Immunoproteomic analysis of the serum IgG response to cell wall-associated proteins of Staphylococcus aureus strains belonging to CC97 and CC151.

CC97 and CC151 are two of the most common Staphylococcus aureus lineages associated with bovine intramammary infection. The genotype of the infecting S. aureus strain influences virulence and the progression of intramammary disease. Strains from CC97 and CC151 encode a distinct array of virulence factors. Identification of proteins elaborated in vivo will provide insights into the molecular mechanism of pathogenesis of these lineages, as well as facilitating the development of tailored treatments and pan-lineage vaccines and diagnostics. The repertoire of genes encoding cell wall-anchored (CWA) proteins was identified for S. aureus strains MOK023 (CC97) and MOK124 (CC151); MOK023 encoded more CWA proteins than MOK124. Serum collected during an in vivo challenge trial was used to investigate whether the humoral response to cell wall proteins was strain-specific. Immunoproteomic analysis demonstrated that the humoral response in MOK023-infected cows predominantly targeted high molecular weight proteins while the response in MOK124-infected cows targeted medium or low molecular weight proteins. Antigenic proteins were identified by two-dimensional serum blotting followed by mass spectometry-based identification of immunoreactive spots, with putative antigens subsequently validated. The CWA proteins ClfB, SdrE/Bbp and IsdA were identified as immunogenic regardless of the infecting strain. In addition, a number of putative strain-specific imunogens were identified. The variation in antigens produced by different strains may indicate that these strains have different strategies for exploiting the intramammary niche. Such variation should be considered when developing novel control strategies including vaccines, therapeutics and diagnostics.

Eating Disorders: Identification and Management in General Medical and Psychiatric Settings.

OBJECTIVE: Eating disorders (EDs) are serious, complex illnesses with both behavioral and physical health features. EDs have high rates of medical and psychiatric morbidity, and a 6% mortality rate, the highest of any mental illness. Early detection of EDs offers the best opportunity for recovery; yet, estimates are that as few as one in 10 individuals with an ED receive treatment. The purpose of this article is to provide an ED identification and management overview for inpatient nurse clinicians in general psychiatric and medical settings, helping to facilitate timely recognition and care. METHOD: An overview of ED diagnostic criteria and two evidence-based ED tools are introduced for consideration. RESULTS: Opportunities to identify and help manage an ED are numerous. Most individuals with an ED make several health care visits in either medical or psychiatric settings without ever being screened for an ED. General ED screening and assessment tool familiarization can facilitate a treatment trajectory for these patients, improve overall quality of life, and may potentially result in a life-saving intervention for this often-deadly cluster of medical and psychiatric disorders. CONCLUSION: Screening and assessment in general clinical settings, identifying patients with undiagnosed EDs, beginning basic treatment plans, and referrals for appropriate follow-up care, have the potential to reduce ED recidivism and related health care costs. Simultaneously, and most important, long-term outcomes for patients with EDs may improve.

The Aspergillus fumigatus transcription factor RglT is important for gliotoxin biosynthesis and self-protection, and virulence.

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.

Quantitative Proteomic Analysis Reveals Yeast Cell Wall Products Influence the Serum Proteome Composition of Broiler Chickens.

With an ever-growing market and continual financial pressures associated with the prohibition of antibiotic growth promoters, the poultry industry has had to rapidly develop non-antibiotic alternatives to increase production yields. A possible alternative is yeast and its derivatives, such as the yeast cell wall (YCW), which have been proposed to confer selected beneficial effects on the host animal. Here, the effect of YCW supplementation on the broiler chicken was investigated using a quantitative proteomic strategy, whereby serum was obtained from three groups of broilers fed with distinct YCW-based Gut Health Products (GHP) or a control basal diet. Development of a novel reagent enabled application of ProteoMiner™ technology for sample preparation and subsequent comparative quantitative proteomic analysis revealed proteins which showed a significant change in abundance (n = 167 individual proteins; p < 0.05); as well as proteins which were uniquely identified (n = 52) in, or absent (n = 37) from, GHP-fed treatment groups versus controls. An average of 7.1% of proteins showed changes in abundance with GHP supplementation. Several effects of these GHPs including immunostimulation (via elevated complement protein detection), potential alterations in the oxidative status of the animal (e.g., glutathione peroxidase and catalase), stimulation of metabolic processes (e.g., differential abundance of glyceraldehyde-3-phosphate dehydrogenase), as well as evidence of a possible hepatoprotective effect (attenuated levels of serum α-glutathione s-transferase) by one GHP feed supplement, were observed. It is proposed that specific protein detection may be indicative of GHP efficacy to stimulate broiler immune status, i.e., may be biomarkers of GHP efficacy. In summary, this work has developed a novel technology for the preparation of high dynamic range proteomic samples for LC-MS/MS analysis, is part of the growing area of livestock proteomics and, importantly, provides evidential support for beneficial effects that GHP supplementation has on the broiler chicken.

