Cell Wall Fragments Research Articles

Effect of vaccination with a novel GnRH-based immunocontraceptive on immune responses and fertility in rats

1. As human-wildlife conflicts increase worldwide, novel methods are required for mitigating these conflicts. Fertility control, based on immunocontraceptives, has emerged as an alternative option to lethal methods for managing wildlife.2. Immunocontraceptives are vaccines that generate an immune response to key components of an animal's reproductive system. Some of these vaccines target the gonadotropin-releasing hormone (GnRH) and have been used successfully as contraceptives for many wildlife species. However, the need to capture animals for treatment limits the field applications of injectable vaccines. The availability of orally delivered immunocontraceptives would increase the breadth of applications of fertility control for wildlife management.3. This study explored a new approach to developing an oral immunocontraceptive, exploiting the bioadhesive and immunologically active properties of killed Mycobacterium avium cell wall fragments (MAF). The MAF was conjugated to a GnRH recombinant protein called IMX294, used as a GnRH-specific immunogen.4. An initial trial using the MAF-IMX294 conjugate provided the first evidence that an orally delivered immunocontraceptive vaccine could generate anti-GnRH antibody titres in laboratory rats.5. Increasing the dose and frequency of vaccine administered to rats, in a second trial, enhanced the immune response, eliciting titres that reduced the proportion of females giving birth. This provided the first evidence of the contraceptive effect of an oral anti-GnRH vaccine.6. Future work is required to further increase the immunogenic effect of the oral vaccine and to establish a dosing schedule that is effective for practical field applications.

Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls

Azo dyes are toxic and carcinogenic synthetic pigments that accumulate as pollutants in aquatic bodies near textile factories. The pigments are structurally diverse, and bioremediation is mostly limited to single dye compounds or related groups. Versatile peroxidase (VP) from Pleurotus eryngii is a heme-containing peroxidase with a broad substrate spectrum that can break down many structurally distinct pollutants, including azo dyes. The utilization of this enzyme could be facilitated by engineering to modify its catalytic activity and substrate range. We used saturation mutagenesis to alter two amino acids in the catalytic tryptophan environment of VP (V160 and A260). Library screening with three azo dyes revealed that these two positions had a significant influence on substrate specificity. We were able to isolate and sequence VP variants with up to 16-fold higher catalytic efficiency for different azo dyes. The same approach could be used to select for VP variants that catalyze the degradation of many other types of pollutants. To allow multiple cycles of dye degradation, we immobilized VP on the surface of yeast cells and used washed cell wall fragments after lysis. VP embedded in the cell wall retained ∼70 % of its initial activity after 10 cycles of dye degradation each lasting 12 h, making this platform ideal for the bioremediation of environments contaminated with azo dyes.

Microarray Strategies for Exploring Bacterial Surface Glycans and Their Interactions With Glycan-Binding Proteins.

Bacterial surfaces are decorated with distinct carbohydrate structures that may substantially differ among species and strains. These structures can be recognized by a variety of glycan-binding proteins, playing an important role in the bacteria cross-talk with the host and invading bacteriophages, and also in the formation of bacterial microcolonies and biofilms. In recent years, different microarray approaches for exploring bacterial surface glycans and their recognition by proteins have been developed. A main advantage of the microarray format is the inherent miniaturization of the method, which allows sensitive and high-throughput analyses with very small amounts of sample. Antibody and lectin microarrays have been used for examining bacterial glycosignatures, enabling bacteria identification and differentiation among strains. In addition, microarrays incorporating bacterial carbohydrate structures have served to evaluate their recognition by diverse host/phage/bacterial glycan-binding proteins, such as lectins, effectors of the immune system, or bacterial and phagic cell wall lysins, and to identify antigenic determinants for vaccine development. The list of samples printed in the arrays includes polysaccharides, lipopoly/lipooligosaccharides, (lipo)teichoic acids, and peptidoglycans, as well as sequence-defined oligosaccharide fragments. Moreover, microarrays of cell wall fragments and entire bacterial cells have been developed, which also allow to study bacterial glycosylation patterns. In this review, examples of the different microarray platforms and applications are presented with a view to give the current state-of-the-art and future prospects in this field.

