Major Wall Component Research Articles

Characterization of the mechanism of action of the Enterococcus faecalis bacteriocin EntV on Candida albicans

Candida albicans shares communal niches with multiple bacterial species. Previous work from our group demonstrated that the Gram-positive bacterium Enterococcus faecalis, a normal constituent of the oral and gut microbiome that is often co-isolated with C. albicans, antagonizes hyphal morphogenesis, biofilm formation, and virulence in C. albicans. These effects are mediated by EntV, a bacteriocin and antimicrobial peptide produced by E. faecalis. The main aim of this work is to unveil the molecular mechanism behind the activity of EntV on C. albicans. Using fluorescence microscopy, we determined that EntV binds to the cell walls of several Candida species, including both yeast and hyphae of C. albicans. Contrary to other antimicrobial peptides, it does not cause cell lysis and does not synergize with cell wall damaging agents. Moreover, we screened a library of C. albicans mutants for strains with altered susceptibility to the peptide; most of the positive hits had functions related to cell wall maintenance and were further screened to ascertain changes in the staining patterns. Furthermore, to identify the target layer on the cell wall, pull-down assays were performed. Mannan was identified as the major wall component able to bind the peptide. Finally, live imaging of macrophages incubated with Candida was carried out in order to assess any change in the phagocytic behaviour in presence of the peptide. Identifying the molecular target of EntV in regard to the anti-virulence mechanisms of C. albicans is an important step in its further development as a therapeutic addition to the classical antifungal agents.

Grass Cell Walls: A Story of Cross-Linking.

Cell wall matrices are complex composites mainly of polysaccharides, phenolics (monomers and polymers), and protein. We are beginning to understand the synthesis of these major wall components individually, but still have a poor understanding of how cell walls are assembled into complex matrices. Valuable insight has been gained by examining intact components to understand the individual elements that make up plant cell walls. Grasses are a prominent group within the plant kingdom, not only for their important roles in global agriculture, but also for the complexity of their cell walls. Ferulate incorporation into grass cell wall matrices (C3 and C4 types) leads to a cross-linked matrix that plays a prominent role in the structure and utilization of grass biomass compared to dicot species. Incorporation of p-coumarates as part of the lignin structure also adds to the complexity of grass cell walls. Feruoylation results in a wall with individual hemicellulosic polysaccharides (arabinoxylans) covalently linked to each other and to lignin. Evidence strongly suggests that ferulates not only cross-link arabinoxylans, but may be important factors in lignification of the cell wall. Therefore, the distribution of ferulates on arabinoxylans could provide a means of structuring regions of the matrix with the incorporation of lignin and have a significant impact upon localized cell wall organization. The role of other phenolics in cell wall formation such as p-coumarates (which can have concentrations higher than ferulates) remains unknown. It is possible that p-coumarates assist in the formation of lignin, especially syringyl rich lignin. The uniqueness of the grass cell wall compared to dicot sepcies may not be so much in the gross composition of the wall, but how the distinctive individual components are organized into a functional wall matrix. These features are discussed and working models are provided to illustrate how changing the organization of feruoylation and p-coumaroylation could lead to differing cell wall properties.

Theoretical evaluation of wall teichoic acids in the cavitation-mediated pores formation in Gram-positive bacteria subjected to an electric field

BackgroundElectroporation is a method of choice to transform living cells. The ability of electroporation to transfer small or large chemicals across the lipid bilayer membrane of eukaryotic cells or Gram-negative bacteria relies on the formation of transient pores across the membrane. To exist, these pores rely on an insulator (the bilayer membrane) and the presence of a potential difference on either side of the membrane mediated by an external electric field. In Gram-positive bacteria, however, the wall is not an insulator but pores can still form when an electric field is applied. Past works have shown that the electrostatic charge of teichoic acids, a major wall component; sensitizes the wall to pore formation when an external electric field is applied. These results suggest that teichoic acids mediate the formation of defects in the wall of Gram-positive bacteria. MethodsWe model the electrostatic repulsion between teichoic acids embedded in the bacterial wall composed of peptidoglycan when an electric field is applied. The repulsion between teichoic acids gives rise to a stress pressure that is able to rupture the wall when a threshold value has been reached. The size of such small defects can diverge leading to the formation of pores. ResultsIt is demonstrated herein that for a bonding energy of about ~1−10kBT between peptidoglycan monomers an intra-wall pressure of about ~5−120kBT/nm3 generates spherical defects of radius ~0.1−1nm diverging in size to create pores. ConclusionThe electrostatic cavitation of the bacterial wall theory has the potential to highlight the role of teichoic acids in the formation pores, providing a new step in the understanding of electroporation in Gram-positive bacteria without requiring the use of an insulator.