Proteomic profiling and glycomic analysis of the yeast cell wall in strains with Aflatoxin B1 elimination ability.

The use of microorganisms for Aflatoxin B1 elimination has been studied as a new alternative tool and it is known that cell wall carried out a critical role. For that reason, cell wall and soluble intracellular fraction of eight yeasts with AFB1 detoxification capability were analysed. The quantitative and qualitative comparative label-free proteomic allowed the identification of diverse common constituent proteins, which revealed that putative cell wall proteins entailed less than 10% of the total proteome. It was possible to characterize different enzymes linked to cell wall polysaccharides biosynthesis as well as other proteins related with the cell wall organization and regulation. Additionally, the concentration of the principal polysaccharides was determined which permitted us to observe that ß-glucans concentration was higher than mannans in most of the samples. In order to better understand the biosorption role of the cell wall against the AFB1 , an antimycotic (Caspofungin) was used to damage the cell wall structure. This assay allowed the observation of an effect on the normal growth of those yeasts with damaged cell walls that were exposed to AFB1 . This effect was not observed in yeast with intact cell walls, which may reveal a protective role of this structure against mycotoxins.

At the metal-metabolite interface in Aspergillus fumigatus: towards untangling the intersecting roles of zinc and gliotoxin.

Cryptic links between apparently unrelated metabolic systems represent potential new drug targets in fungi. Evidence of such a link between zinc and gliotoxin (GT) biosynthesis in Aspergillus fumigatus is emerging. Expression of some genes of the GT biosynthetic gene cluster gli is influenced by the zinc-dependent transcription activator ZafA, zinc may relieve GT-mediated fungal growth inhibition and, surprisingly, GT biosynthesis is influenced by zinc availability. In A. fumigatus, dithiol gliotoxin (DTG), which has zinc-chelating properties, is converted to either GT or bis-dethiobis(methylthio)gliotoxin (BmGT) by oxidoreductase GliT and methyltransferase GtmA, respectively. A double deletion mutant lacking both GliT and GtmA was previously observed to be hypersensitive to exogenous GT exposure. Here we show that compared to wild-type exposure, exogenous GT and the zinc chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) inhibit A. fumigatus ΔgliTΔgtmA growth, specifically under zinc-limiting conditions, which can be reversed by zinc addition. While GT biosynthesis is evident in zinc-depleted medium, addition of zinc (1 µM) suppressed GT and activated BmGT production. In addition, secretion of the unferrated siderophore, triacetylfusarinine C (TAFC), was evident by A. fumigatus wild-type (at >5 µM zinc) and ΔgtmA (at >1 µM zinc) in a low-iron medium. TAFC secretion suggests that differential zinc-sensing between both strains may influence fungal Fe3+ requirement. Label-free quantitative proteomic analysis of both strains under equivalent differential zinc conditions revealed protein abundance alterations in accordance with altered metabolomic observations, in addition to increased GliT abundance in ΔgtmA at 5 µM zinc, compared to wild-type, supporting a zinc-sensing deficiency in the mutant strain. The relative abundance of a range of oxidoreductase- and secondary metabolism-related enzymes was also evident in a zinc- and strain-dependent manner. Overall, we elaborate new linkages between zinc availability, natural product biosynthesis and oxidative stress homeostasis in A. fumigatus.

Effects of antifungal agents on the fungal proteome: informing on mechanisms of sensitivity and resistance.

