Loss Of Cell Wall Integrity Research Articles

A dynamic actin cytoskeleton is required to prevent constitutive VDAC-dependent MAPK signalling and aberrant lipid homeostasis

The dynamic nature of the actin cytoskeleton is required to coordinate many cellular processes, and a loss of its plasticity has been linked to accelerated cell aging and attenuation of adaptive response mechanisms. Cofilin is an actin-binding protein that controls actin dynamics and has been linked to mitochondrial signaling pathways that control drug resistance and cell death. Here we show that cofilin-driven chronic depolarization of the actin cytoskeleton activates cell wall integrity mitogen-activated protein kinase (MAPK) signalling and disrupts lipid homeostasis in a voltage-dependent anion channel (VDAC)-dependent manner. Expression of the cof1-5 mutation, which reduces the dynamic nature of actin, triggers loss of cell wall integrity, vacuole fragmentation, disruption of lipid homeostasis, lipid droplet (LD) accumulation, and the promotion of cell death. The integrity of the actin cytoskeleton is therefore essential to maintain the fidelity of MAPK signaling, lipid homeostasis, and cell health in S.cerevisiae.

On the mechanisms of lysis triggered by perturbations of bacterial cell wall biosynthesis

Inhibition of bacterial cell wall synthesis by antibiotics such as β-lactams is thought to cause explosive lysis through loss of cell wall integrity. However, recent studies on a wide range of bacteria have suggested that these antibiotics also perturb central carbon metabolism, contributing to death via oxidative damage. Here, we genetically dissect this connection in Bacillus subtilis perturbed for cell wall synthesis, and identify key enzymatic steps in upstream and downstream pathways that stimulate the generation of reactive oxygen species through cellular respiration. Our results also reveal the critical role of iron homeostasis for the oxidative damage-mediated lethal effects. We show that protection of cells from oxygen radicals via a recently discovered siderophore-like compound uncouples changes in cell morphology normally associated with cell death, from lysis as usually judged by a phase pale microscopic appearance. Phase paling appears to be closely associated with lipid peroxidation.

Dissecting the roles of peptidoglycan synthetic and autolytic activities in the walled to L-form bacterial transition.

Bacterial cells are surrounded by a peptidoglycan (PG) wall, which is a crucial target for antibiotics. It is well known that treatment with cell wall-active antibiotics occasionally converts bacteria to a non-walled "L-form" state that requires the loss of cell wall integrity. L-forms may have an important role in antibiotic resistance and recurrent infection. Recent work has revealed that inhibition of de novo PG precursor synthesis efficiently induces the L-form conversion in a wide range of bacteria, but the molecular mechanisms remain poorly understood. Growth of walled bacteria requires the orderly expansion of the PG layer, which involves the concerted action not just of synthases but also degradative enzymes called autolysins. Most rod-shaped bacteria have two complementary systems for PG insertion, the Rod and aPBP systems. Bacillus subtilis has two major autolysins, called LytE and CwlO, which are thought to have partially redundant functions. We have dissected the functions of autolysins, relative to the Rod and aPBP systems, during the switch to L-form state. Our results suggest that when de novo PG precursor synthesis is inhibited, residual PG synthesis occurs specifically via the aPBP pathway, and that this is required for continued autolytic activity by LytE/CwlO, resulting in cell bulging and efficient L-form emergence. The failure of L-form generation in cells lacking aPBPs was rescued by enhancing the Rod system and in this case, emergence specifically required LytE but was not associated with cell bulging. Our results suggest that two distinct pathways of L-form emergence exist depending on whether PG synthesis is being supported by the aPBP or RodA PG synthases. This work provides new insights into mechanisms of L-form generation, and specialisation in the roles of essential autolysins in relation to the recently recognised dual PG synthetic systems of bacteria.

Copper Acts Synergistically With Fluconazole in Candida glabrata by Compromising Drug Efflux, Sterol Metabolism, and Zinc Homeostasis.

