Melatonin Mitigates Lead-Induced Oxidative Stress and Modifies Phospholipid Profile in Tobacco BY-2 Suspension Cells.

Many studies have shown that melatonin (an indoleamine) is an important molecule in plant physiology. It is known that this indoleamine is crucial during plant stress responses, especially by counteracting secondary oxidative stress (efficient direct and indirect antioxidant) and switching on different defense plant strategies. In this report, we present exogenous melatonin's potential to protect lipid profile modification and membrane integrity in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) cell culture exposed to lead. There are some reports of the positive effect of melatonin on animal cell membranes; ours is the first to report changes in the lipid profile in plant cells. The experiments were performed in the following variants: LS: cells cultured on unmodified LS medium-control; (ii) MEL: BY-2 cells cultured on LS medium with melatonin added from the beginning of culture; (iii) Pb: BY-2 cells cultured on LS medium with Pb2+ added on the 4th day of culture; (iv) MEL+Pb: BY-2 cells cultured on LS medium with melatonin added from the start of culture and stressed with Pb2+ added on the 4th day of culture. Lipidomic analysis of BY-2 cells revealed the presence of 40 different phospholipids. Exposing cells to lead led to the overproduction of ROS, altered fatty acid composition and increased PLD activity and subsequently elevated the level of phosphatidic acid at the cost of dropping the phosphatidylcholine. In the presence of lead, double-bond index elevation, mainly by higher quantities of linoleic (C18:2) and linolenic (C18:3) acids in the log phase of growth, was observed. In contrast, cells exposed to heavy metal but primed with melatonin showed more similarities with the control. Surprisingly, the overproduction of ROS caused of lipid peroxidation only in the stationary phase of growth, although considerable changes in lipid profiles were observed in the log phase of growth-just 4 h after lead administration. Our results indicate that the pretreatment of BY-2 with exogenous melatonin protected tobacco cells against membrane dysfunctions caused by oxidative stress (lipid oxidation), but also findings on a molecular level suggest the possible role of this indoleamine in the safeguarding of the membrane lipid composition that limited lead-provoked cell death. The presented research indicates a new mechanism of the defense strategy of plant cells generated by melatonin.

The Combined Effects of Azoxystrobin and the Biosurfactant-Producing Bacillus sp. Kol B3 against the Phytopathogenic Fungus Fusarium sambucinum IM 6525.

This study aimed to evaluate how the combined presence of the synthetic fungicide azoxystrobin (AZ) and the biosurfactant-producing Bacillus sp. Kol B3 influences the growth of the phytopathogenic fungus Fusarium sambucinum IM 6525. The results showed a noticeable increase in antifungal effectiveness when biotic and abiotic agents were combined. This effect manifested across diverse parameters, including fungal growth inhibition, changes in hyphae morphology, fungal membrane permeability and levels of intracellular reactive oxygen species (ROS). In response to the presence of Fusarium and AZ in the culture, the bacteria changed the proportions of biosurfactants (surfactin and iturin) produced. The presence of both AZ and/or Fusarium resulted in an increase in iturin biosynthesis. Only in 72 h old bacterial-fungal co-culture a 20% removal of AZ was noted. In the fungal cultures (with and without the addition of the bacteria), the presence of an AZ metabolite named azoxystrobin free acid was detected in the 48th and 72nd hours of the process. The possible involvement of increased iturin and ROS content in antifungal activity of Bacillus sp. and AZ when used together are also discussed. Biosurfactants were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Microscopy techniques and biochemical assays were also used.

Accumulation of pyrethroids induces changes in metabolism of the entomopathogenic fungus Beauveria bassiana-Proteomic and lipidomic background.

