Incapacitating effects of fungal coinfection in a novel pathogen system

As globalization lowers geographic barriers to movement, coinfection with novel and enzootic pathogens is increasingly likely. Novel and enzootic pathogens can interact synergistically or antagonistically, leading to increased or decreased disease severity. Here we examine host immune responses to coinfection with two closely related fungal pathogens: Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). Both pathogens have had detrimental effects on amphibian populations, with Bd now largely enzootic, while Bsal is currently spreading and causing epizootics. Recent experimental work revealed that newts coinfected with Bd and Bsal had significantly higher mortality than those infected with either pathogen alone. Here we characterize host immunogenomic responses to chytrid coinfection relative to single infection. Across several classes of immune genes including pattern recognition receptors, cytokines, and MHC, coinfected host gene expression was weakly upregulated or comparable to that seen in single Bd infection, but significantly decreased when compared to Bsal infection. Combined with strong complement pathway downregulation and keratin upregulation, these results indicate that coinfection with Bd and Bsal compromises immune responses active against Bsal alone. As Bsal continues to invade naïve habitats where Bd is enzootic, coinfection will be increasingly common. If other Bd‐susceptible species in the region have similar responses, interactions between the two pathogens could cause severe population and community‐level declines.     

Localization of protein secretion in fungal colonies using a novel culturing technique; the ring-plate system

A novel culturing technique, called the ring-plate system, is described. Fungal colonies are grown on a polycarbonate membrane placed on a plate with 6 ring-shaped wells that are filled with liquid medium. This culturing technique enables simultaneous monitoring of environmental conditions and secretion in different parts of the fungal colony.     

Continuous Acid Blue 45 decolorization by using a novel open fungal reactor system with ozone as the bactericide

To maintain long-term lignin-degrading enzyme production under non-sterile conditions was a key to the technical application of white rot fungi in wastewater treatment. In this work, a novel open fungal reactor system with ozone as the bactericide, and using immobilized Phanerochaete chrysosporium, was built and operated continuously to produce the manganese peroxidase and decolorize the Acid Blue 45. The results showed that an average of 84% Acid Blue 45 decolorization, the manganese peroxidase production with its activity ranging from 63 U L⁻¹ to 5 U L⁻¹, was achieved during about 25 days system continuous operation. The contaminating bacteria in the reactor can be controlled at a level of 4.65 × 10⁴ CFU ml⁻¹ that did not adversely affect the fungal activity. The result of this study provides a new practical way for future design and operation of white-rot fungi reactor under non-sterile conditions.     

Estimation of fungal growth in a solid state fermentation system

Summary Of four chemical methods for estimating mycelial biomass in koji fermentation which were examined, the modified method of Ride and Drysdale, was found to be most suitable. The observed level of aldehyde, expressed as glucosamine, is related to fungal dry weight. The assay method correlates chitin levels with some enzyme activities in the fermentation. This method may be applicable for detecting the extent of fungal growth in other solid and semi-solid substrates.     

