Metabolomic analysis of Trichophyton rubrum and Microsporum canis during keratin degradation.

Keratin is important and needed for the growth of dermatophytes in the host tissue. In turn, the ability to invade keratinised tissues is defined as a pivotal virulence attribute of this group of medically important fungi. The host-dermatophyte interaction is accompanied by an adaptation of fungal metabolism that allows them to adhere to the host tissue as well as utilize the available nutrients necessary for their survival and growth. Dermatophyte infections pose a significant epidemiological and clinical problem. Trichophyton rubrum is the most common anthropophilic dermatophyte worldwide and its typical infection areas include skin of hands or feet and nail plate. In turn, Microsporum canis is a zoophilic pathogen, and mostly well known for ringworm in pets, it is also known to infect humans. The aim of the study was to compare the intracellular metabolite content in the T. rubrum and M. canis during keratin degradation using liquid chromatography system coupled with tandem mass spectrometer (LC-MS/MS). The metabolite "fingerprints" revealed compounds associated with amino acids metabolism, carbohydrate metabolism related to the glycolysis and the tricarboxylic acid cycle (TCA), as well as nucleotide and energy metabolism. The metabolites such as kynurenic acid, L-alanine and cysteine in case of T. rubrum as well as cysteine and riboflavin in case of M. canis were detected only during keratin degradation what may suggest that these compounds may play a key role in the interactions of T. rubrum and M. canis with the host tissue. The metabolomic results were completed by qPCR gene expression assay. Our findings suggest that metabolomic analysis of T. rubrum and M. canis growing in culture media that mimic the dermatophyte infection could allow the understanding of processes involved in the pathogenesis of dermatophytes.

Atrazine biodegradation by mycoinsecticide Metarhizium robertsii: Insights into its amino acids and lipids profile.

Atrazine, is one of major concern pesticides contaminating agricultural areas and ground water. Its microbial biodegradation seems to be the most efficient in terms of economic and environmental benefits. In the present work the cometabolic biodegradation of atrazine by the fungus Metarhizum robertsii IM 6519 during 10-day batch cultures was characterized. The herbicide was transformed to several hydroxy-, dechlorinated or dealkylated metabolites with the involvement of cytochrome P450 monooxygenases. The obtained metabolomics data revealed that atrazine induced oxidative stress (increased the levels of L-proline, L-ornithine, L-arginine, GABA and L-methionine), disruptions of the carbon and nitrogen metabolism (L-aspartic acid, L-asparagine, L-tyrosine, L-threonine, L-isoleucine, L-phenylalanine, 1-methyl-L-histidine, L-tryptophan, L-valine, L-alanine, O-phospho-L-serine, L-sarcosine or L-lysine) and caused an increase in the membrane fluidity (a rise in the phosphatidylcholines/phosphatidylethanolamines (PC/PE) ratio together with the growth of the taurine level). The increased level of hydroxyl derivatives of linoleic acid (9-HODE and 13-HODE) confirmed that atrazine induced lipid peroxidation. The presented results suggesting that M. robertsii IM 6519 might be applied in atrazine biodegradation and may bring up the understanding of the process of triazine biodegradation by Metarhizum strains.

Oxidation of C-reactive protein by hypochlorous acid leads to the formation of potent platelet activator.

We examined the structural and functional consequences of oxidative modification of C-reactive protein (CRP) by hypochlorous acid (HOCl), which can be generated in vivo via the myeloperoxidase/H2O2/Cl- system. HOCl exposure resulted in the oxidation and chlorination of CRP amino acid residues, leading to protein unfolding, greater surface hydrophobicity and the formation of aggregates. After treatment of isolated platelets with 50µg/ml HOCl-CRP, the modified CRP significantly stimulated platelet activation (over 10-fold increase in the fraction of CD62-positive platelets compared to controls, P<0.008), enhanced deposition of platelets onto immobilized fibrinogen (two-fold rise in platelet adhesion compared to controls, P<0.0001), and induced platelet aggregation by up to 79.5%. The ability of HOCl-CRP to interact with several platelet receptors (TLR-4, GPIIbIIIa) and plasma proteins (C1q, IgG) strongly indicates that HOCl-modification leads to structural changes of CRP resulting in the formation of new ligand binding sites, which is characteristic of the monomeric form of CRP exerting pro-inflammatory effects on a variety of cells. Overall, the oxidation of native CRP by HOCl seems to represent an alternative mechanism of CRP modification, by which CRP reveals its pro-inflammatory and pro-thrombotic properties, and as such, it might be of causal relevance in the pathogenesis of atherosclerosis.

Anticancer agent 3-bromopyruvic acid forms a conjugate with glutathione.