Advances in the agrochemical industry, such as using plant protection products e.g. pyrethroid insecticides, lead to environmental pollution via the accumulation of toxic compounds in soil. An interesting approach to overcoming this threat is using biopreparations based on entomopathogenic fungi that come into contact with the residues of the insecticides in the environment. The aim of this study was to determine whether the soil-dwelling entomopathogenic fungus Beauveria bassiana ARSEF 2860 is capable of accumulating pyrethroids (λ-cyhalothrin, α-cypermethrin and deltamethrin) and to identify the metabolomics and proteomic implications of this process. In this work, we demonstrated for the first time that the tested fungus accumulated pyrethroids as early as on day 2 of incubation with an average efficiency of 90%. Pyrethroids accumulated in large quantities in the mycelium of B. bassiana induced oxidative stress and interacted differently with the enzymes of the basic metabolic pathways, enzymes associated with the organization of the actin cytoskeleton and cell walls, as well as extracellular enzymes responsible for the infectious abilities (α-cypermethrin caused a 61% decrease in PR1, λ-cyhalothrin - a 31% decrease in PR2, which are proteolytic enzymes with a confirmed role in the infectious process). This study also revealed that the accumulated pyrethroids decreased the activity of phospholipase C, which increased the triacylglycerols/diacylglycerols (TAG/DAG) ratio, especially in mycelium in which α-cypermethrin was accumulated. It should be emphasized that the accumulation of pyrethroids in the environment is not fully understood, and current research suggests that entomopathogenic fungi may be part of the process.

Adaptation of the metolachlor-degrading fungus Trichoderma harzianum to the simultaneous presence of low-density polyethylene (LDPE) microplastics.

Although it is known that microplastics (MPs) in soils cause a threat to this complex environment, the actual effects of MPs on soil microorganisms and their catabolic activities, particularly with the biodegradation of herbicides, remain unclear. Hence, the objective of this study was to investigate the effects of a simultaneous presence of metolachlor and low-density polyethylene (LDPE) microplastics on growth inhibition and adaptive responses of Trichoderma harzianum in soil microcosms. Using ergosterol content as an indicator of fungal biomass, it was observed that MPs alone had a marginal inhibitory effect on the growth of the fungus, whereas MET exhibited a dose-dependent inhibitory effect on T. harzianum. However, the presence of MPs did not influence the fungal transforming activity toward the herbicide. Conversely, analysis of lipid profiles in the presence of MPs and herbicides revealed a reduction in the overall fluidity of phospholipid fatty acids, primarily attributed to an increase in lysophospholipids. The activities of six extracellular enzymes in the soil, measured using methylumbelliferone-linked substrates, were significantly enhanced in the presence of MET. These findings contribute to a broader understanding of the alterations in fungal activity in soil resulting from the influence of MPs and MET.

Adjustment in the Composition and Organization of Proteus mirabilis Lipids during the Swarming Process.

Proteus mirabilis, an opportunistic pathogen of the urinary tract, is known for its dimorphism and mobility. A connection of lipid alterations, induced by the rods elongation process, with enhanced pathogenicity of long-form morphotype for the development of urinary tract infections, seems highly probable. Therefore, research on the adjustment in the composition and organization of P. mirabilis lipids forming elongated rods was undertaken. The analyses performed using the ultra-high performance liquid chromatography with tandem mass spectrometry showed that drastic modifications in the morphology of P. mirabilis rods that occur during the swarming process are directly related to deprivation of the long-form cells of PE 33:1 and PG 31:2 and their enrichment with PE 32:1, PE 34:1, PE 34:2, PG 30:2, PG 32:1, and PG 34:1. The analyses conducted by the gas chromatography-mass spectrometry showed negligible effects of the swarming process on fatty acids synthesis. However, the constant proportions between unsaturated and saturated fatty acids confirmed that phenotypic modifications in the P. mirabilis rods induced by motility were independent of the saturation of the phospholipid tails. The method of the Förster resonance energy transfer revealed the influence of the swarming process on the melting of ordered lipid rafts present in the short-form rods, corresponding to the homogeneity of lipid bilayers in the long-form rods of P. mirabilis. Confocal microscope photographs visualized strong Rhod-PE fluorescence of the whole area of swarmer cells, in contrast to weak membrane fluorescence of non-swarmer cells. It suggested an increased permeability of the P. mirabilis bilayers in long-form rods morphologically adapted to the swarming process. These studies clearly demonstrate that swarming motility regulates the lipid composition and organization in P. mirabilis rods.

Combined Application of Aminoglycosides and Ascorbic Acid in the Elimination of Proteus mirabilis Rods Responsible for Causing Catheter-Associated Urinary Tract Infections (CAUTIs)-A Molecular Approach.

