Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics

USDA-ARS?s Scientific Manuscript database

Virion-associated peptidoglycan hydrolases (VAPGH) are phage-encoded lytic enzymes that locally degrade the peptidoglycan (PG) of the bacterial cell wall during infection. Their action usually generates a small hole through which the phage tail crosses the cell envelope to inject the phage genetic m...

Crystal structure of the glycosidase family 73 peptidoglycan hydrolase FlgJ

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashimoto, Wataru; Ochiai, Akihito; Momma, Keiko

Glycoside hydrolase (GH) categorized into family 73 plays an important role in degrading bacterial cell wall peptidoglycan. The flagellar protein FlgJ contains N- and C-terminal domains responsible for flagellar rod assembly and peptidoglycan hydrolysis, respectively. A member of family GH-73, the C-terminal domain (SPH1045-C) of FlgJ from Sphingomonas sp. strain A1 was expressed in Escherichia coli, purified, and characterized. SPH1045-C exhibited bacterial cell lytic activity most efficiently at pH 6.0 and 37 deg. C. The X-ray crystallographic structure of SPH1045-C was determined at 1.74 A resolution by single-wavelength anomalous diffraction. The enzyme consists of two lobes, {alpha} and {beta}. Amore » deep cleft located between the two lobes can accommodate polymer molecules, suggesting that the active site is located in the cleft. Although SPH1045-C shows a structural homology with family GH-22 and GH-23 lysozymes, the arrangement of the nucleophile/base residue in the active site is specific to each peptidoglycan hydrolase.« less

Peptidoglycan synthesis drives an FtsZ-treadmilling-independent step of cytokinesis.

PubMed

Monteiro, João M; Pereira, Ana R; Reichmann, Nathalie T; Saraiva, Bruno M; Fernandes, Pedro B; Veiga, Helena; Tavares, Andreia C; Santos, Margarida; Ferreira, Maria T; Macário, Vânia; VanNieuwenhze, Michael S; Filipe, Sérgio R; Pinho, Mariana G

2018-02-22

Peptidoglycan is the main component of the bacterial wall and protects cells from the mechanical stress that results from high intracellular turgor. Peptidoglycan biosynthesis is very similar in all bacteria; bacterial shapes are therefore mainly determined by the spatial and temporal regulation of peptidoglycan synthesis rather than by the chemical composition of peptidoglycan. The form of rod-shaped bacteria, such as Bacillus subtilis or Escherichia coli, is generated by the action of two peptidoglycan synthesis machineries that act at the septum and at the lateral wall in processes coordinated by the cytoskeletal proteins FtsZ and MreB, respectively. The tubulin homologue FtsZ is the first protein recruited to the division site, where it assembles in filaments-forming the Z ring-that undergo treadmilling and recruit later divisome proteins. The rate of treadmilling in B. subtilis controls the rates of both peptidoglycan synthesis and cell division. The actin homologue MreB forms discrete patches that move circumferentially around the cell in tracks perpendicular to the long axis of the cell, and organize the insertion of new cell wall during elongation. Cocci such as Staphylococcus aureus possess only one type of peptidoglycan synthesis machinery, which is diverted from the cell periphery to the septum in preparation for division. The molecular cue that coordinates this transition has remained elusive. Here we investigate the localization of S. aureus peptidoglycan biosynthesis proteins and show that the recruitment of the putative lipid II flippase MurJ to the septum, by the DivIB-DivIC-FtsL complex, drives peptidoglycan incorporation to the midcell. MurJ recruitment corresponds to a turning point in cytokinesis, which is slow and dependent on FtsZ treadmilling before MurJ arrival but becomes faster and independent of FtsZ treadmilling after peptidoglycan synthesis activity is directed to the septum, where it provides additional force for cell envelope

Immersion Refractometry of Isolated Bacterial Cell Walls

PubMed Central

Marquis, Robert E.

1973-01-01

Immersion-refractometric and light-scattering measurements were adapted to determinations of average refractive indices and physical compactness of isolated bacterial cell walls. The structures were immersed in solutions containing various concentrations of polymer molecules that cannot penetrate into wall pores, and then an estimate was made of the polymer concentration or the refractive index of the polymer solution in which light scattering was reduced to zero. Because each wall preparation was heterogeneous, the refractive index of the medium for zero light scattering had to be estimated by extrapolation. Refractive indices for walls suspended in bovine serum albumin solutions ranged from 1.348 for walls of the rod form of Arthrobacter crystallopoietes to 1.382 for walls of the teichoic acid deficient, 52A5 strain of Staphylococcus aureus. These indices were used to calculate approximate values for solids content per milliliter, and the calculated values agreed closely with those estimated from a knowledge of dextran-impermeable volumes per gram, dry weight, of the walls. When large molecules such as dextrans or serum albumin were used for immersion refractometry, the refractive indices obtained were for entire walls, including both wall polymers and wall water. When smaller molecules that can penetrate wall pores to various extents were used with Micrococcus lysodeikticus walls, the average, apparent refractive index of the structures increased as the molecular size of probing molecules was decreased. It was possible to obtain an estimate of 1.45 to 1.46 for the refractive index of wall polymers, predominantly peptidoglycans in this case, by extrapolating the curve for refractive index versus molecular radius to a value of 0.2 nm, the approximate radius of a water molecule. This relatively low value for polymer refractive index was interpreted as evidence in favor of the amorphous, elastic model of peptidoglycan structure and against the crystalline, rigid

Selection of peptidoglycan-specific aptamers for bacterial cells identification.

PubMed

Ferreira, Iêda Mendes; de Souza Lacerda, Camila Maria; de Faria, Lígia Santana; Corrêa, Cristiane Rodrigues; de Andrade, Antero Silva Ribeiro

2014-12-01

Peptidoglycan is a highly complex and essential macromolecule of bacterial outer cell wall; it is a heteropolymer made up of linear glycan strands cross-linked by peptides. Peptidoglycan has a particular composition which makes it a possible target for specific bacterial recognition. Aptamers are single-stranded DNA or RNA oligonucleotides that bind to target molecules with high affinity and specificity. Aptamers can be labeled with different radioisotopes and possess several properties that make them suitable for molecular imaging. The purpose of this study was to obtain aptamers for use as radiopharmaceutical in bacterial infection diagnosis. Two aptamers (Antibac1 and Antibac2) against peptidoglycan were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology. The dissociation constant (Kd) for Antibac1 was 0.415 + 0.047 μM and for Antibac2 was 1.261 + 0.280 μM. These aptamers labeled with (32)P showed high affinity for Staphylococcus aureus cells. The binding to S. aureus and Escherichia coli in vitro were significantly higher than for Candida albicans and human fibroblasts, demonstrating their specificity for bacterial cells. These results point Antibac1 and Antibac2 as promising tools for bacterial infections identification.

Inhibitors targeting on cell wall biosynthesis pathway of MRSA.

PubMed

Hao, Haihong; Cheng, Guyue; Dai, Menghong; Wu, Qinghua; Yuan, Zonghui

2012-11-01

Methicillin resistant Staphylococcus aureus (MRSA), widely known as a type of new superbug, has aroused world-wide concern. Cell wall biosynthesis pathway is an old but good target for the development of antibacterial agents. Peptidoglycan and wall teichoic acids (WTAs) biosynthesis are two main processes of the cell wall biosynthesis pathway (CWBP). Other than penicillin-binding proteins (PBPs), some key factors (Mur enzymes, lipid I or II precursor, etc.) in CWBP are becoming attractive molecule targets for the discovery of anti-MRSA compounds. A number of new compounds, with higher affinity for PBPs or with inhibitory activity on such molecule targets in CWBP of MRSA, have been in the pipeline recently. This review concludes recent research achievements and provides a complete picture of CWBP of MRSA, including the peptidoglycan and wall teichoic acids synthesis pathway. The potential inhibitors targeting on CWBP are subsequently presented to improve development of novel therapeutic strategies for MRSA.

Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

PubMed

Munshi, Tulika; Gupta, Antima; Evangelopoulos, Dimitrios; Guzman, Juan David; Gibbons, Simon; Keep, Nicholas H; Bhakta, Sanjib

2013-01-01

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

Characterisation of ATP-Dependent Mur Ligases Involved in the Biogenesis of Cell Wall Peptidoglycan in Mycobacterium tuberculosis

PubMed Central

Munshi, Tulika; Gupta, Antima; Evangelopoulos, Dimitrios; Guzman, Juan David; Gibbons, Simon; Keep, Nicholas H.; Bhakta, Sanjib

2013-01-01

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery. PMID:23555903

Peptidoglycan microarray as a novel tool to explore protein-ligand recognition.

PubMed

Wang, Ning; Hirata, Akiyoshi; Nokihara, Kiyoshi; Fukase, Koichi; Fujimoto, Yukari

2016-11-04

Peptidoglycan is a giant bag-shaped molecule essential for bacterial cell shape and resistance to osmotic stresses. The activity of a large number of bacterial surface proteins involved in cell growth and division requires binding to this macromolecule. Recognition of peptidoglycan by immune effectors is also crucial for the establishment of the immune response against pathogens. The availability of pure and chemically defined peptidoglycan fragments is a major technical bottleneck that has precluded systematic studies of the mechanisms underpinning protein-mediated peptidoglycan recognition. Here, we report a microarray strategy suitable to carry out comprehensive studies to characterize proteins-peptidoglycan interactions. We describe a method to introduce a functional group on peptidoglycan fragments allowing their stable immobilization on amorphous carbon chip plates to minimize nonspecific binding. Such peptidoglycan microarrays were used with a model peptidoglycan binding protein-the human peptidoglycan recognition protein-S (hPGRP-S). We propose that this strategy could be implemented to carry out high-throughput analyses to study peptidoglycan binding proteins. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 422-429, 2016. © 2016 Wiley Periodicals, Inc.

Atomic model of a cell-wall cross-linking enzyme in complex with an intact bacterial peptidoglycan.

PubMed

Schanda, Paul; Triboulet, Sébastien; Laguri, Cédric; Bougault, Catherine M; Ayala, Isabel; Callon, Morgane; Arthur, Michel; Simorre, Jean-Pierre

2014-12-24

The maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a highly cross-linked biopolymer. The transpeptidases that perform this cross-linking are important targets for antibiotics. Despite this biomedical importance, to date no structure of a protein in complex with an intact bacterial peptidoglycan has been resolved, primarily due to the large size and flexibility of peptidoglycan sacculi. Here we use solid-state NMR spectroscopy to derive for the first time an atomic model of an l,d-transpeptidase from Bacillus subtilis bound to its natural substrate, the intact B. subtilis peptidoglycan. Importantly, the model obtained from protein chemical shift perturbation data shows that both domains-the catalytic domain as well as the proposed peptidoglycan recognition domain-are important for the interaction and reveals a novel binding motif that involves residues outside of the classical enzymatic pocket. Experiments on mutants and truncated protein constructs independently confirm the binding site and the implication of both domains. Through measurements of dipolar-coupling derived order parameters of bond motion we show that protein binding reduces the flexibility of peptidoglycan. This first report of an atomic model of a protein-peptidoglycan complex paves the way for the design of new antibiotic drugs targeting l,d-transpeptidases. The strategy developed here can be extended to the study of a large variety of enzymes involved in peptidoglycan morphogenesis.

Metabolic labelling of the carbohydrate core in bacterial peptidoglycan and its applications

PubMed Central

Liang, Hai; DeMeester, Kristen E.; Hou, Ching-Wen; Parent, Michelle A.; Caplan, Jeffrey L.; Grimes, Catherine L.

2017-01-01

Bacterial cells are surrounded by a polymer known as peptidoglycan (PG), which protects the cell from changes in osmotic pressure and small molecule insults. A component of this material, N-acetyl-muramic acid (NAM), serves as a core structural element for innate immune recognition of PG fragments. We report the synthesis of modifiable NAM carbohydrate derivatives and the installation of these building blocks into the backbone of Gram-positive and Gram-negative bacterial PG utilizing metabolic cell wall recycling and biosynthetic machineries. Whole cells are labelled via click chemistry and visualized using super-resolution microscopy, revealing higher resolution PG structural details and allowing the cell wall biosynthesis, as well as its destruction in immune cells, to be tracked. This study will assist in the future identification of mechanisms that the immune system uses to recognize bacteria, glean information about fundamental cell wall architecture and aid in the design of novel antibiotics. PMID:28425464

Structural studies on molecular interactions between camel peptidoglycan recognition protein, CPGRP-S, and peptidoglycan moieties N-acetylglucosamine and N-acetylmuramic acid.

PubMed

Sharma, Pradeep; Yamini, Shavait; Dube, Divya; Singh, Amar; Sinha, Mau; Dey, Sharmistha; Mitra, Dipendra K; Kaur, Punit; Sharma, Sujata; Singh, Tej P

2012-06-22

Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc, and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3 × 10(-9), 2.6 × 10(-7), and 1.8 × 10(-7) M, respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules, A, B, C, and D, in the asymmetric unit that exists as A-B and C-D units of two neighboring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms, indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α and IL-6 from human peripheral blood mononuclear cells. The introduction of CPGRP-S to the PGN-challenged cultured peripheral blood mononuclear cells reduced the expressions of proinflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and down-regulated macrophage-mediated inflammation, indicating its potential applications as an antibacterial agent.

A new metabolic cell wall labeling method reveals peptidoglycan in Chlamydia trachomatis

PubMed Central

Liechti, G.; Kuru, E.; Hall, E.; Kalinda, A.; Brun, Y. V.; VanNieuwenhze, M.; Maurelli, A. T.

2014-01-01

Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure1. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia encodes genes for PG biosynthesis2–7 and exhibits susceptibility to "anti-PG" antibiotics8,9, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’10). We employed a novel approach to metabolically label chlamydial PG using D-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis was labeled with the probes throughout its biphasic, developmental life cycle, and differential probe incorporation experiments conducted in the presence of ampicillin is consistent with the presence of chlamydial PG modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence to date that chlamydial species possess functional PG. PMID:24336210

An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis.

PubMed

Rueff, Anne-Stéphanie; Chastanet, Arnaud; Domínguez-Escobar, Julia; Yao, Zhizhong; Yates, James; Prejean, Maria-Victoria; Delumeau, Olivier; Noirot, Philippe; Wedlich-Söldner, Roland; Filipe, Sergio R; Carballido-López, Rut

2014-01-01

MreB proteins play a major role during morphogenesis of rod-shaped bacteria by organizing biosynthesis of the peptidoglycan cell wall. However, the mechanisms underlying this process are not well understood. In Bacillus subtilis, membrane-associated MreB polymers have been shown to be associated to elongation-specific complexes containing transmembrane morphogenetic factors and extracellular cell wall assembly proteins. We have now found that an early intracellular step of cell wall synthesis is also associated to MreB. We show that the previously uncharacterized protein YkuR (renamed DapI) is required for synthesis of meso-diaminopimelate (m-DAP), an essential constituent of the peptidoglycan precursor, and that it physically interacts with MreB. Highly inclined laminated optical sheet microscopy revealed that YkuR forms uniformly distributed foci that exhibit fast motion in the cytoplasm, and are not detected in cells lacking MreB. We propose a model in which soluble MreB organizes intracellular steps of peptidoglycan synthesis in the cytoplasm to feed the membrane-associated cell wall synthesizing machineries. © 2013 John Wiley & Sons Ltd.

Structure of Pneumococcal Peptidoglycan Hydrolase LytB Reveals Insights into the Bacterial Cell Wall Remodeling and Pathogenesis*

PubMed Central

Bai, Xiao-Hui; Chen, Hui-Jie; Jiang, Yong-Liang; Wen, Zhensong; Huang, Yubin; Cheng, Wang; Li, Qiong; Qi, Lei; Zhang, Jing-Ren; Chen, Yuxing; Zhou, Cong-Zhao

2014-01-01

Streptococcus pneumoniae causes a series of devastating infections in humans. Previous studies have shown that the endo-β-N-acetylglucosaminidase LytB is critical for pneumococcal cell division and nasal colonization, but the biochemical mechanism of LytB action remains unknown. Here we report the 1.65 Å crystal structure of the catalytic domain (residues Lys-375–Asp-658) of LytB (termed LytBCAT), excluding the choline binding domain. LytBCAT consists of three structurally independent modules: SH3b, WW, and GH73. These modules form a “T-shaped” pocket that accommodates a putative tetrasaccharide-pentapeptide substrate of peptidoglycan. Structural comparison and simulation revealed that the GH73 module of LytB harbors the active site, including the catalytic residue Glu-564. In vitro assays of hydrolytic activity indicated that LytB prefers the peptidoglycan from the lytB-deficient pneumococci, suggesting the existence of a specific substrate of LytB in the immature peptidoglycan. Combined with in vitro cell-dispersing and in vivo cell separation assays, we demonstrated that all three modules are necessary for the optimal activity of LytB. Further functional analysis showed that the full catalytic activity of LytB is required for pneumococcal adhesion to and invasion into human lung epithelial cells. Structure-based alignment indicated that the unique modular organization of LytB is highly conserved in its orthologs from Streptococcus mitis group and Gemella species. These findings provided structural insights into the pneumococcal cell wall remodeling and novel hints for the rational design of therapeutic agents against pneumococcal growth and thereby the related diseases. PMID:25002590

Renew or die: The molecular mechanisms of peptidoglycan recycling and antibiotic resistance in Gram-negative pathogens.

PubMed

Domínguez-Gil, Teresa; Molina, Rafael; Alcorlo, Martín; Hermoso, Juan A

2016-09-01

Antimicrobial resistance is one of the most serious health threats. Cell-wall remodeling processes are tightly regulated to warrant bacterial survival and in some cases are directly linked to antibiotic resistance. Remodeling produces cell-wall fragments that are recycled but can also act as messengers for bacterial communication, as effector molecules in immune response and as signaling molecules triggering antibiotic resistance. This review is intended to provide state-of-the-art information about the molecular mechanisms governing this process and gather structural information of the different macromolecular machineries involved in peptidoglycan recycling in Gram-negative bacteria. The growing body of literature on the 3D structures of the corresponding macromolecules reveals an extraordinary complexity. Considering the increasing incidence and widespread emergence of Gram-negative multidrug-resistant pathogens in clinics, structural information on the main actors of the recycling process paves the way for designing novel antibiotics disrupting cellular communication in the recycling-resistance pathway. Copyright © 2016. Published by Elsevier Ltd.

Cell-wall preparation containing poly-γ-D-glutamate covalently linked to peptidoglycan, a straightforward extractable molecule, protects mice against experimental anthrax infection.

PubMed

Candela, Thomas; Dumetz, Fabien; Tosi-Couture, Evelyne; Mock, Michèle; Goossens, Pierre L; Fouet, Agnès

2012-12-17

Bacillus anthracis is the causative agent of anthrax that is characterized by septicemia and toxemia. Many vaccine strategies were described to counteract anthrax infection. In contrast with veterinary live vaccines, currently human vaccines are acellular with the protective antigen, a toxin component, as the main constituent. However, in animal models this vaccine is less efficient than the live vaccine. In this study, we analyzed the protection afforded by a single extractable surface element. The poly-γ-D-glutamate capsule is covalently linked to the peptidoglycan. A preparation of peptidoglycan-linked poly-γ-D-glutamate (GluPG) was tested for its immunogenicity and its protective effect. GluPG injection, in mice, elicited the production of specific antibodies directed against poly-glutamate and partially protected the animals against lethal challenges with a non-toxinogenic strain. When combined to protective antigen, GluPG immunization conferred full protection against cutaneous anthrax induced with a fully virulent strain. Copyright © 2012 Elsevier Ltd. All rights reserved.

Sex-dependent alterations in motor and anxiety-like behavior of aged bacterial peptidoglycan sensing molecule 2 knockout mice.

PubMed

Arentsen, Tim; Khalid, Roksana; Qian, Yu; Diaz Heijtz, Rochellys

2018-01-01

Peptidoglycan recognition proteins (PGRPs) are key sensing-molecules of the innate immune system that specifically detect bacterial peptidoglycan (PGN) and its derivates. PGRPs have recently emerged as potential key regulators of normal brain development and behavior. To test the hypothesis that PGRPs play a role in motor control and anxiety-like behavior in later life, we used 15-month old male and female peptidoglycan recognition protein 2 (Pglyrp2) knockout (KO) mice. Pglyrp2 is an N-acetylmuramyl-l-alanine amidase that hydrolyzes PGN between the sugar backbone and the peptide chain (which is unique among the mammalian PGRPs). Using a battery of behavioral tests, we demonstrate that Pglyrp2 KO male mice display decreased levels of anxiety-like behavior compared with wild type (WT) males. In contrast, Pglyrp2 KO female mice show reduced rearing activity and increased anxiety-like behavior compared to WT females. In the accelerated rotarod test, however, Pglyrp2 KO female mice performed better compared to WT females (i.e., they had longer latency to fall off the rotarod). Further, Pglyrp2 KO male mice exhibited decreased expression levels of synaptophysin, gephyrin, and brain-derived neurotrophic factor in the frontal cortex, but not in the amygdala. Pglyrp2 KO female mice exhibited increased expression levels of spinophilin and alpha-synuclein in the frontal cortex, while exhibiting decreased expression levels of synaptophysin, gephyrin and spinophilin in the amygdala. Our findings suggest a novel role for Pglyrp2asa key regulator of motor and anxiety-like behavior in late life. Copyright © 2017. Published by Elsevier Inc.

Rhizocola hellebori gen. nov., sp. nov., an actinomycete of the family Micromonosporaceae containing 3,4-dihydroxydiaminopimelic acid in the cell-wall peptidoglycan.

PubMed

Matsumoto, Atsuko; Kawaguchi, Yoko; Nakashima, Takuji; Iwatsuki, Masato; Ōmura, Satoshi; Takahashi, Yōko

2014-08-01

An actinomycete strain, K12-0602(T), was isolated from the root of a Helleborus orientalis plant in Japan. The 16S rRNA gene sequence of strain K12-0602(T) showed that it had a close relationship with members of the family Micromonosporaceae and the 16S rRNA gene sequence similarity values between strain K12-0602(T) and type strains of type species of 27 genera belonging to the family Micromonosporaceae were below 96.2%. MK-9 (H4) and MK-9 (H6) were detected as major menaquinones, and galactose, xylose, mannose and ribose were present in the whole-cell hydrolysate. The acyl type of the peptidoglycan was glycolyl. Major fatty acids were iso-C(15 : 0), iso-C(16 : 0), C(17 : 1)ω9c and anteiso-C(17 : 0). Phosphatidylethanolamine was detected as the phospholipid corresponding to phospholipid type II. The G+C content of the genomic DNA was 67 mol%. Analyses of the cell-wall peptidoglycan by TLC and LC/MS showed that it was composed of alanine, glycine, hydroxylglutamic acid and an unknown amino acid, which was subsequently determined to be 3,4-dihydroxydiaminopimelic acid using instrumental analyses, including NMR and mass spectrometry. On the basis of the phylogenetic analysis and chemotaxonomic characteristics, strain K12-0602(T) represents a novel species of a new genus in the family Micromonosporaceae, for which the name Rhizocola hellebori gen. nov., sp. nov. is proposed. The type strain of the type species is K12-0602(T) ( = NBRC 109834(T) = DSM 45988(T)). This is the first report, to our knowledge, of 3,4-dihydroxydiaminopimelic acid being found as a diamino acid in bacterial cell-wall peptidoglycan. © 2014 IUMS.

Peptidoglycan architecture can specify division planes in Staphylococcus aureus.

PubMed

Turner, Robert D; Ratcliffe, Emma C; Wheeler, Richard; Golestanian, Ramin; Hobbs, Jamie K; Foster, Simon J

2010-06-15

Division in Staphylococci occurs equatorially and on specific sequentially orthogonal planes in three dimensions, resulting, after incomplete cell separation, in the 'bunch of grapes' cluster organization that defines the genus. The shape of Staphylococci is principally maintained by peptidoglycan. In this study, we use Atomic Force Microscopy (AFM) and fluorescence microscopy with vancomycin labelling to examine purified peptidoglycan architecture and its dynamics in Staphylococcus aureus and correlate these with the cell cycle. At the presumptive septum, cells were found to form a large belt of peptidoglycan in the division plane before the centripetal formation of the septal disc; this often had a 'piecrust' texture. After division, the structures remain as orthogonal ribs, encoding the location of past division planes in the cell wall. We propose that this epigenetic information is used to enable S. aureus to divide in sequentially orthogonal planes, explaining how a spherical organism can maintain division plane localization with fidelity over many generations.

Bacterial cell-wall recycling

PubMed Central

Johnson, Jarrod W.; Fisher, Jed F.; Mobashery, Shahriar

2012-01-01

Many Gram-negative and Gram-positive bacteria recycle a significant proportion of the peptidoglycan components of their cell walls during their growth and septation. In many—and quite possibly all—bacteria, the peptidoglycan fragments are recovered and recycled. While cell-wall recycling is beneficial for the recovery of resources, it also serves as a mechanism to detect cell-wall–targeting antibiotics and to regulate resistance mechanisms. In several Gram-negative pathogens, anhydro-MurNAc-peptide cell-wall fragments regulate AmpC β-lactamase induction. In some Gram-positive organisms, short peptides derived from the cell wall regulate the induction of both β-lactamase and β-lactam-resistant penicillin-binding proteins. The involvement of peptidoglycan recycling with resistance regulation suggests that inhibitors of the enzymes involved in the recycling might synergize with cell-wall-targeted antibiotics. Indeed, such inhibitors improve the potency of β-lactams in vitro against inducible AmpC β-lactamase-producing bacteria. We describe the key steps of cell-wall remodeling and recycling, the regulation of resistance mechanisms by cell-wall recycling, and recent advances toward the discovery of cell-wall recycling inhibitors. PMID:23163477

Genetic basis for the resistance of Staphylococcus aureus to peptidoglycan hydrolase by comparative transcriptome and whole genome sequence analysis

USDA-ARS?s Scientific Manuscript database

Background: Lysostaphin is a glycyl-glycine bacteriocin peptidoglycan hydrolase secreted by Staphylococcus simulans for degrading the peptidoglycan moieties in Staphylococcus aureus cell walls which result in cell lysis. There are known mechanisms of resistance to lysostaphin, e.g. serine in place...

