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.

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.

Lipidomic adaptations of the Metarhizium robertsii strain in response to the presence of butyltin compounds.

Metarhizium robertsii, a butyltin-resistant filamentous fungus, can rapid and complete biodegradation of di- (DBT) and tributyltin (TBT) under conditions of intensive aeration and ascorbic acid supplementation. In this paper, lipidomic investigations were performed to find the membrane adaptations necessary for effective butyltins degradation. HPLC-MS/MS analysis showed that the phospholipid profile was greatly modified during M. robertsii batch cultivation (pO2 ≥ 20%), contributing to increased membrane fluidity and facilitated mass transfer, which could enhance butyltins biodegradation. Intensified biosynthesis of phospholipids, sphingolipids and ergosterol by the mycelia exposed to butyltins was noted. DIOC6(3) fluorescence intensity for TBT-treated mycelium increased 9-fold pointing to membrane hyperpolarization. Fluorescent studies showed improved membrane rigidity and integrity in response to butyltins presence. Vitamin C supplementation restored membrane composition and dynamic properties, followed by supposed acceleration of transport of monobutyltin and its biodegradation thus protecting the M. robertsii cells against oxidative and nitrosative stress.

Trichoderma harzianum diminished oxidative stress caused by 2,4- dichlorophenoxyacetic acid (2,4-D) in wheat, with insights from lipidomics.

2,4-dichlorophenoxyacetic acid (2,4-D) is among the most commonly used herbicides applied for weed control during wheat cultivation. However, its application could affect wheat growth. The present study investigates the effect of the ascomycetous fungus Trichoderma harzianum on lipid peroxidation, phospholipids, signaling lipids and phospholipase D in the seedlings of wheat (Triticum aestivum L.) treated with 2,4-D (2.5 mg L-1). In the group of 4-day-old seedlings exposed to the herbicide, increased lipid peroxidation and inhibition of growth were observed in shoots and roots. Moreover, elevated levels of oxylipins were noted. Among them, the amount of 13-HOTrE oxygenated from linolenic acid (18:3) increased the most significantly. Concurrently, in the seedlings inoculated with T. harzianum, growth was stimulated when the level of phosphatidylcholine (PC) increased. Moreover, in wheat seedlings treated with 2,4-D and T. harzianum, the level of lipid peroxidation was similar to that in the control and there was no increase observed in oxylipins and phospholipase D activity. T. harzianum might have partly alleviated the toxic effect of 2,4-D on wheat seedlings.

2,4-dichlorophenoxyacetic acid-induced oxidative stress: Metabolome and membrane modifications in Umbelopsis isabellina, a herbicide degrader.

The study reports the response to herbicide of the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading fungal strain Umbelopsis isabellina. A comparative analysis covered 41 free amino acids as well as 140 lipid species of fatty acids, phospholipids, acylglycerols, sphingolipids, and sterols. 2,4-D presence led to a decrease in fungal catalase activity, associated with a higher amount of thiobarbituric acid-reactive substances (TBARS). Damage to cells treated with the herbicide resulted in increased membrane permeability and decreased membrane fluidity. Detailed lipidomic profiling showed changes in the fatty acids composition such as an increase in the level of linoleic acid (C18:2). Moreover, an increase in the phosphatidylethanolamine/phosphatidylcholine ratio was observed. Analysis of fungal lipid profiles revealed that the presence of 2,4-D was accompanied by the accumulation of triacylglycerols, a decrease in ergosterol content, and a considerable rise in the level of sphingolipid ceramides. In the exponential phase of growth, increased levels of leucine, glycine, serine, asparagine, and hydroxyproline were found. The results obtained in our study confirmed that in the cultures of U. isabellina oxidative stress was caused by 2,4-D. The herbicide itself forced changes not only to membrane lipids but also to neutral lipids and amino acids, as the difference of tested compounds profiles between 2,4-D-containing and control samples was consequently lower as the pesticide degradation progressed. The presented findings may have a significant impact on the basic understanding of 2,4-D biodegradation and may be applied for process optimization on metabolomic and lipidomic levels.

Elimination and detoxification of 2,4-D by Umbelopsis isabellina with the involvement of cytochrome P450.

The chemical 2,4-dichlorophenoxyacetic acid (2,4-D) is used in agriculture as a herbicide. Its intensive use has an adverse effect on the environment. This study involved examining the degradation of 2,4-D compound by the filamentous fungus Umbelopsis isabellina. After 5 days of incubation, 98% of the herbicide (added at 25 mg L-1) was found to be removed. The elimination of 2,4-D by U. isabellina was connected with the formation of 2,4-dichlorophenol (2,4-DCP), which resulted in a 60% decrease in the sample toxicity toward Artemia franciscana larvae. The metabolism of 2,4-D was inhibited by the addition of metyrapone, a known cytochrome P450 inhibitor. It provides evidence that cytochrome P450 system is involved in 2,4-D metabolism in U. isabellina.