INTRODUCTION: Antifungal agents are essential in the fight against serious fungal disease, however emerging resistance is threatening an already limited collection of therapeutics. Proteomic analyses of effects of antifungal agents can expand our understanding of multifactorial mechanisms of action and have also proven valuable to elucidate proteomic changes associated with antifungal resistance. AREAS COVERED: This review covers the application of proteomic techniques to examine sensitivity and resistance to antifungals including commonly used therapeutics, amphotericin B, echinocandins and the azoles, based predominantly on studies involving Aspergillus fumigatus, Candida albicans and Candida glabrata from the last 10 years. In addition, non-clinical antimicrobial agents are also discussed, which highlight the potential of proteomics to identify new antifungal targets. EXPERT COMMENTARY: Fungal proteomics has evolved in the last decade with increased genome availability and developments in mass spectrometry. Collectively, these have led to the advancement of proteomic techniques, allowing increased coverage of the proteome. Gel-based proteomics laid the foundation for these types of studies, which has now shifted to the more powerful gel-free proteomics. This has resulted in the identification of key mediators and potential biomarkers of antifungal resistance, as well as elucidating the mechanisms of action of novel and established antifungal agents.

Differential distribution and proteomic response of Saccharomyces cerevisiae and non-model yeast species to zinc.

Zinc surplus in yeast cells has been previously investigated thanks to transcriptomic studies by using traditionally Saccharomyces cerevisiae as a model. However, proteome response under zinc-replete conditions needs to be further studied in yeast. For that reason, eight yeast strains from seven different species were inoculated in zinc-depleted and zinc-replete media. The quantitative and qualitative comparative label-free proteomic analysis enabled the identification of between 2000 and 3000 proteins from each strain, and changes to the proteome ranged from 2.5% to 43.7% of identified proteins. Functional analysis (Blast2Go) has allowed the characterization of differentially abundant proteins. Common zinc-responsive proteins have been detected for the eight strains such as oxidoreductases and transferases (increased in abundance) although more of the changes detected were not shared by all the strains tested. Zinc distribution under replete conditions has been analysed in cell wall fractions, and cytoplasm plus organelles (intracellular fraction), with the latter identified to be the main zinc reservoir. Additionally, the energy dispersive spectroscopy coupled to the scanning electron microscopy technique has permitted the visualization of zinc in the whole cell. Proteomic analysis revealed that while there were some shared responses, the non-model yeast species also showed distinct proteomic profiles in zinc-replete conditions, compared to S. cerevisiae, revealing new zinc-responsive proteins in yeast.

An Exploratory Study of a 3-Minute Mindfulness Intervention on Compassion Fatigue in Nurses.

This study shows that breathing mindfully for 3 minutes over a period of 4 weeks, positively affects compassion fatigue in nurses. A nonrandomized, pre/postintervention study was conducted using a 3-minute attentional breathing intervention. Thirty-two nurses participated over 4 weeks. The intervention demonstrated statistically significant reductions in compassion fatigue measures.

Infective juveniles of entomopathogenic nematodes (Steinernema and Heterorhabditis) secrete ascarosides and respond to interspecific dispersal signals.

Ascarosides are a modular series of signalling molecules that are widely conserved in nematodes where they function as pheromones with both behavioural and developmental effects. Here we show that the developmentally arrested infective juvenile (IJ) stage of entomopathogenic nematodes (EPN) secrete ascarosides into the surrounding medium. The exometabolome of Steinernema carpocapsae and Heterorhabditis megidis was examined at 0, 1, 7 and 21 days of storage. The concentration of several ascarosides (ascr#11, ascr#9, ascr#12, ascr#1 and ascr#14 for both species, plus ascr#10 for H. megidis) showed a progressive increase over this period, while the concentration of longer chain ascarosides increased up to day 7, with an apparent decline thereafter. Ascr #9 was the main ascaroside produced by both species. Similar ascarosides were found over a 7-day period for Steinernema longicaudum and S. feltiae. Ascaroside blends have previously been shown to promote nematode dispersal. S. carpocapsae and H. megidis IJs were stored for up to 12 weeks and assayed at intervals. IJs where exometabolome was allowed to accumulate showed higher dispersal rates than those where water was changed frequently, indicating that IJ exometabolome maintained high dispersal. Infectivity was not affected. IJ exometabolome accumulated over 7 days promoted dispersal of freshly harvested IJs, both of their own and other EPN species. Similarly, extracts of nematode-infected cadavers promoted dispersal of con- and heterospecific IJs. Thus, IJs are encouraged to disperse from a source cadaver or from other crowded conditions by public information cues, a finding that may have application in enhancing biocontrol. However, the complexity of the ascaroside blend produced by IJs suggests that it may have ecological functions other than dispersal.