BACKGROUND: 3-Bromopyruvic acid (3-BP), a glycolytic inhibitor and a promising anticancer compound, induces oxidative stress and depletes cells of glutathione (GSH). The causes of GSH loss remain unclear. The aim of this study was to ascertain whether 3-BP forms a conjugate with glutathione. METHODS: GSH was incubated with various amounts of 3-BP and the extent of reaction was titrated with (1)H NMR and (1)H-(1)H NMR. The reaction outcome was identified by MS/MS. Intracellular formation of the conjugate was assessed in cells treated with 3-BP and 3-BP((13)C) and analyzed using the targeted LC-MS/MS method in negative ionization MRM mode. RESULTS: 3-BP was found to react with GSH in a 1:1 ratio forming an S-conjugate. The same conjugate was formed intracellularly in erythrocytes and MCF-7 cells. CONCLUSIONS: 3-BP reacts with GSH in the absence of cells and intracellularly. This reaction appears to be the main cause of GSH loss in 3-BP treated cells.

Nitroxides protect against peroxynitrite-induced nitration and oxidation.

Nitroxides are promising compounds for prevention of undesired protein modifications. The aim of this study was to compare the efficiency of 11 nitroxides, derivatives of 2,2,6,6-tetramethylpiperidine-1-oxide (TEMPO) and 2,2,5,5-tetramethylpirrolidine-1-oxyl (PROXYL) in prevention of nitration and oxidation of model compounds and human serum albumin (HSA). Most nitroxides were very efficient in preventing loss of fluorescein fluorescence induced by peroxynitrite (PN) (IC50 in the nanomolar range) and preventing HSA nitration. The loss of fluorescein fluorescence was demonstrated to be due to nitration. Nitroxides were more effective in prevention nitration than oxidation reactions. They showed a concentration window for preventing dihydrorhodamine (DHR) 123 oxidation but exerted a prooxidant effect at both high and low concentrations. No prooxidant effect of nitroxides was seen in prevention of DHR123 oxidation induced by SIN-1. In all essays hydrophobic nitroxides (especially 4-nonylamido-TEMPO and 3-carbamolyl-dehydroPROXYL) showed the lowest efficiency. An exception was the prevention of thiol group oxidation by PN and SIN-1 where hydrophobic nitroxides were the most effective, apparently due to binding to the protein. Nitroxides showed low toxicity to MCF-7 cells. Most nitroxides, except for the most hydrophobic ones, protected cells from the cytotoxic action of SIN-1 and SIN-1-induced protein nitration. These results point to potential usefulness of nitroxides for prevention of PN-induced oxidation and, especially, nitration.

Antimycobacterial action of a new glycolipid-peptide complex obtained from extracellular metabolites of Raoultella ornithinolytica.

In this paper, an antimycobacterial component of extracellular metabolites of a gut bacterium Raoultella ornithinolytica from D. veneta earthworms was isolated and its antimycobacterial action was tested using Mycobacterium smegmatis. After incubation with the complex obtained, formation of pores and furrows in cell walls was observed using microscopic techniques. The cells lost their shape, stuck together and formed clusters. Surface-enhanced Raman spectroscopy analysis showed that, after incubation, the complex was attached to the cell walls of the Mycobacterium. Analyses of the component performed with Fourier transform infrared spectroscopy demonstrated high similarity to a bacteriocin nisin, but energy dispersive X-ray spectroscopy analysis revealed differences in the elemental composition of this antimicrobial peptide. The component with antimycobacterial activity was identified using mass spectrometry techniques as a glycolipid-peptide complex. As it exhibits no cytotoxicity on normal human fibroblasts, the glycolipid-peptide complex appears to be a promising compound for investigations of its activity against pathogenic mycobacteria.

Efficient alachlor degradation by the filamentous fungus Paecilomyces marquandii with simultaneous oxidative stress reduction.

The acceleration of alachlor degradation by Paecilomyces marquandii under controlled and optimized conditions of fungal cultivation in liquid batches was observed (by ca. 20% in comparison to the flask cultures). Acidic environment and oxygen limitation resulted in deterioration of herbicide elimination. Efficient xenobiotic degradation did not correlate with free radicals formation, but some conditions of bioreactor cultivation such as neutral pH and oxygen enriched atmosphere (pO2⩾30%) caused a decrease in the reactive oxygen species (ROS) accumulation in mycelia. The changes in the glutathione (GSH) and ascorbic acid (AA) levels, also in the dismutase (SOD) and catalase (CAT) activities showed active response of the tested fungus against alachlor induced oxidative stress. These results will contribute to the improvement of chloroacetanilides elimination by fungi and extend the knowledge concerning oxidative stress induction and fungal cellular defense.