Comparison of plant and fungal gravitropic responses using imitational modelling

The mechanisms of gravity perception are still hypothetical, but there are sufficient data from experiments with plants to enable mathematical modelling to imitate the behaviour of gravitropic response systems. We have a much less complete picture of gravitropic kinetics in agaric mushrooms. However, some existing mathematical models which imitate plant responses are in principle universal because their conceptual components are not limited to any specific cellular entities. In this work we have used such models to compare plant and fungal gravitropism, using recently acquired kinetic data from the agarics Coprinus cinereius and Flammulina velutipes. The results show striking similarities between plants and fungi. First, it is evident that the basic assumptions of the plant models are logically applicable to fungi. Secondly, the mechanism of bending is the same (differential growth of opposite flanks of the growing organ). Thirdly, the distribution of growth seems very similar: in both plants and fungi growth of the organ is most intensive just behind the apex and is almost absent at the apex and at the base. Fourthly, in both fungi and plants the gravitropic response exhibits a substantial time delay suggesting that many time-consuming processes are involved in reception, transduction and realization of gravitropic stimuli. Important differences in plant and fungal gravitropism kinetics were: (i) the agaric stem apex always returned to the vertical, whereas some plant organs show stable plagiogravitropic growth; (ii) inflections were usually seen in C. cinereus stem gravitropism time courses suggesting that a curvature compensation process delayed bending for a time; (iii) C. cinercus stems very rarely overshot or oscillated around the vertical although many plant subjects oscillate and the (limited) data for F. velutipes showed a single, exaggerated overshoot and oscillation. In this latter case, experimental modelling with parameters characteristic of a low level of perception improved the fit to the F. velutipes data, indicating that the two fungi may differ in this factor. Application of the plant models focused future research attention on the urgent need for data bearing on angle-response and acceleration–response relationships in fungi, and their detection–level thresholds for gravitational acceleration. Since the modelling also highlighted some fundamental kinetic differences between the only two fungi for which sufficient data are available at the moment, it is also clear that detailed observations need to be made of gravitropism kinetics in a larger number and wider range of fungi.     

Tumor growth inhibition by sonodynamic therapy using a novel sonosensitizer

Sonodynamic therapy (SDT) with low-intensity ultrasound combined with a sonosensitizer may be a promising approach to cancer therapy. Use of ultrasound has the advantage of being noninvasive, with deep-penetration properties, and convenient because of the low or no sensitivity of sonosensitizers to light. In this study, SDT with a novel sonosensitizer (a porphyrin derivative) was evaluated in vitro and in vivo. Ultrasound irradiation with a sonosensitizer elicited potent sonotoxicity in vitro without the danger of phototoxicity. The sonotoxic effect was mediated by reactive oxygen species (ROS) and was reduced by ROS scavengers. Cell membrane lipid peroxidation increased significantly just after ultrasound irradiation with a sonosensitizer, but there was no increase in apoptosis. In an in vivo mouse xenograft model, SDT with a sonosensitizer markedly inhibited tumor cell growth. The skin hypersensitivity after light exposure was not observed in a sonosensitizer-treatment group, consistent with the in vitro findings. These results suggest that ROS generated by SDT with a sensitizer can damage tumor cells, resulting in necrosis and prevention of tumor growth. This noninvasive treatment with no adverse effects such as skin sensitivity is therefore promising for therapy of cancers located deep within patients.     

Functional insights from the molecular modelling of a novel two-component system

Two-component systems (TCSs) are the major signalling pathway in bacteria and represent potential drug targets. Among the 11 paired TCS proteins present in Mycobacterium tuberculosis H37Rv, the histidine kinases (HKs) Rv0600c (HK1) and Rv0601c (HK2) are annotated to phosphorylate one response regulator (RR) Rv0602c (TcrA). We wanted to establish the sequence-structure-function relationship to elucidate the mechanism of phosphotransfer using in silico methods. Sequence alignments and codon usage analysis showed that the two domains encoded by a single gene in homologous HKs have been separated into individual open-reading frames in M. tuberculosis. This is the first example where two incomplete HKs are involved in phosphorylating a single RR. The model shows that HK2 is a unique histidine phosphotransfer (HPt)-mono-domain protein, not found as lone protein in other bacteria. The secondary structure of HKs was confirmed using "far-UV" circular dichroism study of purified proteins. We propose that HK1 phosphorylates HK2 at the conserved H131 and the phosphoryl group is then transferred to D73 of TcrA.     

