SMAD2 and SMAD3 differentially regulate adiposity and the growth of subcutaneous white adipose tissue.

Adipose tissue is the primary site of energy storage, playing important roles in health. While adipose research largely focuses on obesity, fat also has other critical functions, producing adipocytokines and contributing to normal nutrient metabolism, which in turn play important roles in satiety and total energy homeostasis. SMAD2/3 proteins are downstream mediators of activin signaling, which regulate critical preadipocyte and mature adipocyte functions. Smad2 global knockout mice exhibit embryonic lethality, whereas global loss of Smad3 protects mice against diet-induced obesity. The direct contributions of Smad2 and Smad3 in adipose tissues, however, are unknown. Here, we sought to determine the primary effects of adipocyte-selective reduction of Smad2 or Smad3 on diet-induced adiposity using Smad2 or Smad3 "floxed" mice intercrossed with Adiponectin-Cre mice. Additionally, we examined visceral and subcutaneous preadipocyte differentiation efficiency in vitro. Almost all wild type subcutaneous preadipocytes differentiated into mature adipocytes. In contrast, visceral preadipocytes differentiated poorly. Exogenous activin A suppressed differentiation of preadipocytes from both depots. Smad2 conditional knockout (Smad2cKO) mice did not exhibit significant effects on weight gain, irrespective of diet, whereas Smad3 conditional knockout (Smad3cKO) male mice displayed a trend of reduced body weight on high-fat diet. On both diets, Smad3cKO mice displayed an adipose depot-selective phenotype, with a significant reduction in subcutaneous fat mass but not visceral fat mass. Our data suggest that Smad3 is an important contributor to the maintenance of subcutaneous white adipose tissue in a sex-selective fashion. These findings have implications for understanding SMAD-mediated, depot selective regulation of adipocyte growth and differentiation.

Immune checkpoint blockade reprograms systemic immune landscape and tumor microenvironment in obesity-associated breast cancer.

Immune checkpoint blockade (ICB) has improved outcomes in some cancers. A major limitation of ICB is that most patients fail to respond, which is partly attributable to immunosuppression. Obesity appears to improve immune checkpoint therapies in some cancers, but impacts on breast cancer (BC) remain unknown. In lean and obese mice, tumor progression and immune reprogramming were quantified in BC tumors treated with anti-programmed death-1 (PD-1) or control. Obesity augments tumor incidence and progression. Anti-PD-1 induces regression in lean mice and potently abrogates progression in obese mice. BC primes systemic immunity to be highly responsive to obesity, leading to greater immunosuppression, which may explain greater anti-PD-1 efficacy. Anti-PD-1 significantly reinvigorates antitumor immunity despite persistent obesity. Laminin subunit beta-2 (Lamb2), downregulated by anti-PD-1, significantly predicts patient survival. Lastly, a microbial signature associated with anti-PD-1 efficacy is identified. Thus, anti-PD-1 is highly efficacious in obese mice by reinvigorating durable antitumor immunity. VIDEO ABSTRACT.

TGR5 signaling mitigates parenteral nutrition-associated liver disease.

Bile acid receptors regulate the metabolic and immune functions of circulating enterohepatic bile acids. This process is disrupted by administration of parenteral nutrition (PN), which may induce progressive hepatic injury for unclear reasons, especially in the newborn, leading to PN-associated liver disease. To explore the role of bile acid signaling on neonatal hepatic function, we initially observed that Takeda G protein receptor 5 (TGR5)-specific bile acids were negatively correlated with worsening clinical disease markers in the plasma of human newborns with prolonged PN exposure. To test our resulting hypothesis that TGR5 regulates critical liver functions to PN exposure, we used TGR5 receptor deficient mice (TGR5-/-). We observed PN significantly increased liver weight, cholestasis, and serum hepatic stress enzymes in TGR5-/- mice compared with controls. Mechanistically, PN reduced bile acid synthesis genes in TGR5-/-. Serum bile acid composition revealed that PN increased unconjugated primary bile acids and secondary bile acids in TGR5-/- mice, while increasing conjugated primary bile acid levels in TGR5-competent mice. Simultaneously, PN elevated hepatic IL-6 expression and infiltrating macrophages in TGR5-/- mice. However, the gut microbiota of TGR5-/- mice compared with WT mice following PN administration displayed highly elevated levels of Bacteroides and Parabacteroides, and possibly responsible for the elevated levels of secondary bile acids in TGR5-/- animals. Intestinal bile acid transporters expression was unchanged. Collectively, this suggests TGR5 signaling specifically regulates fundamental aspects of liver bile acid homeostasis during exposure to PN. Loss of TGR5 is associated with biochemical evidence of cholestasis in both humans and mice on PN.NEW & NOTEWORTHY Parenteral nutrition is associated with deleterious metabolic outcomes in patients with prolonged exposure. Here, we demonstrate that accelerated cholestasis and parental nutrition-associated liver disease (PNALD) may be associated with deficiency of Takeda G protein receptor 5 (TGR5) signaling. The microbiome is responsible for production of secondary bile acids that signal through TGR5. Therefore, collectively, these data support the hypothesis that a lack of established microbiome in early life or under prolonged parenteral nutrition may underpin disease development and PNALD.