The minor wall-networks between monolignols and interlinked-phenolics predominantly affect biomass enzymatic digestibility in Miscanthus.

Plant lignin is one of the major wall components that greatly contribute to biomass recalcitrance for biofuel production. In this study, total 79 representative Miscanthus germplasms were determined with wide biomass digestibility and diverse monolignol composition. Integrative analyses indicated that three major monolignols (S, G, H) and S/G ratio could account for lignin negative influence on biomass digestibility upon NaOH and H2SO4 pretreatments. Notably, the biomass enzymatic digestions were predominately affected by the non-KOH-extractable lignin and interlinked-phenolics, other than the KOH-extractable ones that cover 80% of total lignin. Furthermore, a positive correlation was found between the monolignols and phenolics at p<0.05 level in the non-KOH-extractable only, suggesting their tight association to form the minor wall-networks against cellulases accessibility. The results indicated that the non-KOH-extractable lignin-complex should be the target either for cost-effective biomass pretreatments or for relatively simply genetic modification of plant cell walls in Miscanthus.

Spatial gradients in cell wall composition and transcriptional profiles along elongating maize internodes

BackgroundThe elongating maize internode represents a useful system for following development of cell walls in vegetative cells in the Poaceae family. Elongating internodes can be divided into four developmental zones, namely the basal intercalary meristem, above which are found the elongation, transition and maturation zones. Cells in the basal meristem and elongation zones contain mainly primary walls, while secondary cell wall deposition accelerates in the transition zone and predominates in the maturation zone.ResultsThe major wall components cellulose, lignin and glucuronoarabinoxylan (GAX) increased without any abrupt changes across the elongation, transition and maturation zones, although GAX appeared to increase more between the elongation and transition zones. Microarray analyses show that transcript abundance of key glycosyl transferase genes known to be involved in wall synthesis or re-modelling did not match the increases in cellulose, GAX and lignin. Rather, transcript levels of many of these genes were low in the meristematic and elongation zones, quickly increased to maximal levels in the transition zone and lower sections of the maturation zone, and generally decreased in the upper maturation zone sections. Genes with transcript profiles showing this pattern included secondary cell wall CesA genes, GT43 genes, some β-expansins, UDP-Xylose synthase and UDP-Glucose pyrophosphorylase, some xyloglucan endotransglycosylases/hydrolases, genes involved in monolignol biosynthesis, and NAM and MYB transcription factor genes.ConclusionsThe data indicated that the enzymic products of genes involved in cell wall synthesis and modification remain active right along the maturation zone of elongating maize internodes, despite the fact that corresponding transcript levels peak earlier, near or in the transition zone.

Molecular Mapping of Lipoarabinomannans on Mycobacteria

Although the chemical composition of mycobacterial cell walls is well known, the 3D organization of the various constituents is not fully understood. In particular, it is unclear whether the major wall component lipoarabinomannan (LAM) is exposed on the outermost surface or hindered by other constituents such as mycolic acids. To address this pertinent question, we used atomic force microscopy (AFM) with tips bearing anti-LAM antibodies to detect single LAM molecules on Mycobacterium bovis BCG cells. First, we showed the ability of anti-LAM tips to detect isolated, purified LAM molecules. We then mapped the distribution of LAM on mycobacteria, prior to and after treatment with the drug isoniazid. We found that LAM was not exposed on the surface of native cells, pointing to the presence of a homogeneous layer of mycolic acids, whereas it was greatly exposed on isoniazid-treated cells, in agreement with the action mode of the drug. This single-molecule study provides novel insight into the architecture of mycobacterial cell walls and offers promising perspectives for understanding the action modes of antimycobacterial drugs.