Wall teichoic acids prevent antibody binding to epitopes within the cell wall of Staphylococcus aureus.

PubMed

Gautam, Samir; Kim, Taehan; Lester, Evan; Deep, Deeksha; Spiegel, David A

2016-01-15

Staphylococcus aureus is a Gram-positive bacterial pathogen that produces a range of infections including cellulitis, pneumonia, and septicemia. The principle mechanism in antistaphylococcal host defense is opsonization with antibodies and complement proteins, followed by phagocytic clearance. Here we use a previously developed technique for installing chemical epitopes in the peptidoglycan cell wall to show that surface glycopolymers known as wall teichoic acids conceal cell wall epitopes, preventing their recognition and opsonization by antibodies. Thus, our results reveal a previously unrecognized immunoevasive role for wall teichoic acids in S. aureus: repulsion of peptidoglycan-targeted antibodies.

Structure of Bordetella pertussis peptidoglycan

DOE Office of Scientific and Technical Information (OSTI.GOV)

Folkening, W.J.; Nogami, W.; Martin, S.A.

1987-09-01

Bordetella pertussis Tohama phases I and III were grown to the late-exponential phase in liquid medium containing (/sup 3/H)diaminopimelic acid and treated by a hot (96/sup 0/C) sodium dodecyl sulfate extraction procedure. Washed sodium dodecyl sulfate-insoluble residue from phases I and III consisted of complexes containing protein (ca. 40%) and peptidoglycan (60/sup 6/). Subsequent treatment with proteinase K yielded purified peptidoglycan which contained N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, and diaminopimelic acid in molar ratios of 1:1:2:1:1 and <2% protein. Radiochemical analyses indicated that /sup 3/H added in diaminopimelic acid was present in peptidoglycan-protein complexes and purified peptidoglycan as diaminopimelicmore » acid exclusively and that pertussis peptidoglycan was not O acetylated, consistent with it being degraded completely by hen egg white lysozyme. Muramidase-derived disaccharide peptide monomers and peptide-cross-linked dimers and higher oligomers were isolated by molecular-sieve chromatography; from the distribution of these peptidoglycan fragments, the extent of peptide cross-linking of both phase I and III peptidoglycan was calculated to be ca. 48%. Unambiguous determination of the structure of muramidase-derived pepidoglycan fragments by fast atom bombardment-mass spectrometry and tandem mass spectrometry indicated that the pertussis peptidoglycan monomer fraction was surprisingly homogeneous, consisting of >95% N-acetylglucosaminyl-N-acetylmuramyl-alanyl-glutamyl-diaminopimelyl-alanine.« less

Bacterial autolysins trim cell surface peptidoglycan to prevent detection by the Drosophila innate immune system

PubMed Central

Atilano, Magda Luciana; Pereira, Pedro Matos; Vaz, Filipa; Catalão, Maria João; Reed, Patricia; Grilo, Inês Ramos; Sobral, Rita Gonçalves; Ligoxygakis, Petros; Pinho, Mariana Gomes; Filipe, Sérgio Raposo

2014-01-01

Bacteria have to avoid recognition by the host immune system in order to establish a successful infection. Peptidoglycan, the principal constituent of virtually all bacterial surfaces, is a specific molecular signature recognized by dedicated host receptors, present in animals and plants, which trigger an immune response. Here we report that autolysins from Gram-positive pathogenic bacteria, enzymes capable of hydrolyzing peptidoglycan, have a major role in concealing this inflammatory molecule from Drosophila peptidoglycan recognition proteins (PGRPs). We show that autolysins trim the outermost peptidoglycan fragments and that in their absence bacterial virulence is impaired, as PGRPs can directly recognize leftover peptidoglycan extending beyond the external layers of bacterial proteins and polysaccharides. The activity of autolysins is not restricted to the producer cells but can also alter the surface of neighboring bacteria, facilitating the survival of the entire population in the infected host. DOI: http://dx.doi.org/10.7554/eLife.02277.001 PMID:24692449

In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA)

DOE PAGES

Sychantha, David; Jones, Carys S.; Little, Dustin J.; ...

2017-10-27

The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain.more » We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.« less

In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sychantha, David; Jones, Carys S.; Little, Dustin J.

The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain.more » We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.« less

In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA)

PubMed Central

Sychantha, David; Jones, Carys S.; Little, Dustin J.; Howell, P. Lynne

2017-01-01

The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens. PMID:29077761

The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli.

PubMed

Lai, Ghee Chuan; Cho, Hongbaek; Bernhardt, Thomas G

2017-07-01

Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes

Toll-like receptor 2-mediated peptidoglycan uptake by immature intestinal epithelial cells from apical side and exosome-associated transcellular transcytosis

PubMed Central

Bu, Heng-Fu; Wang, Xiao; Tang, Yi; Koti, Viola; Tan, Xiao-Di

2015-01-01

Peptidoglycan is a potent immune adjuvant derived from bacterial cell walls. Previous investigations suggest that intestinal epithelium may absorb peptidoglycan from the lumen. Nonetheless, how peptidoglycan is taken up and crosses intestinal epithelium remains largely unclear. Here, we first characterized peptidoglycan transport in vitro using IEC-18 and HT29-CL19A cells, which represent less mature epithelial cells in intestinal crypts. With fluorescent microscopy, we visualized internalization of dual-labeled peptidoglycan by enterocytes. Engulfed peptidoglycan was found to form a complex with peptidoglycan recognition protein-3, which may facilitate delivering peptidoglycan in vivo. Utilizing electronic microscopy, we revealed that uptake of apical peptidoglycan across intestinal epithelial monolayers was involved in phagocytosis, multivesicular body formation, and exosome secretion. We also studied transport of peptidoglycan using the transwell system. Our data indicated that apically loaded peptidoglycan was exocytosed to the basolateral compartment with exosomes by HT29-CL19A cells. The peptidoglycan-contained basolateral exosome extracts induced macrophage activation. Through gavaging mice with labeled peptidoglycan, we found that luminal peptidoglycan was taken up by columnar epithelial cells in crypts of the small intestine. Furthermore, we showed that pre-confluent immature but not post-confluent mature C2BBe1 cells engulfed peptidoglycan via a toll-like receptor 2-dependent manner. Together, our findings suggest that (1) crypt-based immature intestinal epithelial cells play an important role in transport of luminal peptidoglycan over the intestinal epithelium; and (2) luminal peptidoglycan is transcytosed across intestinal epithelia via a toll-like receptor 2-meciated phagocytosis-multivesicular body-exosome pathway. The absorbed peptidoglycan and its derivatives may facilitate maintenance of intestinal immune homeostasis. PMID:20020500

Escherichia coli peptidoglycan structure and mechanics as predicted by atomic-scale simulations.

PubMed

Gumbart, James C; Beeby, Morgan; Jensen, Grant J; Roux, Benoît

2014-02-01

Bacteria face the challenging requirement to maintain their shape and avoid rupture due to the high internal turgor pressure, but simultaneously permit the import and export of nutrients, chemical signals, and virulence factors. The bacterial cell wall, a mesh-like structure composed of cross-linked strands of peptidoglycan, fulfills both needs by being semi-rigid, yet sufficiently porous to allow diffusion through it. How the mechanical properties of the cell wall are determined by the molecular features and the spatial arrangement of the relatively thin strands in the larger cellular-scale structure is not known. To examine this issue, we have developed and simulated atomic-scale models of Escherichia coli cell walls in a disordered circumferential arrangement. The cell-wall models are found to possess an anisotropic elasticity, as known experimentally, arising from the orthogonal orientation of the glycan strands and of the peptide cross-links. Other features such as thickness, pore size, and disorder are also found to generally agree with experiments, further supporting the disordered circumferential model of peptidoglycan. The validated constructs illustrate how mesoscopic structure and behavior emerge naturally from the underlying atomic-scale properties and, furthermore, demonstrate the ability of all-atom simulations to reproduce a range of macroscopic observables for extended polymer meshes.

Human SAP is a novel peptidoglycan recognition protein that induces complement- independent phagocytosis of Staphylococcus aureus

PubMed Central

An, Jang-Hyun; Kurokawa, Kenji; Jung, Dong-Jun; Kim, Min-Jung; Kim, Chan-Hee; Fujimoto, Yukari; Fukase, Koichi; Coggeshall, K. Mark; Lee, Bok Luel

2014-01-01

The human pathogen Staphylococcus aureus is responsible for many community-acquired and hospital-associated infections and is associated with high mortality. Concern over the emergence of multidrug-resistant strains has renewed interest in the elucidation of host mechanisms that defend against S. aureus infection. We recently demonstrated that human serum mannose-binding lectin (MBL) binds to S. aureus wall teichoic acid (WTA), a cell wall glycopolymer, a discovery that prompted further screening to identify additional serum proteins that recognize S. aureus cell wall components. In this report, we incubated human serum with 10 different S. aureus mutants and determined that serum amyloid P component (SAP) bound specifically to a WTA-deficient S. aureus ΔtagO mutant, but not to tagO-complemented, WTA-expressing cells. Biochemical characterization revealed that SAP recognizes bacterial peptidoglycan as a ligand and that WTA inhibits this interaction. Although SAP binding to peptidoglycan was not observed to induce complement activation, SAP-bound ΔtagO cells were phagocytosed by human polymorphonuclear leukocytes in an Fcγ receptor-dependent manner. These results indicate that SAP functions as a host defense factor, similar to other peptidoglycan recognition proteins and nucleotide-binding oligomerization domain (NOD)-like receptors. PMID:23966633

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes*

PubMed Central

Chan, Yvonne Gar-Yun; Kim, Hwan Keun; Schneewind, Olaf; Missiakas, Dominique

2014-01-01

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan. PMID:24753256

Adamantoylated biologically active small peptides and glycopeptides structurally related to the bacterial peptidoglycan.

PubMed

Frkanec, Ruža; Vranešić, Branka; Tomić, Srdjanka

2013-01-01

A large number of novel synthetic compounds representing smaller parts of original peptidoglycan molecules have been synthesized and found to possess versatile biological activity, particularly immunomodulating properties. A series of compounds containing the adamantyl residues coupled to peptides and glycopeptides characteristic for bacterial peptidoglycan was described. The new adamantylpeptides and adamantylglycopeptides were prepared starting from N-protected racemic adamantylglycine and dipeptide L-Ala-D-isoglutamine. The adamantyl glycopeptides were obtained by coupling the adamantyltripeptides with alpha-D-mannose moiety through spacer molecule of fixed chirality. Since the starting material was D,L-(adamantyl-glycine) the condensation products with the dipeptide were mixtures of diastereoisomers. The obtained diastereoisomers were separated, characterized, and tested for immunostimulating activity. An HPLC method for purity testing was developed and adapted for the particular compounds.

Peptidoglycan and Teichoic Acid Levels and Alterations in Staphylococcus aureus by Cell-Wall and Whole-Cell Nuclear Magnetic Resonance.

PubMed

Romaniuk, Joseph A H; Cegelski, Lynette

2018-06-11

Gram-positive bacteria surround themselves with a multilayered macromolecular cell wall that is essential to cell survival and serves as a major target for antibiotics. The cell wall of Staphylococcus aureus is composed of two major structural components, peptidoglycan (PG) and wall teichoic acid (WTA), together creating a heterogeneous and insoluble matrix that poses a challenge to quantitative compositional analysis. Here, we present 13 C cross polarization magic angle spinning solid-state nuclear magnetic resonance (NMR) spectra of intact cell walls, purified PG, and purified WTA. The spectra reveal the clear molecular differences in the two polymers and enable quantification of PG and WTA in isolated cell walls, an attractive alternative to estimating teichoic acid content from a phosphate analysis of completely pyrolyzed cell walls. Furthermore, we discovered that unique PG and WTA spectral signatures could be identified in whole-cell NMR spectra and used to compare PG and WTA levels among intact bacterial cell samples. The distinguishing whole-cell 13 C NMR contributions associated with PG include the GlcNAc-MurNAc sugar carbons and glycyl α-carbons. WTA contributes carbons from the phosphoribitol backbone. Distinguishing 15 N spectral signatures include glycyl amide nitrogens in PG and the esterified d-alanyl amine nitrogens in WTA. 13 C NMR analysis was performed with samples at natural abundance and included 10 whole-cell sample comparisons. Changes consistent with altered PG and WTA content were detected in whole-cell spectra of bacteria harvested at different growth times and in cells treated with tunicamycin. This use of whole-cell NMR provides quantitative parameters of composition in the context of whole-cell activity.

Targeting the permeability barrier and peptidoglycan recycling pathways to disarm Pseudomonas aeruginosa against the innate immune system

PubMed Central

Moya, Bartolomé; Munar-Bestard, Marta; Zamorano, Laura; Cabot, Gabriel; Blázquez, Jesús; Ayala, Juan A.; Oliver, Antonio

2017-01-01

Antimicrobial resistance is a continuously increasing threat that severely compromises our antibiotic arsenal and causes thousands of deaths due to hospital-acquired infections by pathogens such as Pseudomonas aeruginosa, situation further aggravated by the limited development of new antibiotics. Thus, alternative strategies such as those targeting bacterial resistance mechanisms, virulence or potentiating the activity of our immune system resources are urgently needed. We have recently shown that mutations simultaneously causing the peptidoglycan recycling blockage and the β-lactamase AmpC overexpression impair the virulence of P.aeruginosa. These findings suggested that peptidoglycan metabolism might be a good target not only for fighting antibiotic resistance, but also for the attenuation of virulence and/or potentiation of our innate immune weapons. Here we analyzed the activity of the innate immune elements peptidoglycan recognition proteins (PGRPs) and lysozyme against P. aeruginosa. We show that while lysozyme and PGRPs have a very modest basal effect over P. aeruginosa, their bactericidal activity is dramatically increased in the presence of subinhibitory concentrations of the permeabilizing agent colistin. We also show that the P. aeruginosa lysozyme inhibitors seem to play a very residual protective role even in permeabilizing conditions. In contrast, we demonstrate that, once the permeability barrier is overpassed, the activity of lysozyme and PGRPs is dramatically enhanced when inhibiting key peptidoglycan recycling components (such as the 3 AmpDs, AmpG or NagZ), indicating a decisive protective role for cell-wall recycling and that direct peptidoglycan-binding supports, at least partially, the activity of these enzymes. Finally, we show that recycling blockade when occurring simultaneously with AmpC overexpression determines a further decrease in the resistance against PGRP2 and lysozyme, linked to quantitative changes in the cell-wall. Thus, our

Peptidoglycan from Fermentation By-Product Triggers Defense Responses in Grapevine

PubMed Central

Chen, Yang; Takeda, Taito; Aoki, Yoshinao; Fujita, Keiko; Suzuki, Shunji; Igarashi, Daisuke

2014-01-01

Plants are constantly under attack from a variety of microorganisms, and rely on a series of complex detection and response systems to protect themselves from infection. Here, we found that a by-product of glutamate fermentation triggered defense responses in grapevine, increasing the expression of defense response genes in cultured cells, foliar chitinase activity, and resistance to infection by downy mildew in leaf explants. To identify the molecule that triggered this innate immunity, we fractionated and purified candidates extracted from Corynebacterium glutamicum, a bacterium used in the production of amino acids by fermentation. Using hydrolysis by lysozyme, a silkworm larva plasma detection system, and gel filtration analysis, we identified peptidoglycan as inducing the defense responses. Peptidoglycans of Escherichia coli, Bacillus subtilis, and Staphylococcus aureus also generated similar defensive responses. PMID:25427192

Endogenous pyrogen production by human blood monocytes stimulated by staphylococcal cell wall components.

PubMed

Oken, M M; Peterson, P K; Wilkinson, B J

1981-01-01

To determine the properties of Staphylococcus aureus contributing to its pyrogenicity, we compared, in human monocytes, endogenous pyrogen production stimulated by heat-killed S. aureus with that stimulated by purified S. aureus cell walls or by particulate peptidoglycan prepared from the same strain. Peptidoglycan, but not the purified cell wall preparation, was found comparable to S. aureus as an endogenous pyrogen stimulus. This finding was associated with a more effective monocyte phagocytosis of S. aureus and peptidoglycan as compared with that of purified cell walls. Lysostaphin digestion of peptidoglycan markedly reduced its pyrogenicity. To test whether the chemical composition of the ingested particles is important, latex particles were tested as possible stimuli for monocyte endogenous pyrogen release. Although 40 to 68% of monocytes ingested latex particles during the first hour, there was no evidence of endogenous pyrogen activity in the supernatant even when supernatants equivalent to 5.2 X 10(6) monocytes were tested. This study demonstrates that the pyrogenic moiety of the S. aureus cell wall resides in the peptidoglycan component. Phagocytosis is not in itself a pyrogenic stimulus, but rather serves as an effective mechanism to bring about contact between the chemical stimulus and the monocyte.

Molecular mapping of the cell wall polysaccharides of the human pathogen Streptococcus agalactiae

NASA Astrophysics Data System (ADS)

Beaussart, Audrey; Péchoux, Christine; Trieu-Cuot, Patrick; Hols, Pascal; Mistou, Michel-Yves; Dufrêne, Yves F.

2014-11-01

The surface of many bacterial pathogens is covered with polysaccharides that play important roles in mediating pathogen-host interactions. In Streptococcus agalactiae, the capsular polysaccharide (CPS) is recognized as a major virulence factor while the group B carbohydrate (GBC) is crucial for peptidoglycan biosynthesis and cell division. Despite the important roles of CPS and GBC, there is little information available on the molecular organization of these glycopolymers on the cell surface. Here, we use atomic force microscopy (AFM) and transmission electron microscopy (TEM) to analyze the nanoscale distribution of CPS and GBC in wild-type (WT) and mutant strains of S. agalactiae. TEM analyses reveal that in WT bacteria, peptidoglycan is covered with a very thin (few nm) layer of GBC (the ``pellicle'') overlaid by a 15-45 nm thick layer of CPS (the ``capsule''). AFM-based single-molecule mapping with specific antibody probes shows that CPS is exposed on WT cells, while it is hardly detected on mutant cells impaired in CPS production (ΔcpsE mutant). By contrast, both TEM and AFM show that CPS is over-expressed in mutant cells altered in GBC expression (ΔgbcO mutant), indicating that the production of the two surface glycopolymers is coordinated in WT cells. In addition, AFM topographic imaging and molecular mapping with specific lectin probes demonstrate that removal of CPS (ΔcpsE), but not of GBC (ΔgbcO), leads to the exposure of peptidoglycan, organized into 25 nm wide bands running parallel to the septum. These results indicate that CPS forms a homogeneous barrier protecting the underlying peptidoglycan from environmental exposure, while the presence of GBC does not prevent peptidoglycan detection. This work shows that single-molecule AFM, combined with high-resolution TEM, represents a powerful platform for analysing the molecular arrangement of the cell wall polymers of bacterial pathogens.

Structural and functional features of enzymes of Mycobacterium tuberculosis peptidoglycan biosynthesis as targets for drug development

PubMed Central

Moraes, Gleiciane Leal; Gomes, Guelber Cardoso; de Sousa, Paulo Robson Monteiro; Alves, Cláudio Nahum; Govender, Thavendran; Kruger, Hendrik G.; Maguire, Glenn E.M.; Lamichhane, Gyanu; Lameira, Jerônimo

2015-01-01

SUMMARY Tuberculosis (TB) is the second leading cause of human mortality from infectious diseases worldwide. The WHO reported 1.3 million deaths and 8.6 million new cases of TB in 2012. Mycobacterium tuberculosis (M. tuberculosis), the infectious bacteria that causes TB, is encapsulated by a thick and robust cell wall. The innermost segment of the cell wall is comprised of peptidoglycan, a layer that is required for survival and growth of the pathogen. Enzymes that catalyse biosynthesis of the peptidoglycan are essential and are therefore attractive targets for discovery of novel antibiotics as humans lack similar enzymes making it possible to selectively target bacteria only. In this paper, we have reviewed the structures and functions of enzymes GlmS, GlmM, GlmU, MurA, MurB, MurC, MurD, MurE and MurF from M. tuberculosis that are involved in peptidoglycan biosynthesis. In addition, we report homology modelled 3D structures of those key enzymes from M. tuberculosis of which the structures are still unknown. We demonstrated that natural substrates can be successfully docked into the active sites of the GlmS and GlmU respectively. It is therefore expected that the models and the data provided herein will facilitate translational research to develop new drugs to treat TB. PMID:25701501

Crystallographic and Molecular Dynamics Analysis of Loop Motions Unmasking the Peptidoglycan-Binding Site in Stator Protein MotB of Flagellar Motor

PubMed Central

Nahar, Musammat F.; Buckle, Ashley M.; Roujeinikova, Anna

2011-01-01

Background The C-terminal domain of MotB (MotB-C) shows high sequence similarity to outer membrane protein A and related peptidoglycan (PG)-binding proteins. It is believed to anchor the power-generating MotA/MotB stator unit of the bacterial flagellar motor to the peptidoglycan layer of the cell wall. We previously reported the first crystal structure of this domain and made a puzzling observation that all conserved residues that are thought to be essential for PG recognition are buried and inaccessible in the crystal structure. In this study, we tested a hypothesis that peptidoglycan binding is preceded by, or accompanied by, some structural reorganization that exposes the key conserved residues. Methodology/Principal Findings We determined the structure of a new crystalline form (Form B) of Helicobacter pylori MotB-C. Comparisons with the existing Form A revealed conformational variations in the petal-like loops around the carbohydrate binding site near one end of the β-sheet. These variations are thought to reflect natural flexibility at this site required for insertion into the peptidoglycan mesh. In order to understand the nature of this flexibility we have performed molecular dynamics simulations of the MotB-C dimer. The results are consistent with the crystallographic data and provide evidence that the three loops move in a concerted fashion, exposing conserved MotB residues that have previously been implicated in binding of the peptide moiety of peptidoglycan. Conclusion/Significance Our structural analysis provides a new insight into the mechanism by which MotB inserts into the peptidoglycan mesh, thus anchoring the power-generating complex to the cell wall. PMID:21533052

Impact of peptidoglycan O-acetylation on autolytic activities of the Enterococcus faecalis N-acetylglucosaminidase AtlA and N-acetylmuramidase AtlB.

PubMed

Emirian, Aurélie; Fromentin, Sophie; Eckert, Catherine; Chau, Françoise; Dubost, Lionel; Delepierre, Muriel; Gutmann, Laurent; Arthur, Michel; Mesnage, Stéphane

2009-09-17

Autolysins are potentially lethal enzymes that partially hydrolyze peptidoglycan for incorporation of new precursors and septum cleavage after cell division. Here, we explored the impact of peptidoglycan O-acetylation on the enzymatic activities of Enterococcus faecalis major autolysins, the N-acetylglucosaminidase AtlA and the N-acetylmuramidase AtlB. We constructed isogenic strains with various O-acetylation levels and used them as substrates to assay E. faecalis autolysin activities. Peptidoglycan O-acetylation had a marginal inhibitory impact on the activities of these enzymes. In contrast, removal of cell wall glycopolymers increased the AtlB activity (37-fold), suggesting that these polymers negatively control the activity of this enzyme.

Peptidoglycan Recycling in Gram-Positive Bacteria Is Crucial for Survival in Stationary Phase

PubMed Central

Borisova, Marina; Gaupp, Rosmarie; Duckworth, Amanda; Schneider, Alexander; Dalügge, Désirée; Mühleck, Maraike; Deubel, Denise; Unsleber, Sandra; Yu, Wenqi; Muth, Günther; Bischoff, Markus; Götz, Friedrich

2016-01-01

ABSTRACT Peptidoglycan recycling is a metabolic process by which Gram-negative bacteria reutilize up to half of their cell wall within one generation during vegetative growth. Whether peptidoglycan recycling also occurs in Gram-positive bacteria has so far remained unclear. We show here that three Gram-positive model organisms, Staphylococcus aureus, Bacillus subtilis, and Streptomyces coelicolor, all recycle the sugar N-acetylmuramic acid (MurNAc) of their peptidoglycan during growth in rich medium. They possess MurNAc-6-phosphate (MurNAc-6P) etherase (MurQ in E. coli) enzymes, which are responsible for the intracellular conversion of MurNAc-6P to N-acetylglucosamine-6-phosphate and d-lactate. By applying mass spectrometry, we observed accumulation of MurNAc-6P in MurNAc-6P etherase deletion mutants but not in either the isogenic parental strains or complemented strains, suggesting that MurQ orthologs are required for the recycling of cell wall-derived MurNAc in these bacteria. Quantification of MurNAc-6P in ΔmurQ cells of S. aureus and B. subtilis revealed small amounts during exponential growth phase (0.19 nmol and 0.03 nmol, respectively, per ml of cells at an optical density at 600 nm [OD600] of 1) but large amounts during transition (0.56 nmol and 0.52 nmol) and stationary (0.53 nmol and 1.36 nmol) phases. The addition of MurNAc to ΔmurQ cultures greatly increased the levels of intracellular MurNAc-6P in all growth phases. The ΔmurQ mutants of S. aureus and B. subtilis showed no growth deficiency in rich medium compared to the growth of the respective parental strains, but intriguingly, they had a severe survival disadvantage in late stationary phase. Thus, although peptidoglycan recycling is apparently not essential for the growth of Gram-positive bacteria, it provides a benefit for long-term survival. PMID:27729505

Structural and functional features of enzymes of Mycobacterium tuberculosis peptidoglycan biosynthesis as targets for drug development.