Differential requirements of protein geranylgeranylation for the virulence of human pathogenic fungi.

Protein prenylation is a crucial post-translational modification largely mediated by two heterodimeric enzyme complexes, farnesyltransferase and geranylgeranyltransferase type-I (GGTase-I), each composed of a shared α-subunit and a unique ß-subunit. GGTase-I enzymes are validated drug targets that contribute to virulence in Cryptococcus neoformans and to the yeast-to-hyphal transition in Candida albicans. Therefore, we sought to investigate the importance of the α-subunit, RamB, and the ß-subunit, Cdc43, of the A. fumigatus GGTase-I complex to hyphal growth and virulence. Deletion of cdc43 resulted in impaired hyphal morphogenesis and thermo-sensitivity, which was exacerbated during growth in rich media. The Δcdc43 mutant also displayed hypersensitivity to cell wall stress agents and to cell wall synthesis inhibitors, suggesting alterations of cell wall biosynthesis or stress signaling. In support of this, analyses of cell wall content revealed decreased amounts of ß-glucan in the Δcdc43 strain. Despite strong in vitro phenotypes, the Δcdc43 mutant was fully virulent in two models of murine invasive aspergillosis, similar to the control strain. We further found that a strain expressing the α-subunit gene, ramB, from a tetracycline-inducible promoter was inviable under non-inducing in vitro growth conditions and was virtually avirulent in both mouse models. Lastly, virulence studies using C. albicans strains with tetracycline-repressible RAM2 or CDC43 expression revealed reduced pathogenicity associated with downregulation of either gene in a murine model of disseminated infection. Together, these findings indicate a differential requirement for protein geranylgeranylation for fungal virulence, and further inform the selection of specific prenyltransferases as promising antifungal drug targets for each pathogen.

SH3-class Ras guanine nucleotide exchange factors are essential for Aspergillus fumigatus invasive growth.

Proper hyphal morphogenesis is essential for the establishment and progression of invasive disease caused by filamentous fungi. In the human pathogen Aspergillus fumigatus, signalling cascades driven by Ras and Ras-like proteins orchestrate a wide variety of cellular processes required for hyphal growth. For activation, these proteins require interactions with Ras-subfamily-specific guanine nucleotide exchange factors (RasGEFs). Although Ras-protein networks are essential for virulence in all pathogenic fungi, the importance of RasGEF proteins is largely unexplored. A. fumigatus encodes four putative RasGEFs that represent three separate classes of RasGEF proteins (SH3-, Ras guanyl nucleotide-releasing protein [RasGRP]-, and LTE-class), each with fungus-specific attributes. Here, we show that the SH3-class and RasGRP-class RasGEFs are required for properly timed polarity establishment during early growth and branch emergence as well as for cell wall stability. Further, we show that SH3-class RasGEF activity is essential for polarity establishment and maintenance, a phenotype that is, at least, partially independent of the major A. fumigatus Ras proteins, RasA and RasB. Finally, loss of both SH3-class RasGEFs resulted in avirulence in multiple models of invasive aspergillosis. Together, our findings suggest that RasGEF activity is essential for the integration of multiple signalling networks to drive invasive growth in A. fumigatus.