Human beta-secretase activity in yeast detected by a novel cellular growth selection system

Sequential processing of the transmembrane amyloid precursor protein (APP) by the beta-secretase BACE and by the gamma-secretase causes secretion of Abeta peptides. Extracellular aggregation of these peptides in the brain is a major hallmark of Alzheimer's disease. For therapeutic purposes and the development of specific inhibitors, it is important to characterize these secretases. We have established a cellular growth selection system for functional expression of human BACE in the yeast Saccharomyces cerevisiae. A fragment of APP bearing the beta-site, the transmembrane domain and the cytosolic tail was fused to the C-terminus of the yeast enzyme invertase, which is normally secreted to allow cell growth in the presence of sucrose as the sole carbon source. The resulting invertase-APP fusion protein was expressed as a type-I transmembrane protein in intracellular compartments of yeast cells lacking endogenous invertase. In these cells, co-expression of human BACE restored cell growth on selective plates upon cleavage of the invertase-APP fusion protein. The cellular growth selection system presented here can be generally applied to screen for secretases that specifically cleave membrane-bound substrates. Furthermore, this system provides the basis for a high-throughput screen for identifying secretase inhibitors that are active in eukaryotic cells.     

Statistical modelling of growth using a mixed model with orthogonal polynomials

In statistical modelling, the effects of single-nucleotide polymorphisms (SNPs) are often regarded as time-independent. However, for traits recorded repeatedly, it is very interesting to investigate the behaviour of gene effects over time. In the analysis, simulated data from the 13th QTL-MAS Workshop (Wageningen, The Netherlands, April 2009) was used and the major goal was the modelling of genetic effects as time-dependent. For this purpose, a mixed model which describes each effect using the third-order Legendre orthogonal polynomials, in order to account for the correlation between consecutive measurements, is fitted. In this model, SNPs are modelled as fixed, while the environment is modelled as random effects. The maximum likelihood estimates of model parameters are obtained by the expectation–maximisation (EM) algorithm and the significance of the additive SNP effects is based on the likelihood ratio test, with p-values corrected for multiple testing. For each significant SNP, the percentage of the total variance contributed by this SNP is calculated. Moreover, by using a model which simultaneously incorporates effects of all of the SNPs, the prediction of future yields is conducted. As a result, 179 from the total of 453 SNPs covering 16 out of 18 true quantitative trait loci (QTL) were selected. The correlation between predicted and true breeding values was 0.73 for the data set with all SNPs and 0.84 for the data set with selected SNPs. In conclusion, we showed that a longitudinal approach allows for estimating changes of the variance contributed by each SNP over time and demonstrated that, for prediction, the pre-selection of SNPs plays an important role.     

Development of a novel enzyme-redox-mediator system based on a fungal laccase and ruthenium complexes

The laccase produced by the fungus Coriolus hirsutus has been coordinatively modified with ruthenium complexes [Ru(phpy)(phen)(MeCN)2]PF6 and Ru(bpy)2CO3 under aerobic and anaerobic conditions. The amount of the complexes per enzyme molecule does not depend on the oxygen concentration, equaling 5 for [Ru(phpy)(phen)(MeCN)2]PF6 and 3 for Ru(bpy)2CO3. The pH dependence of the enzymatic activity, thermostability, and catalytical and electrocatalytical properties of the modified laccase are reported. It has been shown that, during the modification, at least one molecule of the ruthenium compound was coordinated near the T1 active center of the laccase, being directly involved in the catalysis and enhancing its efficiency.     

Development of a novel enzyme-redox-mediator system based on a fungal laccase and ruthenium complexes

The laccase produced by the fungus Coriolus hirsutus has been coordinatively modified with ruthenium complexes [Ru(phpy)(phen)(MeCN)₂]PF₆ and Ru(bpy)₂CO₃ under aerobic and anaerobic conditions. The amount of the complexes per enzyme molecule does not depend on the oxygen concentration, equaling 5 for [Ru(phpy)(phen)(MeCN)₂]PF₆ and 3 for Ru(bpy)₂CO₃. The pH dependence of the enzymatic activity, thermostability, and catalytical and electrocatalytical properties of the modified laccase are reported. It has been shown that, during the modification, at least one molecule of the ruthenium compound was coordinated near the T1 active center of the laccase, being directly involved in the catalysis and enhancing its efficiency.     