PubMed

Moraes, Gleiciane Leal; Gomes, Guelber Cardoso; Monteiro de Sousa, Paulo Robson; Alves, Cláudio Nahum; Govender, Thavendran; Kruger, Hendrik G; Maguire, Glenn E M; Lamichhane, Gyanu; Lameira, Jerônimo

2015-03-01

Tuberculosis (TB) is the second leading cause of human mortality from infectious diseases worldwide. The WHO reported 1.3 million deaths and 8.6 million new cases of TB in 2012. Mycobacterium tuberculosis (M. tuberculosis), the infectious bacteria that causes TB, is encapsulated by a thick and robust cell wall. The innermost segment of the cell wall is comprised of peptidoglycan, a layer that is required for survival and growth of the pathogen. Enzymes that catalyse biosynthesis of the peptidoglycan are essential and are therefore attractive targets for discovery of novel antibiotics as humans lack similar enzymes making it possible to selectively target bacteria only. In this paper, we have reviewed the structures and functions of enzymes GlmS, GlmM, GlmU, MurA, MurB, MurC, MurD, MurE and MurF from M. tuberculosis that are involved in peptidoglycan biosynthesis. In addition, we report homology modelled 3D structures of those key enzymes from M. tuberculosis of which the structures are still unknown. We demonstrated that natural substrates can be successfully docked into the active sites of the GlmS and GlmU respectively. It is therefore expected that the models and the data provided herein will facilitate translational research to develop new drugs to treat TB. Copyright © 2015. Published by Elsevier Ltd.

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

PubMed Central

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne

2014-01-01

Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids, but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis. PMID:25402772

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

PubMed

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne

2015-01-01

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

Resolving Single Molecule Lysozyme Dynamics with a Carbon Nanotube Electronic Circuit

NASA Astrophysics Data System (ADS)

Choi, Yongki; Moody, Issa S.; Perez, Israel; Sheps, Tatyana; Weiss, Gregory A.; Collins, Philip G.

2011-03-01

High resolution, real-time monitoring of a single lysozyme molecule is demonstrated by fabricating nanoscale electronic devices based on single-walled carbon nanotubes (SWCNT). In this sensor platform, a biomolecule of interest is attached to a single SWCNT device. The electrical conductance transduces chemical events with single molecule sensitivity and 10 microsecond resolution. In this work, enzymatic turnover by lysozyme is investigated, because the mechanistic details for its processivity and dynamics remain incompletely understood. Stochastically distributed binding events between a lysozyme and its binding substrate, peptidoglycan, are monitored via the sensor conductance. Furthermore, the magnitude and repetition rate of these events varies with pH and the presence of inhibitors or denaturation agents. Changes in the conductance signal are analyzed in terms of lysozyme's internal hinge motion, binding events, and enzymatic processing.

Single-molecule electrocatalysis by single-walled carbon nanotubes.

PubMed

Xu, Weilin; Shen, Hao; Kim, Yoon Ji; Zhou, Xiaochun; Liu, Guokun; Park, Jiwoong; Chen, Peng

2009-12-01

We report a single-molecule fluorescence study of electrocatalysis by single-walled carbon nanotubes (SWNTs) at single-reaction resolution. Applying super-resolution optical imaging, we find that the electrocatalysis occurs at discrete, nanometer-dimension sites on SWNTs. Single-molecule kinetic analysis leads to an electrocatalytic mechanism, allowing quantification of the reactivity and heterogeneity of individual reactive sites. Combined with conductivity measurements, this approach will be powerful to interrogate how the electronic structure of SWNTs affects the electrocatalytic interfacial charge transfer, a process fundamental to photoelectrochemical cells.

Peptidoglycan precursor from Fusobacterium nucleatum contains lanthionine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fredriksen, A.; Vasstrand, E.N.; Jensen, H.B.

1991-01-01

Fusobacterium nucleatum was grown in the presence of ({sup 14}C)UDP. By means of sequential precipitation and chromatographic separation of the cytoplasmic content, a peptidoglycan ({sup 14}C)UDP pentapeptide containing lanthionine was isolated. This finding indicates that lanthionine is synthesized and incorporated as such during the assembly of the peptidoglycan.

An endogenous nanomineral chaperones luminal antigen and peptidoglycan to intestinal immune cells

NASA Astrophysics Data System (ADS)

Powell, Jonathan J.; Thomas-McKay, Emma; Thoree, Vinay; Robertson, Jack; Hewitt, Rachel E.; Skepper, Jeremy N.; Brown, Andy; Hernandez-Garrido, Juan Carlos; Midgley, Paul A.; Gomez-Morilla, Inmaculada; Grime, Geoffrey W.; Kirkby, Karen J.; Mabbott, Neil A.; Donaldson, David S.; Williams, Ifor R.; Rios, Daniel; Girardin, Stephen E.; Haas, Carolin T.; Bruggraber, Sylvaine F. A.; Laman, Jon D.; Tanriver, Yakup; Lombardi, Giovanna; Lechler, Robert; Thompson, Richard P. H.; Pele, Laetitia C.

2015-05-01

In humans and other mammals it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer's patches, small areas of the intestine concentrated with particle-scavenging immune cells. In wild-type mice, intestinal immune cells containing these naturally formed nanoparticles expressed the immune tolerance-associated molecule ‘programmed death-ligand 1’, whereas in NOD1/2 double knockout mice, which cannot recognize peptidoglycan, programmed death-ligand 1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and show how this helps to shape intestinal immune homeostasis.

Preservation of protein globules and peptidoglycan in the mineralized cell wall of nitrate-reducing, iron(II)-oxidizing bacteria: a cryo-electron microscopy study.

PubMed

Miot, J; Maclellan, K; Benzerara, K; Boisset, N

2011-11-01

Iron-oxidizing bacteria are important actors of the geochemical cycle of iron in modern environments and may have played a key role all over Earth's history. However, in order to better assess that role on the modern and the past Earth, there is a need for better understanding the mechanisms of bacterial iron oxidation and for defining potential biosignatures to be looked for in the geologic record. In this study, we investigated experimentally and at the nanometre scale the mineralization of iron-oxidizing bacteria with a combination of synchrotron-based scanning transmission X-ray microscopy (STXM), scanning transmission electron microscopy (STEM) and cryo-transmission electron microscopy (cryo-TEM). We show that the use of cryo-TEM instead of conventional microscopy provides detailed information of the successive iron biomineralization stages in anaerobic nitrate-reducing iron-oxidizing bacteria. These results suggest the existence of preferential Fe-binding and Fe-oxidizing sites on the outer face of the plasma membrane leading to the nucleation and growth of Fe minerals within the periplasm of these cells that eventually become completely encrusted. In contrast, the septa of dividing cells remain nonmineralized. In addition, the use of cryo-TEM offers a detailed view of the exceptional preservation of protein globules and the peptidoglycan within the Fe-mineralized cell walls of these bacteria. These organic molecules and ultrastructural details might be protected from further degradation by entrapment in the mineral matrix down to the nanometre scale. This is discussed in the light of previous studies on the properties of Fe-organic interactions and more generally on the fossilization of mineral-organic assemblies. © 2011 Blackwell Publishing Ltd.

Shear-induced desorption of isolated polymer molecules from a planar wall

NASA Astrophysics Data System (ADS)

Dutta, Sarit; Dorfman, Kevin; Kumar, Satish

2014-03-01

Shear-induced desorption of isolated polymer molecules is studied using Brownian dynamics simulations. The polymer molecules are modeled as freely jointed bead-spring chains interacting with a planar wall via a short-range potential. The simulations include both intrachain and chain-wall hydrodynamic interactions. Shear flow is found to cause chain flattening, resulting at low shear rates in an increased fraction of chain segments bound to the wall. However, above a critical shear rate the chains desorb completely. The desorption process is nucleated by random protrusions in the shear gradient direction which evolve under the combined effect of drag, hydrodynamic interaction, and vorticity-induced rotation, and subsequently lead to recapture. Above the critical shear rate, these protrusions grow in length until the entire chain is peeled off the wall. For free-draining chains, the protrusions are not sustained and no desorption is observed even at shear rates much higher than the critical value. These simulations can help in interpreting experiments on shear-induced desorption of polymer films and brushes.

Lyme disease and relapsing fever Borrelia elongate through zones of peptidoglycan synthesis that mark division sites of daughter cells.

PubMed

Jutras, Brandon Lyon; Scott, Molly; Parry, Bradley; Biboy, Jacob; Gray, Joe; Vollmer, Waldemar; Jacobs-Wagner, Christine

2016-08-16

Agents that cause Lyme disease, relapsing fever, leptospirosis, and syphilis belong to the phylum Spirochaetae-a unique lineage of bacteria most known for their long, spiral morphology. Despite the relevance to human health, little is known about the most fundamental aspects of spirochete growth. Here, using quantitative microscopy to track peptidoglycan cell-wall synthesis, we found that the Lyme disease spirochete Borrelia burgdorferi displays a complex pattern of growth. B. burgdorferi elongates from discrete zones that are both spatially and temporally regulated. In addition, some peptidoglycan incorporation occurs along the cell body, with the notable exception of a large region at the poles. Newborn cells inherit a highly active zone of peptidoglycan synthesis at midcell that contributes to elongation for most of the cell cycle. Concomitant with the initiation of nucleoid separation and cell constriction, second and third zones of elongation are established at the 1/4 and 3/4 cellular positions, marking future sites of division for the subsequent generation. Positioning of elongation zones along the cell is robust to cell length variations and is relatively precise over long distances (>30 µm), suggesting that cells ‟sense" relative, as opposed to absolute, cell length to establish zones of peptidoglycan synthesis. The transition from one to three zones of peptidoglycan growth during the cell cycle is also observed in relapsing fever Borrelia. However, this mode of growth does not extend to representative species from other spirochetal genera, suggesting that this distinctive growth mode represents an evolutionary divide in the spirochete phylum.

Robust peptidoglycan growth by dynamic and variable multi-protein complexes.

PubMed

Pazos, Manuel; Peters, Katharina; Vollmer, Waldemar

2017-04-01

In Gram-negative bacteria such as Escherichia coli the peptidoglycan sacculus resides in the periplasm, a compartment that experiences changes in pH value, osmolality, ion strength and other parameters depending on the cell's environment. Hence, the cell needs robust peptidoglycan growth mechanisms to grow and divide under different conditions. Here we propose a model according to which the cell achieves robust peptidoglycan growth by employing dynamic multi-protein complexes, which assemble with variable composition from freely diffusing sets of peptidoglycan synthases, hydrolases and their regulators, whereby the composition of the active complexes depends on the cell cycle state - cell elongation or division - and the periplasmic growth conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structures of the Peptidoglycan N-Acetylglucosamine Deacetylase Bc1974 and Its Complexes with Zinc Metalloenzyme Inhibitors.

PubMed

Giastas, Petros; Andreou, Athena; Papakyriakou, Athanasios; Koutsioulis, Dimitris; Balomenou, Stavroula; Tzartos, Socrates J; Bouriotis, Vassilis; Eliopoulos, Elias E

2018-02-06

The cell wall peptidoglycan is recognized as a primary target of the innate immune system, and usually its disintegration results in bacterial lysis. Bacillus cereus, a close relative of the highly virulent Bacillus anthracis, contains 10 polysaccharide deacetylases. Among these, the peptidoglycan N-acetylglucosamine deacetylase Bc1974 is the highest homologue to the Bacillus anthracis Ba1977 that is required for full virulence and is involved in resistance to the host's lysozyme. These metalloenzymes belong to the carbohydrate esterase family 4 (CE4) and are attractive targets for the development of new anti-infective agents. Herein we report the first X-ray crystal structures of the NodB domain of Bc1974, the conserved catalytic core of CE4s, in the unliganded form and in complex with four known metalloenzyme inhibitors and two amino acid hydroxamates that target the active site metal. These structures revealed the presence of two conformational states of a catalytic loop known as motif-4 (MT4), which were not observed previously for peptidoglycan deacetylases, but were recently shown in the structure of a Vibrio clolerae chitin deacetylase. By employing molecular docking of a substrate model, we describe a catalytic mechanism that probably involves initial binding of the substrate in a receptive, more open state of MT4 and optimal catalytic activity in the closed state of MT4, consistent with the previous observations. The ligand-bound structures presented here, in addition to the five Bc1974 inhibitors identified, provide a valuable basis for the design of antibacterial agents that target the peptidoglycan deacetylase Ba1977.

An Endogenous Nanomineral Chaperones Luminal Antigen and Peptidoglycan to Intestinal Immune Cells

PubMed Central

Powell, Jonathan J; Thomas-McKay, Emma; Thoree, Vinay; Robertson, Jack; Hewitt, Rachel E; Skepper, Jeremy N; Brown, Andy; Hernandez-Garrido, Juan Carlos; Midgley, Paul A; Gomez-Morilla, Inmaculada; Grime, Geoffrey W; Kirkby, Karen J; Mabbott, Neil A; Donaldson, David S; Williams, Ifor R; Rios, Daniel; Girardin, Stephen E; Haas, Carolin T; Bruggraber, Sylvaine FA; Laman, Jon D; Tanriver, Yakup; Lombardi, Giovanna; Lechler, Robert; Thompson, Richard P H; Pele, Laetitia C

2015-01-01

In humans and other mammals, it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally-fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer’s patches - small areas of the intestine concentrated with particle-scavenging immune cells. In wild type mice, intestinal immune cells containing these naturally-formed nanoparticles expressed the immune tolerance-associated molecule ‘programmed death-ligand 1 (PD-L1)’, whereas in NOD1/2 double knock-out mice, which cannot recognize peptidoglycan, PD-L1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and how this helps to shape intestinal immune homeostasis. PMID:25751305

Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria.

PubMed

Domínguez-Escobar, Julia; Chastanet, Arnaud; Crevenna, Alvaro H; Fromion, Vincent; Wedlich-Söldner, Roland; Carballido-López, Rut

2011-07-08

The peptidoglycan cell wall and the actin-like MreB cytoskeleton are major determinants of cell shape in rod-shaped bacteria. The prevailing model postulates that helical, membrane-associated MreB filaments organize elongation-specific peptidoglycan-synthesizing complexes along sidewalls. We used total internal reflection fluorescence microscopy to visualize the dynamic relation between MreB isoforms and cell wall synthesis in live Bacillus subtilis cells. During exponential growth, MreB proteins did not form helical structures. Instead, together with other morphogenetic factors, they assembled into discrete patches that moved processively along peripheral tracks perpendicular to the cell axis. Patch motility was largely powered by cell wall synthesis, and MreB polymers restricted diffusion of patch components in the membrane and oriented patch motion.

Equilibrium binding behavior of magnesium to wall teichoic acid.

PubMed

Thomas, Kieth J; Rice, Charles V

2015-10-01

Peptidoglycan and teichoic acids are the major cell wall components of Gram-positive bacteria that obtain and sequester metal ions required for biochemical processes. The delivery of metals to the cytoplasmic membrane is aided by anionic binding sites within the peptidoglycan and along the phosphodiester polymer of teichoic acid. The interaction with metals is a delicate balance between the need for attraction and ion diffusion to the membrane. Likewise, metal chelation from the extracellular fluid must initially have strong binding energetics that weaken within the cell wall to enable ion release. We employed atomic absorption and equilibrium dialysis to measure the metal binding capacity and metal binding affinity of wall teichoic acid and Mg2+. Data show that Mg2+ binds to WTA with a 1:2Mg2+ to phosphate ratio with a binding capacity of 1.27 μmol/mg. The affinity of Mg2+ to WTA was also found to be 41×10(3) M(-1) at low metal concentrations and 1.3×10(3) M(-1) at higher Mg2+ concentrations due to weakening electrostatic effects. These values are lower than the values describing Mg2+ interactions with peptidoglycan. However, the binding capacity of WTA is 4 times larger than peptidoglycan. External WTA initially binds metals with positive cooperativity, but metal binding switches to negative cooperativity, whereas interior WTA binds metals with only negative cooperativity. The relevance of this work is to describe changes in metal binding behavior depending on environment. When metals are sparse, chelation is strong to ensure survival yet the binding weakens when essential minerals are abundant. Copyright © 2015 Elsevier B.V. All rights reserved.

Multiparametric AFM reveals turgor-responsive net-like peptidoglycan architecture in live streptococci

NASA Astrophysics Data System (ADS)

Saar Dover, Ron; Bitler, Arkady; Shimoni, Eyal; Trieu-Cuot, Patrick; Shai, Yechiel

2015-05-01

Cell-wall peptidoglycan (PG) of Gram-positive bacteria is a strong and elastic multi-layer designed to resist turgor pressure and determine the cell shape and growth. Despite its crucial role, its architecture remains largely unknown. Here using high-resolution multiparametric atomic force microscopy (AFM), we studied how the structure and elasticity of PG change when subjected to increasing turgor pressure in live Group B Streptococcus. We show a new net-like arrangement of PG, which stretches and stiffens following osmotic challenge. The same structure also exists in isogenic mutants lacking surface appendages. Cell aging does not alter the elasticity of the cell wall, yet destroys the net architecture and exposes single segmented strands with the same circumferential orientation as predicted for intact glycans. Together, we show a new functional PG architecture in live Gram-positive bacteria.

The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes.

PubMed

Divakaruni, Arun V; Baida, Cyril; White, Courtney L; Gober, James W

2007-10-01

MreB, the bacterial actin homologue, is thought to function in spatially co-ordinating cell morphogenesis in conjunction with MreC, a protein that wraps around the outside of the cell within the periplasmic space. In Caulobacter crescentus, MreC physically associates with penicillin-binding proteins (PBPs) which catalyse the insertion of intracellularly synthesized precursors into the peptidoglycan cell wall. Here we show that MreC is required for the spatial organization of components of the peptidoglycan-synthesizing holoenzyme in the periplasm and MreB directs the localization of a peptidoglycan precursor synthesis protein in the cytosol. Additionally, fluorescent vancomycin (Van-FL) labelling revealed that the bacterial cytoskeletal proteins MreB and FtsZ, as well as MreC and RodA, were required for peptidoglycan synthetic activity. MreB and FtsZ were found to be required for morphogenesis of the polar stalk. FtsZ was required for a cell cycle-regulated burst of peptidoglycan synthesis early in the cell cycle resulting in the synthesis of cross-band structures, whereas MreB was required for lengthening of the stalk. Thus, the bacterial cytoskeleton and cell shape-determining proteins such as MreC, function in concert to orchestrate the localization of cell wall synthetic complexes resulting in spatially co-ordinated and efficient peptidoglycan synthetic activity.

Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ

NASA Astrophysics Data System (ADS)

Pilhofer, Martin; Aistleitner, Karin; Biboy, Jacob; Gray, Joe; Kuru, Erkin; Hall, Edward; Brun, Yves V.; Vannieuwenhze, Michael S.; Vollmer, Waldemar; Horn, Matthias; Jensen, Grant J.

2013-12-01

Chlamydiae are important pathogens and symbionts with unique cell biological features. They lack the cell-division protein FtsZ, and the existence of peptidoglycan (PG) in their cell wall has been highly controversial. FtsZ and PG together function in orchestrating cell division and maintaining cell shape in almost all other bacteria. Using electron cryotomography, mass spectrometry and fluorescent labelling dyes, here we show that some environmental chlamydiae have cell wall sacculi consisting of a novel PG type. Treatment with fosfomycin (a PG synthesis inhibitor) leads to lower infection rates and aberrant cell shapes, suggesting that PG synthesis is crucial for the chlamydial life cycle. Our findings demonstrate for the first time the presence of PG in a member of the Chlamydiae. They also present a unique example of a bacterium with a PG sacculus but without FtsZ, challenging the current hypothesis that it is the absence of a cell wall that renders FtsZ non-essential.

The cell wall components peptidoglycan and lipoteichoic acid from Staphylococcus aureus act in synergy to cause shock and multiple organ failure.

PubMed Central

De Kimpe, S J; Kengatharan, M; Thiemermann, C; Vane, J R

1995-01-01

Although the incidence of Gram-positive sepsis has risen strongly, it is unclear how Gram-positive organisms (without endotoxin) initiate septic shock. We investigated whether two cell wall components from Staphylococcus aureus, peptidoglycan (PepG) and lipoteichoic acid (LTA), can induce the inflammatory response and multiple organ dysfunction syndrome (MODS) associated with septic shock caused by Gram-positive organisms. In cultured macrophages, LTA (10 micrograms/ml), but not PepG (100 micrograms/ml), induces the release of nitric oxide measured as nitrite. PepG, however, caused a 4-fold increase in the production of nitrite elicited by LTA. Furthermore, PepG antibodies inhibited the release of nitrite elicited by killed S. aureus. Administration of both PepG (10 mg/kg; i.v.) and LTA (3 mg/kg; i.v.) in anesthetized rats resulted in the release of tumor necrosis factor alpha and interferon gamma and MODS, as indicated by a decrease in arterial oxygen pressure (lung) and an increase in plasma concentrations of bilirubin and alanine aminotransferase (liver), creatinine and urea (kidney), lipase (pancreas), and creatine kinase (heart or skeletal muscle). There was also the expression of inducible nitric oxide synthase in these organs, circulatory failure, and 50% mortality. These effects were not observed after administration of PepG or LTA alone. Even a high dose of LTA (10 mg/kg) causes only circulatory failure but no MODS. Thus, our results demonstrate that the two bacterial wall components, PepG and LTA, work together to cause systemic inflammation and multiple systems failure associated with Gram-positive organisms. Images Fig. 2 PMID:7479784

Diversity of Innate Immune Recognition Mechanism for Bacterial Polymeric meso-Diaminopimelic Acid-type Peptidoglycan in Insects

PubMed Central

Yu, Yang; Park, Ji-Won; Kwon, Hyun-Mi; Hwang, Hyun-Ok; Jang, In-Hwan; Masuda, Akiko; Kurokawa, Kenji; Nakayama, Hiroshi; Lee, Won-Jae; Dohmae, Naoshi; Zhang, Jinghai; Lee, Bok Luel

2010-01-01

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects. PMID:20702416

The lantibiotic mersacidin is a strong inducer of the cell wall stress response of Staphylococcus aureus

PubMed Central

Sass, Peter; Jansen, Andrea; Szekat, Christiane; Sass, Vera; Sahl, Hans-Georg; Bierbaum, Gabriele

2008-01-01

Background The lantibiotic mersacidin is an antimicrobial peptide of 20 amino acids that is ribosomally produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin acts by complexing the sugar phosphate head group of the peptidoglycan precursor lipid II, thereby inhibiting the transglycosylation reaction of peptidoglycan biosynthesis. Results Here, we studied the growth of Staphylococcus aureus in the presence of subinhibitory concentrations of mersacidin. Transcriptional data revealed an extensive induction of the cell wall stress response, which is partly controlled by the two-component regulatory system VraSR. In contrast to other cell wall-active antibiotics such as vancomycin, very low concentrations of mersacidin (0.15 × MIC) were sufficient for induction. Interestingly, the cell wall stress response was equally induced in vancomycin intermediately resistant S. aureus (VISA) and in a highly susceptible strain. Since the transcription of the VraDE ABC transporter genes was induced up to 1700-fold in our experiments, we analyzed the role of VraDE in the response to mersacidin. However, the deletion of the vraE gene did not result in an increased susceptibility to mersacidin compared to the wild type strain. Moreover, the efficacy of mersacidin was not affected by an increased cell wall thickness, which is part of the VISA-type resistance mechanism and functions by trapping the vancomycin molecules in the cell wall before they reach lipid II. Therefore, the relatively higher concentration of mersacidin at the membrane might explain why mersacidin is such a strong inducer of VraSR compared to vancomycin. Conclusion In conclusion, mersacidin appears to be a strong inducer of the cell wall stress response of S. aureus at very low concentrations, which reflects its general mode of action as a cell wall-active peptide as well as its use of a unique target site on lipid II. Additionally, mersacidin does not seem to be a substrate for the resistance transporter Vra

Supramolecular organization of pi-conjugated molecules monitored by single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Alvarez, Laurent; Almadori, Yann; Belhboub, Anouar; Le Parc, Rozenn; Aznar, Raymond; Dieudonné-George, Philippe; Rahmani, Abdelali; Hermet, Patrick; Fossard, Frédéric; Loiseau, Annick; Jousselme, Bruno; Campidelli, Stéphane; Saito, Takeshi; Wang, Guillaume; Bantignies, Jean-Louis

2016-03-01

Photoactive pi-conjugated molecules (quaterthiophene and phthalocyanine) are either encapsulated into the hollow core of single-walled carbon nanotubes or noncovalently stacked at their outer surface in order to elaborate hybrid nanosystems with new physical properties, providing practical routes to fit different requirements for potential applications. We are interested in the relationship between the structure and the optoelectronic properties. The structural properties are investigated mainly by x-ray diffraction and/or transmission electron microscopy and Raman spectroscopy. We show that the supramolecular organizations of confined quaterthiophenes depend on the nanocontainer size, whereas phthalocyanine encapsulation leads to the formation of a one-dimensional phase for which the angle between the molecule ring and the nanotube axis is close to 32 deg. Confined phthalocyanine molecules display Raman spectra hardly altered with respect to the bulk phase, suggesting a rather weak interaction with the tubes. In contrast, the vibrational properties of the molecules stacked at the outer surface of tubes display important modifications. We assume a significant curvature of the phthalocyanine induced by the interaction with the tube walls and a change of the central atom position within the molecular ring, in good agreement with our density functional theory calculations.

Structure of the Cell Wall of Bacillus stearothermophilus: Mode of Action of a Thermophilic Bacteriophage Lytic Enzyme

PubMed Central

Welker, N. E.