Whole-genome sequencing reveals highly specific gene targeting by in vitro assembled Cas9-ribonucleoprotein complexes in Aspergillus fumigatus.

BACKGROUND: CRISPR/Cas9-based genome editing is quickly becoming a powerful tool within the field of fungal genetics. Adaptation of CRISPR/Cas9 systems are allowing for rapid and highly efficient gene targeting within fungi. We recently reported the adaptation of a simple CRISPR/Cas9 system for gene deletion that is effective across multiple genetic backgrounds of Aspergillus fumigatus. This system employs in vitro assembly of Cas9 ribonucleoproteins (RNPs) coupled with micro-homology repair templates for gene deletion. Although highly efficient at gene targeting in wild type genetic backgrounds of A. fumigatus, the potential for our system to produce unwanted off-target mutations has not been addressed. RESULTS: Next-generation Illumina sequencing was used to identify genome mutations among transformants isolated from standard (no Cas9) and Cas9-mediated integration of a hygromycin deletion cassette. Two different concentrations of Cas9 were utilized to examine the association of Cas9 concentration with total numbers and types of genomic mutations. For each of the three test groups (zero, low, and high Cas9), three transformants were sequenced and compared to the parent strain. Bioinformatics analyses revealed the average number of total mutations to be similar among all three test groups. A. fumigatus transformation using standard, non-Cas9-mediated methods resulted in an average of 373 ± 28 mutations. In comparison, transformation with in vitro assembled Cas9-RNPs using either high (1 µg/µl) or low (0.5 µg/µl) levels of Cas9 resulted in an average of 326 ± 19 and 395 ± 69 mutations, respectively. In all cases, the vast majority of mutations identified were intergenic. No correlation between the amount of Cas9 utilized for transformation and the overall number of mutations was found. Finally, the specific type of mutation introduced during the transformation process was not Cas9-dependent, as both single-nucleotide polymorphisms and insertion/deletion events were not significantly different between the experimental groups. CONCLUSIONS: CRISPR/Cas9-based genome editing in A. fumigatus using in vitro assembled RNPs coupled with microhomology templates is a reliable method of gene targeting. This system is highly efficient and is not associated with increased off-target mutations caused by introduction of the Cas9 nuclease.

C-terminus Proteolysis and Palmitoylation Cooperate for Optimal Plasma Membrane Localization of RasA in Aspergillus fumigatus.

RasA is a major regulator of fungal morphogenesis and virulence in Aspergillus fumigatus. The proper localization of RasA to the plasma membrane is essential for the formation of invasive hyphae during infection. In yeast, the localization of Ras2p to the plasma membrane is orchestrated by several post-translational modifications (PTM) at the C-terminal CAAX box that are thought to occur in sequential order. These PTMs include: (1) CAAX motif farnesylation by the farnesyltransferase complex composed of Ram1p and Ram2p; (2) proteolysis of the -AAX residues by Rce1p or Ste24p; (3) methylation of the remaining prenylated cysteine residue by Ste14p, and; (4) palmitoylation at a single conserved cysteine residue mediated by the Erf2p/Erf4p palmitoyltransferase. We previously reported that homologs of each RasA PTM enzyme are conserved in A. fumigatus. Additionally, we delineated a major role for protein farnesylation in A. fumigatus growth and virulence. In this work, we characterize the post-prenylation processing enzymes of RasA in A. fumigatus. The genes encoding the RasA post-prenylation enzymes were first deleted and examined for their roles in growth and regulation of RasA. Only when strains lacked cppB, the A. fumigatus homologue of yeast RCE1, there was a significant reduction in fungal growth and conidial germination. In addition, cppB-deletion mutants displayed hypersensitivity to the cell wall-perturbing agents Calcofluor White and Congo Red and the cell wall biosynthesis inhibitor Caspofungin. In contrast to the previously published data in yeast, the deletion of post-prenylation modifying enzymes did not alter the plasma membrane localization or activation of RasA. To delineate the molecular mechanisms underlying these differences, we investigated the interplay between dual-palmitoylation of the RasA hypervariable region and CAAX proteolysis for stabilization of RasA at the plasma membrane. Our data indicate that, in the absence of proper CAAX proteolysis, RasA accumulation at the plasma membrane is stabilized by dual palmitoyl groups on the dual cysteine residues. Therefore, we conclude CAAX proteolysis and dual-palmitoylation of the hypervariable region is important for maintaining a stable attachment association of RasA with the plasma membrane to support optimal fungal growth and development.