Nonclinical pharmacology and toxicology of the first biosimilar insulin glargine drug product (BASAGLAR®/ABASAGLAR®) approved in the European Union.

Basaglar®/Abasaglar® (Lilly insulin glargine [LY IGlar]) is a long-acting human insulin analogue drug product granted marketing authorisation as a biosimilar to Lantus® (Sanofi insulin glargine [SA IGlar]) by the European Medicines Agency. We assessed the similarity of LY IGlar to the reference drug product, European Union-sourced SA IGlar (EU-SA IGlar), using nonclinical in vitro and in vivo studies. No biologically relevant differences were observed for receptor binding affinity at either the insulin or insulin-like growth factor-1 (IGF-1) receptors, or in assays of functional or de novo lipogenic activity. The mitogenic potential of LY IGlar and EU-SA IGlar was similar when tested in both insulin- and IGF-1 receptor dominant cell systems. Repeated subcutaneous daily dosing of rats for 4 weeks with 0, 0.3, 1.0, or 2.0 mg/kg LY IGlar and EU-SA IGlar produced mortalities and clinical signs consistent with severe hypoglycaemia. Glucodynamic profiles of LY IGlar and EU-SA IGlar in satellite animals showed comparable dose-related hypoglycaemia. Severe hypoglycaemia was associated with axonal degeneration of the sciatic nerve; the incidence and severity were low and did not differ between LY IGlar and EU-SA IGlar. These results demonstrated no biologically relevant differences in toxicity between LY IGlar and EU-SA IGlar.

Quantitative proteomics reveals new insights into calcium-mediated resistance mechanisms in Aspergillus flavus against the antifungal protein PgAFP in cheese.

The ability of Aspergillus flavus to produce aflatoxins in dairy products presents a potential hazard. The antifungal protein PgAFP from Penicillium chrysogenum inhibits various foodborne toxigenic fungi, including Aspergillus flavus. However, PgAFP did not inhibit A. flavus growth in cheese, which was related to the associated cation content. CaCl2 increased A. flavus permeability and prevented PgAFP-mediated inhibition in potato dextrose broth (PDB). PgAFP did not elicit any additional increase in permeability of CaCl2-incubated A. flavus. Furthermore, PgAFP did not alter metabolic capability, chitin deposition, or hyphal viability of A. flavus grown with CaCl2. Comparative proteomic analysis after PgAFP treatment of A. flavus in calcium-enriched PDB revealed increased abundance of 125 proteins, including oxidative stress-related proteins, as determined by label-free mass spectrometry (MS)-based proteomics. Seventy proteins were found at lower abundance, with most involved in metabolic pathways and biosynthesis of secondary metabolites. These changes do not support the blockage of potential PgAFP receptors in A. flavus by calcium as the main cause of the protective role. A. flavus resistance appears to be mediated by calcineurin, G-protein, and γ-glutamyltranspeptidase that combat oxidative stress and impede apoptosis. These findings could serve to design strategies to improve PgAFP activity against aflatoxigenic moulds in dairy products.

In Vivo and In Vitro Characterization of Basal Insulin Peglispro: A Novel Insulin Analog.