1971-01-01

The mode of action of a bacteriophage lytic enzyme on cell walls of Bacillus stearothermophilus (NCA 1503-4R) has been investigated. The enzyme is an endopeptidase which catalyzes the hydrolysis of the l-alanyl-d-glutamyl linkage in peptide subunits of the cell wall peptidoglycan. Preliminary studies on the soluble components in lytic cell wall digests indicate that the glycan moiety is composed of alternating glucosamine and muramic acid; one half of the muramic acid residues contain the tripeptide, l-alanyl-d-glutamyldiaminopimelic acid, and the remaining residues contain the tetrapeptide, l-alanyl-d-glutamyldiaminopimeyl-d-alanine. Almost one half of the peptide subunits are involved in cross-linkages of chemotype I. A structure for the cell wall peptidoglycan is proposed in the light of these findings. PMID:4255338

Peptidoglycan sensing by octopaminergic neurons modulates Drosophila oviposition

PubMed Central

Kurz, C Leopold; Charroux, Bernard; Chaduli, Delphine; Viallat-Lieutaud, Annelise; Royet, Julien

2017-01-01

As infectious diseases pose a threat to host integrity, eukaryotes have evolved mechanisms to eliminate pathogens. In addition to develop strategies reducing infection, animals can engage in behaviors that lower the impact of the infection. The molecular mechanisms by which microbes impact host behavior are not well understood. We demonstrate that bacterial infection of Drosophila females reduces oviposition and that peptidoglycan, the component that activates Drosophila antibacterial response, is also the elicitor of this behavioral change. We show that peptidoglycan regulates egg-laying rate by activating NF-κB signaling pathway in octopaminergic neurons and that, a dedicated peptidoglycan degrading enzyme acts in these neurons to buffer this behavioral response. This study shows that a unique ligand and signaling cascade are used in immune cells to mount an immune response and in neurons to control fly behavior following infection. This may represent a case of behavioral immunity. DOI: http://dx.doi.org/10.7554/eLife.21937.001 PMID:28264763

A novel mechanism of programmed cell death in bacteria by toxin-antitoxin systems corrupts peptidoglycan synthesis.

PubMed

Mutschler, Hannes; Gebhardt, Maike; Shoeman, Robert L; Meinhart, Anton

2011-03-01

Most genomes of bacteria contain toxin-antitoxin (TA) systems. These gene systems encode a toxic protein and its cognate antitoxin. Upon antitoxin degradation, the toxin induces cell stasis or death. TA systems have been linked with numerous functions, including growth modulation, genome maintenance, and stress response. Members of the epsilon/zeta TA family are found throughout the genomes of pathogenic bacteria and were shown not only to stabilize resistance plasmids but also to promote virulence. The broad distribution of epsilon/zeta systems implies that zeta toxins utilize a ubiquitous bacteriotoxic mechanism. However, whereas all other TA families known to date poison macromolecules involved in translation or replication, the target of zeta toxins remained inscrutable. We used in vivo techniques such as microscropy and permeability assays to show that pneumococcal zeta toxin PezT impairs cell wall synthesis and triggers autolysis in Escherichia coli. Subsequently, we demonstrated in vitro that zeta toxins in general phosphorylate the ubiquitous peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) and that this activity is counteracted by binding of antitoxin. After identification of the product we verified the kinase activity in vivo by analyzing metabolite extracts of cells poisoned by PezT using high pressure liquid chromatograpy (HPLC). We further show that phosphorylated UNAG inhibitis MurA, the enzyme catalyzing the initial step in bacterial peptidoglycan biosynthesis. Additionally, we provide what is to our knowledge the first crystal structure of a zeta toxin bound to its substrate. We show that zeta toxins are novel kinases that poison bacteria through global inhibition of peptidoglycan synthesis. This provides a fundamental understanding of how epsilon/zeta TA systems stabilize mobile genetic elements. Additionally, our results imply a mechanism that connects activity of zeta toxin PezT to virulence of pneumococcal infections

Staphylococcus aureus Peptidoglycan Stem Packing by Rotational-Echo Double Resonance NMR Spectroscopy

PubMed Central

Kim, Sung Joon; Singh, Manmilan; Preobrazhenskaya, Maria; Schaefer, Jacob

2013-01-01

Staphylococcus aureus grown in the presence of an alanine-racemase inhibitor was labeled with D-[1-13C]alanine and L-[15N]alanine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance NMR of intact whole cells was used to measure internuclear distances between 13C and 15N of labeled amino acids incorporated in the peptidoglycan, and from those labels to 19F of a glycopeptide drug specifically bound to the peptidoglycan. The observed 13C-15N average distance of 4.1 to 4.4 Å between D- and L-alanines in nearest-neighbor peptide stems is consistent with a local, tightly packed, parallel-stem architecture for a repeating structural motif within the peptidoglycan of S. aureus. PMID:23617832

A comparative modeling and molecular docking study on Mycobacterium tuberculosis targets involved in peptidoglycan biosynthesis.

PubMed

Fakhar, Zeynab; Naiker, Suhashni; Alves, Claudio N; Govender, Thavendran; Maguire, Glenn E M; Lameira, Jeronimo; Lamichhane, Gyanu; Kruger, Hendrik G; Honarparvar, Bahareh

2016-11-01

An alarming rise of multidrug-resistant Mycobacterium tuberculosis strains and the continuous high global morbidity of tuberculosis have reinvigorated the need to identify novel targets to combat the disease. The enzymes that catalyze the biosynthesis of peptidoglycan in M. tuberculosis are essential and noteworthy therapeutic targets. In this study, the biochemical function and homology modeling of MurI, MurG, MraY, DapE, DapA, Alr, and Ddl enzymes of the CDC1551 M. tuberculosis strain involved in the biosynthesis of peptidoglycan cell wall are reported. Generation of the 3D structures was achieved with Modeller 9.13. To assess the structural quality of the obtained homology modeled targets, the models were validated using PROCHECK, PDBsum, QMEAN, and ERRAT scores. Molecular dynamics simulations were performed to calculate root mean square deviation (RMSD) and radius of gyration (Rg) of MurI and MurG target proteins and their corresponding templates. For further model validation, RMSD and Rg for selected targets/templates were investigated to compare the close proximity of their dynamic behavior in terms of protein stability and average distances. To identify the potential binding mode required for molecular docking, binding site information of all modeled targets was obtained using two prediction algorithms. A docking study was performed for MurI to determine the potential mode of interaction between the inhibitor and the active site residues. This study presents the first accounts of the 3D structural information for the selected M. tuberculosis targets involved in peptidoglycan biosynthesis.

Molecular cloning and characterization of a short peptidoglycan recognition protein from silkworm Bombyx mori.

PubMed

Yang, P-J; Zhan, M-Y; Ye, C; Yu, X-Q; Rao, X-J

2017-12-01

Peptidoglycan is the major bacterial component recognized by the insect immune system. Peptidoglycan recognition proteins (PGRPs) are a family of pattern-recognition receptors that recognize peptidoglycans and modulate innate immune responses. Some PGRPs retain N-acetylmuramoyl-L-alanine amidase (Enzyme Commission number: 3.5.1.28) activity to hydrolyse bacterial peptidoglycans. Others have lost the enzymatic activity and work only as immune receptors. They are all important modulators for innate immunity. Here, we report the cloning and functional analysis of PGRP-S4, a short-form PGRP from the domesticated silkworm, Bombyx mori. The PGRP-S4 gene encodes a protein of 199 amino acids with a signal peptide and a PGRP domain. PGRP-S4 was expressed in the fat body, haemocytes and midgut. Its expression level was significantly induced by bacterial challenges in the midgut. The recombinant PGRP-S4 bound bacteria and different peptidoglycans. In addition, it inhibited bacterial growth and hydrolysed an Escherichia coli peptidoglycan in the presence of Zn 2+ . Scanning electron microscopy showed that PGRP-S4 disrupted the bacterial cell surface. PGRP-S4 further increased prophenoloxidase activation caused by peptidoglycans. Taken together, our data suggest that B. mori PGRP-S4 has multiple functions in immunity. © 2017 The Royal Entomological Society.

Peptidoglycan Cross-Linking in Glycopeptide-Resistant Actinomycetales

PubMed Central

Hugonnet, Jean-Emmanuel; Haddache, Nabila; Veckerlé, Carole; Dubost, Lionel; Marie, Arul; Shikura, Noriyasu; Mainardi, Jean-Luc; Rice, Louis B.

2014-01-01

Synthesis of peptidoglycan precursors ending in d-lactate (d-Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by l,d-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in d-Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of d-Lac into cytoplasmic precursors. This was due to a d,d-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of d-Lac for d-Ala and Gly. The contribution of l,d-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-d,d-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal d-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by d,d-transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of l,d-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-d,d-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that l,d-transpeptidases merely act as a tolerance mechanism in this bacterium. PMID:24395229

Peptidoglycan cross-linking in glycopeptide-resistant Actinomycetales.

PubMed

Hugonnet, Jean-Emmanuel; Haddache, Nabila; Veckerlé, Carole; Dubost, Lionel; Marie, Arul; Shikura, Noriyasu; Mainardi, Jean-Luc; Rice, Louis B; Arthur, Michel

2014-01-01

Synthesis of peptidoglycan precursors ending in D-lactate (D-Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by L,D-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in D-Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of D-Lac into cytoplasmic precursors. This was due to a D,D-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of D-Lac for D-Ala and Gly. The contribution of L,D-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-D,D-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal D-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by D,D-transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of L,D-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-D,D-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that L,D-transpeptidases merely act as a tolerance mechanism in this bacterium.

Structure-activity relationships of new cyanothiophene inhibitors of the essential peptidoglycan biosynthesis enzyme MurF.

PubMed

Hrast, Martina; Turk, Samo; Sosič, Izidor; Knez, Damijan; Randall, Christopher P; Barreteau, Hélène; Contreras-Martel, Carlos; Dessen, Andréa; O'Neill, Alex J; Mengin-Lecreulx, Dominique; Blanot, Didier; Gobec, Stanislav

2013-08-01

Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosynthesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular peptidoglycan biosynthesis step: the addition of D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc-L-Ala-γ-D-Glu-meso-DAP (or L-Lys). As MurF has no human counterpart, it represents an attractive target for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6. These findings, together with two new co-crystal structures, represent an excellent starting point for further optimization toward effective novel antibacterials. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

A density functional theory for association of fluid molecules with a functionalized surface: fluid-wall single and double bonding.

PubMed

Haghmoradi, Amin; Wang, Le; Chapman, Walter G

2017-02-01

In this manuscript we extend Wertheim's two-density formalism beyond its first order to model a system of fluid molecules with a single association site close to a planar hard wall with association sites on its surface in a density functional theory framework. The association sites of the fluid molecules are small enough that they can form only one bond, while the wall association sites are large enough to bond with more than one fluid molecule. The effects of temperature and of bulk fluid and wall site densities on the fluid density profile, extent of association, and competition between single and double bonding of fluid segments at the wall sites versus distance from the wall are presented. The theory predictions are compared with new Monte Carlo simulation results and they are in good agreement. The theory captures the surface coverage over wide ranges of temperature and bulk density by introducing the effect of steric hindrance in fluid association at a wall site.

Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage [psi]29 tail

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiang, Ye; Morais, Marc C.; Cohen, Daniel N.

2009-08-28

The small bacteriophage {phi}29 must penetrate the {approx}250-{angstrom} thick external peptidoglycan cell wall and cell membrane of the Gram-positive Bacillus subtilis, before ejecting its dsDNA genome through its tail into the bacterial cytoplasm. The tail of bacteriophage {phi}29 is noncontractile and {approx}380 {angstrom} long. A 1.8-{angstrom} resolution crystal structure of gene product 13 (gp13) shows that this tail protein has spatially well separated N- and C-terminal domains, whose structures resemble lysozyme-like enzymes and metallo-endopeptidases, respectively. CryoEM reconstructions of the WT bacteriophage and mutant bacteriophages missing some or most of gp13 shows that this enzyme is located at the distal endmore » of the {phi}29 tail knob. This finding suggests that gp13 functions as a tail-associated, peptidoglycan-degrading enzyme able to cleave both the polysaccharide backbone and peptide cross-links of the peptidoglycan cell wall. Comparisons of the gp13{sup -} mutants with the {phi}29 mature and emptied phage structures suggest the sequence of events that occur during the penetration of the tail through the peptidoglycan layer.« less

Functional characterization of a short peptidoglycan recognition protein, PGRP5 in grass carp Ctenopharyngodon idella.

PubMed

Li, Jun Hua; Chang, Ming Xian; Xue, Na Na; Nie, P

2013-08-01

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved from insects to mammals, recognize bacterial peptidoglycan (PGN) and function in antibacterial innate immunity. In this study, a short-form PGRP, designated as gcPGRP5 was identified from grass carp Ctenopharyngodon idella. The deduced amino acid sequence of gcPGRP5 is composed of 180 residues with a conserved PGRP domain at the C-terminus. The gcPGRP5 gene consists of four exons and three introns, spacing approximately 2.3 kb in genomic sequence. Phylogenetic analysis demonstrated that the gcPGRP5 is clustered with other PGRP-S identified in teleost fish. The gcPGRP5 is constitutively expressed in all organs/tissues examined, and its expression was significantly induced in CIK cells treated with lipoteichoic acid (LTA), polyinosinic polycytidylic acid (Poly I:C) and PGN. Fluorescence analysis showed that gcPGRP5 is distributed in cytoplasm of CIK cells, and cell lysates from CIK cells transfected with pTurbo-gcPGRP5-GFP and ptGFP1-gcPGRP5 plasmids display the binding activity and peptidoglycan-lytic amidase activity toward Lys-PGN from Staphylococcus aureus and Dap-PGN from Bacillus subtilis. Furthermore, heat-shock protein70 (Hsp70), and MyD88, an adaptor molecule in Toll-like receptor pathway, had an increased expression in CIK cells overexpressed with gcPGRP5. It is thus indicated that gcPGRP5 exhibits amidase activity, and also possesses roles in anti-stress, and in Toll-like receptor signaling pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

Entrapment of peptidoglycans and adamantyltripeptides into liposomes: an HPLC assay for determination of encapsulation efficiency.

PubMed

Frkanec, Ruza; Travas, Dijana; Krstanović, Marina; Spoljar, Beata Halassy; Ljevaković, Durdica; Vranesić, Branka; Frkanec, Leo; Tomasić, Jelka

2003-11-01

The encapsulation of different immunomodulating peptides, the peptidoglycan monomer, its semisynthetic derivatives (Adamant-1-yl)-acetyl-peptidoglycan monomer and Boc-Tyr-peptidoglycan monomer, respectively, and of two diastereoisomers of adamantyltripeptides into the large negatively charged multilamellar liposomes was investigated. The reproducible quantitative method using HPLC was established for the determination of the entrapped compounds. It was shown that the tested compounds could be efficiently incorporated into liposomes using either the film or modified film method. The results confirmed that the peptidoglycans with lipophilic substituents and particularly the adamantyltripeptides were incorporated into liposomes with higher efficiency than the peptidoglycan monomer using either of the described methods. Liposome preparations were stable at 4 degrees C up to seven days as shown by minimal leaking of the entrapped material.

The cell wall hydrolase Pmp23 is important for assembly and stability of the division ring in Streptococcus pneumoniae.

PubMed

Jacq, Maxime; Arthaud, Christopher; Manuse, Sylvie; Mercy, Chryslène; Bellard, Laure; Peters, Katharina; Gallet, Benoit; Galindo, Jennifer; Doan, Thierry; Vollmer, Waldemar; Brun, Yves V; VanNieuwenhze, Michael S; Di Guilmi, Anne Marie; Vernet, Thierry; Grangeasse, Christophe; Morlot, Cecile

2018-05-15

Bacterial division is intimately linked to synthesis and remodeling of the peptidoglycan, a cage-like polymer that surrounds the bacterial cell, providing shape and mechanical resistance. The bacterial division machinery, which is scaffolded by the cytoskeleton protein FtsZ, includes proteins with enzymatic, structural or regulatory functions. These proteins establish a complex network of transient functional and/or physical interactions which preserve cell shape and cell integrity. Cell wall hydrolases required for peptidoglycan remodeling are major contributors to this mechanism. Consistent with this, their deletion or depletion often results in morphological and/or division defects. However, the exact function of most of them remains elusive. In this work, we show that the putative lysozyme activity of the cell wall hydrolase Pmp23 is important for proper morphology and cell division in the opportunistic human pathogen Streptococcus pneumoniae. Our data indicate that active Pmp23 is required for proper localization of the Z-ring and the FtsZ-positioning protein MapZ. In addition, Pmp23 localizes to the division site and interacts directly with the essential peptidoglycan synthase PBP2x. Altogether, our data reveal a new regulatory function for peptidoglycan hydrolases.

Nisin, alone and combined with peptidoglycan-modulating antibiotics: activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci.

PubMed

Brumfitt, W; Salton, M R J; Hamilton-Miller, J M T

2002-11-01

We have sought ways to circumvent resistance, by combining nisin with other antibiotics known to target bacterial cell wall biosynthesis. Twenty strains each of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) were tested in vitro by standardized methods against nisin alone and combined with bacitracin, ramoplanin and chloramphenicol. Ramoplanin was the most potent compound, and bacitracin had the least activity. Two-way synergy was observed with nisin and ramoplanin. However, chloramphenicol was clearly antagonistic to the activity of nisin. Observations of synergy between nisin and ramoplanin against MRSA and VRE offer a promising approach to the concept of combining nisin with inhibitors of cell wall peptidoglycan. Further investigations are needed in order to develop this approach as a clinical possibility.

Subpolar addition of new cell wall is directed by DivIVA in mycobacteria

PubMed Central

Meniche, Xavier; Otten, Renee; Siegrist, M. Sloan; Baer, Christina E.; Murphy, Kenan C.; Bertozzi, Carolyn R.; Sassetti, Christopher M.

2014-01-01

Mycobacteria are surrounded by a complex multilayered envelope and elongate at the poles. The principles that organize the coordinated addition of chemically diverse cell wall layers during polar extension remain unclear. We show that enzymes mediating the terminal cytosolic steps of peptidoglycan, arabinogalactan, and mycolic acid synthesis colocalize at sites of cell growth or division. The tropomyosin-like protein, DivIVA, is targeted to the negative curvature of the pole, is enriched at the growing end, and determines cell shape from this site. In contrast, cell wall synthetic complexes are concentrated at a distinct subpolar location. When viewed at subdiffraction resolution, new peptidoglycan is deposited at this subpolar site, and inert cell wall covers the DivIVA-marked tip. The differentiation between polar tip and cell wall synthetic complexes is also apparent at the biochemical level. Enzymes that generate mycolate precursors interact with DivIVA, but the final condensation of mycolic acids occurs in a distinct protein complex at the site of nascent cell wall addition. We propose an ultrastructural model of mycobacterial polar growth where new cell wall is added in an annular zone below the cell tip. This model may be broadly applicable to other bacterial and fungal organisms that grow via polar extension. PMID:25049412

Molecular modeling of Gram-positive bacteria peptidoglycan layer, selected glycopeptide antibiotics and vancomycin derivatives modified with sugar moieties.

PubMed

Ślusarz, Rafał; Szulc, Monika; Madaj, Janusz

2014-05-07

Proper understanding of the mechanisms of binding to Gram-positive bacteria cell wall layers-especially to the peptidoglycan (PG) layer, seems to be crucial for proper development of new drug candidates which are effective against these bacteria. In this work we have constructed two different models of the Gram-positive bacteria PG layer: the layered and the scaffold models. PG conformational changes during geometry optimization, models relaxation, and molecular dynamics were described and discussed. We have found that the border surface of both PG layer models differs from the surface located away from the edge of models and the chains formed by disaccharide units prefer helix-like conformation. This curling of PG chains significantly affects the shape of antibiotic-accessible surface and the process is thus crucial for new drug development. Glycopeptide antibiotics effective against Gram-positive bacteria, such as vancomycin and its semisynthetic derivatives-oritavancin and telavancin, bind to d-alanyl-d-alanine stem termini on the peptidoglycan precursors of the cell wall. This binding inhibits cross-linking between the peptides and subsequently prevents cell wall synthesis. In this study some of the aspects of conformational freedom of vancomycin and restrictions from the modifications of vancomycin structure introduced into oritavancin and telavancin and five other vancomycin derivatives (with addition of 2-acetamido-2-deoxy-β-d-galactopyranosylamine, 2-acetamido-2-deoxy-β-d-glucopyranosylamine, 1-amine-1-deoxy-d-glucitol, 2-amino-2-deoxy-d-galactitol, or 2-amino-2-deoxy-d-glucitol to the C-terminal amino acid group in the vancomycin) are presented and discussed. The resulting molecular dynamics trajectories, root mean square deviation changes of aglycon and saccharide moieties as well as a comparative study of possible interactions with cyclic and chain forms of modified groups have been carried out, measured, and analyzed. Energetically advantageous

The Surface Layer Homology Domain-Containing Proteins of Alkaliphilic Bacillus pseudofirmus OF4 Play an Important Role in Alkaline Adaptation via Peptidoglycan Synthesis.

PubMed

Fujinami, Shun; Ito, Masahiro

2018-01-01

It is well known that the Na + cycle and the cell wall are essential for alkaline adaptation of Na + -dependent alkaliphilic Bacillus species. In Bacillus pseudofirmus OF4, surface layer protein A (SlpA), the most abundant protein in the surface layer (S-layer) of the cell wall, is involved in alkaline adaptation, especially under low Na + concentrations. The presence of a large number of genes that encode S-layer homology (SLH) domain-containing proteins has been suggested from the genome sequence of B. pseudofirmus OF4. However, other than SlpA, the functions of SLH domain-containing proteins are not well known. Therefore, a deletion mutant of the csaB gene, required for the retention of SLH domain-containing proteins on the cell wall, was constructed to investigate its physiological properties. The csaB mutant strain of B. pseudofirmus OF4 had a chained morphology and alkaline sensitivity even under a 230 mM Na + concentration at which there is no growth difference between the parental strain and the slpA mutant strain. Ultra-thin section transmission electron microscopy showed that a csaB mutant strain lacked an S-layer part, and its peptidoglycan (PG) layer was disturbed. The slpA mutant strain also lacked an S-layer part, although its PG layer was not disturbed. These results suggested that the surface layer homology domain-containing proteins of B. pseudofirmus OF4 play an important role in alkaline adaptation via peptidoglycan synthesis.

Principal coordinate analysis assisted chromatographic analysis of bacterial cell wall collection: A robust classification approach.

PubMed

Kumar, Keshav; Cava, Felipe

2018-04-10

In the present work, Principal coordinate analysis (PCoA) is introduced to develop a robust model to classify the chromatographic data sets of peptidoglycan sample. PcoA captures the heterogeneity present in the data sets by using the dissimilarity matrix as input. Thus, in principle, it can even capture the subtle differences in the bacterial peptidoglycan composition and can provide a more robust and fast approach for classifying the bacterial collection and identifying the novel cell wall targets for further biological and clinical studies. The utility of the proposed approach is successfully demonstrated by analysing the two different kind of bacterial collections. The first set comprised of peptidoglycan sample belonging to different subclasses of Alphaproteobacteria. Whereas, the second set that is relatively more intricate for the chemometric analysis consist of different wild type Vibrio Cholerae and its mutants having subtle differences in their peptidoglycan composition. The present work clearly proposes a useful approach that can classify the chromatographic data sets of chromatographic peptidoglycan samples having subtle differences. Furthermore, present work clearly suggest that PCoA can be a method of choice in any data analysis workflow. Copyright © 2018 Elsevier Inc. All rights reserved.

The ng_ζ1 toxin of the gonococcal epsilon/zeta toxin/antitoxin system drains precursors for cell wall synthesis.

PubMed

Rocker, Andrea; Peschke, Madeleine; Kittilä, Tiia; Sakson, Roman; Brieke, Clara; Meinhart, Anton

2018-04-27

Bacterial toxin-antitoxin complexes are emerging as key players modulating bacterial physiology as activation of toxins induces stasis or programmed cell death by interference with vital cellular processes. Zeta toxins, which are prevalent in many bacterial genomes, were shown to interfere with cell wall formation by perturbing peptidoglycan synthesis in Gram-positive bacteria. Here, we characterize the epsilon/zeta toxin-antitoxin (TA) homologue from the Gram-negative pathogen Neisseria gonorrhoeae termed ng_ɛ1 / ng_ζ1. Contrary to previously studied streptococcal epsilon/zeta TA systems, ng_ɛ1 has an epsilon-unrelated fold and ng_ζ1 displays broader substrate specificity and phosphorylates multiple UDP-activated sugars that are precursors of peptidoglycan and lipopolysaccharide synthesis. Moreover, the phosphorylation site is different from the streptococcal zeta toxins, resulting in a different interference with cell wall synthesis. This difference most likely reflects adaptation to the individual cell wall composition of Gram-negative and Gram-positive organisms but also the distinct involvement of cell wall components in virulence.

LytN, a Murein Hydrolase in the Cross-wall Compartment of Staphylococcus aureus, Is Involved in Proper Bacterial Growth and Envelope Assembly*

PubMed Central

Frankel, Matthew B.; Hendrickx, Antoni P. A.; Missiakas, Dominique M.; Schneewind, Olaf

2011-01-01

Cell cycle progression for the spherical microbe Staphylococcus aureus requires the coordinated synthesis and remodeling of peptidoglycan. The majority of these rearrangements takes place at the mid-cell, in a compartment designated the cross-wall. Secreted polypeptides endowed with a YSIRK-G/S signal peptide are directly delivered to the cross-wall compartment. One such YSIRK-containing protein is the murein hydrolase LytN. lytN mutations precipitate structural damage to the cross-wall and interfere with staphylococcal growth. Overexpression of lytN also affects growth and triggers rupture of the cross-wall. The lytN phenotype can be reversed by the controlled expression of lytN but not by adding purified LytN to staphylococcal cultures. LytN harbors LysM and CHAP domains, the latter of which functions as both an N-acetylmuramoyl-l-alanine amidase and d-alanyl-glycine endopeptidase. Thus, LytN secretion into the cross-wall promotes peptidoglycan separation and completion of the staphylococcal cell cycle. PMID:21784864

Structure-Function Analysis of Staphylococcus aureus Amidase Reveals the Determinants of Peptidoglycan Recognition and Cleavage*

PubMed Central

Büttner, Felix Michael; Zoll, Sebastian; Nega, Mulugeta; Götz, Friedrich; Stehle, Thilo

2014-01-01

The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen. PMID:24599952

Structure-function analysis of Staphylococcus aureus amidase reveals the determinants of peptidoglycan recognition and cleavage.