A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates.

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is a novel genome-editing system that has been successfully established in Aspergillus fumigatus. However, the current state of the technology relies heavily on DNA-based expression cassettes for delivering Cas9 and the guide RNA (gRNA) to the cell. Therefore, the power of the technology is limited to strains that are engineered to express Cas9 and gRNA. To overcome such limitations, we developed a simple and universal CRISPR-Cas9 system for gene deletion that works across different genetic backgrounds of A. fumigatus. The system employs in vitro assembly of dual Cas9 ribonucleoproteins (RNPs) for targeted gene deletion. Additionally, our CRISPR-Cas9 system utilizes 35 to 50 bp of flanking regions for mediating homologous recombination at Cas9 double-strand breaks (DSBs). As a proof of concept, we first tested our system in the ΔakuB (ΔakuBku80 ) laboratory strain and generated high rates (97%) of gene deletion using 2 µg of the repair template flanked by homology regions as short as 35 bp. Next, we inspected the portability of our system across other genetic backgrounds of A. fumigatus, namely, the wild-type strain Af293 and a clinical isolate, A. fumigatus DI15-102. In the Af293 strain, 2 µg of the repair template flanked by 35 and 50 bp of homology resulted in highly efficient gene deletion (46% and 74%, respectively) in comparison to classical gene replacement systems. Similar deletion efficiencies were also obtained in the clinical isolate DI15-102. Taken together, our data show that in vitro-assembled Cas9 RNPs coupled with microhomology repair templates are an efficient and universal system for gene manipulation in A. fumigatus. IMPORTANCE Tackling the multifactorial nature of virulence and antifungal drug resistance in A. fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. Our simple and universal CRISPR-Cas9 system for gene manipulation generates efficient gene targeting across different genetic backgrounds of A. fumigatus. We anticipate that our system will simplify genome editing in A. fumigatus, allowing for the generation of single- and multigene knockout libraries. In addition, our system will facilitate the delineation of virulence factors and antifungal drug resistance genes in different genetic backgrounds of A. fumigatus.

The Aspergillus fumigatus farnesyltransferase ß-subunit, RamA, mediates growth, virulence, and antifungal susceptibility.

Post-translational prenylation mechanisms, including farnesylation and geranylgeranylation, mediate both subcellular localization and protein-protein interaction in eukaryotes. The prenyltransferase complex is an αß heterodimer in which the essential α-subunit is common to both the farnesyltransferase and the geranylgeranyltransferase type-I enzymes. The ß-subunit is unique to each enzyme. Farnesyltransferase activity is an important mediator of protein localization and subsequent signaling for multiple proteins, including Ras GTPases. Here, we examined the importance of protein farnesylation in the opportunistic fungal pathogen Aspergillus fumigatus through generation of a mutant lacking the farnesyltransferase ß-subunit, ramA. Although farnesyltransferase activity was found to be non-essential in A. fumigatus, diminished hyphal outgrowth, delayed polarization kinetics, decreased conidial viability, and irregular distribution of nuclei during polarized growth were noted upon ramA deletion (ΔramA). Although predicted to be a target of the farnesyltransferase enzyme complex, we found that localization of the major A. fumigatus Ras GTPase protein, RasA, was only partially regulated by farnesyltransferase activity. Furthermore, the farnesyltransferase-deficient mutant exhibited attenuated virulence in a murine model of invasive aspergillosis, characterized by decreased tissue invasion and development of large, swollen hyphae in vivo. However, loss of ramA also led to a Cyp51A/B-independent increase in resistance to triazole antifungal drugs. Our findings indicate that protein farnesylation underpins multiple cellular processes in A. fumigatus, likely due to the large body of proteins affected by ramA deletion.

A Fungus-Specific Protein Domain Is Essential for RasA-Mediated Morphogenetic Signaling in Aspergillus fumigatus.

