Antibacterial efficacy and membrane mechanism of action of the Serratia-derived non-ionic lipopeptide, serrawettin W2-FL10.

The study aimed to investigate the antibacterial activity, cytotoxicity, and mechanism of action of the non-ionic, cyclic lipopeptide, serrawettin W2-FL10 against Staphylococcus aureus. W2-FL10 exhibited potent activity against the Gram-positive bacteria S. aureus, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, and Bacillus subtilis, with minimum inhibitory concentration (MIC) values ranging from 6.3 to 31.3 µg/mL, while no activity was observed against Gram-negative bacteria. Broth microdilution assays showed that W2-FL10 interacted with key cell membrane components, such as lipid phosphatidyl glycerol and lipoteichoic acid of S. aureus. Upon membrane interaction, W2-FL10 dissipated membrane potential within 12 min and increased S. aureus membrane permeability within 28-40 min, albeit at slower rates and higher concentrations than the lytic peptide melittin. The observed membrane permeability, as detected with propidium iodide (PI), may be attributed to transmembrane pores/lesions, possibly dependent on dimer-driven lipopeptide oligomerization in the membrane. Scanning electron microscopy (SEM) imaging also visually confirmed the formation of lesions in the cell wall of one of the S. aureus strains, and cell damage within 1 h of exposure to W2-FL10, corroborating the rapid time-kill kinetics of the S. aureus strains. This bactericidal action against the S. aureus strains corresponded to membrane permeabilization by W2-FL10, indicating that self-promoted uptake into the cytosol may be part of the mode of action. Finally, this lipopeptide exhibited low to moderate cytotoxicity to the Chinese hamster ovarian (CHO) cell line in comparison to the control (emetine) with an optimal lipophilicity range (log D value of 2.5), signifying its potential as an antibiotic candidate. IMPORTANCE: Antimicrobial resistance is a major public health concern, urgently requiring antibacterial compounds exhibiting low adverse health effects. In this study, a novel antibacterial lipopeptide analog is described, serrawettin W2-FL10 (derived from Serratia marcescens), with potent activity displayed against Staphylococcus aureus. Mechanistic studies revealed that W2-FL10 targets the cell membrane of S. aureus, causing depolarization and permeabilization because of transmembrane lesions/pores, resulting in the leakage of intracellular components, possible cytosolic uptake of W2-FL10, and ultimately cell death. This study provides the first insight into the mode of action of a non-ionic lipopeptide. The low to moderate cytotoxicity of W2-FL10 also highlights its application as a promising therapeutic agent for the treatment of bacterial infections.

Unlocking roles of cationic and aromatic residues in peptide amphiphiles in treating drug-resistant gram-positive pathogens.

Multidrug resistance (MDR) is a rising threat to global health because the number of essential antibiotics used for treating MDR infections is increasingly compromised. In this work we report a group of new amphiphilic peptides (AMPs) derived from the well-studied G3 (G(IIKK)3I-NH2) to fight infections from Gram-positive bacteria including susceptible Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), focusing on membrane interactions. Time-dependent killing experiments revealed that substitutions of II by WW (GWK), II by FF (GFK) and KK by RR (GIR) resulted in improved bactericidal efficiencies compared to G3 (GIK) on both S. aureus and MRSA, with the order of GWK > GIR > GFK > GIK. Electronic microscopy imaging revealed structural disruptions of AMP binding to bacterial cell walls. Fluorescence assays including AMP binding to anionic lipoteichoic acids (LTA) in cell-free and cell systems indicated concentration and time-dependent membrane destabilization associated with bacterial killing. Furthermore, AMP's binding to anionic plasma membrane via similar fluorescence assays revealed a different extent of membrane depolarization and leakage. These observations were supported by the penetration of AMPs into the LTA barrier and the subsequent structural compromise to the cytoplasmic membrane as revealed from SANS (small angle neutron scattering). Both experiments and molecular dynamics (MD) simulations revealed that GWK and GIR could make the membrane more rigid but less effective in diffusive efficiency than GIK and GFK through forming intramembrane peptide nanoaggregates associated with hydrophobic mismatch and formation of fluidic and rigid patches. The reported peptide-aggregate-induced phase-separation emerged as a crucial factor in accelerated membrane disintegration and fast bacterial killing. This work has demonstrated the importance of membrane interactions to the development of more effective AMPs and the relevance of the approaches as reported in assisting this area of research.