Cell wall integrity maintenance during plant development and interaction with the environment.

Cell walls are highly dynamic structures that provide mechanical support for plant cells during growth, development and adaptation to a changing environment. Thus, it is important for plants to monitor the state of their cell walls and ensure their functional integrity at all times. This monitoring involves perception of physical forces at the cell wall-plasma membrane interphase. These forces are altered during cell division and morphogenesis, as well as in response to various abiotic and biotic stresses. Mechanisms responsible for the perception of physical stimuli involved in these processes have been difficult to separate from other regulatory mechanisms perceiving chemical signals such as hormones, peptides or cell wall fragments. However, recently developed technologies in combination with more established genetic and biochemical approaches are beginning to open up this exciting field of study. Here, we will review our current knowledge of plant cell wall integrity signalling using selected recent findings and highlight how the cell wall-plasma membrane interphase can act as a venue for sensing changes in the physical forces affecting plant development and stress responses. More importantly, we discuss how these signals may be integrated with chemical signals derived from established signalling cascades to control specific adaptive responses during exposure to biotic and abiotic stresses.

A top-down chemo-enzymatic approach towards N-acetylglucosamine-N-acetylmuramic oligosaccharides: Chitosan as a reliable template

An unprecedented approach towards oligosaccharides containing N-acetylglucosamine-N-acetylmuramic (NAG-NAM) units was developed. These novel bacterial cell wall surrogates were obtained from chitosan via a top down approach involving both chemical and enzymatic reactions. The chemical modification of chitosan using a molecular clamp based strategy, allowed obtaining N-acetylglucosamine-N-acetylmuramic (NAG-NAM) containing oligomers. Intercalation of NAM residues was confirmed through the analysis of oligosaccharide fragments from enzymatic digestion and it was found that this route affords NAG-NAM containing oligosaccharides in 33% yield. These oligosaccharides mimic the carbohydrate basic skeleton of most bacterial cell surfaces. The oligosaccharides prepared are biologically relevant and will serve as a platform for further molecular recognition studies with different receptors and enzymes of both bacterial cell wall and innate immune system. This strategy combining both chemical modification and enzymatic digestion provides a novel and simple route for an easy access to bacterial cell wall fragments – biologically important targets.

Monoclonal Antibody AP3 Binds Galactomannan Antigens Displayed by the Pathogens Aspergillus flavus, A. fumigatus, and A. parasiticus.

Aspergillus fumigatus and A. flavus are the fungal pathogens responsible for most cases of invasive aspergillosis (IA). Early detection of the circulating antigen galactomannan (GM) in serum allows the prompt application of effective antifungal therapy, thus improving the survival rate of IA patients. However, the use of monoclonal antibodies (mAbs) for the diagnosis of IA is often associated with false positives due to cross-reaction with bacterial polysaccharides. More specific antibodies are therefore needed. Here we describe the characterization of the Aspergillus-specific mAb AP3 (IgG1κ), including the precise identification of its corresponding antigen. The antibody was generated using A. parasiticus cell wall fragments and was shown to bind several Aspergillus species. Immunofluorescence microscopy revealed that AP3 binds a cell wall antigen, but immunoprecipitation and enzyme-linked immunosorbent assays showed that the antigen is also secreted into the culture medium. The inability of AP3 to bind the A. fumigatus galactofuranose (Galf )-deficient mutant ΔglfA confirmed that Galf residues are part of the epitope. Several lines of evidence strongly indicated that AP3 recognizes the Galf residues of O-linked glycans on Aspergillus proteins. Glycoarray analysis revealed that AP3 recognizes oligo-[β-D-Galf-1,5] sequences containing four or more residues with longer chains more efficiently. We also showed that AP3 captures GM in serum, suggesting it may be useful as a diagnostic tool for patients with IA.

Damage-Associated Molecular Pattern-Triggered Immunity in Plants.