Ras proteins function as conserved regulators of eukaryotic growth and differentiation and are essential signaling proteins orchestrating virulence in pathogenic fungi. Here, we report the identification of a novel N-terminal domain of the RasA protein in the filamentous fungus Aspergillus fumigatus. Whereas this domain is absent in Ras homologs of higher eukaryotes, the N-terminal extension is conserved among fungi and is characterized by a short string of two to eight amino acids terminating in an invariant arginine. For this reason, we have termed the RasA N-terminal domain the invariant arginine domain (IRD). Through mutational analyses, the IRD was found to be essential for polarized morphogenesis and asexual development, with the invariant arginine residue being most essential. Although IRD truncation resulted in a nonfunctional Ras phenotype, IRD mutation was not associated with mislocalization of the RasA protein or significant changes in steady-state RasA activity levels. Mutation of the RasA IRD diminished protein kinase A (PKA) activation and resulted in decreased interaction with the Rho-type GTPase, Cdc42. Taken together, our findings reveal novel, fungus-specific mechanisms for Ras protein function and signal transduction. IMPORTANCEAspergillus fumigatus is an important fungal pathogen against which limited treatments exist. During invasive disease, A. fumigatus hyphae grow in a highly polarized fashion, forming filaments that invade blood vessels and disseminate to distant sites. Once invasion and dissemination occur, mortality rates are high. We have previously shown that the Ras signaling pathway is an important regulator of the hyphal growth machinery supporting virulence in A. fumigatus. Here, we show that functional Ras signaling in A. fumigatus requires a novel, fungus-specific domain within the Ras protein. This domain is highly conserved among fungi, yet absent in higher eukaryotes, suggesting a potentially crucial difference in the regulation of Ras pathway activity between the human host and the fungal pathogen. Exploration of the mechanisms through which this domain regulates signaling could lead to novel antifungal therapies specifically targeting fungal Ras pathways.

The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps.

Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs.

Exploration of Aspergillus fumigatus Ras pathways for novel antifungal drug targets.

Ras pathway signaling is a critical virulence determinant for pathogenic fungi. Localization of Ras to the plasma membrane (PM) is required for Ras network interactions supporting fungal growth and virulence. For example, loss of Aspergillus fumigatus RasA signaling at the PM via inhibition of palmitoylation leads to decreased growth, altered hyphal morphogenesis, decreased cell wall integrity and loss of virulence. In order to be properly localized and activated, Ras proteins must transit a series of post-translational modification (PTM) steps. These steps include farnesylation, proteolytic cleavage of terminal amino acids, carboxymethylation, and palmitoylation. Because Ras activation drives tumor development, Ras pathways have been extensively studied in mammalian cells as a potential target for anti-cancer therapy. Inhibitors of mammalian Ras interactions and PTM components have been, or are actively being, developed. This review will focus on the potential for building upon existing scaffolds to exploit fungal Ras proteins for therapy, synthesizing data from studies employing both mammalian and fungal systems.

Aspergillus galactosaminogalactan mediates adherence to host constituents and conceals hyphal ß-glucan from the immune system.

Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall ß-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis.

A conserved C-terminal domain of the Aspergillus fumigatus developmental regulator MedA is required for nuclear localization, adhesion and virulence.

MedA is a developmental regulator that is conserved in the genome of most filamentous fungi. In the pathogenic fungus Aspergillus fumigatus MedA regulates conidiogenesis, adherence to host cells, and pathogenicity. The mechanism by which MedA governs these phenotypes remains unknown. Although the nuclear import of MedA orthologues has been reported in other fungi, no nuclear localization signal, DNA-binding domain or other conserved motifs have been identified within MedA. In this work, we performed a deletion analysis of MedA and identified a novel domain within the C-terminal region of the protein, designated MedA(346-557), that is necessary and sufficient for nuclear localization of MedA. We further demonstrate that MedA nuclear localization is required for the function of MedA. Surprisingly, expression of the minimal nuclear localization fragment MedA(346-557) alone was sufficient to restore conidogenesis, biofilm formation and virulence to the medA mutant strain. Collectively these results suggest that MedA functions in the regulation of transcription, and that the MedA(346-557) domain is both necessary and sufficient to mediate MedA function.

