Chytridiomycosis causes catastrophic organism-wide metabolic dysregulation including profound failure of cellular energy pathways

Chytridiomycosis is among several recently emerged fungal diseases of wildlife that have caused decline or extinction of naive populations. Despite recent advances in understanding pathogenesis, host response to infection remains poorly understood. Here we modelled a total of 162 metabolites across skin and liver tissues of 61 frogs from four populations (three long-exposed and one naive to the fungus) of the Australian alpine tree frog (Litoria verreauxii alpina) throughout a longitudinal exposure experiment involving both infected and negative control individuals. We found that chytridiomycosis dramatically altered the organism-wide metabolism of clinically diseased frogs. Chytridiomycosis caused catastrophic failure of normal homeostatic mechanisms (interruption of biosynthetic and degradation metabolic pathways), and pronounced dysregulation of cellular energy metabolism. Key intermediates of the tricarboxylic acid cycle were markedly depleted, including in particular a-ketoglutarate and glutamate that together constitute a key nutrient pathway for immune processes. This study was the first to apply a non-targeted metabolomics approach to a fungal wildlife disease and specifically to dissect the host-pathogen interface of Bd-infected frogs. The patterns of metabolite accumulation we have identified reveal whole-body metabolic dysfunction induced by a fungal skin infection, and these findings have broad relevance for other fungal diseases

The Transcription Factor StuA Regulates Central Carbon Metabolism, Mycotoxin Production, and Effector Gene Expression in the Wheat Pathogen Stagonospora nodorum

The Stagonospora nodorum StuA transcription factor gene SnStuA was identified by homology searching in the genome of the wheat pathogen Stagonospora nodorum. Gene expression analysis revealed that SnStuA transcript abundance increased throughout infection and in vitro growth to peak during sporulation. To investigate its role, the gene was deleted by homologous recombination. The growth of the resulting mutants was retarded on glucose compared to the wild-type growth, and the mutants also failed to sporulate. Glutamateas a sole carbon source restored the growth rate defect observed on glucose, although sporulation remained impaired. The SnstuA strains were essentially nonpathogenic, with only minor growth observed around the point of inoculation. The role of SnstuA was investigated using metabolomics, which revealed that this gene's product played a key role in regulating central carbon metabolism, with glycolysis, the TCA cycle, and amino acid synthesis all affected in the mutants. SnStuA was also found to positively regulate the synthesis of the mycotoxin alternariol. Gene expression studies on the recently identified effectors in Stagonospora nodorum found that SnStuA was a positive regulator of SnTox3 but was not required for the expression of ToxA. This study has uncovered a multitude of novel regulatory targets of SnStuA and has highlighted the critical role of this gene product in the pathogenicity of Stagonospora nodorum

Hepatic iron concentration correlates with insulin sensitivity in non-alcoholic fatty liver disease

Rodent and cell‐culture models support a role for iron‐related adipokine dysregulation and insulin resistance in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); however, substantial human data are lacking. We examined the relationship between measures of iron status, adipokines, and insulin resistance in patients with NAFLD in the presence and absence of venesection. This study forms part of the Impact of Iron on Insulin Resistance and Liver Histology in Nonalcoholic Steatohepatitis (IIRON2) study, a prospective randomized controlled trial of venesection for adults with NAFLD. Paired serum samples at baseline and 6 months (end of treatment) in controls (n = 28) and patients who had venesection (n = 23) were assayed for adiponectin, leptin, resistin, retinol binding protein‐4, tumor necrosis factor α, and interleukin‐6, using a Quantibody, customized, multiplexed enzyme‐linked immunosorbent assay array. Hepatic iron concentration (HIC) was determined using MR FerriScan. Unexpectedly, analysis revealed a significant positive correlation between baseline serum adiponectin concentration and HIC, which strengthened after correction for age, sex, and body mass index (rho = 0.36; P = 0.007). In addition, there were significant inverse correlations between HIC and measures of insulin resistance (adipose tissue insulin resistance (Adipo‐IR), serum insulin, serum glucose, homeostasis model assessment of insulin resistance, hemoglobin A1c, and hepatic steatosis), whereas a positive correlation was noted with the insulin sensitivity index. Changes in serum adipokines over 6 months did not differ between the control and venesection groups. Conclusion: HIC positively correlates with serum adiponectin and insulin sensitivity in patients with NAFLD. Further study is required to establish causality and mechanistic explanations for these associations and their relevance in the pathogenesis of insulin resistance and NAFLD