The aim of this research was to characterize the in vivo and in vitro properties of basal insulin peglispro (BIL), a new basal insulin, wherein insulin lispro was derivatized through the covalent and site-specific attachment of a 20-kDa polyethylene-glycol (PEG; specifically, methoxy-terminated) moiety to lysine B28. Addition of the PEG moiety increased the hydrodynamic size of the insulin lispro molecule. Studies show there is a prolonged duration of action and a reduction in clearance. Given the different physical properties of BIL, it was also important to assess the metabolic and mitogenic activity of the molecule. Streptozotocin (STZ)-treated diabetic rats were used to study the pharmacokinetic and pharmacodynamic characteristics of BIL. Binding affinity and functional characterization of BIL were compared with those of several therapeutic insulins, insulin AspB10, and insulin-like growth factor 1 (IGF-1). BIL exhibited a markedly longer time to maximum concentration after subcutaneous injection, a greater area under the concentration-time curve, and a longer duration of action in the STZ-treated diabetic rat than insulin lispro. BIL exhibited reduced binding affinity and functional potency as compared with insulin lispro and demonstrated greater selectivity for the human insulin receptor (hIR) as compared with the human insulin-like growth factor 1 receptor. Furthermore, BIL showed a more rapid rate of dephosphorylation following maximal hIR stimulation, and reduced mitogenic potential in an IGF-1 receptor-dominant cellular model. PEGylation of insulin lispro with a 20-kDa PEG moiety at lysine B28 alters the absorption, clearance, distribution, and activity profile receptor, but does not alter its selectivity and full agonist receptor properties.

Proteomic analysis of Aspergillus fumigatus - clinical implications.

INTRODUCTION: Aspergillus fumigatus is a ubiquitous saprophytic fungus capable of producing small airborne spores, which are frequently inhaled by humans. In healthy individuals, the fungus is rapidly cleared by innate mechanisms, including immune cells. However, in individuals with impaired lung function or immunosuppression the spores can germinate and prompt severe allergic responses, and disease with limited or extensive invasiveness. AREAS COVERED: The traits that make A. fumigatus a successful colonizer and pathogen of humans are multi-factorial. Thus, a global investigative approach is required to elucidate the mechanisms utilized by the fungus to cause disease. Expert commentary: In doing so, a better understanding of disease pathology can be achieved with improved therapeutic/diagnostic solutions, thereby improving patient outcome. Proteomic analysis permits such investigations and recent work has yielded insight into these mechanisms.

Manuscript title: antifungal proteins from moulds: analytical tools and potential application to dry-ripened foods.

Moulds growing on the surface of dry-ripened foods contribute to their sensory qualities, but some of them are able to produce mycotoxins that pose a hazard to consumers. Small cysteine-rich antifungal proteins (AFPs) from moulds are highly stable to pH and proteolysis and exhibit a broad inhibition spectrum against filamentous fungi, providing new chances to control hazardous moulds in fermented foods. The analytical tools for characterizing the cellular targets and affected pathways are reviewed. Strategies currently employed to study these mechanisms of action include 'omics' approaches that have come to the forefront in recent years, developing in tandem with genome sequencing of relevant organisms. These techniques contribute to a better understanding of the response of moulds against AFPs, allowing the design of complementary strategies to maximize or overcome the limitations of using AFPs on foods. AFPs alter chitin biosynthesis, and some fungi react inducing cell wall integrity (CWI) pathway. However, moulds able to increase chitin content at the cell wall by increasing proteins in either CWI or calmodulin-calcineurin signalling pathways will resist AFPs. Similarly, AFPs increase the intracellular levels of reactive oxygen species (ROS), and moulds increasing G-protein complex ß subunit CpcB and/or enzymes to efficiently produce glutathione may evade apoptosis. Unknown aspects that need to be addressed include the interaction with mycotoxin production by less sensitive toxigenic moulds. However, significant steps have been taken to encourage the use of AFPs in intermediate-moisture foods, particularly for mould-ripened cheese and meat products.

Increased chitin biosynthesis contributes to the resistance of Penicillium polonicum against the antifungal protein PgAFP.