The synergistic combinations of drugs are promising strategies to boost the effectiveness of current antifungals and thus prevent the emergence of resistance. In this work, we show that copper and the antifungal fluconazole act synergistically against Candida glabrata, an opportunistic pathogenic yeast intrinsically tolerant to fluconazole. Analyses of the transcriptomic profile of C. glabrata after the combination of copper and fluconazole showed that the expression of the multidrug transporter gene CDR1 was decreased, suggesting that fluconazole efflux could be affected. In agreement, we observed that copper inhibits the transactivation of Pdr1, the transcription regulator of multidrug transporters and leads to the intracellular accumulation of fluconazole. Copper also decreases the transcriptional induction of ergosterol biosynthesis (ERG) genes by fluconazole, which culminates in the accumulation of toxic sterols. Co-treatment of cells with copper and fluconazole should affect the function of proteins located in the plasma membrane, as several ultrastructural alterations, including irregular cell wall and plasma membrane and loss of cell wall integrity, were observed. Finally, we show that the combination of copper and fluconazole downregulates the expression of the gene encoding the zinc-responsive transcription regulator Zap1, which possibly, together with the membrane transporters malfunction, generates zinc depletion. Supplementation with zinc reverts the toxic effect of combining copper with fluconazole, underscoring the importance of this metal in the observed synergistic effect. Overall, this work, while unveiling the molecular basis that supports the use of copper to enhance the effectiveness of fluconazole, paves the way for the development of new metal-based antifungal strategies.

Synthesis titanium dioxide nanoparticles doped with silver and Novel antibacterial activity

Pure and Ag doped TiO2 nanoparticles were synthesis via a hydrothermal process and have anti-bacterial activity were examined against Escherichia coli, Staphylococcus aureus. The effect of Ag doping on TiO2 to rutile phase transformation was investigated by X-ray diffraction (XRD), Scanning electron microscopy (SEM),(EDX), Fourier transform infrared (FTIR) spectroscopy, and (UV). The average size of the TiO2 was in the range of (12.6)nm, and the doped TiO2+Ag had an average size of (12)nm. The antibacterial activity of the Tio2 and TiO2+Ag were evaluated against E. coli,S. aureus using the release of cellular materials method. The results showed that TiO2+Ag nanoparticles as a novel DNA-mediated antibacterial agent. The TiO2+Ag nanoparticles were observed to destroy the bacterial cells by permeating the bacterial nucleic acid and cytoplasmic membrane, resulting in the loss of cell wall integrity, nucleic acid damage, and increased cell-wall permeability.

Polyethylene Glycol Functionalized Graphene Oxide Nanoparticles Loaded with Nigella sativa Extract: A Smart Antibacterial Therapeutic Drug Delivery System.

Flaky graphene oxide (GO) nanoparticles (NPs) were synthesized using Hummer’s method and then capped with polyethylene glycol (PEG) by an esterification reaction, then loaded with Nigella sativa (N. sativa) seed extract. Aiming to investigate their potential use as a smart drug delivery system against Staphylococcus aureus and Escherichia coli, the spectral and structural characteristics of GO-PEG NPs were comprehensively analyzed by XRD, AFM, TEM, FTIR, and UV- Vis. XRD patterns revealed that GO-PEG had different crystalline structures and defects, as well as a higher interlayer spacing. AFM results showed GONPs with the main grain size of 24.41 nm, while GONPs–PEG revealed graphene oxide aggregation with the main grain size of 287.04 nm after loading N. sativa seed extract, which was verified by TEM examination. A strong OH bond appeared in FTIR spectra. Furthermore, UV- Vis absorbance peaks at (275, 284, 324, and 327) nm seemed to be correlated with GONPs, GO–PEG, N. sativa seed extract, and GO –PEG- N. sativa extract. The drug delivery system was observed to destroy the bacteria by permeating the bacterial nucleic acid and cytoplasmic membrane, resulting in the loss of cell wall integrity, nucleic acid damage, and increased cell-wall permeability.

The damage and tolerance mechanisms of Phaffia rhodozyma mutant strain MK19 grown at 28\xa0\xb0C