PPARα and PPARγ activation is associated with pleural mesothelioma invasion but therapeutic inhibition is ineffective

Mesothelioma is a cancer that typically originates in the pleura of the lungs. It rapidly invades the surrounding tissues, causing pain and shortness of breath. We compared cell lines injected either subcutaneously or intrapleurally and found that only the latter resulted in invasive and rapid growth. Pleural tumors displayed a transcriptional signature consistent with increased activity of nuclear receptors PPARα and PPARγ and with an increased abundance of endogenous PPAR-activating ligands. We found that chemical probe GW6471 is a potent, dual PPARα/γ antagonist with anti-invasive and anti-proliferative activity in vitro. However, administration of GW6471 at doses that provided sustained plasma exposure levels sufficient for inhibition of PPARα/γ transcriptional activity did not result in significant anti-mesothelioma activity in mice. Lastly, we demonstrate that the in vitro anti-tumor effect of GW6471 is off-target. We conclude that dual PPARα/γ antagonism alone is not a viable treatment modality for mesothelioma

A metabolomic analysis of G-protein signalling mutants of Stagonospora nodorum

Stagonospora nodorum is the causal agent of Stagonospora nodorum blotch (SNB) of wheat. This fungus has cost the Australian grains industry upwards of 100 million dollars (AUD) p.a. in recent growing seasons, making it one of the most agriculturally damaging pathogens in Australia. Disease severity is governed by the polycyclic lifecycle of S. nodorum, requiring a succession of spore inoculum arising from the asexual fruiting body of the fungus, known as the pycnidium. The resultant fungal density will determine the level of damage and ultimately influence the grain yield of the plant. G-protein signalling through the heterotrimeric G-protein is a biochemical mechanism used by S. nodorum in the host-pathogen interaction and has been linked to important biological processes including asexual sporulation. In this work, the unique phenotypes of three mutant strains of S. nodorum; each lacking either the Gα (Gna1), Gβ (Gba1), or Gγ (GgaA) subunit of the heterotrimeric G-protein were explored, and the biochemistry underpinning the phenotypes assessed by metabolomics. The mutant strain S. nodorum ggaA was created by homologous recombination of the GgaA gene for comparison with the previously created gna1 and gba1 strains. All strains possessed developmental defects and reduced pathogenicity on the wheat plant. Growth assays uncovered differences in carbon source utilisation between the strains. Asexual sporulation was monitored by light microscopy; with the differentiation of mutant mycelia into pycnidia found to occur only after a comparatively longer culture time than in wild type, and at a reduced temperature. Until this time, asexual sporulation is completely abolished in the mutant strains. The matured pycnidia also possessed an irregular morphology. These results identified an association of all three G-protein subunits in asexual sporulation in S. nodorum. Metabolites were isolated from S. nodorum mycelia for gas chromatography-mass spectrometer (GC-MS) analysis. An assessment of existing metabolomic methods identified some key steps in the sample preparation employed prior to injection into the GC-MS. Quenching the fungal metabolism upon harvesting, drying the fungal mycelia prior to metabolite extraction and isolation, and lyophilisation of the fungal metabolites in preparation for chemical derivatisation; each improved the metabolite recovery and overall reliability of the metabolomic analyses. These methods were applied to the metabolomic characterisations that followed. Metabolite extracts from the in vitro cultured fungal strains were analysed using a single-quadrupole GC-MS and the recorded analytes cross-refereces to purchased metabolite standards for identification. Changes in the accumulation of various carbohydrates were apparent in the mutant metabolomes. Of those, the altered abundances of the metabolites glucose and trehalose are believed to in part explain or be consequential to the sporulation phenomena of these strains. Metabolomic analysis of the mutant strains in differentiating from a non-sporulating to a sporulating phenotype revealed the specific association of a number of metabolites with each of the two phenotypic classifications. Many of which have been targeted for identification in future studies. Among those identified was again trehalose, providing further evidence for it having a role in the asexual sporulation of this fungus. These results have demonstrated the requirement for Gna1, Gba1 and GgaA in regulating developmental processes and the pathogenesis of S. nodorum, and added significantly to the biochemical dissection of asexual sporulation in this fungus