Developmental Expression Patterns of Arabidopsis XTH Genes Reported by Transgenes and Genevestigator

The plant cell wall is the structural basis of cellular form and thus forms a foundation on which morphogenesis builds organs and tissues. Enzymes capable of modifying major wall components are prominent candidates for regulating wall form and function. Xyloglucan endotransglucosylases/hydrolases (XTHs) are predicted to participate in xyloglucan integration and/or restructuring. XTHs are encoded by large gene families in plants; the Arabidopsis genome encodes 33 XTHs. To gain insight into the potential physiological relevance of the distinct members of this family, GUS reporter fusion genes were constructed, and plants expressing these transgenes were characterized to reveal spatial and temporal patterns of expression. In addition, Genevestigator sources were mined for comprehensive and comparative XTH expression regulation analysis. These data reveal that the Arabidopsis XTHs are likely expressed in every developmental stage from seed germination through flowering. All organs show XTH::GUS expression and most, if not all, are found to express multiple XTH::GUS genes. These data suggest that XTHs may contribute to morphogenesis at every developmental stage and in every plant organ. Different XTHs have remarkably diverse and distinct expression patterns indicating that paralogous genes have evolved differential expression regulation perhaps contributing to the maintenance of the large gene family. Extensive overlap in XTH expression patterns is evident; thus, XTHs may act combinatorially in determining wall properties of specific tissues or organs. Knowledge of gene-specific expression among family members yields evidence of where and when gene products may function and provides insights to guide rational approaches to investigate function through reverse genetics.

PEPTIDOGLYCAN OF STAPHYLOCCUS AUREUS INDUCES ENHANCED LEVELS OF MATRIX METALLOPROTEINASE-9 IN HUMAN BLOOD ORIGINATING FROM NEUTROPHILS

Enhanced plasma levels of matrix metalloproteinase 9 (MMP-9) detected in patients with severe sepsis are thought to contribute to the development of organ dysfunction in endotoxemia. We have recently reported that peptidoglycan, the major wall component of gram-positive bacteria, increases MMP-9 levels in lung and liver and organ injury in the rat. Thus far, it is unclear whether MMP-9 is part of the septic response to peptidoglycan in human blood. The aim of the present study was to examine the regulation of MMP-9 by peptidoglycan in human leukocytes. The addition of peptidoglycan to whole human blood caused enhanced levels of MMP-9 after 1 h of incubation (306 vs. 75 ng/mL, P < or = 0.05) and onward, as measured by enzyme-linked immunoabsorbant assay. In neutrophil cultures, MMP-9 values increased significantly after 30 min of incubation with peptidoglycan (242 vs. 121 ng/mL, P < or = 0.05), whereas muramyl dipeptide had no effect. In contrast, adherent monocytes released insignificant amounts of MMP-9. To examine whether the released MMP-9 resulted from de novo synthesis, intracellular and secreted MMP-9 was measured during stimulation of neutrophils. The total MMP-9 values (the sum of intracellular and secreted MMP-9) before and after stimulation were mainly unaltered. The enhanced MMP-9 levels induced by peptidoglycan was attenuated by inhibitors of p38 mitogen-activated protein kinases (MAPK), (SB202190, 25 microM) and ERK1/2 (PD98059, 25 microM) and inhibitors of Src Tyrosine kinase (PP2, 5 microM) and PI3-K (LY294002, 25 microM).

Peptidoglycan and lipoteichoic acid in gram-positive bacterial sepsis: receptors, signal transduction, biological effects, and synergism.