BackgroundPhaffia rhodozyma has many desirable properties for astaxanthin production, including rapid heterotrophic metabolism and high cell densities in fermenter culture. The low optimal temperature range (17–21 °C) for cell growth and astaxanthin synthesis in this species presents an obstacle to efficient industrial-scale astaxanthin production. The inhibition mechanism of cell growth at > 21 °C in P. rhodozyma have not been investigated.ResultsMK19, a mutant P. rhodozyma strain grows well at moderate temperatures, its cell growth was also inhibited at 28 °C, but such inhibition was mitigated, and low biomass 6 g/L was obtained after 100 h culture. Transcriptome analysis indicated that low biomass at 28 °C resulted from strong suppression of DNA and RNA synthesis in MK19. Growth inhibition at 28 °C was due to cell membrane damage with a characteristic of low mRNA content of fatty acid (f.a.) pathway transcripts (acc, fas1, fas2), and consequent low f.a. content. Thinning of cell wall and low mannose content (leading to loss of cell wall integrity) also contributed to reduced cell growth at 28 °C in MK19. Levels of astaxanthin and ergosterol, two end-products of isoprenoid biosynthesis (a shunt pathway of f.a. biosynthesis), reached 2000 µg/g and 7500 µg/g respectively; ~2-fold higher than levels at 21 or 25 °C. Abundance of ergosterol, an important cell membrane component, compensated for lack of f.a., making possible the biomass production of 6 g/L for MK19 at 28 °C.ConclusionsInhibition of growth of P. rhodozyma at 28 °C results from blocking of DNA, RNA, f.a., and cell wall biosynthesis. In MK19, abundant ergosterol made possible biomass production 6 g/L at 28 °C. Significant accumulation of astaxanthin and ergosterol indicated an active MVA pathway in MK19 at 28 °C. Strengthening of the MVA pathway can be a feasible metabolic engineering approach for enhancement of astaxanthin synthesis in P. rhodozyma. The present findings provide useful mechanistic insights regarding adaptation of P. rhodozyma to 28 °C, and improved understanding of feasible metabolic engineering techniques for industrial scale astaxanthin production by this economically important yeast species.

Dry-heat processing at different conditions impact the nutritional composition and in vitro starch and protein digestibility of immature rice-based products.

Immature grain represents a precious nutritional source in many rural Africa areas. To optimize processing of immature rice into pepeta (a traditional rice-flakes produced from immature rice grains), immature rice (TXD306 variety) harvested at 18 and 26 days after 50% heading were processed in the laboratory under different soaking (0 and 12 h) and roasting temperature (80, 100 and 120 °C) regimes. Riboflavin, nicotinic acid, nicotinamide and iron concentration increased with severity of roasting temperature, while thiamine has an opposite trend. Heating promoted the transformation of insoluble into soluble dietary fiber, increased lipid digestibility decreasing protein one, which showed the highest value when rice was roasted at 100 °C. Soaking before roasting significantly increased moisture and iron content while slightly increased riboflavin, nicotinic acid and nicotinamide when compared to unsoaked products. Among roasted products, starch digestibility increased with roasting temperature. Microstructure analysis indicated a complete loss of cell wall integrity in cooked rice, determining a complete starch and protein digestion while this is delayed in raw rice and roasted products. We concluded that roasting at 100 °C is the optimum temperature to produce pepeta of the highest protein digestibility and low starch digestibility. Soaking before roasting at 120 °C is best when retaining micronutrients is considered.

Vegetative desiccation tolerance in Eragrostiella brachyphylla: biochemical and physiological responses

Eragrostiella brachyphylla is an angiosperm desiccation-tolerant resurrection plant, which can survive during desiccation in the air-dry state and recover completely on availability of water. The present study was conducted to understand the vegetative desiccation tolerance of Eragrostiella brachyphylla by evaluating its ability to recover the physiological, biochemical and morphological functions post desiccation. In order to understand the responses of Eragrostiella brachyphylla to desiccation and subsequent rehydration experiments were conducted in the hydrated state (HS), desiccated state (DS) and rehydrated state (RS). Scanning electron microscopy revealed significant changes between the three stages in the internal ultra-structures of leaves and stems. Compared to the other states, photosynthetic parameters such as chlorophyll a, chlorophyll b, total chlorophylland total carotenoid contents decreased significantly in the desiccated state. Superoxide radical (O2•−) content also increased, resulting in an oxidative burst during desiccation. Consequently, antioxidant enzymes such as catalase (CAT) superoxide dismutase (SOD) peroxidase (APX) and glutathione reductase (GR) activities were found to be significantly elevated in the desiccated state to avoid oxidative damage. Increased malondialdehyde (MDA) content and relative electrolyte leakage (REL) during desiccation provide evidence for membrane damage and loss of cell-wall integrity. During desiccation, the contents of osmolytes represented by sucrose and proline were found to increase to maintain cell structure integrity. After rehydration, all physiological, biochemical and morphological properties remain unchanged or slightly changed when compared to the hydrated state. Hence, we believe that these unique adaptations contribute to the remarkable desiccation-tolerance property of this plant.