Investigating the role of hypothetical protein (AAB33144.1) in HIV-1 virus pathogenicity: A comparative study with FDA-Approved inhibitor compounds through In silico analysis and molecular docking.

Aim and objective: Due to the a lot of unexplored proteins in HIV-1, this research aimed to explore the functional roles of a hypothetical protein (AAB33144.1) that might play a key role in HIV-1 pathogenicity. Methods: The homologous protein was identified along with building and validating the 3D structure by searching several bioinformatics tools. Results: Retroviral aspartyl protease and retropepsin like functional domains and motifs, folding pattern (cupredoxins), and subcellular localization in cytoplasmic membrane were determined as biological activity. Besides, the functional annotation revealed that the chosen hypothetical protein possessed protease-like activity. To validate our generated protein 3D structure, molecular docking was performed with five compounds where nelfinavir showed (-8.2 kcal/mol) best binding affinity against HXB2 viral protease (PDB ID: 7SJX) and main protease (PDB ID: 4EYR) protein. Conclusions: This study suggests that the annotated hypothetical protein related to protease action, which may be useful in viral genetics and drug discovery.

Role of cell membrane homeostasis in the pathogenicity of pathogenic filamentous fungi.

The cell membrane forms a fundamental part of all living cells and participates in a variety of physiological processes, such as material exchange, stress response, cell recognition, signal transduction, cellular immunity, apoptosis, and pathogenicity. Here, we review the mechanisms and functions of the membrane structure (lipid components of the membrane and the biosynthesis of unsaturated fatty acids), membrane proteins (transmembrane proteins and proteins contributing to membrane curvature), transcriptional regulation, and cell wall components that influence the virulence and pathogenicity of filamentous fungi.

The Primary Mode of Action of Lippia graveolens Essential Oil on Salmonella enterica subsp. Enterica Serovar Typhimurium.

Essential oils are known to exhibit diverse antimicrobial properties, showing their value as a natural resource. Our work aimed to investigate the primary mode of action of Cuban Lippia graveolens (Kunth) essential oil (EO) against Salmonella enterica subsp. enterica serovar Typhimurium (S. enterica ser. Typhimurium). We assessed cell integrity through various assays, including time-kill bacteriolysis, loss of cell material with absorption at 260 and 280 nm, total protein leakage, and transmission electron microscopy (TEM). The impact of L. graveolens EO on membrane depolarization was monitored and levels of intracellular and extracellular ATP were measured by fluorescence intensity. The minimum inhibitory and bactericidal concentrations (MIC and MBC) of L. graveolens EO were 0.4 and 0.8 mg/mL, respectively. This EO exhibited notable bactericidal effects on treated cells within 15 min without lysis or leakage of cellular material. TEM showed distinct alterations in cellular ultrastructure, including membrane shrinkage and cytoplasmic content redistribution. We also observed disruption of the membrane potential along with reduced intracellular and extracellular ATP concentrations. These findings show that L. graveolens EO induces the death of S. enterica ser. Typhimurium, important information that can be used to combat this foodborne disease-causing agent.

Locations of membrane protein production in a cyanobacterium.