Proteus mirabilis is a common cause of catheter-associated urinary tract infections (CAUTIs). In this study, we verified the effectiveness of amikacin or gentamicin and ascorbic acid (AA) co-therapy in eliminating uropathogenic cells, as well as searched for the molecular basis of AA activity by applying chromatographic and fluorescent techniques. Under simulated physiological conditions, a combined activity of the antibiotic and AA supported the growth (threefold) of the P. mirabilis C12 strain, but reduced catheter colonization (≤30%) in comparison to the drug monotherapy. Slight modifications in the phospholipid and fatty acid profiles, as well as limited (≤62%) 2',7'-dichlorofluorescein fluorescence, corresponding to the hydroxyl radical level, allowed for the exclusion of the hypothesis that the anti-biofilm effect of AA was related to membrane perturbations of the C12 strain. However, the reduced (≤20%) fluorescence intensity of propidium iodide, as a result of a decrease in membrane permeability, may be evidence of P. mirabilis cell defense against AA activity. Quantitative analyses of ascorbic acid over time with a simultaneous measurement of the pH values proved that AA can be an effective urine acidifier, provided that it is devoid of the presence of urease-positive cells. Therefore, it could be useful in a prevention of recurrent CAUTIs, rather than in their treatment.

K143R Amino Acid Substitution in 14-α-Demethylase (Erg11p) Changes Plasma Membrane and Cell Wall Structure of Candida albicans.

The opportunistic pathogen Candida albicans is responsible for life-threating infections in immunocompromised individuals. Azoles and polyenes are two of the most commonly used antifungals and target the ergosterol biosynthesis pathway or ergosterol itself. A limited number of clinically employed antifungals correspond to the development of resistance mechanisms. One resistance mechanism observed in clinical isolates of azole-resistant C. albicans is the introduction of point mutations in the ERG11 gene, which encodes a key enzyme (lanosterol 14-α-demethylase) on the ergosterol biosynthesis pathway. Here, we demonstrate that a point mutation K143R in ERG11 (C. albicans ERG11K143R/K143R) contributes not only to azole resistance, but causes increased gene expression. Overexpression of ERG11 results in increased ergosterol content and a significant reduction in plasma membrane fluidity. Simultaneously, the same point mutation caused cell wall remodeling. This could be facilitated by the unmasking of chitin and ß-glucan on the fungal cell surface, which can lead to recognition of the highly immunogenic ß-glucan, triggering a stronger immunological reaction. For the first time, we report that a frequently occurring azole-resistance strategy makes C. albicans less susceptible to azole treatment while, at the same time, affects its cell wall architecture, potentially leading to exposure of the pathogen to a more effective host immune response.

Microplastic-Induced Oxidative Stress in Metolachlor-Degrading Filamentous Fungus Trichoderma harzianum.

While there has been intensive research on the influence of microplastics (MPs) on aquatic organisms and humans, their effect on microorganisms is relatively little-known. The present study describes the response of the Trichoderma harzianum strain to low-density polyethylene (LDPE) microparticles. MPs, either separately or with metolachlor (MET), were added to the cultures. Initially, MP was not found to have a negative effect on fungal growth and MET degradation. After 72 h of cultivation, the content of fungal biomass in samples with MPs was almost three times higher than that in the cultures without MPs. Additionally, a 75% degradation of the initial MET was observed. However, due to the qualitative and quantitative changes in individual classes of phospholipids, cell membrane permeability was increased. Additionally, MPs induced the overproduction of reactive oxygen species. The activity of superoxide dismutase and catalase was also increased in response to MPs. Despite these defense mechanisms, there was enhanced lipid peroxidation in the cultures containing the LDPE microparticles. The results of the study may fill the knowledge gap on the influence of MPs on filamentous fungi. The findings will be helpful in future research on the biodegradation of contaminants coexisting with MPs in soil.

The Role of Ergosterol and Sphingolipids in the Localization and Activity of Candida albicans' Multidrug Transporter Cdr1p and Plasma Membrane ATPase Pma1p.