PubMed

Büttner, Felix Michael; Zoll, Sebastian; Nega, Mulugeta; Götz, Friedrich; Stehle, Thilo

2014-04-18

The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen.

In Escherichia coli, MreB and FtsZ direct the synthesis of lateral cell wall via independent pathways that require PBP 2.

PubMed

Varma, Archana; Young, Kevin D

2009-06-01

In Escherichia coli, the cytoplasmic proteins MreB and FtsZ play crucial roles in ensuring that new muropeptide subunits are inserted into the cell wall in a spatially correct way during elongation and division. In particular, to retain a constant diameter and overall shape, new material must be inserted into the wall uniformly around the cell's perimeter. Current thinking is that MreB accomplishes this feat through intermediary proteins that tether peptidoglycan synthases to the outer face of the inner membrane. We tested this idea in E. coli by using a DD-carboxypeptidase mutant that accumulates pentapeptides in its peptidoglycan, allowing us to visualize new muropeptide incorporation. Surprisingly, inhibiting MreB with the antibiotic A22 did not result in uneven insertion of new wall, although the cells bulged and lost their rod shapes. Instead, uneven (clustered) incorporation occurred only if MreB and FtsZ were inactivated simultaneously, providing the first evidence in E. coli that FtsZ can direct murein incorporation into the lateral cell wall independently of MreB. Inhibiting penicillin binding protein 2 (PBP 2) alone produced the same clustered phenotype, implying that MreB and FtsZ tether peptidoglycan synthases via a common mechanism that includes PBP 2. However, cell shape was determined only by the presence or absence of MreB and not by the even distribution of new wall material as directed by FtsZ.

On the mechanism of peptidoglycan binding and cleavage by the endo-specific lytic transglycosylase MltE from Escherichia coli.

PubMed

Fibriansah, Guntur; Gliubich, Francesca I; Thunnissen, Andy-Mark W H

2012-11-13

The lytic transglycosylase MltE from Escherichia coli is a periplasmic, outer membrane-attached enzyme that cleaves the β-1,4-glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine residues in the cell wall peptidoglycan, producing 1,6-anhydromuropeptides. Here we report three crystal structures of MltE: in a substrate-free state, in a binary complex with chitopentaose, and in a ternary complex with the glycopeptide inhibitor bulgecin A and the murodipeptide N-acetylglucosaminyl-N-acetylmuramyl-l-Ala-d-Glu. The substrate-bound structures allowed a detailed analysis of the saccharide-binding interactions in six subsites of the peptidoglycan-binding groove (subsites -4 to +2) and, combined with site-directed mutagenesis analysis, confirmed the role of Glu64 as catalytic acid/base. The structures permitted the precise modeling of a short glycan strand of eight saccharide residues, providing evidence for two additional subsites (+3 and +4) and revealing the productive conformational state of the substrate at subsites -1 and +1, where the glycosidic bond is cleaved. Full accessibility of the peptidoglycan-binding groove and preferential binding of an N-acetylmuramic acid residue in a (4)C(1) chair conformation at subsite +2 explain why MltE shows only endo- and no exo-specific activity toward glycan strands. The results further indicate that catalysis of glycosidic bond cleavage by MltE proceeds via distortion toward a sofa-like conformation of the N-acetylmuramic acid sugar ring at subsite -1 and by anchimeric assistance of the sugar's N-acetyl group, as shown previously for the lytic transglycosylases Slt70 and MltB.

Activity Augmentation of Amphioxus Peptidoglycan Recognition Protein BbtPGRP3 via Fusion with a Chitin Binding Domain

PubMed Central

Wang, Wen-Jie; Cheng, Wang; Luo, Ming; Yan, Qingyu; Yu, Hong-Mei; Li, Qiong; Cao, Dong-Dong; Huang, Shengfeng; Xu, Anlong; Mariuzza, Roy A.; Chen, Yuxing; Zhou, Cong-Zhao

2015-01-01

Peptidoglycan recognition proteins (PGRPs), which have been identified in most animals, are pattern recognition molecules that involve antimicrobial defense. Resulting from extraordinary expansion of innate immune genes, the amphioxus encodes many PGRPs of diverse functions. For instance, three isoforms of PGRP encoded by Branchiostoma belcheri tsingtauense, termed BbtPGRP1~3, are fused with a chitin binding domain (CBD) at the N-terminus. Here we report the 2.7 Å crystal structure of BbtPGRP3, revealing an overall structure of an N-terminal hevein-like CBD followed by a catalytic PGRP domain. Activity assays combined with site-directed mutagenesis indicated that the individual PGRP domain exhibits amidase activity towards both DAP-type and Lys-type peptidoglycans (PGNs), the former of which is favored. The N-terminal CBD not only has the chitin-binding activity, but also enables BbtPGRP3 to gain a five-fold increase of amidase activity towards the Lys-type PGNs, leading to a significantly broadened substrate spectrum. Together, we propose that modular evolution via domain shuffling combined with gene horizontal transfer makes BbtPGRP1~3 novel PGRPs of augmented catalytic activity and broad recognition spectrum. PMID:26479246

The MurC ligase essential for peptidoglycan biosynthesis is regulated by the serine/threonine protein kinase PknA in Corynebacterium glutamicum.

PubMed

Fiuza, Maria; Canova, Marc J; Patin, Delphine; Letek, Michal; Zanella-Cléon, Isabelle; Becchi, Michel; Mateos, Luís M; Mengin-Lecreulx, Dominique; Molle, Virginie; Gil, José A

2008-12-26

The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (L-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis.

Bacillus anthracis Cell Wall Peptidoglycan but Not Lethal or Edema Toxins Produces Changes Consistent With Disseminated Intravascular Coagulation in a Rat Model

PubMed Central

Qiu, Ping; Li, Yan; Shiloach, Joseph; Cui, Xizhong; Sun, Junfeng; Trinh, Loc; Kubler-Kielb, Joanna; Vinogradov, Evgeny; Mani, Haresh; Al-Hamad, Mariam; Fitz, Yvonne; Eichacker, Peter Q.

2013-01-01

Background. Disseminated intravascular coagulation (DIC) appears to be important in the pathogenesis of Bacillus anthracis infection, but its causes are unclear. Although lethal toxin (LT) and edema toxin (ET) could contribute, B. anthracis cell wall peptidoglycan (PGN), not the toxins, stimulates inflammatory responses associated with DIC. Methods and Results. To better understand the pathogenesis of DIC during anthrax, we compared the effects of 24-hour infusions of PGN, LT, ET, or diluent (control) on coagulation measures 6, 24, or 48 hours after infusion initiation in 135 rats. No control recipient died. Lethality rates (approximately 30%) did not differ among PGN, LT, and ET recipients (P = .78). Thirty-three of 35 deaths (94%) occurred between 6 and 24 hours after the start of challenge. Among challenge components, PGN most consistently altered coagulation measures. Compared with control at 6 hours, PGN decreased platelet and fibrinogen levels and increased prothrombin and activated partial thromboplastin times and tissue factor, tissue factor pathway inhibitor, protein C, plasminogen activator inhibitor (PAI), and thrombin-antithrombin complex levels, whereas LT and ET only decreased the fibrinogen level or increased the PAI level (P ≤ .05). Nearly all effects associated with PGN infusion significantly differed from changes associated with toxin infusion (P ≤ .05 for all comparisons except for PAI level). Conclusion. DIC during B. anthracis infection may be related more to components such as PGN than to LT or ET. PMID:23737601

Novel Materials Containing Single-Wall Carbon Nanotubes Wrapped in Polymer Molecules

NASA Technical Reports Server (NTRS)

Smalley, Richard E.; O'Connell, Michael J.; Smith, Kenneth; Colbert, Daniel T.

2009-01-01

In this design, single-wall carbon nanotubes (SWNTs) have been coated in polymer molecules to create a new type of material that has low electrical conductivity, but still contains individual nanotubes, and small ropes of individual nanotubes, which are themselves good electrical conductors and serve as small conducting rods immersed in an electrically insulating matrix. The polymer is attached through weak chemical forces that are primarily non-covalent in nature, caused primarily through polarization rather than the sharing of valence electrons. Therefore, the electronic structure of the SWNT involved is substantially the same as that of free, individual (and small ropes of) SWNT. Their high conductivity makes the individual nanotubes extremely electrically polarizable, and materials containing these individual, highly polarizable molecules exhibit novel electrical properties including a high dielectric constant.

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

PubMed Central

Romaniuk, Joseph A. H.; Cegelski, Lynette

2015-01-01

The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms. PMID:26370936

Role of N-acetylglucosaminidase and N-acetylmuramidase activities in Enterococcus faecalis peptidoglycan metabolism.

PubMed

Mesnage, Stéphane; Chau, Françoise; Dubost, Lionel; Arthur, Michel

2008-07-11

Identification of the full complement of peptidoglycan hydrolases detected by zymogram in Enterococcus faecalis extracts led to the characterization of two novel hydrolases that we named AtlB and AtlC. Both enzymes have a similar modular organization comprising a central catalytic domain fused to two LysM peptidoglycan-binding modules. AtlB and AtlC displayed N-acetylmuramidase activity, as demonstrated by tandem mass spectrometry analyses of peptidoglycan fragments generated by the purified enzymes. The genes encoding AtlB and AtlC were deleted either alone or in combination with the gene encoding AtlA, a previously described N-acetylglucosaminidase. No autolytic activity was detected in the triple mutant indicating that AtlA, AtlB, and AtlC account for the major hydrolytic activities in E. faecalis. Analysis of cell size distribution by flow cytometry showed that deletion of atlA resulted in the formation of long chains. Thus, AtlA digests the septum and is required for cell separation after cell division. We found that AtlB could act as a surrogate for AtlA, although the enzyme was less efficient at septum digestion. Deletion of atlC had no impact on cell morphology. Labeling of the peptidoglycan with N-[14C]acetylglucosamine revealed an unusually slow turnover as compared with model organisms, almost completely dependent upon the combined activities of AtlA and AtlB. In contrast to atlA, the atlB and atlC genes are located in putative prophages. Because AtlB and AtlC were produced in the absence of cell lysis or production of phage progeny, these enzymes may have been hijacked by E. faecalis to contribute to peptidoglycan metabolism.

The MurC Ligase Essential for Peptidoglycan Biosynthesis Is Regulated by the Serine/Threonine Protein Kinase PknA in Corynebacterium glutamicum*

PubMed Central

Fiuza, Maria; Canova, Marc J.; Patin, Delphine; Letek, Michal; Zanella-Cléon, Isabelle; Becchi, Michel; Mateos, Luís M.; Mengin-Lecreulx, Dominique; Molle, Virginie; Gil, José A.

2008-01-01

The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (l-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis. PMID:18974047

Wall Teichoic Acids Are Involved in the Medium-Induced Loss of Function of the Autolysin CD11 against Clostridium difficile

PubMed Central

Wu, Xia; Paskaleva, Elena E.; Mehta, Krunal K.; Dordick, Jonathan S.; Kane, Ravi S.

2016-01-01

Bacterial lysins are potent antibacterial enzymes with potential applications in the treatment of bacterial infections. Some lysins lose activity in the growth media of target bacteria, and the underlying mechanism remains unclear. Here we use CD11, an autolysin of Clostridium difficile, as a model lysin to demonstrate that the inability of this enzyme to kill C. difficile in growth medium is not associated with inhibition of the enzyme activity by medium, or the modification of the cell wall peptidoglycan. Rather, wall teichoic acids (WTAs) appear to prevent the enzyme from binding to the cells and cleaving the cell wall peptidoglycan. By partially blocking the biosynthetic pathway of WTAs with tunicamycin, cell binding improved and the lytic efficacy of CD11 was significantly enhanced. This is the first report of the mechanism of lysin inactivation in growth medium, and provides insights into understanding the behavior of lysins in complex environments, including the gastrointestinal tract. PMID:27759081

The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

PubMed

Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

2017-07-15

Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

AtlA functions as a peptidoglycan lytic transglycosylase in the Neisseria gonorrhoeae type IV secretion system.

PubMed

Kohler, Petra L; Hamilton, Holly L; Cloud-Hansen, Karen; Dillard, Joseph P

2007-08-01

Type IV secretion systems require peptidoglycan lytic transglycosylases for efficient secretion, but the function of these enzymes is not clear. The type IV secretion system gene cluster of Neisseria gonorrhoeae encodes two peptidoglycan transglycosylase homologues. One, LtgX, is similar to peptidoglycan transglycosylases from other type IV secretion systems. The other, AtlA, is similar to endolysins from bacteriophages and is not similar to any described type IV secretion component. We characterized the enzymatic function of AtlA in order to examine its role in the type IV secretion system. Purified AtlA was found to degrade macromolecular peptidoglycan and to produce 1,6-anhydro peptidoglycan monomers, characteristic of lytic transglycosylase activity. We found that AtlA can functionally replace the lambda endolysin to lyse Escherichia coli. In contrast, a sensitive measure of lysis demonstrated that AtlA does not lyse gonococci expressing it or gonococci cocultured with an AtlA-expressing strain. The gonococcal type IV secretion system secretes DNA during growth. A deletion of ltgX or a substitution in the putative active site of AtlA severely decreased DNA secretion. These results indicate that AtlA and LtgX are actively involved in type IV secretion and that AtlA is not involved in lysis of gonococci to release DNA. This is the first demonstration that a type IV secretion peptidoglycanase has lytic transglycosylase activity. These data show that AtlA plays a role in type IV secretion of DNA that requires peptidoglycan breakdown without cell lysis.

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

PubMed Central

Navarre, William Wiley; Schneewind, Olaf

1999-01-01

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins. PMID:10066836

Chemogenomics profiling of drug targets of peptidoglycan biosynthesis pathway in Leptospira interrogans by virtual screening approaches.

PubMed

Bhattacharjee, Biplab; Simon, Rose Mary; Gangadharaiah, Chaithra; Karunakar, Prashantha

2013-06-28

Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. The availability of ligand libraries has facilitated the search for novel drug targets using chemogenomics approaches, compared with the traditional method of drug discovery, which is time consuming and yields few leads with little intracellular information for guiding target selection. Recent subtractive genomics studies have revealed the putative drug targets in peptidoglycan biosynthesis pathways in Leptospira interrogans. Aligand library for the murD ligase enzyme in the peptidoglycan pathway has also been identified. Our approach in this research involves screening of the pre-existing ligand library of murD with related protein family members in the putative drug target assembly in the peptidoglycan biosynthesis pathway. A chemogenomics approach has been implemented here, which involves screening of known ligands of a protein family having analogous domain architecture for identification of leads for existing druggable protein family members. By means of this approach, one murC and one murF inhibitor were identified, providing a platform for developing an antileptospirosis drug targeting the peptidoglycan biosynthesis pathway. Given that the peptidoglycan biosynthesis pathway is exclusive to bacteria, the in silico identified mur ligase inhibitors are expected to be broad-spectrum Gram-negative inhibitors if synthesized and tested in in vitro and in vivo assays.

Accumulation of Peptidoglycan O-Acetylation Leads to Altered Cell Wall Biochemistry and Negatively Impacts Pathogenesis Factors of Campylobacter jejuni.

PubMed

Ha, Reuben; Frirdich, Emilisa; Sychantha, David; Biboy, Jacob; Taveirne, Michael E; Johnson, Jeremiah G; DiRita, Victor J; Vollmer, Waldemar; Clarke, Anthony J; Gaynor, Erin C

2016-10-21

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its mechanisms of pathogenesis are poorly understood. Peptidoglycan (PG) is important for helical shape, colonization, and host-pathogen interactions in C. jejuni Therefore, changes in PG greatly impact the physiology of this organism. O-acetylation of peptidoglycan (OAP) is a bacterial phenomenon proposed to be important for proper cell growth, characterized by acetylation of the C6 hydroxyl group of N-acetylmuramic acid in the PG glycan backbone. The OAP gene cluster consists of a PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a PG O-acetyltransferase B (patB) for O-acetylation, and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. In this study, reduced OAP in ΔpatA and ΔpatB had minimal impact on C. jejuni growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 resulted in marked differences in PG biochemistry, including O-acetylation, anhydromuropeptide levels, and changes not expected to result directly from Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG biosynthesis. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibited defects in pathogenesis-associated phenotypes, including cell shape, motility, biofilm formation, cell surface hydrophobicity, and sodium deoxycholate sensitivity. Δape1 was also impaired for chick colonization and adhesion, invasion, intracellular survival, and induction of IL-8 production in INT407 cells in vitro The importance of Ape1 in C. jejuni biology makes it a good candidate as an antimicrobial target. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Adjuvant activity of peptidoglycan monomer and its metabolic products.

PubMed

Halassy, Beata; Krstanović, Marina; Frkanec, Ruza; Tomasić, Jelka

2003-02-14

Peptidoglycan monomer (PGM) is a natural compound of bacterial origin. It is a non-toxic, non-pyrogenic, water-soluble immunostimulator potentiating humoral immune response to ovalbumin (OVA) in mice. It is fast degraded and its metabolic products-the pentapeptide (PP) and the disaccharide (DS)-are excreted from the mammalian organism upon parenteral administration. The present study investigates: (a). whether PGM could influence the long-living memory generation; (b). whether metabolic products retain adjuvant properties of the parent compound and contribute to its adjuvanticity. We report now that mice immunised twice with OVA+PGM had significantly higher anti-OVA IgG levels upon challenge with antigen alone 6 months later in comparison to control group immunised with OVA only. PP and DS were prepared enzymatically in vitro as apyrogenic and chemically pure compounds. When mice were immunised with OVA plus PP and DS, respectively, the level of anti-OVA IgGs in sera was not higher than in mice immunised with OVA alone, while PGM raised the level of specific antibodies. Results implicate that the adjuvant active molecule, capable of enhancing long-living memory generation, is PGM itself, and none of its metabolic products.

Bacillus megaterium sporal peptidoglycan synthesis studied by high-resolution autoradiography.

PubMed

Frehel, C; Ryter, A

1980-11-01

Cells of a Dap- Lys- mutant strain of Bacillus megaterium were pulse labeled with [3H]diaminopimelic acid at different times of growth and sporulation. They were processed for radioactivity measurements and high-resolution autoradiography either just after the pulse or after a chase in a nonradioactive medium until refractile forespores started to appear at time (t)4,5. In the pulse-labeled cells, autoradiographs and radioactivity measurements showed that the radioactivity incorporated during a pulse decreased abruptly after t0 and stayed at a low level until t5, although the forespore wall and cortex were formed between t4 and t5. In the pulse-chased bacteria, the acid-insoluble radioactivity, as well as the number of silver grains on autoradiographs, increased during the chase in cells labeled at t1 to t2, whereas it decreased in those labeled before t0. Furthermore, analysis of silver grain distribution showed that, in stage IV bacteria, grains were distributed at the outside of the forespore, mostly on the sporangium cell wall, when pulse-labeling occurred before or at t0; they were located along the cortex and in the forespore cytoplasm when labeling was made at t1 or t2. These facts show that [3H]diaminopimelic acid necessary for spore envelope synthesis was incorporated before their morphological appearance. Free or small diaminopimelic acid precursors entered the sporangium between t1 and t2. The appearance of silver grains in the forespore cytoplasm suggests that the forespore is implicated in sporal peptidoglycan synthesis.

The Peptidoglycan-Binding Protein SjcF1 Influences Septal Junction Function and Channel Formation in the Filamentous Cyanobacterium Anabaena.

PubMed

Rudolf, Mareike; Tetik, Nalan; Ramos-León, Félix; Flinner, Nadine; Ngo, Giang; Stevanovic, Mara; Burnat, Mireia; Pernil, Rafael; Flores, Enrique; Schleiff, Enrico

2015-06-30

Filamentous, heterocyst-forming cyanobacteria exchange nutrients and regulators between cells for diazotrophic growth. Two alternative modes of exchange have been discussed involving transport either through the periplasm or through septal junctions linking adjacent cells. Septal junctions and channels in the septal peptidoglycan are likely filled with septal junction complexes. While possible proteinaceous factors involved in septal junction formation, SepJ (FraG), FraC, and FraD, have been identified, little is known about peptidoglycan channel formation and septal junction complex anchoring to the peptidoglycan. We describe a factor, SjcF1, involved in regulation of septal junction channel formation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. SjcF1 interacts with the peptidoglycan layer through two peptidoglycan-binding domains and is localized throughout the cell periphery but at higher levels in the intercellular septa. A strain with an insertion in sjcF1 was not affected in peptidoglycan synthesis but showed an altered morphology of the septal peptidoglycan channels, which were significantly wider in the mutant than in the wild type. The mutant was impaired in intercellular exchange of a fluorescent probe to a similar extent as a sepJ deletion mutant. SjcF1 additionally bears an SH3 domain for protein-protein interactions. SH3 binding domains were identified in SepJ and FraC, and evidence for interaction of SjcF1 with both SepJ and FraC was obtained. SjcF1 represents a novel protein involved in structuring the peptidoglycan layer, which links peptidoglycan channel formation to septal junction complex function in multicellular cyanobacteria. Nonetheless, based on its subcellular distribution, this might not be the only function of SjcF1. Cell-cell communication is central not only for eukaryotic but also for multicellular prokaryotic systems. Principles of intercellular communication are well established for eukaryotes, but the

The Disruptive Effect of Lysozyme on the Bacterial Cell Wall Explored by an "In-Silico" Structural Outlook

ERIC Educational Resources Information Center

Primo, Emiliano D.; Otero, Lisandro H.; Ruiz, Francisco; Klinke, Sebastián; Giordano, Walter

2018-01-01

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…

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa - their role in the development of resistance.

PubMed

Dhar, Supurna; Kumari, Hansi; Balasubramanian, Deepak; Mathee, Kalai

2018-01-01

The bacterial cell-wall that forms a protective layer over the inner membrane is called the murein sacculus - a tightly cross-linked peptidoglycan mesh unique to bacteria. Cell-wall synthesis and recycling are critical cellular processes essential for cell growth, elongation and division. Both de novo synthesis and recycling involve an array of enzymes across all cellular compartments, namely the outer membrane, periplasm, inner membrane and cytoplasm. Due to the exclusivity of peptidoglycan in the bacterial cell-wall, these players are the target of choice for many antibacterial agents. Our current understanding of cell-wall biochemistry and biogenesis in Gram-negative organisms stems mostly from studies of Escherichia coli. An incomplete knowledge on these processes exists for the opportunistic Gram-negative pathogen, Pseudomonas aeruginosa. In this review, cell-wall synthesis and recycling in the various cellular compartments are compared and contrasted between E. coli and P. aeruginosa. Despite the fact that there is a remarkable similarity of these processes between the two bacterial species, crucial differences alter their resistance to β-lactams, fluoroquinolones and aminoglycosides. One of the common mediators underlying resistance is the amp system whose mechanism of action is closely associated with the cell-wall recycling pathway. The activation of amp genes results in expression of AmpC β-lactamase through its cognate regulator AmpR which further regulates multi-drug resistance. In addition, other cell-wall recycling enzymes also contribute to antibiotic resistance. This comprehensive summary of the information should spawn new ideas on how to effectively target cell-wall processes to combat the growing resistance to existing antibiotics.

PG-Metrics: A chemometric-based approach for classifying bacterial peptidoglycan data sets and uncovering their subjacent chemical variability

PubMed Central

Kumar, Keshav; Espaillat, Akbar; Cava, Felipe

2017-01-01

Bacteria cells are protected from osmotic and environmental stresses by an exoskeleton-like polymeric structure called peptidoglycan (PG) or murein sacculus. This structure is fundamental for bacteria’s viability and thus, the mechanisms underlying cell wall assembly and how it is modulated serve as targets for many of our most successful antibiotics. Therefore, it is now more important than ever to understand the genetics and structural chemistry of the bacterial cell walls in order to find new and effective methods of blocking it for the treatment of disease. In the last decades, liquid chromatography and mass spectrometry have been demonstrated to provide the required resolution and sensitivity to characterize the fine chemical structure of PG. However, the large volume of data sets that can be produced by these instruments today are difficult to handle without a proper data analysis workflow. Here, we present PG-metrics, a chemometric based pipeline that allows fast and easy classification of bacteria according to their muropeptide chromatographic profiles and identification of the subjacent PG chemical variability between e.g. bacterial species, growth conditions and, mutant libraries. The pipeline is successfully validated here using PG samples from different bacterial species and mutants in cell wall proteins. The obtained results clearly demonstrated that PG-metrics pipeline is a valuable bioanalytical tool that can lead us to cell wall classification and biomarker discovery. PMID:29040278

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE PAGES

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; ...

2015-09-15

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes

Specificity determinants for lysine incorporation in Staphylococcus aureus peptidoglycan as revealed by the structure of a MurE enzyme ternary complex.

PubMed

Ruane, Karen M; Lloyd, Adrian J; Fülöp, Vilmos; Dowson, Christopher G; Barreteau, Hélène; Boniface, Audrey; Dementin, Sébastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Gobec, Stanislav; Dessen, Andréa; Roper, David I

2013-11-15

Formation of the peptidoglycan stem pentapeptide requires the insertion of both L and D amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between L-lysine and D,L-diaminopimelic acid, the predominant amino acid that replaces L-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of L-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for L-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic L-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.

Specificity Determinants for Lysine Incorporation in Staphylococcus aureus Peptidoglycan as Revealed by the Structure of a MurE Enzyme Ternary Complex*

PubMed Central

Ruane, Karen M.; Lloyd, Adrian J.; Fülöp, Vilmos; Dowson, Christopher G.; Barreteau, Hélène; Boniface, Audrey; Dementin, Sébastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Gobec, Stanislav; Dessen, Andréa; Roper, David I.

2013-01-01

Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly. PMID:24064214

Calculation of ground state rotational populations for kinetic gas homonuclear diatomic molecules including electron-impact excitation and wall collisions.