In sepsis and multiple organ dysfunction syndrome (MODS) caused by gram-negative bacteria, lipopolysaccharide (LPS) initiates the early signaling events leading to the deleterious inflammatory response. However, it has become clear that LPS can not reproduce all of the clinical features of sepsis, which emphasize the roles of other contributing factors. Gram-positive bacteria, which lack LPS, are today responsible for a substantial part of the incidents of sepsis with MODS. The major wall components of gram-positive bacteria, peptidoglycan and lipoteichoic acid, are thought to contribute to the development of sepsis and MODS. In this review, the literature underlying our current understanding of how peptidoglycan and lipoteichoic acid activate inflammatory responses will be presented, with a focus on recent advances in this field.

Expression and preferential inhibition of inducible nitric oxide synthase in aortas of endotoxemic rats.

Septic shock is associated with high mortality. There is in vitro evidence that the induction of nitric oxide synthase (iNOS) in vascular smooth muscle cells may be an important mediator of the systemic vasodilation and hypotension associated with sepsis. In this study, an in vivo murine model of sepsis was used to further examine this important question. Lipopolysaccharide (LPS), the major wall component of gram-negative bacteria, was administered to rats. By the use of a selective cDNA probe for iNOS, mRNA for iNOS was demonstrated in the aortas of these rats. The functional significance of this iNOS was then examined with aminoguanidine, a preferential inhibitor of iNOS. Aminoguanidine reversed the blunted phenylephrine-evoked contraction of endothelium-denuded aortic rings from LPS-treated rats or rings exposed to LPS in vitro. Aminoguanidine did not impair the relaxation of aortic rings with endothelium to acetylcholine, a known stimulator of endothelial NOS. The reversal of LPS-induced vascular hyporesponsiveness by aminoguanidine therefore strongly supports the functional importance of iNOS mRNA expression in the aorta of endotoxemic rats. Future clinical trials in treating septic shock should therefore consider the preferential inhibition of iNOS while maintaining the integrity of endothelial NOS.

Enzymatic cell wall proteins in higher plants

Abstract The entire life of the plant cell is intimately related to the biogenesis of cell wall and growing attention has been devoted in last years to the understanding of the biochemical events that lead to the synthesis and cross-linking of the major wall components. In this context it is now clearly assessed that proteins play a key role in the architecture and chemo-rheological properties of growing cell wall thus participating in important ontogenetics events such as growth and differentiation. Wall proteins are mostly glycoprotein accounting up to almost 20% wall dry weight and many of them are structural proteins. Here we are dealing with enzymatic proteins occurring in the cell wall and their physiological significance.

The relationship betweenin vitroenzymatic digestibility of cell walls of wheat internodes and compositional changes during maturation

Analyses of internodes from wheat taken at various times during maturation, and determination of their in vitro dry matter digestibility confirmed that the decrease in digestibility could be attributed both to a progressive loss in cell contents and also to a marked decrease in cell-wall digestibility. The latter was not easily explained by the changes in the proportion of the major wall components (cellulose, arabinoxylan and lignin), but may be explained in terms of the physical and chemical associations between wall polymers. Among these, covalent crosslinking and bridging by hydroxycinnamic acids are important candidates

CHEMICAL COMPOSITION OF THE CELL WALLS OF THE FRESHWATER RED ALGA LEMANEA ANNULATA (BATRACHOSPERMALES)1

ABSTRACTCell walls of the generic phase of the freshwater red alga Lemanea annulata Kütz were mechanically isolated and chemically characterized. Walls consisted mainly of polysaccharide with lesser quantities of associated protein and lipid. The major wall component was alkali‐soluble xylan, comprised mainly of 4‐linked β‐xylopyranosyl residues and small amounts of 3‐O‐substituted β‐xylopyranosyl residues. Hot water extracts yielded non‐sulfated polymers, with 3‐ or 3,4‐linked β‐galactosyl residues alternating with 4‐linked α‐glucuronosyl residues as the predominant structural features. This acid polysaccharide shares many characteristics of the mucilage previously described from the freshwater genus Batrachospermum. Isolated cell walls of L. annulata contained approximately 6% cellulose. Cellobiohydrolase/colloidal gold labelling of cell walls revealed β‐4‐glycan throughout the fibrillar portion of the wall. Wall protein consisted of at least 17 amino acids, of which threonine and alanine were the most abundant. Polysaccharides of the cell walls of L. annulata differ from those of marine red algae and are similar to those described for other Batrachospermales.