As a universal process in multicellular organisms, including animals and plants, cells usually emit danger signals when suffering from attacks of microbes and herbivores, or physical damage. These signals, termed as damage-associated molecular patterns (DAMPs), mainly include cell wall or extracellular protein fragments, peptides, nucleotides, and amino acids. Once exposed on cell surfaces, DAMPs are detected by plasma membrane-localized receptors of surrounding cells to regulate immune responses against the invading organisms and promote damage repair. DAMPs may also act as long-distance mobile signals to mediate systemic wounding responses. Generation, release, and perception of DAMPs, and signaling events downstream of DAMP perception are all rigorously modulated by plants. These processes integrate together to determine intricate mechanisms of DAMP-triggered immunity in plants. In this review, we present an extensive overview on our current understanding of DAMPs in plant immune system.

Production and localization of hydrogen peroxide and nitric oxide in grapevine cells elicited with cyclodextrins and methyl jasmonate

The use of methyl jasmonate, alone or in combination with cyclic oligosaccharides such as cyclodextrins, has proved to be a successful strategy for increasing the production of trans-resveratrol in Vitis vinifera cell cultures. However, understanding the intracellular signalling pathways involved in its production would improve the management of grapevine cells as biofactories of this high-value natural product. The results obtained herein confirm the involvement of hydrogen peroxide and nitric oxide in cyclodextrins and methyl jasmonate-induced trans-resveratrol production in grapevine cell cultures. In fact, methyl jasmonate led to maximal intracellular levels of hydrogen peroxide and nitric oxide after 24 h of treatment, but extracellular hydrogen peroxide was only detected in the culture medium when grapevine cells were treated with cyclodextrins. The results derived from the cytochemical detection of H2O2 in elicited grapevine cell cultures also suggested that the combined treatment with cyclodextrins and methyl jasmonate not only increased the production of H2O2 but also released cell wall fragments with electron-dense deposits.Moreover, nitric oxide was localized in all the cellular compartments, particularly in the nucleus and cytoplasmic organelles, whereas hydrogen peroxide was mainly found in cytoplasmic areas close to the cell wall, and in the nucleoplasm.

2‐Amino Muramyl Dipeptide Derivatives: Chemical probes to assay the stability and activation of NOD2

Mutations of the cytosolic protein nucleotide‐binding oligomerization domain‐containing protein 2 (NOD2), which detects the synthetic bacterial cell wall fragment muramyl dipeptide (MDP), are implicated in making individuals more susceptible to Crohn's disease (CD). However, the mode by which these mutations affect the pathogenesis of this disease is not fully understood. Interestingly, studies suggest NOD2 positively and negatively regulates the expression of toll‐like receptors (TLRs) and the consequent production of inflammatory molecules upon activation. Like NOD2, TLRs can detect bacterial fragments and produce an inflammatory response via the NF‐kB pathway, though through distinct mechanisms. Stabilizing the NOD2 Crohn's mutants through protein‐ligand binding can be a potential therapeutic target for CD. A library of MDP derivatives with altering functionality at the 2‐amino position were synthesized in order to investigate the stabilization imparted to NOD2 upon binding. A simplified 7‐step synthetic route to prepare 2‐amino MDP and subsequent functionalization of the molecule with mild NHS coupling or acetylating conditions is utilized. Compounds are then tested for stabilization with the cellular thermal shift assay (CESTA). In this cell‐based assay, binding of a ligand to a protein stabilizes a protein and protects it against thermal degradation at increased temperatures, resulting in a thermal shift (ΔTm) when compared to the unbound protein. The activation of the NF‐kB pathway by these molecules is tested concurrently, using a luciferase reporter assay to determine if changes in functionalization also yield a modulated immune response.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microbes play role in arthritis

People with aching joints and arthritis might be surprised to learn that their pain might start partly in the gut. Singapore-based researchers have shown that there is a link between autoimmune arthritis and the gut microbiome (Nat. Microbiol. 2019, DOI: 10.1038/s41564-019-0381-1). The human body is home to trillions of bacteria collectively known as microbiota. These bugs coexist with us and can release small molecules that circulate throughout the body, including bacterial peptidoglycans. These fragments of the bacterial cell wall are released when bacteria remodel their cells. Some are involved in immunostimulation, sleep promotion, and fever. Yue Wang’s team at the Institute of Molecular and Cell Biology decided to investigate the role of the circulating peptidoglycans in autoimmune arthritis. They found that increasing the amounts of peptidoglycans in the blood speeds up disease development in mice that are bred to have arthritis. But a single injection of a peptidoglycan antibody that

Multiple iron reduction by methoxylated phenolic lignin structures and the generation of reactive oxygen species by lignocellulose surfaces.