Antibiotics And Bacteria: Mechanisms Of Action And Resistance Strategies

Abstract The resistance of bacteria to antimicrobial substances is one of the most serious epidemiological problems present on a global scale. The widespread use of same classes of antimicrobials in human and veterinary medicine, often without laboratory confirmation of the efficacy of active compounds used, contributes to the selection of resistant bacteria in humans and animals, and their spread in nature. The increasing resistance of pathogenic bacteria leads to serious consequences for both human and animal health. However, the resistance of commensal bacteria is equally important as they constitute a reservoir and vector of resistance determinants in the environment. Exposure to antimicrobials belonging to different classes can lead to cross-resistance and the selection of genes that may spread horizontally on mobile genetic elements. The emergence of plasmid-encoded resistance to critically important antibiotics for human medicine e.g. carbapenems or polymyxins is alarming. On the example of antibiotics classified as critically important for human medicine, it is possible to discuss almost all bacterial mechanisms of antimicrobial resistance. For effective combat against the growing antibiotic resistance of bacteria, it is necessary to know the mechanisms of resistance and the methods of their acquisition by bacteria. The aim of the paper is to review the ways that critically important antimicrobials act on bacterial cells and present complex mechanisms that are responsible for resistance to these substances as well as genes conferring for resistance. 1. Introduction. 2. Antimicrobials that cause loss of cell wall integrity: β-lactams, glycopeptides and phosphonic acid derivatives. 2.1. Mechanisms of antimicrobial action. 2.2. Mechanisms of resistance. 3. Antimicrobials affecting the cell membrane: polymyxins and lipopeptides. 3.1. Mechanisms of antimicrobial action. 3.2. Mechanisms of resistance. 4. Antimicrobial substances that inhibit the synthesis of nucleic acids: quinolones and ansamycins. 4.1. Mechanisms of antimicrobial action. 4.2. Mechanisms of resistance. 5. Antimicrobial substances inhibiting protein synthesis: macrolides, ketolides, aminoglycosides, glycylcyclines, oxazolidinones. 5.1. Mechanisms of antimicrobial action. 5.2. Mechanisms of resistance. 6. Summary

POLYETHYLENE GLYCOL-FUNCTIONALIZED MAGNETIC (Fe3O4) NANOPARTICLES: A GOOD METHOD FOR A SUCCESSFUL ANTIBACTERIAL THERAPEUTIC AGENT VIA DAMAGE DNA MOLECULE

Magnetite (Fe3O4) nanoparticles (MPs) capped with polyethylene glycol (PEG) were prepared by a hydrothermal method, and their antibacterial activity was examined against Staphylococcus aureus, Escherichia coli and Psudomonas aeruginosa. The functionalized NPs were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), Fourier transform infrared (FTIR) spectroscopy, and Thermogravimetry (TG). The average size of the Fe3O4 was in the range 9–20[Formula: see text]nm, while the functionalized PEG–Fe3O4 had an average size of 5–15[Formula: see text]nm. The PEG–Fe3O4 exhibited superparamagnetism and high saturation magnetization at room temperature. The antibacterial activity of the Fe3O4 and PEG–Fe3O4 were evaluated against E. coli, S. aureus, and P. aeruginosa using the agar well diffusion method. The changes in the morphology of the studied bacterial species were observed via SEM, while the mode of action of the studied agents was determined via the detection of reactive oxygen species (ROS) using Acridine orange-ethidium bromide (AO/EtBr) staining method. The results showed that PEG-functionalized magnetic (Fe3O4) NPs as a novel DNA-mediated antibacterial agent. The PEG–Fe3O4 NPs were observed to destroy the bacterial cells by permeating the bacterial nucleic acid and cytoplasmic membrane, resulting in the loss of cell-wall integrity, nucleic acid damage, and increased cell-wall permeability. The PEG–Fe3O4 NPs could serve as a potential antibacterial agent in future biomedical and pharmaceutical applications.

Suppression of Arabidopsis GGLT1 affects growth by reducing the L-galactose content and borate cross-linking of rhamnogalacturonan-II.