Characterization of genetic transformation in Streptococcus mutans by using a novel high-efficiency plasmid marker rescue system.

We developed a marker rescue system for study of competence development and genetic transformation in Streptococcus mutans. The system involved the recombinational rescue of a tetracycline resistance (Tcr) determinant by a homologous, inactive locus (Tcs because of a small deletion). Streptococcal cells harboring this in vitro-prepared Tcs construct (pVA1208) were restored to Tcr when plasmid (pVA981) DNA was used as donor material. pVA981 contained the intact streptococcal Tcr locus and was unable to autonomously replicate in streptococci. Marker rescue with this system followed first-order kinetics and occurred at a frequency 8- or 160-fold higher than did transformation with homologous chromosomal or plasmid DNA, respectively. By using the rescue system, we were able to confirm that competence of S. mutans appeared to be inducible. This was indicated by a sequential increase and then decrease in Tcr transformation frequencies during growth in complex medium. Also, donor DNA binding was not sequence specific, since the recovery of Tcr transformants was reduced by increasing the concentrations of heterologous DNA. We investigated the fate of donor DNA and the kinetics of plasmid establishment in the transformation of S. mutans with plasmid DNA. Monomeric plasmid molecules transformed S. mutans as a second-order process, whereas multimeric plasmid DNA and chromosomal markers were recovered as a first-order process. Approximately 50% of the initially bound donor plasmid DNA was found to remain in a trichloroacetic acid-insoluble form. Our results suggested that molecular cloning in S. mutans would be conducted most efficiently by using helper plasmid systems or shuttle vectors and that gene transfer by transformation of S. mutans occurred in a manner similar to that observed in Streptococcus sanguis.     

Permeabilization of fungal membranes by plant defensins inhibits fungal growth

We used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi. Membrane permeabilization induced by plant defensins in Neurospora crassa was biphasic, depending on the plant defensin dose. At high defensin levels (10 to 40 microM), strong permeabilization was detected that could be strongly suppressed by cations in the medium. This permeabilization appears to rely on direct peptide-phospholipid interactions. At lower defensin levels (0.1 to 1 microM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the inhibition of fungal growth. Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from Raphanus sativus, failed to induce cation-resistant permeabilization in N. crassa. Dm-AMP1, a plant defensin from Dahlia merckii, induced cation-resistant membrane permeabilization in yeast (Saccharomyces cerevisiae) which correlated with its antifungal activity. However, Dm-AMP1 could not induce cation-resistant permeabilization in the Dm-AMP1-resistant S. cerevisiae mutant DM1, which has a drastically reduced capacity for binding Dm-AMP1. We think that cation-resistant permeabilization is binding site mediated and linked to the primary cause of fungal growth inhibition induced by plant defensins.     

Expression of a novel chitinase by the fungal endophyte in Poa ampla

Many wild and cultivated cool-season grass species are naturally infected with fungal endophytes of the genera Neotyphodium and Epichlo?. These associations generally are considered mutualistic with the plants benefiting from reduced herbivory and the fungi benefiting from nutrients supplied by the plants. The fungi secrete proteins that might have a role in the interspecies symbiosis. In the interaction between Poa ampla Merr. and the endophyte Neotyphodium sp., a fungal chitinase was detected in the apoplastic protein fraction. The chitinase was also the major protein secreted in culture. Sequence analysis of the chitinase revealed it has a low level of amino acid sequence identity to other fungal chitinases and one of the conserved active site residues is altered. DNA gel-blot analysis indicated the chitinase was encoded by a single gene. Expression of similar chitinases also was detected in endophyte-infected tall fescue (Festuca arundinacea Schreb.), perennial ryegrass (Lolium perenne L.) and Chewings fescue (Festuca rubra L. subsp. fallax [Thuill] Nyman). This is the first report of an endophyte chitinase expressed in the infected host grass. As a secreted hydrolytic enzyme, the chitinase might have roles in the nutrition, growth or defense of the endophyte.     