Chelator-mediated Fenton chemistry is capable of reducing non-stochiometric amounts of iron via hydroquinone oxidation. These types of reactions have previously been demonstrated to be promoted by some lignocellulose degrading fungi in generating hydroxyl radicals to permit lignified plant cell wall deconstruction. Here we demonstrate that lignocellulose surfaces, when exposed by chemical treatment or fragmentation, can promote a similar multi-oxidative mechanism in the presence of iron. Iron reduction by lignin surfaces permits the generation of hydroxyl radicals in the cell wall to help explain fungal non-enzymatic cell wall deconstruction, and it also provides an explanation for certain phenomenon such as the anthropogenic generation of formaldehyde by wood. The mechanism also provides a basis for the generation of electrons by lignin that are required by certain fungal redox enzymes active in plant cell wall degrading systems. Overall, the data demonstrate that iron found naturally in lignocellulose materials will promote the oxidation of phenolic lignin compounds in the naturally low pH environments occurring within lignified plant cell walls, and that this activity is promoted by cell wall fragmentation.

Existing cell wall fragments modify the thermal properties and hydrolysis of potato starch

The effect of cell wall fragments on the thermal properties and hydrolysis of potato starch were investigated. The relative degree of crystallinity of purified starch and starch with cell wall fragments were 19.7% and 20.7%, respectively. The onset temperature (To) and peak temperature (Tp) of the starch with cell wall fragments were much higher than those of purified starch, whereas the △H value of purified starch (9.5 ± 0.5 J/g) was much higher than that of the starch with cell wall fragments (7.6 ± 0.7 J/g). Moreover, slightly lower equilibrium hydrolysis of starch with cell wall fragments (78.8 ± 6.3%) and different morphologies changes between those two samples during heating were also observed. Our study demonstrated that it might be an alternative way to improve or extend the property of potato starch with the remaining of its cell wall fragments.

Damage-associated molecular patterns (DAMPs) as future plant vaccines that protect crops from pests

Abstract Domestication reduced the resistance of most crops which, therefore, require an ever-increasing application of pesticides. Nevertheless, in most domesticated species, the resistance against herbivores and disease agents can be activated by so-called elicitors. Here, we review various strategies of biological pest control and particularly focus on damage-associated molecular patterns (DAMPs) as a potential novel class of plant vaccines: plant-derived molecules that trigger immunity because they indicate damage when they appear in the extracellular space or, in the case of damage-induced volatile compounds, in the aerial space around the plant. The application of plant or algal extracts, which arguably contain many DAMPs, enhanced the resistance against herbivory in cabbage, tomato and maize and to fungal or bacterial disease in all major grain crops and in banana, apple, grapevine, melon, tomato, cucumber or carrot. Similarly, purified DAMPs such as ATP, fragmented DNA, peptides, cell-wall fragments, or volatiles, elicited resistance-related responses in species such wheat, corn, soybean, grapevine, bean, eggplant, cucumber, potato or tomato. In conclusion, plant-based preparations with different degrees of purity can improve the health of multiple crops and horticultural plants. Commercial products based on plant extracts are available, and leaf extracts can even be home-made, e.g., by low-income farmers. We conclude that plant extracts and their derivatives bear an as-yet underestimated potential to serve as environmentally friendly vaccines of cultivated plants.

Characterization of Bacterial Cell Wall Fragment Recognition by the Yeast Protein CYR1p