Cyanobacteria show an unusually complex prokaryotic cell structure including a distinct intracytoplasmic membrane system, the thylakoid membranes that are the site of the photosynthetic light reactions. The thylakoid and plasma membranes have sharply distinct proteomes, but the mechanisms that target proteins to a specific membrane remain poorly understood. Here, we investigate the locations of translation of thylakoid and plasma membrane proteins in the model unicellular cyanobacterium Synechococcus elongatus PCC 7942. We use fluorescent in situ hybridization to probe the locations of mRNAs encoding membrane-integral proteins, plus Green Fluorescent Protein tagging of the RplL subunit to reveal the location of ribosomes under different conditions. We show that membrane-integral thylakoid and plasma membrane proteins are translated in different locations. Thylakoid membrane proteins are translated in patches at the innermost thylakoid membrane surface facing the nucleoid. However, different proteins are translated in different patches, even when they are subunits of the same multiprotein complex. This implies that translation is distributed over the proximal thylakoid surface, with newly inserted proteins migrating within the membrane prior to incorporation into complexes. mRNAs encoding plasma membrane proteins form patches at the plasma membrane. Ribosomes can be observed at similar locations near the thylakoid and plasma membranes, with more ribosomes near the plasma membrane when conditions force rapid production of plasma membrane proteins. There must be routes for ribosomes and mRNAs past the thylakoids to the plasma membrane. We infer a system to chaperone plasma membrane mRNAs to prevent their translation prior to arrival at the correct membrane. IMPORTANCE Cyanobacteria have a complex and distinct membrane system within the cytoplasm, the thylakoid membranes that house the photosynthetic light reactions. The thylakoid and plasma membranes contain distinct sets of proteins, but the steps that target proteins to the two membranes remain unclear. Knowledge of the protein sorting rules will be crucial for the biotechnological re-engineering of cyanobacterial cells, and for understanding the evolutionary development of the thylakoids. Here, we probe the subcellular locations of the mRNAs that encode cyanobacterial membrane proteins and the ribosomes that translate them. We show that thylakoid and plasma membrane proteins are produced at different locations, providing the first direct evidence for a sorting mechanism that operates prior to protein translation.

Streptomyces albidoflavus Q antifungal metabolites inhibit the ergosterol biosynthesis pathway and yeast growth in fluconazole-resistant Candida glabrata: phylogenomic and metabolomic analyses.

There is an urgent need to develop new antifungals due to the increasing prevalence of multidrug-resistant fungal infections and the recent emergence of COVID-19-associated candidiasis. A good study model for evaluating new antifungal compounds is Candida glabrata, an opportunistic fungal pathogen with intrinsic resistance to azoles (the most common clinical drugs for treating fungal infections). The aim of the current contribution was to conduct in vitro tests of antifungal metabolites produced by the bacteria Streptomyces albidoflavus Q, identify their molecular structures, and utilize several techniques to provide evidence of their therapeutic target. S. albidoflavus was isolated from maize rhizospheric soil in Mexico and identified by phylogenomic analysis using a 92-gene core. Of the 66 metabolites identified in S. albidoflavus Q by a liquid chromatography-high resolution mass spectrometry (LC-HRMS) metabolomic analysis of the lyophilized supernatant, six were selected by the Way2drug server based on their in silico binding to the likely target, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR, the key enzyme in the ergosterol biosynthesis pathway). Molecular modeling studies show a relatively high binding affinity for the CgHMGR enzyme by two secondary metabolites: isogingerenone B (diaryl heptanoid) and notoginsenoside J (polycyclic triterpene). These secondary metabolites were able to inhibit ergosterol synthesis and affect yeast viability in vitro. They also caused alterations in the ultrastructure of the yeast cytoplasmic membrane, as evidenced by transmission electron microscopy. The putative target of isogingerenone B and notoginsenoside J is distinct from that of azole drugs (the most common clinical antifungals). The target for the latter is the lanosterol 14 alpha-demethylase enzyme (Erg11). IMPORTANCE Multidrug resistance has emerged among yeasts of the genus Candida, posing a severe threat to global health. The problem has been exacerbated by the pandemic associated with COVID-19, during which resistant strains of Candida auris and Candida glabrata have been isolated from patients infected with the SARS-CoV-2 virus. To confront this challenge, the World Health Organization has invoked scientists to search for new antifungals with alternative molecular targets. This study identified 66 metabolites produced by the bacteria Streptomyces albidoflavus Q, 6 of which had promising properties for potential antifungal activity. The metabolites were tested in vitro as inhibitors of ergosterol synthesis and C. glabrata growth, with positive results. They were also found to damage the cytoplasmic membrane of the fungus. The corresponding molecular structures and their probable therapeutic target were established. The target is apparently distinct from that of azole drugs.

Evaluation of the Effect of Plectranthus amboinicus L. Leaf Extracts on the Bacterial Antioxidant System and Cell Membrane Integrity of Pseudomonas aeruginosa PA01 and Staphylococcus aureus NCTC8325.