PubMed

Farley, David R

2010-09-07

A model has been developed to calculate the ground state rotational populations of homonuclear diatomic molecules in kinetic gases, including the effects of electron-impact excitation, wall collisions, and gas feed rate. The equations are exact within the accuracy of the cross sections used and of the assumed equilibrating effect of wall collisions. It is found that the inflow of feed gas and equilibrating wall collisions can significantly affect the rotational distribution in competition with nonequilibrating electron-impact effects. The resulting steady-state rotational distributions are generally Boltzmann for N≥3, with a rotational temperature between the wall and feed gas temperatures. The N=0,1,2 rotational level populations depend sensitively on the relative rates of electron-impact excitation versus wall collision and gas feed rates.

Compound-specific nitrogen isotope analysis of D-alanine, L-alanine, and valine: application of diastereomer separation to delta15N and microbial peptidoglycan studies.

PubMed

Takano, Yoshinori; Chikaraishi, Yoshito; Ogawa, Nanako O; Kitazato, Hiroshi; Ohkouchi, Naohiko

2009-01-01

We have developed an analytical method to determine the compound-specific nitrogen isotope compositions of individual amino acid enantiomers using gas chromatography/combustion/isotope ratio mass spectrometry. A novel derivatization of amino acid diastereomers by optically active (R)-(-)-2-butanol or (S)-(+)-2-butanol offers two advantages for nitrogen isotope analysis. First, chromatographic chiral separation can be achieved without the use of chiral stationary-phase columns. Second, the elution order of these compounds on the chromatogram can be switched by a designated esterification reaction. We applied the method to the compound-specific nitrogen isotope analysis of D- and L-alanine in a peptidoglycan derived from the cell walls of cultured bacteria (Firmicutes and Actinobacteria; Enterococcus faecalis, Staphylococcus aureus, Staphylococcus staphylolyticus, Lactobacillus acidophilus, Bacillus subtilis, Micrococcus luteus, and Streptomyces sp.), natural whole bacterial cells (Bacillus subtilis var. natto), (pseudo)-peptidoglycan from archaea (Methanobacterium sp.), and cell wall from eukaryota (Saccharomyces cerevisiae). We observed statistically significant differences in nitrogen isotopic compositions; e.g., delta15N ( per thousand vs air) in Staphylococcus staphylolyticus for d-alanine (19.2 +/- 0.5 per thousand, n = 4) and L-alanine (21.3 +/- 0.8 per thousand, n = 4) and in Bacillus subtilis for D-alanine (6.2 +/- 0.2 per thousand, n = 3) and L-alanine (8.2 +/- 0.4 per thousand, n = 3). These results suggest that enzymatic reaction pathways, including the alanine racemase reaction, produce a nitrogen isotopic difference in amino acid enantiomers, resulting in 15N-depleted D-alanine. This method is expected to facilitate compound-specific nitrogen isotope studies of amino acid stereoisomers.

Growth mechanics of bacterial cell wall and morphology of bacteria

NASA Astrophysics Data System (ADS)

Jiang, Hongyuan; Sun, Sean

2010-03-01

The peptidoglycan cell wall of bacteria is responsible for maintaining the cell shape and integrity. During the bacterial life cycle, the growth of the cell wall is affected by mechanical stress and osmotic pressure internal to the cell. We develop a theory to describe cell shape changes under the influence of mechanical forces. We find that the theory predicts a steady state size and shape for bacterial cells ranging from cocci to spirillum. Moreover, the theory suggest a mechanism by which bacterial cytoskeletal proteins such as MreB and crescentin can maintain the shape of the cell. The theory can also explain the several recent experiments on growing bacteria in micro-environments.

Is the wall of a cellulose fiber saturated with liquid whether or not permeable with CO2 dissolved molecules? Application to bubble nucleation in champagne wines.

PubMed

Liger-Belair, Gérard; Topgaard, Daniel; Voisin, Cédric; Jeandet, Philippe

2004-05-11

In this paper, the transversal diffusion coefficient D perpendicular of CO2 dissolved molecules through the wall of a hydrated cellulose fiber was approached, from the liquid bulk diffusion coefficient of CO2 dissolved molecules modified by an obstruction factor. The porous network between the cellulose microfibrils of the fiber wall was assumed being saturated with liquid. We retrieved information from previous NMR experiments on the self-diffusion of water in cellulose fibers to reach an order of magnitude for the transversal diffusion coefficient of CO2 molecules through the fiber wall. A value of about D perpendicular approximately 0.2D0 was proposed, D0 being the diffusion coefficient of CO2 molecules in the liquid bulk. Because most of bubble nucleation sites in a glass poured with carbonated beverage are cellulose fibers cast off from paper or cloth which floated from the surrounding air, or remaining from the wiping process, this result directly applies to the kinetics of carbon dioxide bubble formation from champagne and sparkling wines. If the cellulose fiber wall was impermeable with regard to CO2 dissolved molecules, it was suggested that the kinetics of bubbling would be about three times less than it is.

Understanding Microbial Sensing in Inflammatory Bowel Disease Using Click Chemistry

DTIC Science & Technology

2017-10-01

pathogens and commensals. However, the technology available to track these molecules in host cells and tissues remains primitive. To address this...live, luminal bacteria into specific host intestinal immune cells and their subsequent degradation in host phagocytes. Notably, this approach also...click-chemistry, bacterial cell wall, bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide, inflammatory

A spin labelling study of immunomodulating peptidoglycan monomer and adamantyltripeptides entrapped into liposomes.

PubMed

Frkanec, Ruza; Noethig-Laslo, Vesna; Vranesić, Branka; Mirosavljević, Krunoslav; Tomasić, Jelka

2003-04-01

The interaction of immunostimulating compounds, the peptidoglycan monomer (PGM) and structurally related adamantyltripeptides (AdTP1 and AdTP2), respectively, with phospholipids in liposomal bilayers were investigated by electron paramagnetic resonance spectroscopy. (1). The fatty acids bearing the nitroxide spin label at different positions along the acyl chain were used to investigate the interaction of tested compounds with negatively charged multilamellar liposomes. Electron spin resonance (ESR) spectra were studied at 290 and 310 K. The entrapment of the adamantyltripeptides affected the motional properties of all spin labelled lipids, while the entrapment of PGM had no effect. (2). Spin labelled PGM was prepared and the novel compound bearing the spin label attached via the amino group of diaminopimelic acid was chromatographically purified and chemically characterized. The rotational correlation time of the spin labelled molecule dissolved in buffer at pH 7.4 was studied as a function of temperature. The conformational change was observed above 300 K. The same effect was observed with the spin labelled PGM incorporated into liposomes. Such effect was not observed when the spin labelled PGM was studied at alkaline pH, probably due to the hydrolysis of PGM molecule. The study of possible interaction with liposomal membrane is relevant to the use of tested compounds incorporated into liposomes, as adjuvants in vivo.

Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria

PubMed Central

Mistou, Michel-Yves; Sutcliffe, Iain C.; van Sorge, Nina M.

2016-01-01

The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications. PMID:26975195

Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria.

PubMed

Mistou, Michel-Yves; Sutcliffe, Iain C; van Sorge, Nina M

2016-07-01

The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications. © FEMS 2016.

Antimicrobial Peptides Targeting Gram-Positive Bacteria

PubMed Central

Malanovic, Nermina; Lohner, Karl

2016-01-01

Antimicrobial peptides (AMPs) have remarkably different structures as well as biological activity profiles, whereupon most of these peptides are supposed to kill bacteria via membrane damage. In order to understand their molecular mechanism and target cell specificity for Gram-positive bacteria, it is essential to consider the architecture of their cell envelopes. Before AMPs can interact with the cytoplasmic membrane of Gram-positive bacteria, they have to traverse the cell wall composed of wall- and lipoteichoic acids and peptidoglycan. While interaction of AMPs with peptidoglycan might rather facilitate penetration, interaction with anionic teichoic acids may act as either a trap for AMPs or a ladder for a route to the cytoplasmic membrane. Interaction with the cytoplasmic membrane frequently leads to lipid segregation affecting membrane domain organization, which affects membrane permeability, inhibits cell division processes or leads to delocalization of essential peripheral membrane proteins. Further, precursors of cell wall components, especially the highly conserved lipid II, are directly targeted by AMPs. Thereby, the peptides do not inhibit peptidoglycan synthesis via binding to proteins like common antibiotics, but form a complex with the precursor molecule, which in addition can promote pore formation and membrane disruption. Thus, the multifaceted mode of actions will make AMPs superior to antibiotics that act only on one specific target. PMID:27657092

Cell Wall Chemical Composition of Enterococcus faecalis in the Viable but Nonculturable State

PubMed Central

Signoretto, Caterina; del Mar Lleò, Maria; Tafi, Maria Carla; Canepari, Pietro

2000-01-01

The viable but nonculturable (VBNC) state is a survival mechanism adopted by many bacteria (including those of medical interest) when exposed to adverse environmental conditions. In this state bacteria lose the ability to grow in bacteriological media but maintain viability and pathogenicity and sometimes are able to revert to regular division upon restoration of normal growth conditions. The aim of this work was to analyze the biochemical composition of the cell wall of Enterococcus faecalis in the VBNC state in comparison with exponentially growing and stationary cells. VBNC enterococcal cells appeared as slightly elongated and were endowed with a wall more resistant to mechanical disruption than dividing cells. Analysis of the peptidoglycan chemical composition showed an increase in total cross-linking, which rose from 39% in growing cells to 48% in VBNC cells. This increase was detected in oligomers of a higher order than dimers, such as trimers (24% increase), tetramers (37% increase), pentamers (65% increase), and higher oligomers (95% increase). Changes were also observed in penicillin binding proteins (PBPs), the enzymes involved in the terminal stages of peptidoglycan assembly, with PBPs 5 and 1 being prevalent, and in autolytic enzymes, with a threefold increase in the activity of latent muramidase-1 in E. faecalis in the VBNC state. Accessory wall polymers such as teichoic acid and lipoteichoic acid proved unchanged and doubled in quantity, respectively, in VBNC cells in comparison to dividing cells. It is suggested that all these changes in the cell wall of VBNC enterococci are specific to this particular physiological state. This may provide indirect confirmation of the viability of these cells. PMID:10788366

Understanding Microbial Sensing in Inflammatory Bowel Disease Using Click Chemistry

DTIC Science & Technology

2017-10-01

both pathogens and commensals. However, the technology available to track these molecules in host cells and tissues remains primitive. To address this...from live, luminal bacteria into specific host intestinal immune cells and their subsequent degradation in host phagocytes. Notably, this approach...Bioorthogonal click-chemistry, bacterial cell wall, bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide

Molecular cloning and functional characterization of a short peptidoglycan recognition protein (HcPGRPS1) from the freshwater mussel, Hyriopsis cumingi.

PubMed

Yang, Ziyan; Li, Junhua; Li, Ying; Wu, Hongjuan; Wang, Xiaoyan

2013-12-01

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved from invertebrates to vertebrates, function as pattern-recognition and effector molecules in innate immunity. In the present study, a short-form PGRP, designated as HcPGRPS1 was identified from freshwater mussel Hyriopsis cumingi. The deduced amino acid sequence of HcPGRPS1 is composed of 235 residues which contains a conserved PGRP domain at the C-terminus. Sequence analysis showed that HcPGRPS1 shared high identities with other known PGRPs. The mRNA of HcPGRPS1 is constitutively expressed in a wide range of all tested tissues, with highest expression level in hepatopancreas, and its expression in tissues (gonad, nephridium, gill and foot) was up-regulated significantly after LPS or PGN stimulation (P<0.05). The recombinant protein of HcPGRPS1 exhibited binding activity and peptidoglycan-lytic amidase activity toward Lys-PGN from Staphylococcus aureus and DAP-PGN from Bacillus subtilis. Furthermore, recombinant HcPGRPS1 displayed strong antibacterial activity to both Gram-negative bacteria Escherichia coli, Aeromonas hydrophila, Aeromonas sobria and Gram-positive bacteria S. aureus in the presence of Zn(2+). These results suggested that HcPGRPS1 plays a multifunctional role in the defense and protection mechanisms of mussel innate immunity against infections. Copyright © 2013 Elsevier Ltd. All rights reserved.

Dislocation-mediated growth of bacterial cell walls

PubMed Central

Amir, Ariel; Nelson, David R.

2012-01-01

Recent experiments have illuminated a remarkable growth mechanism of rod-shaped bacteria: proteins associated with cell wall extension move at constant velocity in circles oriented approximately along the cell circumference [Garner EC, et al., (2011) Science 333:222–225], [Domínguez-Escobar J, et al. (2011) Science 333:225–228], [van Teeffelen S, et al. (2011) PNAS 108:15822–15827]. We view these as dislocations in the partially ordered peptidoglycan structure, activated by glycan strand extension machinery, and study theoretically the dynamics of these interacting defects on the surface of a cylinder. Generation and motion of these interacting defects lead to surprising effects arising from the cylindrical geometry, with important implications for growth. We also discuss how long range elastic interactions and turgor pressure affect the dynamics of the fraction of actively moving dislocations in the bacterial cell wall. PMID:22660931

Phosphorylation of the Peptidoglycan Synthase PonA1 Governs the Rate of Polar Elongation in Mycobacteria

PubMed Central

Kieser, Karen J.; Baer, Christina E.; Barczak, Amy K.; Meniche, Xavier; Chao, Michael C.; Rego, E. Hesper; Sassetti, Christopher M.; Fortune, Sarah M.; Rubin, Eric J.

2015-01-01

Cell growth and division are required for the progression of bacterial infections. Most rod-shaped bacteria grow by inserting new cell wall along their mid-section. However, mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce new cell wall material at their poles. How mycobacteria control this different mode of growth is incompletely understood. Here we find that PonA1, a penicillin binding protein (PBP) capable of transglycosylation and transpeptidation of cell wall peptidoglycan (PG), is a major governor of polar growth in mycobacteria. PonA1 is required for growth of Mycobacterium smegmatis and is critical for M. tuberculosis during infection. In both cases, PonA1’s catalytic activities are both required for normal cell length, though loss of transglycosylase activity has a more pronounced effect than transpeptidation. Mutations that alter the amount or the activity of PonA1 result in abnormal formation of cell poles and changes in cell length. Moreover, altered PonA1 activity results in dramatic differences in antibiotic susceptibility, suggesting that a balance between the two enzymatic activities of PonA1 is critical for survival. We also find that phosphorylation of a cytoplasmic region of PonA1 is required for normal activity. Mutations in a critical phosphorylated residue affect transglycosylase activity and result in abnormal rates of cell elongation. Together, our data indicate that PonA1 is a central determinant of polar growth in mycobacteria, and its governance of cell elongation is required for robust cell fitness during both host-induced and antibiotic stress. PMID:26114871

Imaging the distribution of individual platinum-based anticancer drug molecules attached to single-wall carbon nanotubes

PubMed Central

Bhirde, Ashwin A; Sousa, Alioscka A; Patel, Vyomesh; Azari, Afrouz A; Gutkind, J Silvio; Leapman, Richard D; Rusling, James F

2009-01-01

Aims To image the distribution of drug molecules attached to single-wall carbon nanotubes (SWNTs). Materials & methods Herein we report the use of scanning transmission electron microscopy (STEM) for atomic scale visualization and quantitation of single platinum-based drug molecules attached to SWNTs designed for targeted drug delivery. Fourier transform infrared spectroscopy and energy-dispersive x-ray spectroscopy were used for characterization of the SWNT drug conjugates. Results Z-contrast STEM imaging enabled visualization of the first-line anticancer drug cisplatin on the nanotubes at single molecule level. The identity and presence of cisplatin on the nanotubes was confirmed using energy-dispersive x-ray spectroscopy and Fourier transform infrared spectroscopy. STEM tomography was also used to provide additional insights concerning the nanotube conjugates. Finally, our observations provide a rationale for exploring the use of SWNT bioconjugates to selectively target and kill squamous cancer cells. Conclusion Z-contrast STEM imaging provides a means for direct visualization of heavy metal containing molecules (i.e., cisplatin) attached to surfaces of carbon SWNTs along with distribution and quantitation. PMID:19839812

Pathogenic Chlamydia Lack a Classical Sacculus but Synthesize a Narrow, Mid-cell Peptidoglycan Ring, Regulated by MreB, for Cell Division

PubMed Central

Packiam, Mathanraj; Hsu, Yen-Pang; Tekkam, Srinivas; Hall, Edward; Rittichier, Jonathan T.; VanNieuwenhze, Michael; Brun, Yves V.; Maurelli, Anthony T.

2016-01-01

The peptidoglycan (PG) cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a ≤ 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actin-like protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe’s developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host. PMID:27144308

Pathogenic Chlamydia Lack a Classical Sacculus but Synthesize a Narrow, Mid-cell Peptidoglycan Ring, Regulated by MreB, for Cell Division.

PubMed

Liechti, George; Kuru, Erkin; Packiam, Mathanraj; Hsu, Yen-Pang; Tekkam, Srinivas; Hall, Edward; Rittichier, Jonathan T; VanNieuwenhze, Michael; Brun, Yves V; Maurelli, Anthony T

2016-05-01

The peptidoglycan (PG) cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a ≤ 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actin-like protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe's developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host.

Molecular cloning and functional characterization of peptidoglycan recognition protein 6 in grass carp Ctenopharyngodon idella.

PubMed

Li, Jun Hua; Yu, Zhang Long; Xue, Na Na; Zou, Peng Fei; Hu, Jing Yu; Nie, P; Chang, Ming Xian

2014-02-01

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of innate immunity. In this study, a long-form PGRP, designated as gcPGRP6, was identified from grass carp Ctenopharyngodon idella. The deduced amino acid sequence of gcPGRP6 is composed of 464 residues with a conserved PGRP domain at the C-terminus. The gcPGRP6 gene consists of four exons and three introns, spacing approximately 2.7 kb of genomic sequence. Phylogenetic analysis demonstrated that gcPGRP6 is clustered closely with zebrafish PGLYRP6, and formed a long-type PGRP subfamily together with PGLYRP2 members identified in teleosts and mammals. Real-time PCR and Western blotting analyses revealed that gcPGRP6 is constitutively expressed in organs/tissues examined, and its expression was significantly induced in liver and intestine of grass carp in response to PGN stimulation and in CIK cells treated with lipoteichoic acid (LTA), polyinosinic polycytidylic acid (Poly I:C) and peptidoglycan (PGN). Immunofluorescence microscopy and Western blotting analyses revealed that gcPGRP6 is effectively secreted to the exterior of CIK cells. The over-expression of gcPGRP6 in CIK cells leads to the activation of NF-κB and the inhibition of intracellular bacterial growth. Moreover, cell lysates from CIK cells transfected with pTurbo-gcPGRP6-GFP plasmid display the binding activity towards Lys-type PGN from Staphylococcus aureus and DAP-type PGN from Bacillus subtilis. Furthermore, proinflammatory cytokine IL-2 and intracellular PGN receptor NOD2 had a significantly increased expression in CIK cells overexpressed with gcPGRP6. It is demonstrated that the PGRP6 in grass carp has a role in binding PGN, in inhibiting the growth of intracellular bacteria, and in activating NF-κB, as well as in regulating innate immune genes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis.

PubMed

Garner, Ethan C; Bernard, Remi; Wang, Wenqin; Zhuang, Xiaowei; Rudner, David Z; Mitchison, Tim

2011-07-08

Rod-shaped bacteria elongate by the action of cell wall synthesis complexes linked to underlying dynamic MreB filaments. To understand how the movements of these filaments relate to cell wall synthesis, we characterized the dynamics of MreB and the cell wall elongation machinery using high-precision particle tracking in Bacillus subtilis. We found that MreB and the elongation machinery moved circumferentially around the cell, perpendicular to its length, with nearby synthesis complexes and MreB filaments moving independently in both directions. Inhibition of cell wall synthesis by various methods blocked the movement of MreB. Thus, bacteria elongate by the uncoordinated, circumferential movements of synthetic complexes that insert radial hoops of new peptidoglycan during their transit, possibly driving the motion of the underlying MreB filaments.

A peptidoglycan recognition protein from Sciaenops ocellatus is a zinc amidase and a bactericide with a substrate range limited to Gram-positive bacteria.

PubMed

Li, Mo-Fei; Zhang, Min; Wang, Chun-Lin; Sun, Li

2012-02-01

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRPs are highly conserved in invertebrates and vertebrates including fish. However, the biological function of teleost PGRP remains largely uninvestigated. In this study, we identified a PGRP homologue, SoPGLYRP-2, from red drum (Sciaenops ocellatus) and analyzed its activity and potential function. The deduced amino acid sequence of SoPGLYRP-2 is composed of 482 residues and shares 46-94% overall identities with known fish PGRPs. SoPGLYRP-2 contains at the C-terminus a single zinc amidase domain with conserved residues that form the catalytic site. Quantitative RT-PCR analysis detected SoPGLYRP-2 expression in multiple tissues, with the highest expression occurring in liver and the lowest expression occurring in brain. Experimental bacterial infection upregulated SoPGLYRP-2 expression in kidney, spleen, and liver in time-dependent manners. To examine the biological activity of SoPGLYRP-2, purified recombinant proteins representing the intact SoPGLYRP-2 (rSoPGLYRP-2) and the amidase domain (rSoPGLYRP-AD) were prepared from Escherichia coli. Subsequent analysis showed that rSoPGLYRP-2 and rSoPGLYRP-AD (i) exhibited comparable Zn(2+)-dependent peptidoglycan-lytic activity and were able to recognize and bind to live bacterial cells, (ii) possessed bactericidal effect against Gram-positive bacteria and slight bacteriostatic effect against Gram-negative bacteria, (iii) were able to block bacterial infection into host cells. These results indicate that SoPGLYRP-2 is a zinc-dependent amidase and a bactericide that targets preferentially at Gram-positive bacteria, and that SoPGLYRP-2 is likely to play a role in host innate immune defense during bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

SpyB, a small heme-binding protein, affects the composition of the cell wall in Streptococcus pyogenes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edgar, Rebecca J.; Chen, Jing; Kant, Sashi

Streptococcus pyogenes (Group A Streptococcus or GAS) is a hemolytic human pathogen associated with a wide variety of infections ranging from minor skin and throat infections to life-threatening invasive diseases. The cell wall of GAS consists of peptidoglycan sacculus decorated with a carbohydrate comprising a polyrhamnose backbone with immunodominant N-acetylglucosamine side-chains. All GAS genomes contain the spyBA operon, which encodes a 35-amino-acid membrane protein SpyB, and a membrane-bound C 3-like ADP-ribosyltransferase SpyA. In this study, we addressed the function of SpyB in GAS. Phenotypic analysis of a spyB deletion mutant revealed increased bacterial aggregation, and reduced sensitivity to β-lactams ofmore » the cephalosporin class and peptidoglycan hydrolase PlyC. Glycosyl composition analysis of cell wall isolated from the spyB mutant suggested an altered carbohydrate structure compared with the wild-type strain. Furthermore, we found that SpyB associates with heme and protoporphyrin IX. Heme binding induces SpyB dimerization, which involves disulfide bond formation between the subunits. Furthermore, our data suggest the possibility that SpyB activity is regulated by heme.« less

SpyB, a small heme-binding protein, affects the composition of the cell wall in Streptococcus pyogenes

DOE PAGES

Edgar, Rebecca J.; Chen, Jing; Kant, Sashi; ...

2016-10-13

Streptococcus pyogenes (Group A Streptococcus or GAS) is a hemolytic human pathogen associated with a wide variety of infections ranging from minor skin and throat infections to life-threatening invasive diseases. The cell wall of GAS consists of peptidoglycan sacculus decorated with a carbohydrate comprising a polyrhamnose backbone with immunodominant N-acetylglucosamine side-chains. All GAS genomes contain the spyBA operon, which encodes a 35-amino-acid membrane protein SpyB, and a membrane-bound C 3-like ADP-ribosyltransferase SpyA. In this study, we addressed the function of SpyB in GAS. Phenotypic analysis of a spyB deletion mutant revealed increased bacterial aggregation, and reduced sensitivity to β-lactams ofmore » the cephalosporin class and peptidoglycan hydrolase PlyC. Glycosyl composition analysis of cell wall isolated from the spyB mutant suggested an altered carbohydrate structure compared with the wild-type strain. Furthermore, we found that SpyB associates with heme and protoporphyrin IX. Heme binding induces SpyB dimerization, which involves disulfide bond formation between the subunits. Furthermore, our data suggest the possibility that SpyB activity is regulated by heme.« less

SpyB, a Small Heme-Binding Protein, Affects the Composition of the Cell Wall in Streptococcus pyogenes.

PubMed

Edgar, Rebecca J; Chen, Jing; Kant, Sashi; Rechkina, Elena; Rush, Jeffrey S; Forsberg, Lennart S; Jaehrig, Bernhard; Azadi, Parastoo; Tchesnokova, Veronika; Sokurenko, Evgeni V; Zhu, Haining; Korotkov, Konstantin V; Pancholi, Vijay; Korotkova, Natalia

2016-01-01

Streptococcus pyogenes (Group A Streptococcus or GAS) is a hemolytic human pathogen associated with a wide variety of infections ranging from minor skin and throat infections to life-threatening invasive diseases. The cell wall of GAS consists of peptidoglycan sacculus decorated with a carbohydrate comprising a polyrhamnose backbone with immunodominant N-acetylglucosamine side-chains. All GAS genomes contain the spyBA operon, which encodes a 35-amino-acid membrane protein SpyB, and a membrane-bound C3-like ADP-ribosyltransferase SpyA. In this study, we addressed the function of SpyB in GAS. Phenotypic analysis of a spyB deletion mutant revealed increased bacterial aggregation, and reduced sensitivity to β-lactams of the cephalosporin class and peptidoglycan hydrolase PlyC. Glycosyl composition analysis of cell wall isolated from the spyB mutant suggested an altered carbohydrate structure compared with the wild-type strain. Furthermore, we found that SpyB associates with heme and protoporphyrin IX. Heme binding induces SpyB dimerization, which involves disulfide bond formation between the subunits. Thus, our data suggest the possibility that SpyB activity is regulated by heme.