Opportunistic pathogen Candida albicans causes systemic infections named candidiasis. Due to the increasing number of multi-drug resistant clinical isolates of Candida sp., currently employed antifungals (e.g., azoles) are insufficient for combating fungal infection. One of the resistance mechanisms toward azoles is increased expression of plasma membrane (PM) transporters (e.g., Cdr1p), and such an effect was observed in C. albicans clinical isolates. At the same time, it has been proven that a decrease in PMs sphingolipids (SLs) content correlates with altered sensitivity to azoles and diminished Cdr1p levels. This indicates an important role for SL in maintaining the properties of PM and gaining resistance to antifungal agents. Here, we prove using a novel spot variation fluorescence correlation spectroscopy (svFCS) technique that CaCdr1p localizes in detergent resistant microdomains (DRMs). Immunoblot analysis confirmed the localization of CaCdr1p in DRMs fraction in both the C. albicans WT and erg11Δ/Δ strains after 14 and 24 h of culture. We also show that the C. albicanserg11Δ/Δ strain is more sensitive to the inhibitor of SLs synthesis; aureobasidin A (AbA). AbA treatment leads to a diminished amount of SLs in C. albicans WT and erg11Δ/Δ PM, while, for C. albicanserg11Δ/Δ, the general levels of mannose-inositol-P-ceramide and inositol-P-ceramide are significantly lower than for the C. albicans WT strain. Simultaneously, the level of ergosterol in the C. albicans WT strain after adding of AbA remains unchanged, compared to the control conditions. Analysis of PM permeabilization revealed that treatment with AbA correlates with the disruption of PM integrity in C. albicanserg11Δ/Δ but not in the C. albicans WT strain. Additionally, in the C. albicans WT strain, we observed lower activity of H+-ATPase, correlated with the delocalization of both CaCdr1p and CaPma1p.

Effect of Quinoline on the Phospholipid Profile of Curvularia lunata and Its Microbial Detoxification.

Quinoline is an N-heterocyclic compound commonly found in wastewater, especially that derived from coal processing, chemical, and pharmaceutical industries. In the present study, the microscopic fungus Curvularia lunata IM 4417, which is known to degrade various xenobiotics, was used. The aim of the research was to study the elimination of quinoline and its influence on fungal phospholipids, which are considered to be excellent indicators of environmental monitoring. Quinoline biodegradation products and phospholipid contents were analyzed using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. C. lunata IM 4417 degraded quinoline, which led to the formation of conjugates of glucose with hydroxylated derivatives of the compound. Toxicity tests (Artoxkit M and Microtox assay) indicated that the elimination of lower concentrations of quinoline was efficient and led to a reduction in sample toxicity. The presence of quinoline also significantly affected the profile of fatty acids and phospholipids. The addition of quinoline to a culture of C. lunata IM 4417 caused an increase in the content of phosphatidylcholine (PC) and a decrease in the amount of phosphatidylethanolamine (PE), two major structural lipids. Additionally, decreases in the contents of phosphatidylinositol (PI) and phosphatidylserine (PS), which are responsible for tolerance to toxic substances, cell viability, and signal transduction, were noted. Thus, it can be concluded that the presence of quinoline modifies the membrane composition, and this change may be an important indicator of the presence of N-heterocyclic compounds or other toxins in the environment.

Lactate Like Fluconazole Reduces Ergosterol Content in the Plasma Membrane and Synergistically Kills Candida albicans.

Candida albicans is an opportunistic pathogen that induces vulvovaginal candidiasis (VVC), among other diseases. In the vaginal environment, the source of carbon for C. albicans can be either lactic acid or its dissociated form, lactate. It has been shown that lactate, similar to the popular antifungal drug fluconazole (FLC), reduces the expression of the ERG11 gene and hence the amount of ergosterol in the plasma membrane. The Cdr1 transporter that effluxes xenobiotics from C. albicans cells, including FLC, is delocalized from the plasma membrane to a vacuole under the influence of lactate. Despite the overexpression of the CDR1 gene and the increased activity of Cdr1p, C. albicans is fourfold more sensitive to FLC in the presence of lactate than when glucose is the source of carbon. We propose synergistic effects of lactate and FLC in that they block Cdr1 activity by delocalization due to changes in the ergosterol content of the plasma membrane.

Potential of Trichoderma harzianum and Its Metabolites to Protect Wheat Seedlings against Fusarium culmorum and 2,4-D.