Within the human body are trillions of microorganisms which must maintain a proper balance with the human host as well as amongst each other. These organisms must be able to accurately identify and respond to the bacterial cells that surround them. Pattern recognition receptors (PRRs) can bind pathogen associated molecular patterns (PAMPs) and enable these microorganisms to recognize their surrounding microbiome. Similarly, in yeast species Candida albicans the adenylyl cyclase CYR1p is found to bind muramyl dipeptide (MDP) a fragment of the bacterial cell wall or peptidoglycan. Bacterial cell wall fragments like MDP are interestingly found to trigger a phenotypical change in C. albicans to enter pathogenic hyphal growth through a cascade initiated by the binding of MDP to CYR1p. Another member of the Candida family, Candida glabrata has been found to not experience the same phenotypical switch to hyphal growth observed in C. albicans in the presence of MDP despite both species being of the same family and both commonly involved in yeast infections. The Leucine Rich Repeat (LRR) domain specifically has been shown to be the site of MDP binding within the protein CYR1p in C. albicans. To understand the differences in the mechanism of peptidoglycan recognition by C. albicans and C. glabrata, the LRR domain of C. glabrata was expressed and purified. Analysis of the binding or lack thereof between MDP and the LRR domain of CYR1p from C. glabrata could help to elucidate why C. albicans has hyphae growth triggered by MDP but C. glabrata does not.Support or Funding InformationUniversity of Delaware Department of Chemistry and BiochemistryThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Instrumental analyses of nanostructures and interactions with bound water of superheated steam treated plant materials

Using multi-scale instrumental analyses, nanostructural changes due to superheated steam treatments were compared between Japanese cedar (Cryptomeria japonica) and bamboo (Phyllostachys pubescens Mazel). The lower amounts of aromatic carboxylic acids contained in lignin and hemicellulose/lignin in Japanese cedar suppressed not only the formation of pyrolysis products from hemicellulose but also fragmentation of cell walls. Variable temperature solid-state NMR spectra and relaxation time analyses were carried out on the nanostructures of the plant materials in order to examine molecular motions and interaction with bound water for the biomass constituents of the plant materials. The untreated and steam treated bamboos strongly interacted with bound water to change the 1H spin-lattice relaxation time (T1H) values depending on the content of bound water. Because the interaction between the biomass constituents and bound water in Japanese cedar was weaker than that in bamboo, the T1H values of Japanese cedar were reduced less than those of bamboo in the humid conditions. The difference in the interaction with bound water between bamboo and Japanese cedar also affected the amount of the pyrolysis products and the decomposition rate of hemicellulose in the superheated steam treated materials. Furthermore, the pyrolysis products caused little change in the molecular mobility of Japanese cedar although they restrained that of bamboo.

Multiple effects of grape seed polyphenolics to prevent metabolic diseases

Obesity is increasing in developing countries. Population studies show a relationship between affluence and obesity. Changing food intake patterns with affluence such as preference for foods with less astringent polyphenolic compounds and dietary fibers may increase risk of metabolic dysfunctions due to caloric imbalance. Animal models of obesity consistently show that grape seed procyanidins prevent increases in body and abdo- minal adipose weight gain, plasma cholesterol, liver weight gain and inflammation in animals on high fat diets. The mechanisms are not clear because the oral intake of procyanidins results in pleiotropic interactions with proteins in the mouth, stomach, small intestine, cecum and colon that affect the rate of digestion of bioavailability of macronutrients, sterols, and dietary fiber. Procyanidins also bind bile acids and reduce intestinal permeability to inflammatory bacterial cell wall fragment. Procyanidins are not degraded or metabolized until reaching the lower gut where they can be metabolized into phenolic acids by gut bacteria. While they are metabolized by gut bacteria, they also alter total numbers and distribution of phyla and species of gut bacteria. Gut bacteria are recognized as significant contributors to obesity and obesity related metabolic diseases. The review examines the different pleiotropic effects of grape seed procyanidins that have a significant effect on metabolic disease in animal models of obesity.

The disruptive effect of lysozyme on the bacterial cell wall explored by an in-silico structural outlook.

The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N-acetylglucosamine (NAG) and N-acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall and disrupt the bacterial life cycle by cleaving the linkage between the NAG and NAM carbohydrates. Lab exercises focused on the effects of lysozyme on the bacterial cell wall are frequently incorporated in biochemistry classes designed for undergraduate students in diverse fields as biology, microbiology, chemistry, agronomy, medicine, and veterinary medicine. Such exercises typically do not include structural data. We describe here a sequence of computer tasks designed to illustrate and reinforce both physiological and structural concepts involved in lysozyme effects on the bacterial cell-wall structure. This lab class usually lasts 3.5 hours. First, the instructor presents introductory concepts of the bacterial cell wall and the effect of lysozyme on its structure. Then, students are taught to use computer modeling to visualize the three-dimensional structure of a lysozyme in complex with bacterial cell-wall fragments. Finally, the lysozyme inhibitory effect on a bacterial culture is optionally proposed as a simple microbiological assay. The computer lab exercises described here give students a realistic understanding of the disruptive effect of lysozymes on the bacterial cell wall, a crucial component in bacterial survival. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):83-90, 2018.