Plectranthus amboinicus (Indian borage) has been extensively studied for its medicinal properties, which can be exploited to develop new antimicrobial therapeutics. The current study investigated the effect of Plectranthus amboinicus leaf extracts on the catalase activity, reactive oxygen species, lipid peroxidation, cytoplasmic membrane permeability, and efflux pump activity in S. aureus NCTC8325 and P. aeruginosa PA01. As the enzyme catalase protects bacteria against oxidative stress, disruption of its activity creates an imbalance in reactive oxygen species (ROS) levels, which subsequently oxidizes lipid chains, leading to lipid peroxidation. In addition, bacterial cell membranes are a potential target for new antibacterial agents, as efflux pump systems play a crucial role in antimicrobial resistance. Upon exposure of the microorganisms to Indian borage leaf extracts, the observed catalase activity decreased by 60% and 20% in P. aeruginosa and S. aureus, respectively. The generation of ROS can cause oxidation reactions to occur within the polyunsaturated fatty acids of the lipid membranes and induce lipid peroxidation. To investigate these phenomena, the increase in ROS activity in P. aeruginosa and S. aureus was studied using H2DCFDA, which is oxidized to 2',7'-dichlorofluorescein (DCF) by ROS. Furthermore, the concentration of lipid peroxidation product (malondialdehyde) was assessed using the Thiobarbituric acid assay and was shown to increase by 42.4% and 42.5% in P. aeruginosa and S. aureus, respectively. The effect of the extracts on the cell membrane permeability was monitored using diSC3-5 dye and it was observed that the cell membrane permeability of P. aeruginosa increased by 58% and of S. aureus by 83%. The effect on efflux pump activity was investigated using Rhodamine-6-uptake assay, which displayed a decrease in efflux activity of 25.5% in P. aeruginosa and 24.2% in S. aureus after treatment with the extracts. This combination of different methods to study various bacterial virulence factors provides a more robust, mechanistic understanding of the effect of P. amboinicus extracts on P. aeruginosa and S. aureus. This study thus represents the first report of the assessment of the effect of Indian borage leaf extracts on bacterial antioxidant systems and bacterial cell membranes, and can facilitate the future development of bacterial resistance modifying agents derived from P. amboinicus.

Bacterial extracellular vesicles: an emerging avenue to tackle diseases.

A growing body of research, especially in recent years, has shown that bacterial extracellular vesicles (bEVs) are one of the key underlying mechanisms behind the pathogenesis of various diseases like pulmonary fibrosis, sepsis, systemic bone loss, and Alzheimer's disease. Given these new insights, bEVs are proposed as an emerging vehicle that can be used as a diagnostic tool or to tackle diseases when used as a therapeutic target. To further boost the understanding of bEVs in health and disease we thoroughly discuss the contribution of bEVs in disease pathogenesis and the underlying mechanisms. In addition, we speculate on their potential as novel diagnostic biomarkers and how bEV-related mechanisms can be exploited as therapeutic targets.

Structural and mechanical properties of the red blood cell's cytoplasmic membrane seen through the lens of biophysics.

Red blood cells (RBCs) are the most abundant cell type in the human body and critical suppliers of oxygen. The cells are characterized by a simple structure with no internal organelles. Their two-layered outer shell is composed of a cytoplasmic membrane (RBC cm ) tethered to a spectrin cytoskeleton allowing the cell to be both flexible yet resistant against shear stress. These mechanical properties are intrinsically linked to the molecular composition and organization of their shell. The cytoplasmic membrane is expected to dominate the elastic behavior on small, nanometer length scales, which are most relevant for cellular processes that take place between the fibrils of the cytoskeleton. Several pathologies have been linked to structural and compositional changes within the RBC cm and the cell's mechanical properties. We review current findings in terms of RBC lipidomics, lipid organization and elastic properties with a focus on biophysical techniques, such as X-ray and neutron scattering, and Molecular Dynamics simulations, and their biological relevance. In our current understanding, the RBC cm 's structure is patchy, with nanometer sized liquid ordered and disordered lipid, and peptide domains. At the same time, it is surprisingly soft, with bending rigidities κ of 2-4 kBT. This is in strong contrast to the current belief that a high concentration of cholesterol results in stiff membranes. This extreme softness is likely the result of an interaction between polyunsaturated lipids and cholesterol, which may also occur in other biological membranes. There is strong evidence in the literature that there is no length scale dependence of κ of whole RBCs.