Dissecting the role of G-protein signalling in primary metabolism in the wheat pathogen Stagonospora nodorum

Mutants of the wheat pathogenic fungus Stagonospora nodorum lacking G-protein subunits display a variety of phenotypes including melanization defects, primary metabolic changes and a decreased ability to sporulate. To better understand the causes of these phenotypes, Stagonospora nodorum strains lacking a Gα, Gβ or Gγ subunit were compared to a wild-type strain using metabolomics. Agar plate growth at 22 °C revealed a number of fundamental metabolic changes and highlighted the influential role of these proteins in glucose utilization. A further characterization of the mutants was undertaken during prolonged storage at 4 °C, conditions known to induce sporulation in these sporulation-deficient signalling mutants. The abundance of several compounds positively correlated with the onset of sporulation including the dissacharide trehalose, the tryptophan degradation product tryptamine and the secondary metabolite alternariol; metabolites all previously associated with sporulation. Several other compounds decreased or were absent during sporulation. The levels of one such compound (Unknown_35.27_2194_319) decreased from being one of the more abundant compounds to absence during pycnidial maturation. This study has shed light on the role of G-protein subunits in primary metabolism during vegetative growth and exploited the cold-induced sporulation phenomenon in these mutants to identify some key metabolic changes that occur during asexual reproduction

Matrix-assisted laser desorption ionization mass spectrometry imaging by freeze-spot deposition of the matrix

Imaging mass spectrometry has emerged as a powerful metabolite measurement approach to capture the spatial dimension of metabolite distribution in a biological sample. In matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI), deposition of the chemical-matrix onto the sample serves to simultaneously extract biomolecules to the sample surface and concurrently render the sample amenable to MALDI. However, matrix application may mobilize sample metabolites and will dictate the efficiency of matrix crystallization, together limiting the lateral resolution which may be optimally achieved by MSI. Here, we describe a matrix application technique, herein referred to as the freeze-spot method, conceived as a low-cost preparative approach requiring minimal amounts of chemical matrix while maintaining the spatial dimension of sample metabolites for MALDI-MSI. Matrix deposition was achieved by pipette spot application of the matrix-solubilized within a solvent solution with a freezing point above that of a chilled sample stage to which the sample section is mounted. The matrix solution freezes on contact with the sample and the solvent is removed by sublimation, leaving a fine crystalline matrix on the sample surface. Freeze-spotting is quick to perform, found particularly useful for MALDI-MSI of small sample sections, and well suited to efficient and cost-effective method development pipelines, while capable of maintaining the lateral resolution required by MSI

Factors driving the compositional diversity of Apis mellifera bee venom from a Corymbia calophylla (marri) ecosystem, Southwestern Australia

Bee venom (BV) is the most valuable product harvested from honeybees ($30 -$300 USD per gram) but marginally produced in apiculture. Though widely studied and used in alternative medicine, recent efforts in BV research have focused on its therapeutic and cosmetic applications, for the treatment of degenerative and infectious diseases. The protein and peptide composition of BV is integral to its bioactivity, yet little research has investigated the ecological factors influencing the qualitative and quantitative variations in the BV composition. Bee venom from Apis mellifera ligustica (Apidae), collected over one flowering season of Corymbia calophylla (Myrtaceae; marri) was characterized to test if the protein composition and amount of BV variation between sites is influenced by i) ecological factors (temperature, relative humidity, flowering index and stage, nectar production); ii) management (nutritional supply and movement of hives); and/or iii) behavioural factors. BV samples from 25 hives across a 200 km-latitudinal range in Southwestern Australia were collected using stimulatory devices. We studied the protein composition of BV by mass spectrometry, using a bottom-up proteomics approach. Peptide identification utilised sequence homology to the A. mellifera reference genome, assembling a BV peptide profile representative of 99 proteins, including a number of previously uncharacterised BV proteins. Among ecological factors, BV weight and protein diversity varied by temperature and marri flowering stage but not by index, this latter suggesting that inter and intra-year flowering index should be further explored to better appreciate this influence. Site influenced BV protein diversity and weight difference in two sites. Bee behavioural response to the stimulator device impacted both the protein profile and weight, whereas management factors did not. Continued research using a combination of proteomics, and bio-ecological approaches is recommended to further understand causes of BV variation in order to standardise and improve the harvest practice and product quality attributes