Structural model of the full-length Ser/Thr protein kinase StkP from S. pneumoniae and its recognition of peptidoglycan fragments

The unique eukaryotic-like Ser/Thr protein kinases of Streptococcus pneumoniae, StkP, plays a primary role in the cell division process. It is composed of an intracellular kinase domain, a transmembrane helix and four extracellular PASTA subunits. PASTA domains were shown to interact with cell wall fragments but the key questions related to the molecular mechanism governing ligand recognition remain unclear. To address this issue, the full-length structural model of StkP was generated by combining small-angle X-ray scattering data with the results of computer simulations. Docking and molecular dynamics studies on the generated three-dimensional model structure reveal the possibility of peptidoglycan fragment binding at the hinge regions between PASTA subunits with a preference for a bent hinge between PASTA3 and PASTA4.

Damage-associated responses of the host contribute to defence against cyst nematodes but not root-knot nematodes.

When nematodes invade and subsequently migrate within plant roots, they generate cell wall fragments (in the form of oligogalacturonides; OGs) that can act as damage-associated molecular patterns and activate host defence responses. However, the molecular mechanisms mediating damage responses in plant-nematode interactions remain unexplored. Here, we characterized the role of a group of cell wall receptor proteins in Arabidopsis, designated as polygalacturonase-inhibiting proteins (PGIPs), during infection with the cyst nematode Heterodera schachtii and the root-knot nematode Meloidogyne incognita. PGIPs are encoded by a family of two genes in Arabidopsis, and are involved in the formation of active OG elicitors. Our results show that PGIP gene expression is strongly induced in response to cyst nematode invasion of roots. Analyses of loss-of-function mutants and overexpression lines revealed that PGIP1 expression attenuates infection of host roots by cyst nematodes, but not root-knot nematodes. The PGIP1-mediated attenuation of cyst nematode infection involves the activation of plant camalexin and indole-glucosinolate pathways. These combined results provide new insights into the molecular mechanisms underlying plant damage perception and response pathways during infection by cyst and root-knot nematodes, and establishes the function of PGIP in plant resistance to cyst nematodes.

Enhanced capture of bacteria and endotoxin by antimicrobial WLBU2 peptide tethered on polyethylene oxide spacers.

Severe sepsis is a life threatening immune response that may be caused by endotoxins (lipopolysaccharides) in circulating bacterial cell wall fragments. Hemoperfusion through a sorbent column coated with the antimicrobial peptide polymyxin B (PMB) is a promising treatment for sepsis. However, PMB is cytotoxic and neurotoxic, and is a membrane disruptor that may fragment endotoxin vesicles. In addition, the blood is not protected from nonspecific interactions with the synthetic surface of the solid support. These effects may be responsible for the variety of undesirable clinical outcomes, including nonspecific adsorption of proteins, blood cell damage, platelet activation, and a lack of clear evidence of efficacy of the current hemoperfusion products. An alternative endotoxin-binding agent is WLBU2, a synthetic cationic amphiphilic peptide that exhibits better selectivity for bacterial cell membranes and reduced host cell cytotoxicity. Tethering the peptide at the periphery of a hydrophilic polyethylene oxide (PEO) brush should also mask the underlying surface, preventing cell and protein adsorption, and is expected to increase the solvent accessibility and molecular mobility of the tethered peptides. WLBU2 tethered on pendant PEO chains exhibited significantly greater capture of intact bacterial cells and endotoxin than surface-immobilized WLBU2. Tethered WLBU2 also captured amounts of endotoxin comparable to PMB. These results suggest that PEO-tethered WLBU2 coatings may be safer and more effective than the state-of-the-art PMB-based technology.