PGRP-SD, an Extracellular Pattern-Recognition Receptor, Enhances Peptidoglycan-Mediated Activation of the Drosophila Imd Pathway.

PubMed

Iatsenko, Igor; Kondo, Shu; Mengin-Lecreulx, Dominique; Lemaitre, Bruno

2016-11-15

Activation of the innate immune response in Metazoans is initiated through the recognition of microbes by host pattern-recognition receptors. In Drosophila, diaminopimelic acid (DAP)-containing peptidoglycan from Gram-negative bacteria is detected by the transmembrane receptor PGRP-LC and by the intracellular receptor PGRP-LE. Here, we show that PGRP-SD acted upstream of PGRP-LC as an extracellular receptor to enhance peptidoglycan-mediated activation of Imd signaling. Consistent with this, PGRP-SD mutants exhibited impaired activation of the Imd pathway and increased susceptibility to DAP-type bacteria. PGRP-SD enhanced the localization of peptidoglycans to the cell surface and hence promoted signaling. Moreover, PGRP-SD antagonized the action of PGRP-LB, an extracellular negative regulator, to fine-tune the intensity of the immune response. These data reveal that Drosophila PGRP-SD functions as an extracellular receptor similar to mammalian CD14 and demonstrate that, comparable to lipopolysaccharide sensing in mammals, Drosophila relies on both intra- and extracellular receptors for the detection of bacteria. Copyright © 2016 Elsevier Inc. All rights reserved.

Degradation changes in plant root cell wall structural molecules during extended decomposition of important agricultural crop and forage species

USDA-ARS?s Scientific Manuscript database

Little is known about the changes in the cell wall structural molecules lignin, cellulose and hemicellulose as plant roots decompose, despite their importance in the formation of soil organic matter. The objectives of this study were to quantify changes in root composition during 270 d incubations o...

Neither Lys- and DAP-type peptidoglycans stimulate mouse or human innate immune cells via Toll-like receptor 2

PubMed Central

Langer, Marybeth; Girton, Alanson W.; Popescu, Narcis I.; Burgett, Tarea; Metcalf, Jordan P.

2018-01-01

Peptidoglycan (PGN), a major component of bacterial cell walls, is a pathogen-associated molecular pattern (PAMP) that causes innate immune cells to produce inflammatory cytokines that escalate the host response during infection. In order to better understand the role of PGN in infection, we wanted to gain insight into the cellular receptor for PGN. Although the receptor was initially identified as Toll-like receptor 2 (TLR2), this receptor has remained controversial and other PGN receptors have been reported. We produced PGN from live cultures of Bacillus anthracis and Staphylococcus aureus and tested samples of PGN isolated during the purification process to determine at what point TLR2 activity was removed, if at all. Our results indicate that although live B. anthracis and S. aureus express abundant TLR2 ligands, highly-purified PGN from either bacterial source is not recognized by TLR2. PMID:29474374

A novel type of peptidoglycan-binding domain highly specific for amidated D-Asp cross-bridge, identified in Lactobacillus casei bacteriophage endolysins.

PubMed

Regulski, Krzysztof; Courtin, Pascal; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre

2013-07-12

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase, whereas Lc-Lys-2 is a γ-D-glutamyl-L-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with D-Ala(4)→D-Asx-L-Lys(3) in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting D-Ala(4)→L-Ala-(L-Ala/L-Ser)-L-Lys(3); moreover, they do not lyse the L. lactis mutant containing only the nonamidated D-Asp cross-bridge, i.e. D-Ala(4)→D-Asp-L-Lys(3). In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 L-Lys(3)-D-Asn-L-Lys(3) bridges replacing the wild-type 4→3 D-Ala(4)-D-Asn-L-Lys(3) bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly D-Asn but not PG with only the nonamidated D-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the D-Asn interpeptide bridge of PG.

The bacterial actin MreB rotates, and rotation depends on cell-wall assembly

PubMed Central

van Teeffelen, Sven; Wang, Siyuan; Furchtgott, Leon; Huang, Kerwyn Casey; Wingreen, Ned S.; Shaevitz, Joshua W.; Gitai, Zemer

2011-01-01

Bacterial cells possess multiple cytoskeletal proteins involved in a wide range of cellular processes. These cytoskeletal proteins are dynamic, but the driving forces and cellular functions of these dynamics remain poorly understood. Eukaryotic cytoskeletal dynamics are often driven by motor proteins, but in bacteria no motors that drive cytoskeletal motion have been identified to date. Here, we quantitatively study the dynamics of the Escherichia coli actin homolog MreB, which is essential for the maintenance of rod-like cell shape in bacteria. We find that MreB rotates around the long axis of the cell in a persistent manner. Whereas previous studies have suggested that MreB dynamics are driven by its own polymerization, we show that MreB rotation does not depend on its own polymerization but rather requires the assembly of the peptidoglycan cell wall. The cell-wall synthesis machinery thus either constitutes a novel type of extracellular motor that exerts force on cytoplasmic MreB, or is indirectly required for an as-yet-unidentified motor. Biophysical simulations suggest that one function of MreB rotation is to ensure a uniform distribution of new peptidoglycan insertion sites, a necessary condition to maintain rod shape during growth. These findings both broaden the view of cytoskeletal motors and deepen our understanding of the physical basis of bacterial morphogenesis. PMID:21903929

The bacterial actin MreB rotates, and rotation depends on cell-wall assembly.

PubMed

van Teeffelen, Sven; Wang, Siyuan; Furchtgott, Leon; Huang, Kerwyn Casey; Wingreen, Ned S; Shaevitz, Joshua W; Gitai, Zemer

2011-09-20

Bacterial cells possess multiple cytoskeletal proteins involved in a wide range of cellular processes. These cytoskeletal proteins are dynamic, but the driving forces and cellular functions of these dynamics remain poorly understood. Eukaryotic cytoskeletal dynamics are often driven by motor proteins, but in bacteria no motors that drive cytoskeletal motion have been identified to date. Here, we quantitatively study the dynamics of the Escherichia coli actin homolog MreB, which is essential for the maintenance of rod-like cell shape in bacteria. We find that MreB rotates around the long axis of the cell in a persistent manner. Whereas previous studies have suggested that MreB dynamics are driven by its own polymerization, we show that MreB rotation does not depend on its own polymerization but rather requires the assembly of the peptidoglycan cell wall. The cell-wall synthesis machinery thus either constitutes a novel type of extracellular motor that exerts force on cytoplasmic MreB, or is indirectly required for an as-yet-unidentified motor. Biophysical simulations suggest that one function of MreB rotation is to ensure a uniform distribution of new peptidoglycan insertion sites, a necessary condition to maintain rod shape during growth. These findings both broaden the view of cytoskeletal motors and deepen our understanding of the physical basis of bacterial morphogenesis.

An Unusual Mutation Results in the Replacement of Diaminopimelate with Lanthionine in the Peptidoglycan of a Mutant Strain of Mycobacterium smegmatis†

PubMed Central

Consaul, Sandra A.; Wright, Lori F.; Mahapatra, Sebabrata; Crick, Dean C.; Pavelka, Martin S.

2005-01-01

Mycobacterial peptidoglycan contains l-alanyl-d-iso-glutaminyl-meso-diaminopimelyl-d-alanyl-d-alanine peptides, with the exception of the peptidoglycan of Mycobacterium leprae, in which glycine replaces the l-alanyl residue. The third-position amino acid of the peptides is where peptidoglycan cross-linking occurs, either between the meso-diaminopimelate (DAP) moiety of one peptide and the penultimate d-alanine of another peptide or between two DAP residues. We previously described a collection of spontaneous mutants of DAP-auxotrophic strains of Mycobacterium smegmatis that can grow in the absence of DAP. The mutants are grouped into seven classes, depending on how well they grow without DAP and whether they are sensitive to DAP, temperature, or detergent. Furthermore, the mutants are hypersusceptible to β-lactam antibiotics when grown in the absence of DAP, suggesting that these mutants assemble an abnormal peptidoglycan. In this study, we show that one of these mutants, M. smegmatis strain PM440, utilizes lanthionine, an unusual bacterial metabolite, in place of DAP. We also demonstrate that the abilities of PM440 to grow without DAP and use lanthionine for peptidoglycan biosynthesis result from an unusual mutation in the putative ribosome binding site of the cbs gene, encoding cystathionine β-synthase, an enzyme that is a part of the cysteine biosynthetic pathway. PMID:15716431

Revised Model of Calcium and Magnesium Binding to the Bacterial Cell Wall

PubMed Central

Thomas, Kieth J.; Rice, Charles V.

2014-01-01

Metals bind to the bacterial cell wall yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane (lipoteichoic acid) or covalently bound to the cell wall (wall teichoic acid). The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, contradicting previous reports that calcium binding is 100% dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger and previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, meaning that the binding affinity decreases as more ions become bound to the sample. For Ca2+, Region I has a KA = (1.0 ± 0.2) × 106 M−1 and Region II has a KA = (0.075 ± 0.058) × 106 M−1. For Mg2+, KA1 = (1.5 ± 0.1) × 106 and KA2 = (0.17 ± 0.10) × 106. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η2, which are 0.70 ± 0.04 µmol/mg and 0.67 ± 0.03 µmol/mg for Ca2+ and Mg2+ respectively. These data contradict the current paradigm of there being a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent, suggesting a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations. PMID:25315444

Control of Lung Inflammation by Microbiome and Peptidoglycan Recognition Protein

DTIC Science & Technology

2017-07-01

AWARD NUMBER: W81XWH-16-1-0230 TITLE: Control of Lung Inflammation by Microbiome and Peptidoglycan Recognition Protein PRINCIPAL INVESTIGATOR...the Army position, policy or decision unless so designated by other documentation. REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188...for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO

Roles of tRNA in cell wall biosynthesis

PubMed Central

Dare, Kiley; Ibba, Michael

2013-01-01

Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families responsible for cell wall modifications, aminoacyl-phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids to phosphatidylglycerol (PG) by aaPGSs neutralizes the lipid bilayer making the bacteria less susceptible to positively charged antimicrobial agents. Fem transferases utilize aa-tRNA to form peptide bridges that link strands of peptidoglycan. These bridges vary among the bacterial species in which they are present and play a role in resistance to antibiotics that target the cell wall. Additionally, the formation of truncated peptides results in shorter peptide bridges and loss of branched linkages which makes bacteria more susceptible to antimicrobials. A greater understanding of the structure and substrate specificity of this diverse enzymatic family is necessary to aid current efforts in designing potential bactericidal agents. These two enzyme families are linked only by the substrate with which they modify the cell wall, aa-tRNA; their structure, cell wall modification processes and the physiological changes they impart on the bacterium differ greatly. PMID:22262511

Life in the "old bag" yet: structure of peptidoglycan L,D-carboxypeptidases.

PubMed

Cadby, Ian T; Lovering, Andrew L

2014-07-08

In this issue of Structure, Hoyland and colleagues describe the structure of a peptidoglycan L,D-carboxypeptidase in both substrate-bound and apoenzyme forms. These studies reveal the basis for enzyme specificity and assist greatly in a field where form and function overlap. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

PubMed Central

Butler, Emily K.; Davis, Rebecca M.; Bari, Vase; Nicholson, Paul A.

2013-01-01

Gram-negative bacteria such as Escherichia coli build a peptidoglycan (PG) cell wall in their periplasm using the precursor known as lipid II. Lipid II is a large amphipathic molecule composed of undecaprenyl diphosphate and a disaccharide-pentapeptide that PG-synthesizing enzymes use to build the PG sacculus. During PG biosynthesis, lipid II is synthesized at the cytoplasmic face of the inner membrane and then flipped across the membrane. This translocation of lipid II must be assisted by flippases thought to shield the disaccharide-pentapeptide as it crosses the hydrophobic core of the membrane. The inner membrane protein MurJ is essential for PG biogenesis and homologous to known and putative flippases of the MOP (multidrug/oligo-saccharidyl-lipid/polysaccharide) exporter superfamily, which includes flippases that translocate undecaprenyl diphosphate-linked oligosaccharides across the cytoplasmic membranes of bacteria. Consequently, MurJ has been proposed to function as the lipid II flippase in E. coli. Here, we present a three-dimensional structural model of MurJ generated by the I-TASSER server that suggests that MurJ contains a solvent-exposed cavity within the plane of the membrane. Using in vivo topological studies, we demonstrate that MurJ has 14 transmembrane domains and validate features of the MurJ structural model, including the presence of a solvent-exposed cavity within its transmembrane region. Furthermore, we present functional studies demonstrating that specific charged residues localized in the central cavity are essential for function. Together, our studies support the structural homology of MurJ to MOP exporter proteins, suggesting that MurJ might function as an essential transporter in PG biosynthesis. PMID:23935042

Rate and topography of peptidoglycan synthesis during cell division in Escherichia coli: Concept of a leading edge

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wientjes, F.B.; Nanninga, N.

1989-06-01

The rate at which the peptidoglycan of Escherichia coli is synthesized during the division cycle was studied with two methods. One method involved synchronization of E. coli MC4100 lysA cultures by centrifugal elutriation and subsequent pulse-labeling of the synchronously growing cultures with (meso-{sup 3}H)diaminopimelic acid (({sup 3}H)Dap). The second method was autoradiography of cells pulse-labeled with ({sup 3}H)Dap. It was found that the peptidoglycan is synthesized at a more or less exponentially increasing rate during the division cycle with a slight acceleration in this rate as the cells start to constrict. Apparently, polar cap formation requires synthesis of extra surfacemore » components, presumably to accommodate for a change in the surface-to-volume ratio. Furthermore, it was found that the pool size of Dap was constant during the division cycle. Close analysis of the topography of ({sup 3}H)Dap incorporation at the constriction site revealed that constriction proceeded by synthesis of peptidoglycan at the leading edge of the invaginating cell envelope. During constriction, no reallocation of incorporation occurred, i.e., the incorporation at the leading edge remained high throughout the process of constriction. Impairment of penicillin-binding protein 3 by mutation or by the specific {beta}-lactam antibiotic furazlocillin did not affect ({sup 3}H)Dap incorporation during initiation of constriction. However, the incorporation at the constriction site was inhibited in later stages of the constriction process. It is concluded that during division at least two peptidoglycan-synthesizing systems are operating sequentially.« less

Endogenous Molecules Induced by a Pathogen-Associated Molecular Pattern (PAMP) Elicit Innate Immunity in Shrimp

PubMed Central

Chen, Yu-Yuan; Chen, Jiann-Chu; Lin, Yong-Chin; Kitikiew, Suwaree; Li, Hui-Fang; Bai, Jia-Chin; Tseng, Kuei-Chi; Lin, Bo-Wei; Liu, Po-Chun; Shi, Yin-Ze; Kuo, Yi-Hsuan; Chang, Yu-Hsuan

2014-01-01

Invertebrates rely on an innate immune system to combat invading pathogens. The system is initiated in the presence of cell wall components from microbes like lipopolysaccharide (LPS), β-1,3-glucan (βG) and peptidoglycan (PG), altogether known as pathogen-associated molecular patterns (PAMPs), via a recognition of pattern recognition protein (PRP) or receptor (PRR) through complicated reactions. We show herein that shrimp hemocytes incubated with LPS, βG, and PG caused necrosis and released endogenous molecules (EMs), namely EM-L, EM-β, and EM-P, and found that shrimp hemocytes incubated with EM-L, EM-β, and EM-P caused changes in cell viability, degranulation and necrosis of hemocytes, and increased phenoloxidase (PO) activity and respiratory burst (RB) indicating activation of immunity in vitro. We found that shrimp receiving EM-L, EM-β, and EM-P had increases in hemocyte count and other immune parameters as well as higher phagocytic activity toward a Vibrio pathogen, and found that shrimp receiving EM-L had increases in proliferation cell ratio and mitotic index of hematopoietic tissues (HPTs). We identified proteins of EMs deduced from SDS-PAGE and LC-ESI-MS/MS analyses. EM-L and EM-P contained damage-associated molecular patterns (DAMPs) including HMGBa, HMGBb, histone 2A (H2A), H2B, and H4, and other proteins including proPO, Rab 7 GPTase, and Rab 11 GPTase, which were not observed in controls (EM-C, hemocytes incubated in shrimp salt solution). We concluded that EMs induced by PAMPs contain DAMPs and other immune molecules, and they could elicit innate immunity in shrimp. Further research is needed to identify which individual molecule or combined molecules of EMs cause the results, and determine the mechanism of action in innate immunity. PMID:25517999

Processive motions of MreB micro-filaments coordinate cell wall growth

NASA Astrophysics Data System (ADS)

Garner, Ethan

2012-02-01

Rod-shaped bacteria elongate by the action of cell-wall synthesis complexes linked to underlying dynamic MreB filaments, but how these proteins function to allow continued elongation as a rod remains unknown. To understand how the movement of these filaments relates to cell wall synthesis, we characterized the dynamics of MreB and the cell wall elongation machinery using high-resolution particle tracking in Bacillus subtilis. We found that both MreB and the elongation machinery move in linear paths across the cell, moving at similar rates (˜20nm / second) and angles to the cell body, suggesting they function as single complexes. These proteins move circumferentially around the cell, principally perpendicular to its length. We find that the motions of these complexes are independent, as they can pause and reverse,and also as nearby complexes move independently in both directions across one surface of the cell. Inhibition of cell wall synthesis with antibiotics or depletions in the cell wall synthesis machinery blocked MreB movement, suggesting that the cell wall synthetic machinery is the motor in this system. We propose that bacteria elongate by the uncoordinated, circumferential movements of synthetic complexes that span the plasma membrane and insert radial hoops of new peptidoglycan during their transit.

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

PubMed Central

Verbree, Carolin T.; Dätwyler, Steven M.; Meile, Susanne; Eichenseher, Fritz; Donovan, David M.; Loessner, Martin J.

2017-01-01

ABSTRACT Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have screened a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk, using a microtiter plate-based protocol. Nine suitable PGH constructs were identified by this approach and further compared in time-kill assays for their efficacy against S. aureus in heat-treated milk. The three most active enzymes (lysostaphin, Ami2638A, and CHAPK_CWT-LST) reduced S. aureus in milk to undetectable numbers within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity in most combinations, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The most effective PGH combination completely eradicated S. aureus from milk, with no more bacteria being detected within 24 h after addition of the enzymes, corresponding to a reduction of >9 log units compared to the control. Efficacy was also retained at different inoculum levels (3 versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas both Ami2638A and lysostaphin retained their activity, reducing bacterial numbers by >3.5 log units within 3 h. IMPORTANCE Staphylococci and S. aureus in particular are a major cause of bovine mastitis, an inflammation of the mammary gland in cows associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary

Evolutionarily distinct bacteriophage endolysins featuring conserved peptidoglycan cleavage sites protect mice from MRSA infection

USDA-ARS?s Scientific Manuscript database

Staphylococcus aureus is a Gram-positive pathogen relevant for both human and animal health. With multi-drug resistant S. aureus strains becoming increasingly prevalent, alternative therapeutics are urgently needed. Bacteriophage endolysins (peptidoglycan hydrolases, PGH) are capable of killing Gra...

Downregulation of the Musca domestica peptidoglycan recognition protein SC (PGRP-SC) leads to overexpression of antimicrobial peptides and tardy pupation.

PubMed

Gao, Yifu; Tang, Ting; Gu, Jihai; Sun, Lingling; Gao, Xiaobin; Ma, Xianyong; Wang, Xiaochun; Liu, Fengsong; Wang, Jianhui

2015-10-01

PGRP (peptidoglycan recognition protein) is a conserved protein family that recognizes the peptidoglycan in bacterial cell wall and causes the activation of various innate immune responses. Previous studies have reported that PGRP-SCs in Drosophila dampen the activation of Immune Deficiency (Imd) pathway to microbial infection, and participate in the lifespan extension of the insects. To facilitate understanding the function of PGRP-SCs from an evolutionary angle, we identified and functionally characterized the PGRP-SC gene in the housefly Musca domestica, a species that has adapted to a septic environment much harsher than the natural habitat of Drosophila. The gene designated as MdPGRP-SC was found most abundantly expressed in the 3rd instar larvae, and is expressed at this developmental stage predominantly in the gut. MdPGRP-SC was virtually unchanged in whole larvae after a septic injury at the second larval instar, while two antimicrobial peptides (AMPs), diptericin and attacin, were upregulated in the first 24h but not later. Through dsRNA microinjection, MdPGRP-SC was knocked down by RNA interference (RNAi), and caused the significant increased expression of diptericin and attacin. The pupation of MdPGRP-SC-depleted larvae was severely suppressed compared to controls. Opposite to the expression trend of MdPGRP-SC, a spontaneous active expression of diptericin and attacin was found in pre-pupae but not in third instar larvae. Taken together, our study reveals that downregulation of MdPGRP-SC leads to the overexpression of the AMPs, and is involved in the larvae-to-pupa transition of housefly. Copyright © 2015 Elsevier Ltd. All rights reserved.

Drosophila immunity: analysis of PGRP-SB1 expression, enzymatic activity and function.

PubMed

Zaidman-Rémy, Anna; Poidevin, Mickael; Hervé, Mireille; Welchman, David P; Paredes, Juan C; Fahlander, Carina; Steiner, Hakan; Mengin-Lecreulx, Dominique; Lemaitre, Bruno

2011-02-18

Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and able to bind PGN (non-catalytic PGRPs) and, in some cases, to efficiently degrade it (catalytic PGRPs). In Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.

Structural basis of recognition of pathogen-associated molecular patterns and inhibition of proinflammatory cytokines by camel peptidoglycan recognition protein.

PubMed

Sharma, Pradeep; Dube, Divya; Singh, Amar; Mishra, Biswajit; Singh, Nagendra; Sinha, Mau; Dey, Sharmistha; Kaur, Punit; Mitra, Dipendra K; Sharma, Sujata; Singh, Tej P

2011-05-06

Peptidoglycan recognition proteins (PGRPs) are involved in the recognition of pathogen-associated molecular patterns. The well known pathogen-associated molecular patterns include LPS from Gram-negative bacteria and lipoteichoic acid (LTA) from Gram-positive bacteria. In this work, the crystal structures of two complexes of the short form of camel PGRP (CPGRP-S) with LPS and LTA determined at 1.7- and 2.1-Å resolutions, respectively, are reported. Both compounds were held firmly inside the complex formed with four CPGRP-S molecules designated A, B, C, and D. The binding cleft is located at the interface of molecules C and D, which is extendable to the interface of molecules A and C. The interface of molecules A and B is tightly packed, whereas that of molecules B and D forms a wide channel. The hydrophilic moieties of these compounds occupy a common region, whereas hydrophobic chains interact with distinct regions in the binding site. The binding studies showed that CPGRP-S binds to LPS and LTA with affinities of 1.6 × 10(-9) and 2.4 × 10(-8) M, respectively. The flow cytometric studies showed that both LPS- and LTA-induced expression of the proinflammatory cytokines TNF-α and IL-6 was inhibited by CPGRP-S. The results of animal studies using mouse models indicated that both LPS- and LTA-induced mortality rates decreased drastically when CPGRP-S was administered. The recognition of both LPS and LTA, their high binding affinities for CPGRP-S, the significant decrease in the production of LPS- and LTA-induced TNF-α and IL-6, and the drastic reduction in the mortality rates in mice by CPGRP-S indicate its useful properties as an antibiotic agent.

OmpA: A Flexible Clamp for Bacterial Cell Wall Attachment.

PubMed

Samsudin, Firdaus; Ortiz-Suarez, Maite L; Piggot, Thomas J; Bond, Peter J; Khalid, Syma

2016-12-06

The envelope of Gram-negative bacteria is highly complex, containing separate outer and inner membranes and an intervening periplasmic space encompassing a peptidoglycan (PGN) cell wall. The PGN scaffold is anchored non-covalently to the outer membrane via globular OmpA-like domains of various proteins. We report atomically detailed simulations of PGN bound to OmpA in three different states, including the isolated C-terminal domain (CTD), the full-length monomer, or the complete full-length dimeric form. Comparative analysis of dynamics of OmpA CTD from different bacteria helped to identify a conserved PGN-binding mode. The dynamics of full-length OmpA, embedded within a realistic representation of the outer membrane containing full-rough (Ra) lipopolysaccharide, phospholipids, and cardiolipin, suggested how the protein may provide flexible mechanical support to the cell wall. An accurate model of the heterogeneous bacterial cell envelope should facilitate future efforts to develop antibacterial agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila

DOE Office of Scientific and Technical Information (OSTI.GOV)

Butler, C.A.; Hoffman, P.S.

1990-05-01

A 31-kilodalton (kDa) protein was solubilized from the peptidoglycan (PG) fraction of Legionella pneumophila after treatment with either N-acetylmuramidase from the fungus Chalaropsis sp. or with mutanolysin from Streptomyces globisporus. The protein exhibited a ladderlike banding pattern by autoradiography when radiolabeled ((35S)cysteine or (35S)methionine) PG material was extensively treated with hen lysozyme. The banding patterns ranging between 31 and 45 kDa and between 55 and 60 kDa resolved as a single 31-kDa protein when the material was subsequently treated with N-acetylmuramidase. Analysis of the purified 31-kDa protein for diaminopimelic acid by gas chromatography revealed 1 mol of diaminopimelic acid permore » mol of protein. When outer membrane PG material containing the major outer membrane porin protein was treated with N-acetylmuramidase or mutanolysin, both the 28.5-kDa major outer membrane protein and the 31-kDa protein were solubilized from the PG material under reducing conditions. In the absence of 2-mercaptoethanol, a high-molecular-mass complex (100 kDa) was resolved. The results of this study indicate that a 31-kDa PG-bound protein is a major component of the cell wall of L. pneumophila whose function may be to anchor the major outer membrane protein to PG. Finally, a survey of other Legionella species and other serogroups of L. pneumophila suggested that PG-bound proteins may be a common feature of this genus.« less

Determination of Cu Environments in the Cyanobacterium Anabaena flos-aquae by X-Ray Absorption Spectroscopy

PubMed Central

Kretschmer, X. C.; Meitzner, G.; Gardea-Torresdey, J. L.; Webb, R.