Wheat is a critically important crop. The application of fungi, such as Trichoderma harzianum, to protect and improve crop yields could become an alternative solution to synthetic chemicals. However, the interaction between the fungus and wheat in the presence of stress factors at the molecular level has not been fully elucidated. In the present work, we exposed germinating seeds of wheat (Triticum aestivum) to the plant pathogen Fusarium culmorum and the popular herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of T. harzianum or its extracellular metabolites. Then, the harvested roots and shoots were analyzed using spectrometry, 2D-PAGE, and MALDI-TOF/MS techniques. Although F. culmorum and 2,4-D were found to disturb seed germination and the chlorophyll content, T. harzianum partly alleviated these negative effects and reduced the synthesis of zearalenone by F. culmorum. Moreover, T. harzianum decreased the activity of oxidoreduction enzymes (CAT and SOD) and the contents of the oxylipins 9-Hode, 13-Hode, and 13-Hotre induced by stress factors. Under the influence of various growth conditions, changes were observed in over 40 proteins from the wheat roots. Higher volumes of proteins and enzymes performing oxidoreductive functions, such as catalase, ascorbate peroxidase, cytochrome C peroxidase, and Cu/Zn superoxide dismutase, were found in the Fusarium-inoculated and 2,4-D-treated wheat roots. Additionally, observation of the level of 12-oxo-phytodienoic acid reductase involved in the oxylipin signaling pathway in wheat showed an increase. Trichoderma and its metabolites present in the system leveled out the mentioned proteins to the control volumes. Among the 30 proteins examined in the shoots, the expression of the proteins involved in photosynthesis and oxidative stress response was found to be induced in the presence of the herbicide and the pathogen. In summary, these proteomic and metabolomic studies confirmed that the presence of T. harzianum results in the alleviation of oxidative stress in wheat induced by 2,4-D or F. culmorum.

Phospholipids and Fatty Acids Affect the Colonization of Urological Catheters by Proteus mirabilis.

Proteus mirabilis-mediated CAUTIs are usually initiated by the adherence of bacteria to a urinary catheter surface. In this paper, three isolates of different origin and exhibiting different adhesion abilities were investigated in search of any changes in lipidome components which might contribute to P. mirabilis adhesion to catheters. Using GC-MS and LC-MS/MS techniques, 21 fatty acids and 27 phospholipids were identified in the examined cells. The comparison of the profiles of phospholipids and fatty acids obtained for catheter-attached cells and planktonic cells of the pathogens indicated C11:0 and PE 37:2 levels as values which could be related to P. mirabilis adhesion to a catheter, as well as cis C16:1, PE 32:0, PE 33:0, PE 38:2, PG 33:1, PG 34:0, PE 30:1, PE 32:1 and PG 30:2 levels as values which could be associated with cell hydrophobicity. Based on DiBAC4 (3) fluorescence intensity and an affinity to p-xylene, it was found that the inner membrane depolarization, as well as strong cell-surface hydrophobicity, were important for P. mirabilis adhesion to a silicone catheter. A generalized polarization of Laurdan showed lower values for P. mirabilis cells attached to the catheter surface than for planktonic cells, suggesting lower packing density of membrane components of the adherent cells compared with tightly packed, stiffened membranes of the planktonic cells. Taken together, these data indicate that high surface hydrophobicity, fluidization and depolarization of P. mirabilis cell membranes enable colonization of a silicone urinary catheter surface.

Synthesis of Dolichols in Candida albicans Is Co-Regulated with Elongation of Fatty Acids.

Protein glycosylation requires dolichyl phosphate as a carbohydrate carrier. Dolichols are α-saturated polyprenols, and their saturation in S. cerevisiae is catalyzed by polyprenyl reductase Dfg10 together with some other unknown enzymes. The aim of this study was to identify such enzymes in Candida. The Dfg10 polyprenyl reductase from S. cerevisiae comprises a C-terminal 3-oxo-5-alpha-steroid 4-dehydrogenase domain. Alignment analysis revealed such a domain in two ORFs (orf19.209 and orf19.3293) from C. albicans, which were similar, respectively, to Dfg10 polyprenyl reductase and Tsc13 enoyl-transferase from S. cerevisiae. Deletion of orf19.209 in Candida impaired saturation of polyprenols. The Tsc13 homologue turned out not to be capable of saturating polyprenols, but limiting its expression reduce the cellular level of dolichols and polyprenols. This reduction was not due to a decreased expression of genes encoding cis-prenyltransferases from the dolichol branch but to a lower expression of genes encoding enzymes of the early stages of the mevalonate pathway. Despite the resulting lower consumption of acetyl-CoA, the sole precursor of the mevalonate pathway, it was not redirected towards fatty acid synthesis or elongation. Lowering the expression of TSC13 decreased the expression of the ACC1 gene encoding acetyl-CoA carboxylase, the key regulatory enzyme of fatty acid synthesis and elongation.