Antifungal proteins from molds have been proposed as a valuable tool against unwanted molds, but the resistance of some fungi limits their use. Resistance to antimicrobial peptides has been suggested to be due to lack of interaction with the mold or to a successful response. The antifungal protein PgAFP produced by Penicillium chrysogenum inhibits the growth of various ascomycetes, but not Penicillium polonicum. To study the basis for resistance to this antifungal protein, localization of PgAFP and metabolic, structural, and morphological changes were investigated in P. polonicum. PgAFP bound the outer layer of P. polonicum but not regenerated chitin, suggesting an interaction with specific molecules. Comparative two-dimensional gel electrophoresis (2D-PAGE) and comparative quantitative proteomics revealed changes in the relative abundance of several proteins from ribosome, spliceosome, metabolic, and biosynthesis of secondary metabolite pathways. The proteome changes and an altered permeability reveal an active reaction of P. polonicum to PgAFP. The successful response of the resistant mold seems to be based on the higher abundance of protein Rho GTPase Rho1 that would lead to the increased chitin deposition via cell wall integrity (CWI) signaling pathway. Thus, combined treatment with chitinases could provide a complementary means to combat resistance to antifungal proteins.

Interplay between Gliotoxin Resistance, Secretion, and the Methyl/Methionine Cycle in Aspergillus fumigatus.

Mechanistic studies on gliotoxin biosynthesis and self-protection in Aspergillus fumigatus, both of which require the gliotoxin oxidoreductase GliT, have revealed a rich landscape of highly novel biochemistries, yet key aspects of this complex molecular architecture remain obscure. Here we show that an A. fumigatus ΔgliA strain is completely deficient in gliotoxin secretion but still retains the ability to efflux bisdethiobis(methylthio)gliotoxin (BmGT). This correlates with a significant increase in sensitivity to exogenous gliotoxin because gliotoxin trapped inside the cell leads to (i) activation of the gli cluster, as disabling gli cluster activation, via gliZ deletion, attenuates the sensitivity of an A. fumigatus ΔgliT strain to gliotoxin, thus implicating cluster activation as a factor in gliotoxin sensitivity, and (ii) increased methylation activity due to excess substrate (dithiol gliotoxin) for the gliotoxin bis-thiomethyltransferase GtmA. Intracellular dithiol gliotoxin is oxidized by GliT and subsequently effluxed by GliA. In the absence of GliA, gliotoxin persists in the cell and is converted to BmGT, with levels significantly higher than those in the wild type. Similarly, in the ΔgliT strain, gliotoxin oxidation is impeded, and methylation occurs unchecked, leading to significant S-adenosylmethionine (SAM) depletion and S-adenosylhomocysteine (SAH) overproduction. This in turn significantly contributes to the observed hypersensitivity of gliT-deficient A. fumigatus to gliotoxin. Our observations reveal a key role for GliT in preventing dysregulation of the methyl/methionine cycle to control intracellular SAM and SAH homeostasis during gliotoxin biosynthesis and exposure. Moreover, we reveal attenuated GliT abundance in the A. fumigatus ΔgliK strain, but not the ΔgliG strain, following exposure to gliotoxin, correlating with relative sensitivities. Overall, we illuminate new systems interactions that have evolved in gliotoxin-producing, compared to gliotoxin-naive, fungi to facilitate their cellular presence.

Impact of the antifungal protein PgAFP from Penicillium chrysogenum on the protein profile in Aspergillus flavus.

Antifungal proteins produced by molds are generally small, highly basic, and cysteine-rich. The best known effects of these proteins include morphological changes, metabolic inactivation, and membrane perturbation on sensitive fungi. Reactive oxygen species (ROS) generation leads to apoptosis, with G -protein playing a key role in transduction of cell death signals. The antifungal protein PgAFP from Penicillium chrysogenum inhibits growth of some toxigenic molds. Here we analyzed the effect of the antifungal protein PgAFP on the growth of Aspergillus flavus. For this, comparative proteomic analysis was used to identify the whole protein profile and protein change in abundance after PgAFP treatment. PgAFP provoked metabolic changes related to reduced energy metabolism, cell wall integrity alteration, and increased stress response due to higher levels of ROS. The observed changes in protein abundance, favoring a higher glutathione concentration as well as the increased abundance in heat shock proteins, do not seem to be enough to avoid necrosis. The decreased chitin deposition observed in PgAFP-treated A. flavus is attributed to a lower relative quantity of Rho1. The reduced relative abundance of a ß subunit of G -protein seems to be the underlying reason for modulation of apoptosis in PgAFP-treated A. flavus hyphae. We propose Rho1 and G -protein subunit ß CpcB to be the main factors in the mode of action of PgAFP in A. flavus. Additionally, enzymes essential for the biosynthesis of aflatoxin were no longer detectable in A. flavus hyphae at 24 h, following treatment with PgAFP. This presents a promising effect of PgAFP, which may prevent A. flavus from producing mycotoxins. However, the impact of PgAFP on actual aflatoxin production requires further study.