Regulatory network modelling of iron acquisition by a fungal pathogen in contact with epithelial cells

Background  

Reverse engineering of gene regulatory networks can be used to predict regulatory interactions of an organism faced with environmental changes, but can prove problematic, especially when focusing on complicated multi-factorial processes. Candida albicans is a major human fungal pathogen. During the infection process, this fungus is able to adapt to conditions of very low iron availability. Such adaptation is an important virulence attribute of virtually all pathogenic microbes. Understanding the regulation of iron acquisition genes will extend our knowledge of the complex regulatory changes during the infection process and might identify new potential drug targets. Thus, there is a need for efficient modelling approaches predicting key regulatory events of iron acquisition genes during the infection process.     

Changes to water repellence of soil caused by the growth of white-rot fungi: studies using a novel microcosm system

A microcosm system is described which permits assessment of the progressive growth of filamentous fungi through soil. We report on its application to measure the effects of Coriolus versicolor and Phanerochaete chrysosporium upon the sorptivity and water repellence of a mineral soil, measured using a miniature infiltration device. Both fungal species caused moderate sub-critical repellence. Since the pore structure was unaffected, the repellence was probably due to hydrophobic substances of fungal origin. This is the first report of changes in soil repellence caused by the growth of potential xenobiotic bioremediating fungi. The potential consequences are discussed.     

Prediction of promiscuous p-glycoprotein inhibition using a novel machine learning scheme

Background

P-glycoprotein (P-gp) is an ATP-dependent membrane transporter that plays a pivotal role in eliminating xenobiotics by active extrusion of xenobiotics from the cell. Multidrug resistance (MDR) is highly associated with the over-expression of P-gp by cells, resulting in increased efflux of chemotherapeutical agents and reduction of intracellular drug accumulation. It is of clinical importance to develop a P-gp inhibition predictive model in the process of drug discovery and development.

Methodology/Principal Findings

An in silico model was derived to predict the inhibition of P-gp using the newly invented pharmacophore ensemble/support vector machine (PhE/SVM) scheme based on the data compiled from the literature. The predictions by the PhE/SVM model were found to be in good agreement with the observed values for those structurally diverse molecules in the training set (n = 31, r ² = 0.89, q ² = 0.86, RMSE = 0.40, s = 0.28), the test set (n = 88, r ² = 0.87, RMSE = 0.39, s = 0.25) and the outlier set (n = 11, r ² = 0.96, RMSE = 0.10, s = 0.05). The generated PhE/SVM model also showed high accuracy when subjected to those validation criteria generally adopted to gauge the predictivity of a theoretical model.

Conclusions/Significance

This accurate, fast and robust PhE/SVM model that can take into account the promiscuous nature of P-gp can be applied to predict the P-gp inhibition of structurally diverse compounds that otherwise cannot be done by any other methods in a high-throughput fashion to facilitate drug discovery and development by designing drug candidates with better metabolism profile.     

Production physiology and properties of a novel fungal fibrinolytic enzyme.

A potent extracellular fibrinolytic enzyme was obtained from cultures of the imperfect fungus Fusarium semitectum under certain growth conditions. Nitrate addition to cultures increased enzyme production. The enzyme showed a versatile proteolytic activity against several protein substrates including casein, gelatin, haemoglobin, bovine serum albumin, and fibrin from both buffalo and human sources. Optimal fibrinolysis occurred at pH values around 7.0. The fibrinolytic activity exhibited marked heat stability in enzyme samples heated at 60 degrees C, and retained more than 40% of its activity in samples heated to 100 degrees C for 10 min. Fibrinolysis proceeded optimally in the temperature range between 50--60 degrees C. Copper ions significantly activated the enzyme. Other biochemical properties are also reported.