SummaryBoron is a micronutrient that is required for the normal growth and development of vascular plants, but its precise functions remain a subject of debate. One established role for boron is in the cell wall where it forms a diester cross‐link between two monomers of the low‐abundance pectic polysaccharide rhamnogalacturonan‐II (RG‐II). The inability of RG‐II to properly assemble into a dimer results in the formation of cell walls with abnormal biochemical and biomechanical properties and has a severe impact on plant productivity. Here we describe the effects on RG‐II structure and cross‐linking and on the growth of plants in which the expression of a GDP‐sugar transporter (GONST3/GGLT1) has been reduced. In the GGLT1‐silenced plants the amount of L‐galactose in side‐chain A of RG‐II is reduced by up to 50%. This leads to a reduction in the extent of RG‐II cross‐linking in the cell walls as well as a reduction in the stability of the dimer in the presence of calcium chelators. The silenced plants have a dwarf phenotype, which is rescued by growth in the presence of increased amounts of boric acid. Similar to the mur1 mutant, which also disrupts RG‐II cross‐linking, GGLT1‐silenced plants display a loss of cell wall integrity under salt stress. We conclude that GGLT1 is probably the primary Golgi GDP‐L‐galactose transporter, and provides GDP‐L‐galactose for RG‐II biosynthesis. We propose that the L‐galactose residue is critical for RG‐II dimerization and for the stability of the borate cross‐link.

Bacillus anthracis Peptidoglycan Integrity Is Disrupted by the Chemokine CXCL10 through the FtsE/X Complex

The antimicrobial activity of the chemokine CXCL10 against vegetative cells of Bacillus anthracis occurs via both bacterial FtsE/X-dependent and-independent pathways. Previous studies established that the FtsE/X-dependent pathway was mediated through interaction of the N-terminal region(s) of CXCL10 with a functional FtsE/X complex, while the FtsE/X-independent pathway was mediated through the C-terminal α-helix of CXCL10. Both pathways result in cell lysis and death of B. anthracis. In other bacterial species, it has been shown that FtsE/X is involved in cellular elongation though activation of complex-associated peptidoglycan hydrolases. Thus, we hypothesized that the CXCL10-mediated killing of vegetative cells of B. anthracis through the FtsE/X-dependent pathway resulted from the disruption of peptidoglycan processing. Immunofluorescence microscopy studies using fluorescent peptidoglycan probes revealed that incubation of B. anthracis Sterne (parent) strain with CXCL10 or a C-terminal truncated CXCL10 (CTTC) affected peptidoglycan processing and/or incorporation of precursors into the cell wall. B. anthracis ΔftsX or ftsE(K123A/D481N) mutant strains, which lacked a functional FtsE/X complex, exhibited little to no evidence of disruption in peptidoglycan processing by either CXCL10 or CTTC. Additional studies demonstrated that the B. anthracis parent strain exhibited a statistically significant increase in peptidoglycan release in the presence of either CXCL10 or CTTC. While B. anthracis ΔftsX strain showed increased peptidoglycan release in the presence of CXCL10, no increase was observed with CTTC, suggesting that the FtsE/X-independent pathway was responsible for the activity observed with CXCL10. These results indicate that FtsE/X-dependent killing of vegetative cells of B. anthracis results from a loss of cell wall integrity due to disruption of peptidoglycan processing and suggest that FtsE/X may be an important antimicrobial target to study in the search for alternative microbial therapeutics.

Magnesium Uptake by CorA Transporters Is Essential for Growth, Development and Infection in the Rice Blast Fungus Magnaporthe oryzae.

Magnaporthe oryzae, the causative organism of rice blast, infects cereal crops and grasses at various stages of plant development. A comprehensive understanding of its metabolism and the implications on pathogenesis is necessary for countering this devastating crop disease. We present the role of the CorA magnesium transporters, MoAlr2 and MoMnr2, in development and pathogenicity of M. oryzae. The MoALR2 and MoMNR2 genes individually complement the Mg2+ uptake defects of a S. cerevisiae CorA transporter double mutant. MoALR2 and MoMNR2 respond to extracellular Mg2+ and Ca2+ levels and their expression is elevated under Mg2+ scarce conditions. RNA silencing mediated knockdown of MoALR2 (WT+siALR2, Δmnr2+siALR2 and ALR2+MNR2 simultaneous silencing) drastically alters intracellular cation concentrations and sensitivity to metal ions. MoALR2 silencing is detrimental to vegetative growth and surface hydrophobicity of mycelia, and the transformants display loss of cell wall integrity. MoALR2 is required for conidiogenesis and appressorium development, and is essential for infection. Investigation of knockdown transformants reveal low cAMP levels and altered expression of genes encoding proteins involved in MoMps1 cell wall integrity and cAMP MoPmk1 driven MAP Kinase signaling pathways. In contrast to MoALR2 knockdowns, the MoMNR2 deletion (Δmnr2) shows increased sensitivity to CorA inhibitors as well as altered cation sensitivity, but has limited effect on surface hydrophobicity and severity of plant infection. Interestingly, MoALR2 expression is elevated in Δmnr2. Impairment of development and infectivity of knockdown transformants and altered intracellular cation composition suggest that CorA transporters are essential for Mg2+ homeostasis within the cell, and are crucial to maintaining normal gene expression associated with cell structure, signal transduction and surface hydrophobicity in M. oryzae. We suggest that CorA transporters, and especially MoALR2, constitute an attractive target for the development of antifungal agents against this pathogen.