RNA-seq reveals the pan-transcriptomic impact of attenuating the gliotoxin self-protection mechanism in Aspergillus fumigatus.

BACKGROUND: Aspergillus fumigatus produces a number of secondary metabolites, one of which, gliotoxin, has been shown to exhibit anti-fungal activity. Thus, A. fumigatus must be able to protect itself against gliotoxin. Indeed one of the genes in the gliotoxin biosynthetic gene cluster in A. fumigatus, gliT, is required for self-protection against the toxin- however the global self-protection mechanism deployed is unclear. RNA-seq was employed to identify genes differentially regulated upon exposure to gliotoxin in A. fumigatus wild-type and A. fumigatus ∆gliT, a strain that is hypersensitive to gliotoxin. RESULTS: Deletion of A. fumigatus gliT resulted in altered expression of 208 genes (log2 fold change of 1.5) when compared to A. fumigatus wild-type, of which 175 genes were up-regulated and 33 genes were down-regulated. Expression of 164 genes was differentially regulated (log2 fold change of 1.5) in A. fumigatus wild-type when exposed to gliotoxin, consisting of 101 genes with up-regulated expression and 63 genes with down-regulated expression. Interestingly, a much larger number of genes, 1700, were found to be differentially regulated (log2 fold change of 1.5) in A. fumigatus ∆gliT when challenged with gliotoxin. These consisted of 508 genes with up-regulated expression, and 1192 genes with down-regulated expression. Functional Catalogue (FunCat) classification of differentially regulated genes revealed an enrichment of genes involved in both primary metabolic functions and secondary metabolism. Specifically, genes involved in gliotoxin biosynthesis, helvolic acid biosynthesis, siderophore-iron transport genes and also nitrogen metabolism genes and ribosome biogenesis genes underwent altered expression. It was confirmed that gliotoxin biosynthesis is induced upon exposure to exogenous gliotoxin, production of unrelated secondary metabolites is attenuated in A. fumigatus ∆gliT, while quantitative proteomic analysis confirmed disrupted translation in A. fumigatus ∆gliT challenged with exogenous gliotoxin. CONCLUSIONS: This study presents the first global investigation of the transcriptional response to exogenous gliotoxin in A. fumigatus wild-type and the hyper-sensitive strain, ∆gliT. Our data highlight the global and extensive affects of exogenous gliotoxin on a sensitive strain devoid of a self-protection mechanism and infer that GliT functionality is required for the optimal biosynthesis of selected secondary metabolites in A. fumigatus.

Molecular characterization of an adaptive response to alkylating agents in the opportunistic pathogen Aspergillus fumigatus.

An adaptive response to alkylating agents based upon the conformational change of a methylphosphotriester (MPT) DNA repair protein to a transcriptional activator has been demonstrated in a number of bacterial species, but this mechanism appears largely absent from eukaryotes. Here, we demonstrate that the human pathogen Aspergillus fumigatus elicits an adaptive response to sub-lethal doses of the mono-functional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We have identified genes that encode MPT and O(6)-alkylguanine DNA alkyltransferase (AGT) DNA repair proteins; deletions of either of these genes abolish the adaptive response and sensitize the organism to MNNG. In vitro DNA repair assays confirm the ability of MPT and AGT to repair methylphosphotriester and O(6)-methylguanine lesions respectively. In eukaryotes, the MPT protein is confined to a select group of fungal species, some of which are major mammalian and plant pathogens. The evolutionary origin of the adaptive response is bacterial and rooted within the Firmicutes phylum. Inter-kingdom horizontal gene transfer between Firmicutes and Ascomycete ancestors introduced the adaptive response into the Fungal kingdom. Our data constitute the first detailed characterization of the molecular mechanism of the adaptive response in a lower eukaryote and has applications for development of novel fungal therapeutics targeting this DNA repair system.