Role of Aspergillus niger acrA in arsenic resistance and its use as the basis for an arsenic biosensor.

Arsenic contamination of groundwater sources is a major issue worldwide, since exposure to high levels of arsenic has been linked to a variety of health problems. Effective methods of detection are thus greatly needed as preventive measures. In an effort to develop a fungal biosensor for arsenic, we first identified seven putative arsenic metabolism and transport genes in Aspergillus niger, a widely used industrial organism that is generally regarded as safe (GRAS). Among the genes tested for RNA expression in response to arsenate, acrA, encoding a putative plasma membrane arsenite efflux pump, displayed an over 200-fold increase in gene expression in response to arsenate. We characterized the function of this A. niger protein in arsenic efflux by gene knockout and confirmed that AcrA was located at the cell membrane using an enhanced green fluorescent protein (eGFP) fusion construct. Based on our observations, we developed a putative biosensor strain containing a construct of the native promoter of acrA fused with egfp. We analyzed the fluorescence of this biosensor strain in the presence of arsenic using confocal microscopy and spectrofluorimetry. The biosensor strain reliably detected both arsenite and arsenate in the range of 1.8 to 180 µg/liter, which encompasses the threshold concentrations for drinking water set by the World Health Organization (10 and 50 µg/liter).

ParAB-mediated intermolecular association of plasmid P1 parS sites.

The P1 plasmid partition system depends on ParA-ParB proteins acting on centromere-like parS sites for a faithful plasmid segregation during the Escherichia coli cell cycle. In vivo we placed parS into host E. coli chromosome and on a Sop(+) F plasmid and found that the stability of a P1 plasmid deleted for parA-parB could be partially restored when parB was expressed in trans. In vitro, parS, conjugated to magnetic beads could capture free parS DNA fragment in presence of ParB. In vitro, ParA stimulated ParB-mediated association of intermolecular parS sites in an ATP-dependent manner. However, in the presence of ADP, ParA reduced ParB-mediated pairing to levels below that seen by ParB alone. ParB of P1 pairs the parS sites of plasmids in vivo and fragments in vitro. Our findings support a model whereby ParB complexes P1 plasmids, ParA-ATP stimulates this interaction and ParA-ADP inhibits ParB pairing activity in a parS-independent manner.

The CCAAT-binding complex coordinates the oxidative stress response in eukaryotes.

The heterotrimeric CCAAT-binding complex is evolutionary conserved in eukaryotic organisms. The corresponding Aspergillus nidulans CCAAT-binding factor (AnCF) consists of the subunits HapB, HapC and HapE. All of the three subunits are necessary for DNA binding. Here, we demonstrate that AnCF senses the redox status of the cell via oxidative modification of thiol groups within the histone fold motif of HapC. Mutational and in vitro interaction analyses revealed that two of these cysteine residues are indispensable for stable HapC/HapE subcomplex formation and high-affinity DNA binding of AnCF. Oxidized HapC is unable to participate in AnCF assembly and localizes in the cytoplasm, but can be recycled by the thioredoxin system in vitro and in vivo. Furthermore, deletion of the hapC gene led to an impaired oxidative stress response. Therefore, the central transcription factor AnCF is regulated at the post-transcriptional level by the redox status of the cell serving for a coordinated activation and deactivation of antioxidative defense mechanisms including the specific transcriptional activator NapA, production of enzymes such as catalase, thioredoxin or peroxiredoxin, and maintenance of a distinct glutathione homeostasis. The underlying fine-tuned mechanism very likely represents a general feature of the CCAAT-binding complexes in eukaryotes.

Aspects on evolution of fungal beta-lactam biosynthesis gene clusters and recruitment of trans-acting factors.