Dysregulation of Cell Envelope Homeostasis in Staphylococcus aureus Exposed to Solvated Lignin.

Lignin is an aromatic plant cell wall polymer that facilitates water transport through the vasculature of plants and is generated in large quantities as an inexpensive by-product of pulp and paper manufacturing and biorefineries. Although lignin's ability to reduce bacterial growth has been reported previously, its hydrophobicity complicates the ability to examine its biological effects on living cells in aqueous growth media. We recently described the ability to solvate lignin in Good's buffers with neutral pH, a breakthrough that allowed examination of lignin's antimicrobial effects against the human pathogen Staphylococcus aureus. These analyses showed that lignin damages the S. aureus cell membrane, causes increased cell clustering, and inhibits growth synergistically with tunicamycin, a teichoic acid synthesis inhibitor. In the present study, we examined the physiological and transcriptomic responses of S. aureus to lignin. Intriguingly, lignin restored the susceptibility of genetically resistant S. aureus isolates to penicillin and oxacillin, decreased intracellular pH, impaired normal cell division, and rendered cells more resistant to detergent-induced lysis. Additionally, transcriptome sequencing (RNA-Seq) differential expression (DE) analysis of lignin-treated cultures revealed significant gene expression changes (P < 0.05 with 5% false discovery rate [FDR]) related to the cell envelope, cell wall physiology, fatty acid metabolism, and stress resistance. Moreover, a pattern of concurrent up- and downregulation of genes within biochemical pathways involved in transmembrane transport and cell wall physiology was observed, which likely reflects an attempt to tolerate or compensate for lignin-induced damage. Together, these results represent the first comprehensive analysis of lignin's antibacterial activity against S. aureus. IMPORTANCE S. aureus is a leading cause of skin and soft tissue infections. The ability of S. aureus to acquire genetic resistance to antibiotics further compounds its ability to cause life-threatening infections. While the historical response to antibiotic resistance has been to develop new antibiotics, bacterial pathogens are notorious for rapidly acquiring genetic resistance mechanisms. As such, the development of adjuvants represents a viable way of extending the life span of current antibiotics to which pathogens may already be resistant. Here, we describe the phenotypic and transcriptomic response of S. aureus to treatment with lignin. Our results demonstrate that lignin extracted from sugarcane and sorghum bagasse restores S. aureus susceptibility to ß-lactams, providing a premise for repurposing these antibiotics in treatment of resistant S. aureus strains, possibly in the form of topical lignin/ß-lactam formulations.

The tremendous biomedical potential of bacterial extracellular vesicles.

Bacterial extracellular vesicles (bEVs) are nano-sized, lipid membrane-delimited particles filled with bacteria-derived components. They have important roles in the physiology and pathogenesis of bacteria, and in bacteria-bacteria and bacteria-host interactions. Interestingly, recent advances in biotechnology have made it possible to engineer the bEV surface and decorate it with diverse biomolecules and nanoparticles (NPs). bEVs have been the focus of significant interest in a range of biomedical fields and are being evaluated as vaccines, cancer immunotherapy agents, and drug delivery vehicles. However, significant hurdles in terms of their safety, efficacy, and mass production need to be addressed to enable their full clinical potential. Here, we review recent advances and remaining obstacles regarding the use of bEVs in different biomedical applications and discuss paths toward clinical translation.

Extracellular vesicles-mediated interaction within intestinal microenvironment in inflammatory bowel disease.