Fine-Tuning of the Cpx Envelope Stress Response Is Required for Cell Wall Homeostasis in Escherichia coli.

ABSTRACTThe envelope of Gram-negative bacteria is an essential compartment that constitutes a protective and permeability barrier between the cell and its environment. The envelope also hosts the cell wall, a mesh-like structure made of peptidoglycan (PG) that determines cell shape and provides osmotic protection. Since the PG must grow and divide in a cell-cycle-synchronized manner, its synthesis and remodeling are tightly regulated. Here, we discovered that PG homeostasis is intimately linked to the levels of activation of the Cpx system, an envelope stress response system traditionally viewed as being involved in protein quality control in the envelope. We first show that Cpx is activated when PG integrity is challenged and that this activation provides protection to cells exposed to antibiotics inhibiting PG synthesis. By rerouting the outer membrane lipoprotein NlpE, a known Cpx activator, to a different envelope subcompartment, we managed to manipulate Cpx activation levels. We found that Cpx overactivation leads to aberrant cellular morphologies, to an increased sensitivity to β-lactams, and to dramatic division and growth defects, consistent with a loss of PG homeostasis. Remarkably, these phenotypes were largely abrogated by the deletion of ldtD, a Cpx-induced gene involved in noncanonical PG cross-linkage, suggesting that this transpeptidase is an important link between PG homeostasis and the Cpx system. Altogether our data show that fine-tuning of an envelope quality control system constitutes an important layer of regulation of the highly organized cell wall structure.

Antibacterial properties and mode of action of new triaryl butene citrate compounds

The aim of this study was to evaluate the antibacterial activity of newly synthesized triaryl butene analogues of tamoxifen. Several compounds were synthesized and converted to citrate salts to ensure greater solubility. Four compounds showed significant antibacterial activity at micromolar concentrations against Gram-positive and Gram-negative foodborne pathogens including Listeria monocytogenes, Listeria ivanovii, Enterococcus faecalis, Staphylococcus aureus and Escherichia coli. Two compounds at 50 μM, caused only 7.8 and 11% hemolysis. One of these as well as the remaining two caused high K+ and Na+ efflux from bacterial cells. Ultrastructural alterations were also visible using transmission electron microscopy, which revealed severe damage of the inner or outer membrane of E. coli. L. ivanovii showed swelling, corrugations and similar damage indicating a loss of cell-wall integrity. Organometallic compounds may offer interesting opportunities for the design of novel classes of antimicrobial compounds.

ANXUR Receptor-Like Kinases Coordinate Cell Wall Integrity with Growth at the Pollen Tube Tip Via NADPH Oxidases

Author SummaryTip-growing cells, such as plant root hairs and pollen tubes or fungal hyphae, are characterized by a tip-focused Ca2+ gradient. These tip-growing cells tightly coordinate the loosening and pressure-driven deformation of their extracellular matrix (ECM)—the cell wall in plant cells—by locally adding new membrane and cell wall materials. In pollen tubes, which grow at amazing speeds to effect fertilization in plants, a class of kinases called the ANXUR receptor-like kinases (RLKs) sense perturbations in cell wall integrity, and their loss leads to pollen tube rupture. Here, we gain new insights into the mechanism of cell wall surveillance by these RLKs in the model plant Arabidopsis. We show that over-expressing ANXUR RLKs over-activates exocytosis, causing an over-accumulation of secreted cell wall material that eventually leads to growth arrest. Moreover, we find that the ANXUR RLKs function upstream of NADPH oxidases, which are membrane-anchored enzymes that produce reactive oxygen species (ROS). Using H2O2- and Ca2+-sensitive reporters, we show that NADPH oxidases generate tip-localized H2O2 production, which is required to maintain a steady, tip-focused Ca2+ gradient that is essential for pollen tube growth. We postulate that ECM-sensing receptors, such as the ANXUR RLKs, regulate ROS production, Ca2+ homeostasis, and exocytosis to coordinate the status of the ECM with the cell's internal growth machinery.

