physically associated with the proteins FlbB, FlbC, BrlA, and RasB during this process, suggesting another level of cross talk between the CWIP and asexual development pathways. In summary, our results support the conclusion that one function of the CWIP is the regulation of asexual development in filamentous fungi. IMPORTANCE A remarkable feature of the human pathogen Aspergillus fumigatus is its ability to produce impressive amounts of infectious propagules known as conidia. These particles reach immunocompromised patients and may initiate a life-threatening mycosis. The conidiation process in Aspergillus is governed by a sequence of proteins that coordinate the development of conidiophores. This process requires the remodeling of the cell wall so that the conidiophores can rise and withstand the chains of conidia. The events regulating cell wall remodeling during conidiation are currently unknown. Here, we show that the cell wall integrity pathway (CWIP) components RlmA and MpkA directly contribute to the activation of the conidiation cascade by enabling transcription or phosphorylation of critical proteins involved in asexual development. This study points to an essential role for the CWIP during conidiation and provides further insights into the complex regulation of asexual development in filamentous fungi., (Copyright © 2020 American Society for Microbiology.)

The cell wall (CW) and plasma membrane are fundamental structures that define cell shape and support different cellular functions. In pathogenic fungi, such as Aspegillus fumigatus, they not only play structural roles but are also important for virulence and immune recognition. Both the CW and the plasma membrane remain as attractive drug targets to treat fungal infections, such as the Invasive Pulmonary Aspergillosis (IPA), a disease associated with high morbimortality in immunocompromised individuals. The low efficiency of echinocandins that target the fungal CW biosynthesis, the occurrence of environmental isolates resistant to azoles such as voriconazole and the known drawbacks associated with amphotericin toxicity foster the urgent need for fungal-specific drugable targets and/or more efficient combinatorial therapeutic strategies. Reverse genetic approaches in fungi unveil that perturbations of the CW also render cells with increased susceptibility to membrane disrupting agents and vice-versa. However, how the fungal cells simultaneously cope with perturbation in CW polysaccharides and cell membrane proteins to allow morphogenesis is scarcely known. Here, we focus on current information on how the main signaling pathways that maintain fungal cell wall integrity, such as the Cell Wall Integrity and the High Osmolarity Glycerol pathways, in different species often cross-talk to regulate the synthesis of molecules that comprise the plasma membrane, especially sphingolipids, ergosterol and phospholipids to promote functioning of both structures concomitantly and thus, cell viability. We propose that the conclusions drawn from other organisms are the foundations to point out experimental lines that can be endeavored in A. fumigatus., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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., Competing Interests: The authors have declared that no competing interests exist.

[This corrects the article DOI: 10.3389/fmicb.2019.02008.]., (Copyright © 2019 Souza, Baltazar, Carregal, Gouveia-Eufrasio, Oliveira, Dias, Rocha, Miranda, Malavazi, Santos, Frézard, Souza, Teixeira and Soriani.)

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.

Beta-thalassaemia (BT) is classified according to blood transfusion requirement as minor (BTMi), intermedia (BTI) and major (BTM). BTM is the most severe form, requiring regular transfusions while transfusion need is only occasional in BTI. Differential gene expression between patients has not been assessed so far. Here, we evaluated the global gene expression profiles during differentiation of human erythroid cells of two patients carrying the same mutation [CD39, (C → T)], though displaying different phenotypes (BTI and BTM). Considering the role of reactive oxygen species (ROS) in the pathophysiology of thalassaemia, we focused on differentially expressed genes involved in metabolic pathways triggered by ROS, such as inflammation and apoptosis, and, from these, we selected the Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) and High Mobility Group Box1 (HMGB1) genes, whose role in BT is not well established. An in-depth expression analysis of transcriptional and protein levels in patients carrying a range of mutations associated with BT phenotypes indicated that APEX1 was increased in both BTI and BTM. Furthermore, higher amounts of HMGB1 was found in the plasma of BTI patients. Our findings suggest that these proteins have important roles in BT and could represent new targets for further studies aiming to improve the management of the disease., (© 2019 British Society for Haematology and John Wiley & Sons Ltd.)

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 Δ msbA 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., (Copyright © 2019 Gurgel et al.)