Background: The intestinal tract is a complicated ecosystem with dynamic homeostasis via interaction of intestine and microbiota. Inflammatory bowel disease (IBD) is chronic intestinal inflammation involving dysbiosis of intestinal microenvironment. Extracellular vesicles (EVs), as vital characteristics of cell-cell and cell-organism communication, contribute to homeostasis in intestine. Recently, EVs showed excellent potential for clinical applications in disease diagnoses and therapies. Aim of Review: Our current review discusses the modulatory functions of EVs derived from different sources in intestine, especially their effects and applications in IBD clinical therapy. EV-mediated interaction systems between host intestine and microbiota were established to describe possible mechanisms of IBD pathogenesis and its cure. Key Scientific Concepts of Review: EVs are excellent vehicles for delivering molecules containing genetic information to recipient cells. Multiple pieces of evidence have illustrated that EVs participate the interaction between host and microbiota in intestinal microenvironment. In inflammatory intestine with dysbiosis of microbiota, EVs as regulators target promoting immune response and microbial reconstruction. EVs-based immunotherapy could be a promising therapeutic approach for the treatment of IBD in the near future.

Polyribosome-Dependent Clustering of Membrane-Anchored RNA Degradosomes To Form Sites of mRNA Degradation in Escherichia coli.

The essential endoribonuclease RNase E, which is a component of the Escherichia coli multienzyme RNA degradosome, has a global role in RNA processing and degradation. RNase E localizes to the inner cytoplasmic membrane in small, short-lived clusters (puncta). Rifampin, which arrests transcription, inhibits RNase E clustering and increases its rate of diffusion. Here, we show that inhibition of clustering is due to the arrest of transcription using a rifampin-resistant control strain. Two components of the RNA degradosome, the 3' exoribonuclease polynucleotide phosphorylase (PNPase) and the DEAD box RNA helicase RhlB, colocalize with RNase E in puncta. Clustering of PNPase and RhlB is inhibited by rifampin, and their diffusion rates increase, as evidenced by in vivo photobleaching measurements. Results with rifampin treatment reported here show that RNA degradosome diffusion is constrained by interaction with RNA substrate. Kasugamycin, which arrests translation initiation, inhibits formation of puncta and increases RNA degradosome diffusion rates. Since kasugamycin treatment results in continued synthesis and turnover of ribosome-free mRNA but inhibits polyribosome formation, RNA degradosome clustering is therefore polyribosome dependent. Chloramphenicol, which arrests translation elongation, results in formation of large clusters (foci) of RNA degradosomes that are distinct from puncta. Since chloramphenicol-treated ribosomes are stable, the formation of RNA degradosome foci could be part of a stress response that protects inactive polyribosomes from degradation. Our results strongly suggest that puncta are sites where translationally active polyribosomes are captured by membrane-associated RNA degradosomes. These sites could be part of a scanning process that is an initial step in mRNA degradation. IMPORTANCE Here, we show that RNase E, RhlB, and PNPase act together as components of the multienzyme RNA degradosome in polyribosome-dependent clustering to form puncta on the inner cytoplasmic membrane. Our results support the hypothesis that RNA degradosome puncta are sites of mRNA degradation. We propose that clustering of RNA degradosomes is a pre-RNase E cleavage step in which polyribosomes are scanned in a search for ribosome-free mRNA. This work is part of an emerging view that posttranscriptional events such as tRNA maturation, late steps in ribosome assembly, and mRNA degradation are membrane associated and partitioned from translation in the cytoplasm and transcription in the nucleoid. This separation could protect newly synthesized transcripts from premature destructive interactions with the RNA degradosome. The scanning of ribosomes and polyribosomes could be part of a general mechanism in which defective stable RNA or ribosome-free mRNA is targeted for destruction by the RNA degradosome.

d-Amino acids in antimicrobial peptides: a potential approach to treat and combat antimicrobial resistance.