Plant Cell Wall Homeostasis Is Mediated by Brassinosteroid Feedback Signaling

Brassinosteroid (BR) signaling is required for normal plant growth as shown by the dwarf phenotype of loss-of-function BR biosynthetic or perception mutants. Despite a detailed understanding of the BR signaling network, it is not clear how exactly BRs control growth. For instance, genetic sector analysis shows that BRs, in contrast to most other growth regulators, act locally, presumably in an autocrine and/or paracrine mode, suggesting that they have some role in feedback regulation. Here, we show that at least one role for BRs in growth control is to ensure pectin-dependent cell wall homeostasis. Pectins are complex block cell wall polymers, which can be modified in the wall by the enzyme pectin methylesterase (PME). Genetic or pharmacological interference with PME activity causes dramatic changes in growth behavior, which are primarily the result of the activation of the BR signaling pathway. We propose that this activation of BR signaling is part of a compensatory response, which protects the plant against the loss of cell wall integrity caused by the imbalance in pectin modification. Thus, feedback signaling from the cell wall is integrated by the BR signaling module to ensure homeostasis of cell wall biosynthesis and remodeling.

RNA interference ofWdFKS1mRNA expression causes slowed growth, incomplete septation and loss of cell wall integrity in yeast cells of the polymorphic, pathogenic fungusWangiella (Exophiala) dermatitidis

As one of the main components of the fungal cell wall, β-1,3-glucan provides the mechanical strength to protect fungal protoplasts. The enzyme responsible for the synthesis of β-1,3-glucans in fungi is β-1,3-glucan synthase. Here we report the cloning, sequencing and characterization of the WdFKS1 gene, which in the pathogenic fungus Wangiella dermatitidis encodes the catalytic domain of its β-1, 3-glucan synthase. Because our research suggested that WdFKS1 is a single copy essential gene, we used RNA interference to reduce its expression. Reduction of the WdFKS1 messenger retarded growth and caused the loss of cell wall integrity of yeast cells, but not hyphae or sclerotic cells. We suggest that the WdFKS1 in this polymorphic agent of phaeohyphomycosis is not only required for cell wall construction and maintenance, but also is involved in septum formation.

Synthetic arylquinuclidine derivatives exhibit antifungal activity against Candida albicans, Candida tropicalis and Candida parapsilopsis

BackgroundSterol biosynthesis is an essential pathway for fungal survival, and is the biochemical target of many antifungal agents. The antifungal drugs most widely used to treated fungal infections are compounds that inhibit cytochrome P450-dependent C14α-demethylase (CYP51), but other enzymes of this pathway, such as squalene synthase (SQS) which catalyses the first committed step in sterol biosynthesis, could be viable targets. The aim of this study was to evaluate the antifungal activity of SQS inhibitors on Candida albicans, Candida tropicalis and Candida parapsilopsis strains.MethodsTen arylquinuclidines that act as SQS inhibitors were tested as antiproliferative agents against three ATCC strains and 54 clinical isolates of Candida albicans, Candida tropicalis and Candida parapsilopsis. Also, the morphological alterations induced in the yeasts by the experimental compounds were evaluated by fluorescence and transmission electron microscopy.ResultsThe most potent arylquinuclidine derivative (3-[1'-{4'-(benzyloxy)-phenyl}]-quinuclidine-2-ene) (WSP1267) had a MIC50 of 2 μg/ml for all species tested and MIC90 varying from 4 μg/ml to 8 μg/ml. Ultrathin sections of C. albicans treated with 1 μg/ml of WSP1267 showed several ultrastructural alterations, including (a) loss of cell wall integrity, (b) detachment of the plasma membrane from the fungal cell wall, (c) accumulation of small vesicles in the periplasmic region, (d) presence of large electron-dense vacuoles and (e) significantly increased cell size and cell wall thickness. In addition, fluorescence microscopy of cells labelled with Nile Red showed an accumulation of lipid droplets in the cytoplasm of treated yeasts. Nuclear staining with DAPI revealed the appearance of uncommon yeast buds without a nucleus or with two nuclei.ConclusionTaken together, our data demonstrate that arylquinuclidine derivatives could be useful as lead compounds for the rational synthesis of new antifungal drugs.