Bacterial cell walls and membranes. Discovery of the teichoic acids.

Teichoic acids are major wall components of most Gram-positive bacteria. Their discovery followed that of their nucleotide precursors. Lipoteichoic acids associated with the cell membrane were discovered at the same time. Events leading to these discoveries and the probable function of teichoic acids in cation control are described.

Cell wall composition of the aquatic fungus Blastocladiella emersonii

Cell walls from Blastocladiella emersonii were isolated by repeated washing and centrifugation. Purity and uniformity of cell wall preparations were assessed by light and electron microscopy and chemical reproducibility. Electron microscopy showed the cell walls to consist of an inner microfibrillar network and an outer amorphous layer. Analyses by X-ray and infrared spectroscopy were consistent with chitin as the major wall component. Gross chemical analysis indicated that the cell walls were composed of 74.7% amino sugar (as anhydro N-acetylhexosamine), 10.7% neutral sugar (as anhydro hexose), 10.6% protein, and 4.2% lipid. Analysis of the neutral sugars showed that isolated cell walls contain 1.5% mannose, 3.0% galactose, and 3.0% glucose. Isolated cell walls were fractionated using a hot sodium dodecyl sulfate (SDS) extraction followed by either Pronase digestion or hot KOH extraction. The hot SDS extract was found to contain two polymer types, galactose- and/or glucose-containing polymers and glycoprotein. However, the residue from the hot SDS extraction still contained most of the neutral sugars and protein present in the isolated walls. Both Pronase digestion and the hot potassium hydroxide extraction removed all of the neutral sugars except glucose. The cell wall fractionation results indicate that the major wall component is microfibrillar chitin. The results further suggest that the SDS-solubilized glycoproteins and neutral sugar polymers may represent an outer amorphous layer.

Effect of tunicamycin treatment on the adherence ofCandida albicansto human buccal epithelial cells

The adherence of microorganisms to mucosal surfaces is believed to involve specific interactions between microbial ligands (or adhesins) and complementary host-cell receptors. Preliminary information on the chemical nature of these surface components is sometimes obtained by testing potential analogues of the ligand or receptor for their ability to inhibit adherence in vitro [ 1 ]. Several investigators have described the adherence of Candida albicans to buccal or vaginal epithelial cells [2-6] and recent evidence from inhibition experiments with sugars and lectins implicates a mannose-containing yeast component in the attachment process [7]. However, analogous studies suggest a possible role for cell-wall chitin [8]. The work described here represents an alternative approach to the identification of the yeast ligand. It involved treating C. albicans with tunicamycin, an antibiotic which, in yeasts, specifically inhibits the synthesis of mannoprotein and not that of the other major wall components, glucan and chitin [9]. Our results suggest that yeast mannoprotein is of primary importance in the interaction with buccal epithelial cells. 2.1. Organism and growth conditions

Rapid Identification of Cell Wall Components As A Guide to The Classification of Aerobic Coryneform Bacteria from Human Skin

In a survey of over 1000 isolates of aerobic skin coryneforms from a wide variety of sources, chromatographic methods were used to identify the major cell-wall components in whole-cell hydrolysates. Most of the skin isolates-like members of the genus Corynebacterium--possessed meso-diaminopimelic acid and arabinose. However, substantial numbers of coryneforms apparently resident on the skin did not have this pattern; the sites from which they were isolated suggested that some were derived from the environment whilst others (possessing meso-DAP and galactose but not arabinose as the major wall components) were members of the resident skin flora.

Mechanism and control of cell wall synthesis in bacteria

Abstract

An Assessment of Infrared Spectra as Indicators of Fungal Cell Wall Composition

Infrared spectroscopy is assessed as a technique for identifying polymers derived from fungal cell walls, both as isolated materials and in mixtures with one another. The technique is then applied to a study of the composition of fungal cell walls and the conclusion reached that infrared spectra provide a rapid and valuable indication of the major components of such walls. They can also be used to follow the effect of chemical treatments designed to separate major wall components.