Antimicrobial resistance is one of the leading challenges in the human healthcare segment. Advances in antimicrobial resistance have triggered exploration of natural alternatives to stabilize its seriousness. Antimicrobial peptides are small, positively charged oligopeptides that are as potent as commercially available antibiotics against a wide spectrum of organisms, such as Gram-positive bacteria, Gram-negative bacteria, viruses, and fungal strains. In addition to their antibiotic capabilities, these peptides possess anticancer activity, activate the immune response, and regulate inflammation. Peptides have distinct modes of action and fall into various categories due to their amino acid composition. Although antimicrobial peptides specifically target the bacterial cytoplasmic membrane, they can also target the cell nucleus and protein synthesis. Owing to the increasing demand for novel treatments against the threat of antimicrobial resistance, naturally synthesized peptides are a beneficial development concept. Antimicrobial peptides are pervasive and can easily be modified using de-novo synthesis technology. Antimicrobial peptides can be isolated from natural resources such as humans, plants, bacteria, and fungi. This review gives a brief overview of antimicrobial peptides and their diastereomeric composition. Other current trends, the future scope of antimicrobial peptides, and the role of d-amino acids are also discussed, with a specific emphasis on the design and development of new drugs.

Ultrastructure of Cells and Microanalysis in Malus domestica Borkh. 'Szampion' Fruit in Relation to Varied Calcium Foliar Feeding.

Calcium is one of the most poorly reutilized nutrients. Its deficiencies cause various physiological disturbances and, consequently, reduce the quantity and quality of yields. Reduced content of Ca2+ ions in cells leads to development of, e.g., bitter pit in apples. Efficient and instantaneous mitigation of Ca2+ deficiencies is provided by foliar feeding. There are no detailed data on the effect of foliar feeding with various calcium forms on the cell structure or on the microanalysis and mapping of this element in apple fruit cells. Therefore, we carried out comparative studies of the ultrastructure of epidermis and hypodermis cells, to assess the content and distribution of calcium in the cell wall, cytoplasmic membrane, cytoplasm, and precipitates of Malus domestica Borkh. 'Szampion' fruit exposed to four Ca treatments, including the control with no additional Ca supplementation (I) and foliar applications of Ca(NO3)2 (II), CaCl2 (III), and Ca chelated with EDTA (IV). Light and transmission electron microscopy and an X-ray microanalyzer were used and showed a beneficial effect of calcium preparations on the ultrastructure of fruit epidermis and hypodermis cells, manifested in the presence of a normally developed cell wall with a regular middle lamella, preserved continuity of cytoplasmic membranes, and stabilized cell structure. In the selected elements of apical epidermis cells, the highest level of Ca2+ ions was detected in the middle lamella, cell wall, plasmalemma, and cytoplasm. The highest increase in the Ca2+ content in these cell constituents was recorded in treatment IV, whereas the lowest value of the parameters was noted in variant III.

Poplar Tree Response to Feeding by the Petiole Gall Aphid Pemphigus spyrothecae Pass.

Pemphigus spyrothecae Pass. which is a member of the subfamily Pemphiginae is one of the gall-inducing aphids that occurs on poplar trees. Phloem feeding of a founding mother on leaf petiole results in the formation of a new organ, i.e., the spiral gall. This study documents aphid development inside the galls during the growing season and the effect of their feeding on leaf architecture and physiology of the host plant. In particular, leaf length, width, and area were measured, as well as hydrogen peroxide (H2O2) content, electrolyte leakage (EL), malondialdehyde (MDA) concentration, and the activity of ascorbate (APX) and guaiacol peroxidase (GPX) were determined in galls and galled leaves. The presence of petiole galls significantly decreased the length, width, and leaf area. Aphid activity increased H2O2 concentration in galls and EL from galls and leaf tissues, which was accompanied by a strong decrease in MDA content and both peroxidase activities, especially in gall tissues. It can be suggested that P. spyrothecae can manipulate physiological machinery of the host plant for its own benefit.

Interactions of Tea-Derived Catechin Gallates with Bacterial Pathogens.