An In Vitro Study of the Effect of Viburnum opulus Extracts on Key Processes in the Development of Staphylococcal Infections.

Staphylococcus aureus is still one of the leading causes of both hospital- and community-acquired infections. Due to the very high percentage of drug-resistant strains, the participation of drug-tolerant biofilms in pathological changes, and thus the limited number of effective antibiotics, there is an urgent need to search for alternative methods of prevention or treatment for S. aureus infections. In the present study, biochemically characterized (HPLC/UPLC-QTOF-MS) acetonic, ethanolic, and water extracts from fruits and bark of Viburnum opulus L. were tested in vitro as diet additives that potentially prevent staphylococcal infections. The impacts of V. opulus extracts on sortase A (SrtA) activity (Fluorimetric Assay), staphylococcal protein A (SpA) expression (FITC-labelled specific antibodies), the lipid composition of bacterial cell membranes (LC-MS/MS, GC/MS), and biofilm formation (LIVE/DEAD BacLight) were assessed. The cytotoxicity of V. opulus extracts to the human fibroblast line HFF-1 was also tested (MTT reduction). V. opulus extracts strongly inhibited SrtA activity and SpA expression, caused modifications of S. aureus cell membrane, limited biofilm formation by staphylococci, and were non-cytotoxic. Therefore, they have pro-health potential. Nevertheless, their usefulness as diet supplements that are beneficial for the prevention of staphylococcal infections should be confirmed in animal models in the future.

Surfactants of microbial origin as antibiofilm agents.

The microbial world provides new energy sources and many various 'green' chemicals. One type of chemicals produced by microorganisms is the biosurfactant group. Biosurfactants are universal molecules, exhibiting surface properties often accompanied by desired biological activity. Biosurfactants are considered to be environmentally 'friendly' due to their low toxicity and biodegradable nature. These compounds have unique features and therefore they can find potential applications in many different industries, ranging from biotechnology to environmental remediation technologies. Antibacterial and antifungal activities make them relevant for applications as inhibitory agents against microbial biofilm. This review covers the current knowledge and the recent advances in the field of biosurfactants as antibiofilm agents.

Increased activity of the sterol branch of the mevalonate pathway elevates glycosylation of secretory proteins and improves antifungal properties of Trichoderma atroviride.

Some Trichoderma spp. have an ability to inhibit proliferation of fungal plant pathogens in the soil. Numerous compounds with a proven antifungal activity are synthesized via the terpene pathway. Here, we stimulated the activity of the mevalonate pathway in T. atroviride P1 by expressing the Saccharomyces cerevisiae ERG20 gene coding for farnesyl pyrophosphate (FPP) synthase, a key enzyme of this pathway. ERG20-expressing Trichoderma strains showed higher activities of FPP synthase and squalene synthase, the principal recipient of FPP in the mevalonate pathway. We also observed activation of dolichyl phosphate mannose (DPM) synthase, an enzyme in protein glycosylation, and significantly increased O- and N-glycosylation of secreted proteins. The hyper-glycosylation of secretory hydrolases could explain their increased activity observed in the ERG20 transformants. Analysis of the antifungal properties of the new strains revealed that the hydrolases secreted by the transformants inhibited growth of a plant pathogen, Pythium ultimum more efficiently compared to the control strain. Consequently, the biocontrol activity of the transgenic strains, determined as their ability to protect bean seeds and seedlings against harmful action of P. ultimum, was also improved substantially.

Lipid composition and cell surface hydrophobicity of Candida albicans influence the efficacy of fluconazole-gentamicin treatment.