Penicillins and cephalosporins are beta-lactam antibiotics. The formation of hydrophobic penicillins has been reported in fungi only, notably Penicillium chrysogenum and Aspergillus (Emericella) nidulans, whereas the hydrophilic cephalosporins are produced by both fungi, e.g., Acremonium chrysogenum (cephalosporin C), and bacteria. The producing bacteria include Gram-negatives and Gram-positives, e.g., Streptomyces clavuligerus (cephamycin C) and Lysobacter lactamgenus (cephabacins), respectively. The evolutionary origin of beta-lactam biosynthesis genes has been the subject of discussion for many years, and two main hypotheses have been proposed: (i) horizontal gene transfer (HGT) from bacteria to fungi or (ii) vertical decent. There are strong arguments in favour of HGT, e.g., unlike most other fungal genes, beta-lactam biosynthesis genes are clustered and some of these genes lack introns. In contrast to S. clavuligerus, all regulators of fungal beta-lactam biosynthesis genes represent wide-domain regulators that are not part of the gene cluster. If bacterial regulators were co-transferred with the gene cluster from bacteria to fungi, most likely they would have been non-functional in eukaryotes and lost during evolution. Recently, the penicillin biosynthesis gene aatB was discovered, which is not part of the penicillin biosynthesis gene cluster and is even located on a different chromosome. The aatB gene is regulated by the same regulators AnCF and AnBH1 as the penicillin biosynthesis gene aatA (penDE). Data suggest that aatA and aatB are paralogues derived by duplication of a common ancestor gene. This data supports a model in which part of the beta-lactam biosynthesis gene cluster was transferred to some fungi, i.e., the acvA and ipnA gene without a regulatory gene. We propose that during the assembly of aatA and acvA-ipnA into a single gene cluster, recruitment of transcriptional regulators occurred along with acquisition of the duplicated aatA ancestor gene and its cis-acting sites.

A chimeric plum pox virus shows reduced spread and cannot compete with its parental wild-type viruses in a mixed infection.

The effect of a recombination event in the genomic 3' end on the biological properties and competitiveness of plum pox virus (PPV) was investigated. Therefore, a fragment spanning the coat protein (CP) coding region and a part of the 3' non-translated region of a non-aphid-transmissible strain of PPV (PPV-NAT) was replaced by the corresponding region of a PPV sour cherry isolate (PPV-SoC). The resulting chimera (PPV-NAT/SoC) caused severe symptoms in Nicotiana benthamiana, resembling those of PPV-NAT. In mixed infections with either of the parental viruses, the chimera PPV-NAT/SoC was less competitive. Labelling experiments with DsRed showed that PPV-NAT/SoC (PPV-NAT/SoC-red) moved more slowly from cell to cell than PPV-NAT (PPV-NAT-red). In mixed infections of PPV-NAT/SoC-red with a green fluorescent protein-expressing PPV-NAT (PPV-NAT-AgfpS), spatial separation of the viruses was observed. These data suggest that, in PPV infections, symptom severity and competitiveness are independent aspects and that spatial separation may contribute to the displacement of a recombinant virus.

The thioredoxin system of the filamentous fungus Aspergillus nidulans: impact on development and oxidative stress response.

Redox regulation has been shown to be of increasing importance for many cellular processes. Here, redox homeostasis was addressed in Aspergillus nidulans, an important model organism for fundamental biological questions such as development, gene regulation or the regulation of the production of secondary metabolites. We describe the characterization of a thioredoxin system from the filamentous fungus A. nidulans. The A. nidulans thioredoxin A (AnTrxA) is an 11.6-kDa protein with a characteristic thioredoxin active site motif (WCGPC) encoded by the trxA gene. The corresponding thioredoxin reductase (AnTrxR), encoded by the trxR gene, represents a homodimeric flavoprotein with a native molecular mass of 72.2 kDa. When combined in vitro, the in Escherichia coli overproduced recombinant proteins AnTrxA and AnTrxR were able to reduce insulin and oxidized glutathione in an NADPH-dependent manner indicating that this in vitro redox system is functional. Moreover, we have created a thioredoxin A deletion strain that shows decreased growth, an increased catalase activity, and the inability to form reproductive structures like conidiophores or cleistothecia when cultivated under standard conditions. However, addition of GSH at low concentrations led to the development of sexual cleistothecia, whereas high GSH levels resulted in the formation of asexual conidiophores. Furthermore, by applying the principle of thioredoxin-affinity chromatography we identified several novel putative targets of thioredoxin A, including a hypothetical protein with peroxidase activity and an aldehyde dehydrogenase.