Green tea-derived galloylated catechins have weak direct antibacterial activity against both Gram-positive and Gram-negative bacterial pathogens and are able to phenotypically transform, at moderate concentrations, methicillin-resistant Staphylococcus aureus (MRSA) clonal pathogens from full ß-lactam resistance (minimum inhibitory concentration 256-512 mg/L) to complete susceptibility (~1 mg/L). Reversible conversion to susceptibility follows intercalation of these compounds into the bacterial cytoplasmic membrane, eliciting dispersal of the proteins associated with continued cell wall peptidoglycan synthesis in the presence of ß-lactam antibiotics. The molecules penetrate deep within the hydrophobic core of the lipid palisade to force a reconfiguration of cytoplasmic membrane architecture. The catechin gallate-induced staphylococcal phenotype is complex, reflecting perturbation of an essential bacterial organelle, and includes prevention and inhibition of biofilm formation, disruption of secretion of virulence-related proteins, dissipation of halotolerance, cell wall thickening and cell aggregation and poor separation of daughter cells during cell division. These features are associated with the reduction of capacity of potential pathogens to cause lethal, difficult-to-treat infections and could, in combination with ß-lactam agents that have lost therapeutic efficacy due to the emergence of antibiotic resistance, form the basis of a new approach to the treatment of staphylococcal infections.

Effect of Neuroterus quercusbaccarum (L.) galls on physiological and biochemical response of Quercus robur leaves.

Gall formation is associated with multiple changes in plant cells, which still requires a better understanding. In this study, galls caused by sexual generation (♀♂) of Neuroterus quercusbaccarum (L.) (Hymenoptera: Cynipidae) on pedunculate oak trees (Quercus robur L.) were used as a model. Cytoplasmic membrane condition, concentration of hydrogen peroxide (H2O2), the activity of antioxidant enzymes and amino acid decarboxylase as well as chlorophyll fluorescence parameters were determined. Changes in physiological and biochemical parameters were analyzed in foliar tissues with galls and gall tissues themselves and compared to control. The presence of galls on oak leaves caused an increase of lipid peroxidation level. A significant decline in H2O2 and TBARS content with the reduction of guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) activity were observed in gall tissues. The activity amino acid decarboxylase, i.e., LDC, ODC and TyDC varied between samples, which may affect the content of amino acids. The presence of N. quercusbaccarum galls caused an insignificant increase of the chlorophylls, carotenoids and anthocyanin contents, while the content of pigments and their ratios in gall tissues was extremely low. Moreover, photosynthetic parameters (F0, Fm, Fv/Fm, Y, qP) were significantly decreased. Data generated in this study indicate that the development of N. quercusbaccarum galls on pedunculate oak leaves has a negative effect on host plant related to the disruption of cell membrane integrity, disturbance of photosynthesis and reduction of the antioxidant potential of the host plant.

Physiological mechanism of improved tolerance of Saccharomyces cerevisiae to lignin-derived phenolic acids in lignocellulosic ethanol fermentation by short-term adaptation.

BACKGROUND: Phenolic acids are lignin-derived fermentation inhibitors formed during many pretreatment processes of lignocellulosic biomass. In this study, vanillic, p-hydroxybenzoic, and syringic acids were selected as the model compounds of phenolic acids, and the effect of short-term adaptation strategies on the tolerance of S. cerevisiae to phenolic acids was investigated. The mechanism of phenolic acids tolerance in the adapted yeast strains was studied at the morphological and physiological levels. RESULTS: The multiple phenolic acids exerted the synergistic inhibitory effect on the yeast cell growth. In particular, a significant interaction between vanillic and hydroxybenzoic acids was found. The optimal short-term adaptation strategies could efficiently improve the growth and fermentation performance of the yeast strain not only in the synthetic media with phenolic acids, but also in the simultaneous saccharification and ethanol fermentation of corncob residue. Morphological analysis showed that phenolic acids caused the parental strain to generate many cytoplasmic membrane invaginations with crack at the top of these sites and some mitochondria gathered around. The adapted strain presented the thicker cell wall and membrane and smaller cell size than those of the parental strain. In particular, the cytoplasmic membrane generated many little protrusions with regular shape. The cytoplasmic membrane integrity was analyzed by testing the relative electrical conductivity, leakage of intracellular substance, and permeation of fluorescent probe. The results indicated that the short-term adaptation improved the membrane integrity of yeast cell. CONCLUSION: The inhibition mechanism of phenolic acid might be attributed to the combined effect of the cytoplasmic membrane damage and the intracellular acidification. The short-term adaptation strategy with varied stressors levels and adaptive processes accelerated the stress response of yeast cell structure to tolerate phenolic acids. This strategy will contribute to the development of robust microbials for biofuel production from lignocellulosic biomass.