Adherence of the fungus, Candida albicans, to biotic (e.g. human tissues) and abiotic (e.g. catheters) surfaces can lead to emergence of opportunistic infections in humans. The process of adhesion and further biofilm development depends, in part, on cell surface hydrophobicity (CSH). In this study, we compared the resistance of C. albicans strains with different CSH to the most commonly prescribed antifungal drug, fluconazole, and the newly described synergistic combination, fluconazole and gentamicin. The hydrophobic strain was more resistant to fluconazole due to, among others, overexpression of the ERG11 gene encoding the fluconazole target protein (CYP51A1, Erg11p), which leads to overproduction of ergosterol in this strain. Additionally, the hydrophobic strain displayed high efflux activity of the multidrug resistance Cdr1 pump due to high expression of the CDR1 gene. On the other hand, the hydrophobic C. albicans strain was more susceptible to fluconazole-gentamicin combination because of its different effect on lipid content in the two strains. The combination resulted in ergosterol depletion with subsequent Cdr1p mislocalization and loss of activity in the hydrophobic strain. We propose that C. albicans strains with different CSH may possess altered lipid metabolism and consequently may differ in their response to treatment.

Lipids, proteins and extracellular metabolites of Trichoderma harzianum modifications caused by 2,4-dichlorophenoxyacetic acid as a plant growth stimulator.

Strains of Trichoderma harzianum are well-known producers of bioactive secondary metabolites and have a beneficial effect on plants. However, to the best of our knowledge, the effect of the commonly used pesticides on the activity of this fungus is not yet investigated. Therefore, in the present study, the effect of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the lipidome and selected extracellular compounds synthesized by T. harzianum IM 0961 was examined. It was observed that the herbicide 2,4-D caused changes in the lipid composition of the mycelium and that the herbicide exhibited lipophilic properties. In addition, the herbicide disturbed the phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio and increased membrane permeability. The higher amount of cardiolipin CL 72:7 and the lower amount of CL 72:8 could have been associated with a decreased ratio of 18:2 and 18:1 fatty acids in the herbicide-treated samples. Moreover, in the presence of 2,4-D, an increased lipid peroxidation (twofold), as well as a higher content of oxylipin (9-HODE and 13-HODE) and phosphatidic acid (PA), was noted, confirming that 2,4-D induced lipid peroxidation in the mycelium. The herbicide also exerted its toxic effect on the production of 14-aminoacid peptaibols and two compounds, harzianic acid and t22-azaphilone, exhibiting antibiotic and plant growth-promoting activity. During proteomic analysis, the synthesis of some proteins, such as calcineurin-like phosphoesterase metallophosphatases (MPPs), which modulate the properties of cell walls, was found to be inhibited by the herbicide. These presented findings may be of significant value in understanding the effect of 2,4-D on the activity of T. harzianum.

Biotransformation and detoxification of chloroacetanilide herbicides by Trichoderma spp. with plant growth-promoting activities.

Due to the increasing use of chlorinated organic compounds, environmental pollution is a key issue in agricultural and industrial areas. In this study, biodegradation of chloroacetanilide herbicides, such as alachlor and metolachlor, by eight fungal strains of Trichoderma spp. originating from different microorganism collections was investigated. The tested fungi converted 80-99% of alachlor and 40-79% of metolachlor after 7 days of incubation. Biotransformation of herbicides was performed mainly by dechlorination and hydroxylation reactions. Eight alachlor metabolites and four byproducts of metolachlor conversion were detected in Trichoderma cultures, including two metolachlor intermediates for the first time identified in fungi. Moreover, in the cultures of six Trichoderma strains supplemented with chloroacetanilides, a decrease in toxicity was observed toward tested Artemia franciscana crustaceans. Simultaneously, 7 days after the application of the spores of T. koningii IM 0956, T. citrinoviride IM 6325, T. harzianum KKP 534, T. viride KKP 792 and T. virens DSM 1963 the length of roots and shoots of rapeseed seedlings treated with alachlor or metolachlor significantly increased. All tested strains exhibited plant growth-promoting traits, such as siderophore production, 1-aminocyclopropane-1-carboxylate deaminase (ACCD) activity, and phosphate solubilization, even in the presence of chloroacetanilide herbicides.