2004-01-01

Whole cells and peptidoglycan isolated from cell walls of the cyanobacterium Anabaena flos-aquae were lyophilized and used at pH 2 and pH 5 in Cu(II) binding studies. X-ray absorption spectra measured at the Cu K-edge were used to determine the oxidation states and chemical environments of Cu species in the whole-cell and peptidoglycan samples. In the whole-cell samples, most of the Cu retained at both pH values was coordinated by phosphate ligands. The whole-cell fractions contained significant concentrations of Cu(I) as well as Cu(II). An X-ray absorption near-edge spectrum analysis suggested that Cu(I) was coordinated by amine and thiol ligands. An analysis of the peptidoglycan fractions found that more Cu was adsorbed by the peptidoglycan fraction prepared at pH 5, due to increased chelation by amine and carboxyl ligands. The peptidoglycan fractions, also referred to as the cell wall fractions, contained little or no Cu(I). The Cu loading level was 30 times higher in the cell wall sample prepared at pH 5 than in the sample prepared at pH 2. Amine and bidentate carboxyl ligands had similar relative levels of importance in cell wall peptidoglycan samples prepared at both pH values, but phosphate coordination was insignificant. PMID:14766554

Anchoring of LPXTG-Like Proteins to the Gram-Positive Cell Wall Envelope.

PubMed

Siegel, Sara D; Reardon, Melissa E; Ton-That, Hung

2017-01-01

In Gram-positive bacteria, protein precursors with a signal peptide and a cell wall sorting signal (CWSS)-which begins with an LPXTG motif, followed by a hydrophobic domain and a tail of positively charged residues-are targeted to the cell envelope by a transpeptidase enzyme call sortase. Evolution and selective pressure gave rise to six classes of sortase, i.e., SrtA-F. Only class C sortases are capable of polymerizing substrates harboring the pilin motif and CWSS into protein polymers known as pili or fimbriae, whereas the others perform cell wall anchoring functions. Regardless of the products generated from these sortases, the basic principle of sortase-catalyzed transpeptidation is the same. It begins with the cleavage of the LPXTG motif, followed by the cross-linking of this cleaved product at the threonine residue to a nucleophile, i.e., an active amino group of the peptidoglycan stem peptide or the lysine residue of the pilin motif. This chapter will summarize the efforts to identify and characterize sortases and their associated pathways with emphasis on the cell wall anchoring function.

Cell-wall deficient L. monocytogenes L-forms feature abrogated pathogenicity

PubMed Central

Schnell, Barbara; Staubli, Titu; Harris, Nicola L.; Rogler, Gerhard; Kopf, Manfred; Loessner, Martin J.; Schuppler, Markus

2014-01-01

Stable L-forms are cell wall-deficient bacteria which are able to multiply and propagate indefinitely, despite the absence of a rigid peptidoglycan cell wall. We investigated whether L-forms of the intracellular pathogen L. monocytogenes possibly retain pathogenicity, and if they could trigger an innate immune response. While phagocytosis of L. monocytogenes L-forms by non-activated macrophages sometimes resulted in an unexpected persistence of the bacteria in the phagocytes, they were effectively eliminated by IFN-γ preactivated or bone marrow-derived macrophages (BMM). These findings were in line with the observed down-regulation of virulence factors in the cell-wall deficient L. monocytogenes. Absence of Interferon-β (IFN-β) triggering indicated inability of L-forms to escape from the phagosome into the cytosol. Moreover, abrogated cytokine response in MyD88-deficient dendritic cells (DC) challenged with L. monocytogenes L-forms suggested an exclusive TLR-dependent host response. Taken together, our data demonstrate a strong attenuation of Listeria monocytogenes L-form pathogenicity, due to diminished expression of virulence factors and innate immunity recognition, eventually resulting in elimination of L-form bacteria from phagocytes. PMID:24904838

Promoting acid resistance and nisin yield of Lactococcus lactis F44 by genetically increasing D-Asp amidation level inside cell wall.

PubMed

Hao, Panlong; Liang, Dongmei; Cao, Lijie; Qiao, Bin; Wu, Hao; Caiyin, Qinggele; Zhu, Hongji; Qiao, Jianjun

2017-08-01

Nisin fermentation by Lactococcus lactis requires a low pH to maintain a relatively higher nisin activity. However, the acidic environment will result in cell arrest, and eventually decrease the relative nisin production. Hence, constructing an acid-resistant L. lactis is crucial for nisin harvest in acidic nisin fermentation. In this paper, the first discovery of the relationship between D-Asp amidation-associated gene (asnH) and acid resistance was reported. Overexpression of asnH in L. lactis F44 (F44A) resulted in a sevenfold increase in survival capacity during acid shift (pH 3) and enhanced nisin desorption capacity compared to F44 (wild type), which subsequently contributed to higher nisin production, reaching 5346 IU/mL, 57.0% more than that of F44 in the fed-batch fermentation. Furthermore, the engineered F44A showed a moderate increase in D-Asp amidation level (from 82 to 92%) compared to F44. The concomitant decrease of the negative charge inside the cell wall was detected by a newly developed method based on the nisin adsorption amount onto cell surface. Meanwhile, peptidoglycan cross-linkage increased from 36.8% (F44) to 41.9% (F44A), and intracellular pH can be better maintained by blocking extracellular H + due to the maintenance of peptidoglycan integrity, which probably resulted from the action of inhibiting hydrolases activity. The inference was further supported by the acmC-overexpression strain F44C, which was characterized by uncontrolled peptidoglycan hydrolase activity. Our results provided a novel strategy for enhancing nisin yield through cell wall remodeling, which contributed to both continuous nisin synthesis and less nisin adsorption in acidic fermentation (dual enhancement).

Interactions between late acting proteins required for peptidoglycan synthesis during sporulation

PubMed Central

Fay, Allison; Meyer, Pablo; Dworkin, Jonathan

2010-01-01

The requirement of peptidoglycan synthesis for growth complicates the analysis of interactions between proteins involved in this pathway. In particular, the later steps that involve membrane-linked substrates have proven largely recalcitrant to in vivo analysis. Here we have taken advantage of the peptidoglycan synthesis that occurs during sporulation in Bacillus subtilis to examine the interactions between SpoVE, a non-essential, sporulation-specific homolog of the well-conserved and essential SEDS proteins, and SpoVD, a non-essential class B penicillin binding protein (PBP). We found that localization of SpoVD is dependent on SpoVE and that SpoVD protects SpoVE from in vivo proteolysis. Co-immunoprecipitations and Fluorescence Resonance Energy Transfer experiments indicated that SpoVE and SpoVD interact and co-affinity purification in E. coli demonstrated that this interaction is direct. Finally, we generated a functional protein consisting of a SpoVE-SpoVD fusion and found that a loss-of-function point mutation in either part of the fusion resulted in a loss of function of the entire fusion that was not complemented by a wild type protein. Thus, SpoVE has a direct and functional interaction with SpoVD and this conclusion will facilitate understanding the essential function SpoVE and related SEDS proteins such as FtsW and RodA play in bacterial growth and division. PMID:20417640

Understanding Mircrobial Sensing in Inflammatory Bowel Disease Using Click Chemistry

DTIC Science & Technology

2016-10-01

limitation, we have developed an expanded metabolic labeling approach that chemically tags lipopolysaccharide, capsular polysaccharide , and peptidoglycan...click-chemistry, bacterial cell wall, bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide , inflammatory...bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide , inflammatory bowel disease, microbiome, microbiota

Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consistingmore » of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling

Reversed-phase high-performance liquid chromatographic method for the determination of peptidoglycan monomers and structurally related peptides and adamantyltripeptides.

PubMed

Krstanović, Marina; Frkanec, Ruza; Vranesić, Branka; Ljevaković, Durdica; Sporec, Vesna; Tomasić, Jelka

2002-06-25

The reversed-phase HPLC method using UV detection was developed for the determination of (a) immunostimulating peptidoglycan monomers represented by the basic structure GlcNAc-MurNAc-L-Ala-D-isoGln-meso-DAP(omegaNH(2))-D-Ala-D-Ala (PGM) and two more lipophilic derivatives, Boc-Tyr-PGM and (Ada-1-yl)-CH(2)-CO-PGM, (b) two diastereomeric immunostimulating adamantyltripeptides L- and D-(adamant-2-yl)-Gly-L-Ala-D-isoGln and (c) peptides obtained by the enzyme hydrolyses of peptidoglycans and related peptides. The enzymes used, N-acetylmuramyl-L-alanine amidase and an L,D-aminopeptidase are present in mammalian sera and are involved in the metabolism of peptidoglycans and related peptides. Appropriate solvent systems were chosen with regard to structure and lipophilicity of each compound. As well, different gradient systems within the same solvent system had to be applied in order to achieve satisfactory separation and retention time. HPLC separation was developed with the aim to use this method for the study of the stability of the tested compounds, the purity during preparation and isolation and for following the enzyme hydrolyses.

MreB and MurG as scaffolds for the cytoplasmic steps of peptidoglycan biosynthesis.

PubMed

Favini-Stabile, Sandy; Contreras-Martel, Carlos; Thielens, Nicole; Dessen, Andréa

2013-12-01

Peptidoglycan is a major determinant of cell shape in bacteria, and its biosynthesis involves the concerted action of cytoplasmic, membrane-associated and periplasmic enzymes. Within the cytoplasm, Mur enzymes catalyse the first steps leading to peptidoglycan precursor biosynthesis, and have been suggested as being part of a multicomponent complex that could also involve the transglycosylase MurG and the cytoskeletal protein MreB. In order to initialize the characterization of a potential Mur interaction network, we purified MurD, MurE, MurF, MurG and MreB from Thermotoga maritima and characterized their interactions using membrane blotting and surface plasmon resonance. MurD, MurE and MurF all recognize MurG and MreB, but not each other, while the two latter proteins interact. In addition, we solved the crystal structures of MurD, MurE and MurF, which indicate that their C-termini display high conformational flexibilities. The differences in Mur conformations could be important parameters for the stability of an intracytoplasmic murein biosynthesis complex. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Structure and Function of the First Full-Length Murein Peptide Ligase (Mpl) Cell Wall Recycling Protein

PubMed Central

Das, Debanu; Hervé, Mireille; Feuerhelm, Julie; Farr, Carol L.; Chiu, Hsiu-Ju; Elsliger, Marc-André; Knuth, Mark W.; Klock, Heath E.; Miller, Mitchell D.; Godzik, Adam; Lesley, Scott A.; Deacon, Ashley M.; Mengin-Lecreulx, Dominique; Wilson, Ian A.

2011-01-01

Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In Gram-negative bacteria, ∼30–60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships. PMID:21445265

Structure and function of the first full-length murein peptide ligase (Mpl) cell wall recycling protein.

PubMed

Das, Debanu; Hervé, Mireille; Feuerhelm, Julie; Farr, Carol L; Chiu, Hsiu-Ju; Elsliger, Marc-André; Knuth, Mark W; Klock, Heath E; Miller, Mitchell D; Godzik, Adam; Lesley, Scott A; Deacon, Ashley M; Mengin-Lecreulx, Dominique; Wilson, Ian A

2011-03-18

Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In gram-negative bacteria, ∼30-60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships.

Development of a one-pot assay for screening and identification of Mur pathway inhibitors in Mycobacterium tuberculosis

PubMed Central

Eniyan, Kandasamy; Kumar, Anuradha; Rayasam, Geetha Vani; Perdih, Andrej; Bajpai, Urmi

2016-01-01

The cell wall of Mycobacterium tuberculosis (Mtb) consists of peptidoglycan, arabinogalactan and mycolic acids. The cytoplasmic steps in the peptidoglycan biosynthetic pathway, catalyzed by the Mur (A-F) enzymes, involve the synthesis of UDP-n-acetylmuramyl pentapeptide, a key precursor molecule required for the formation of the peptidoglycan monomeric building blocks. Mur enzymes are indispensable for cell integrity and their lack of counterparts in eukaryotes suggests them to be promising Mtb drug targets. However, the caveat is that most of the current assays utilize a single Mur enzyme, thereby identifying inhibitors against only one of the enzymes. Here, we report development of a one-pot assay that reconstructs the entire Mtb Mur pathway in vitro and has the advantage of eliminating the requirement for nucleotide intermediates in the pathway as substrates. The MurA-MurF enzymes were purified and a one-pot assay was developed through optimization of successive coupled enzyme assays using UDP-n-acetylglucosamine as the initial sugar substrate. The assay is biochemically characterized and optimized for high-throughput screening of molecules that could disrupt multiple targets within the pathway. Furthermore, we have validated the assay by performing it to identify D-Cycloserine and furan-based benzene-derived compounds with known Mur ligase inhibition as inhibitors of Mtb MurE and MurF. PMID:27734910

One and two hydrogen molecules in the large cage of the structure II clathrate hydrate: quantum translation-rotation dynamics close to the cage wall.

PubMed

Sebastianelli, Francesco; Xu, Minzhong; Kanan, Dalal K; Bacić, Zlatko

2007-07-19

We have performed a rigorous theoretical study of the quantum translation-rotation (T-R) dynamics of one and two H2 and D2 molecules confined inside the large hexakaidecahedral (5(12)6(4)) cage of the sII clathrate hydrate. For a single encapsulated H2 and D2 molecule, accurate quantum five-dimensional calculations of the T-R energy levels and wave functions are performed that include explicitly, as fully coupled, all three translational and the two rotational degrees of freedom of the hydrogen molecule, while the cage is taken to be rigid. In addition, the ground-state properties, energetics, and spatial distribution of one and two p-H2 and o-D2 molecules in the large cage are calculated rigorously using the diffusion Monte Carlo method. These calculations reveal that the low-energy T-R dynamics of hydrogen molecules in the large cage are qualitatively different from that inside the small cage, studied by us recently. This is caused by the following: (i) The large cage has a cavity whose diameter is about twice that of the small cage for the hydrogen molecule. (ii) In the small cage, the potential energy surface (PES) for H2 is essentially flat in the central region, while in the large cage the PES has a prominent maximum at the cage center, whose height exceeds the T-R zero-point energy of H2/D2. As a result, the guest molecule is excluded from the central part of the large cage, its wave function localized around the off-center global minimum. Peculiar quantum dynamics of the hydrogen molecule squeezed between the central maximum and the cage wall manifests in the excited T-R states whose energies and wave functions differ greatly from those for the small cage. Moreover, they are sensitive to the variations in the hydrogen-bonding topology, which modulate the corrugation of the cage wall.

Peptidoglycan Hydrolases of Escherichia coli

PubMed Central

van Heijenoort, Jean

2011-01-01

Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

DBIO Best Thesis Award: Mechanics, Dynamics, and Organization of the Bacterial Cytoskeleton and Cell Wall

NASA Astrophysics Data System (ADS)

Wang, Siyuan

2012-02-01

Bacteria come in a variety of shapes. While the peptidoglycan (PG) cell wall serves as an exoskeleton that defines the static cell shape, the internal bacterial cytoskeleton mediates cell shape by recruiting PG synthesis machinery and thus defining the pattern of cell-wall synthesis. While much is known about the chemistry and biology of the cytoskeleton and cell wall, much of their biophysics, including essential aspects of the functionality, dynamics, and organization, remain unknown. This dissertation aims to elucidate the detailed biophysical mechanisms of cytoskeleton guided wall synthesis. First, I find that the bacterial cytoskeleton MreB contributes nearly as much to the rigidity of an Escherichia coli cell as the cell wall. This conclusion implies that the cytoskeletal polymer MreB applies meaningful force to the cell wall, an idea favored by theoretical modeling of wall growth, and suggests an evolutionary origin of cytoskeleton-governed cell rigidity. Second, I observe that MreB rotates around the long axis of E. coli, and the motion depends on wall synthesis. This is the first discovery of a cell-wall assembly driven molecular motor in bacteria. Third, I prove that both cell-wall synthesis and the PG network have chiral ordering, which is established by the spatial pattern of MreB. This work links the molecular structure of the cytoskeleton and of the cell wall with organismal-scale behavior. Finally, I develop a mathematical model of cytoskeleton-cell membrane interactions, which explains the preferential orientation of different cytoskeleton components in bacteria.

Living with an imperfect cell wall: compensation of femAB inactivation in Staphylococcus aureus.

PubMed

Hübscher, Judith; Jansen, Andrea; Kotte, Oliver; Schäfer, Juliane; Majcherczyk, Paul A; Harris, Llinos G; Bierbaum, Gabriele; Heinemann, Matthias; Berger-Bächi, Brigitte

2007-09-04

Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore beta-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors.

Living with an imperfect cell wall: compensation of femAB inactivation in Staphylococcus aureus

PubMed Central

Hübscher, Judith; Jansen, Andrea; Kotte, Oliver; Schäfer, Juliane; Majcherczyk, Paul A; Harris, Llinos G; Bierbaum, Gabriele; Heinemann, Matthias; Berger-Bächi, Brigitte

2007-01-01

Background Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore β-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. Results In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. Conclusion Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors. PMID:17784943

Indistinguishability and identifiability of kinetic models for the MurC reaction in peptidoglycan biosynthesis.

PubMed

Hattersley, J G; Pérez-Velázquez, J; Chappell, M J; Bearup, D; Roper, D; Dowson, C; Bugg, T; Evans, N D

2011-11-01

An important question in Systems Biology is the design of experiments that enable discrimination between two (or more) competing chemical pathway models or biological mechanisms. In this paper analysis is performed between two different models describing the kinetic mechanism of a three-substrate three-product reaction, namely the MurC reaction in the cytoplasmic phase of peptidoglycan biosynthesis. One model involves ordered substrate binding and ordered release of the three products; the competing model also assumes ordered substrate binding, but with fast release of the three products. The two versions are shown to be distinguishable; however, if standard quasi-steady-state assumptions are made distinguishability cannot be determined. Once model structure uniqueness is ensured the experimenter must determine if it is possible to successfully recover rate constant values given the experiment observations, a process known as structural identifiability. Structural identifiability analysis is carried out for both models to determine which of the unknown reaction parameters can be determined uniquely, or otherwise, from the ideal system outputs. This structural analysis forms an integrated step towards the modelling of the full pathway of the cytoplasmic phase of peptidoglycan biosynthesis. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion associated peptidoglycan hydrolase: fusions, deletions and synergy with LysH5

USDA-ARS?s Scientific Manuscript database

Virion-associated peptidoglycan hydrolases have a potential as antimicrobial agents due to their ability to lyse Gram positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriopha...

Solid-state NMR on bacterial cells: selective cell wall signal enhancement and resolution improvement using dynamic nuclear polarization.

PubMed

Takahashi, Hiroki; Ayala, Isabel; Bardet, Michel; De Paëpe, Gaël; Simorre, Jean-Pierre; Hediger, Sabine

2013-04-03

Dynamic nuclear polarization (DNP) enhanced solid-state nuclear magnetic resonance (NMR) has recently emerged as a powerful technique for the study of material surfaces. In this study, we demonstrate its potential to investigate cell surface in intact cells. Using Bacillus subtilis bacterial cells as an example, it is shown that the polarizing agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL) has a strong binding affinity to cell wall polymers (peptidoglycan). This particular interaction is thoroughly investigated with a systematic study on extracted cell wall materials, disrupted cells, and entire cells, which proved that TOTAPOL is mainly accumulating in the cell wall. This property is used on one hand to selectively enhance or suppress cell wall signals by controlling radical concentrations and on the other hand to improve spectral resolution by means of a difference spectrum. Comparing DNP-enhanced and conventional solid-state NMR, an absolute sensitivity ratio of 24 was obtained on the entire cell sample. This important increase in sensitivity together with the possibility of enhancing specifically cell wall signals and improving resolution really opens new avenues for the use of DNP-enhanced solid-state NMR as an on-cell investigation tool.

How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function

PubMed Central

Otero, Lisandro H.; Rojas-Altuve, Alzoray; Llarrull, Leticia I.; Carrasco-López, Cesar; Kumarasiri, Malika; Lastochkin, Elena; Fishovitz, Jennifer; Dawley, Matthew; Hesek, Dusan; Lee, Mijoon; Johnson, Jarrod W.; Fisher, Jed F.; Chang, Mayland; Mobashery, Shahriar; Hermoso, Juan A.

2013-01-01

The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the dd-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain—a remarkable 60 Å distant from the dd-transpeptidase active site—discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design. PMID:24085846

De novo transcriptome assembly and analysis of differential gene expression following peptidoglycan (PGN) challenge in Antheraea pernyi.

PubMed

Liu, Yu; Xin, Zhao-Zhe; Zhang, Dai-Zhen; Zhu, Xiao-Yu; Wang, Ying; Chen, Li; Tang, Bo-Ping; Zhou, Chun-Lin; Chai, Xin-Yue; Tian, Ji-Wu; Liu, Qiu-Ning

2018-06-01

Antheraea pernyi is not only an important economic insect, it is increasingly employed as a model organism due to a variety of advantages, including ease of rearing and experimental manipulation compared with other Lepidoptera. Peptidoglycan (PGN) is a major component of the bacterial cell wall, and interactions between PGN and A. pernyi cause a series of physiological changes in the insect. In the present study, we constructed cDNA libraries from a A. pernyi PGN-infected group and a control group stimulated with phosphate-buffered saline (PBS). The transcriptome was de novo assembled using the Trinity platform, and 1698 differentially expressed genes (DEGs) were identified, comprising 894 up-regulated and 804 down-regulated genes. To further investigate immune-related DEGs, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed. GO analysis identified major immune-related GO terms and KEGG enrichment indicated gene responses to three pathways related to the insect immune system. Several homologous genes related to the immune response of the A. pernyi fat body post-PGN infection were identified and categorised. Taken together, the results provide insight into the complex molecular mechanisms of the responses to bacterial infection at the transcriptional level. Copyright © 2018 Elsevier B.V. All rights reserved.

Inhibition of bacterial DD-peptidases (penicillin-binding proteins) in membranes and in vivo by peptidoglycan-mimetic boronic acids.

PubMed

Dzhekieva, Liudmila; Kumar, Ish; Pratt, R F

2012-04-03

The DD-peptidases or penicillin-binding proteins (PBPs) catalyze the final steps of bacterial peptidoglycan biosynthesis and are inhibited by the β-lactam antibiotics. There is at present a question of whether the active site structure and activity of these enzymes is the same in the solubilized (truncated) DD-peptidase constructs employed in crystallographic and kinetics studies as in membrane-bound holoenzymes. Recent experiments with peptidoglycan-mimetic boronic acids have suggested that these transition state analogue-generating inhibitors may be able to induce reactive conformations of these enzymes and thus inhibit strongly. We have now, therefore, measured the dissociation constants of peptidoglycan-mimetic boronic acids from Escherichia coli and Bacillus subtilis PBPs in membrane preparations and, in the former case, in vivo, by means of competition experiments with the fluorescent penicillin Bocillin Fl. The experiments showed that the boronic acids bound measurably (K(i) < 1 mM) to the low-molecular mass PBPs but not to the high-molecular mass enzymes, both in membrane preparations and in whole cells. In two cases, E. coli PBP2 and PBP5, the dissociation constants obtained were very similar to those obtained with the pure enzymes in homogeneous solution. The boronic acids, therefore, are unable to induce tightly binding conformations of these enzymes in vivo. There is no evidence from these experiments that DD-peptidase inhibitors are more or less effective in vivo than in homogeneous solution.

Transpeptidase activity of penicillin-binding protein SpoVD in peptidoglycan synthesis conditionally depends on the disulfide reductase StoA.

PubMed

Bukowska-Faniband, Ewa; Hederstedt, Lars

2017-07-01

Endospore cortex peptidoglycan synthesis is not required for bacterial growth but essential for endospore heat resistance. It therefore constitutes an amenable system for research on peptidoglycan biogenesis. The Bacillus subtilis sporulation-specific class B penicillin-binding protein (PBP) SpoVD and many homologous PBPs contain two conserved cysteine residues of unknown function in the transpeptidase domain - one as residue x in the SxN catalytic site motif and the other in a flexible loop near the catalytic site. A disulfide bond between these residues blocks the function of SpoVD in cortex synthesis. With a combination of experiments with purified proteins and B. subtilis mutant cells, it was shown that in active SpoVD the two cysteine residues most probably interact by hydrogen bonding and that this is important for peptidoglycan synthesis in vivo. It was furthermore demonstrated that the sporulation-specific thiol-disulfide oxidoreductase StoA reduces SpoVD and that requirement of StoA for cortex synthesis can be suppressed by two completely different types of structural alterations in SpoVD. It is concluded that StoA plays a critical role mainly during maturation of SpoVD in the forespore outer membrane. The findings advance our understanding of essential PBPs and redox control of extra-cytoplasmic protein disulfides in bacterial cells. © 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

Evidence for a Structural Role for Acid-Fast Lipids in Oocyst Walls of Cryptosporidium, Toxoplasma, and Eimeria

PubMed Central

Bushkin, G. Guy; Motari, Edwin; Carpentieri, Andrea; Dubey, Jitender P.; Costello, Catherine E.; Robbins, Phillips W.; Samuelson, John

2013-01-01

ABSTRACT Coccidia are protozoan parasites that cause significant human disease and are of major agricultural importance. Cryptosporidium spp. cause diarrhea in humans and animals, while Toxoplasma causes disseminated infections in fetuses and untreated AIDS patients. Eimeria is a major pathogen of commercial chickens. Oocysts, which are the infectious form of Cryptosporidium and Eimeria and one of two infectious forms of Toxoplasma (the other is tissue cysts in undercooked meat), have a multilayered wall. Recently we showed that the inner layer of the oocyst walls of Toxoplasma and Eimeria is a porous scaffold of fibers of β-1,3-glucan, which are also present in fungal walls but are absent from Cryptosporidium oocyst walls. Here we present evidence for a structural role for lipids in the oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. Briefly, oocyst walls of each organism label with acid-fast stains that bind to lipids in the walls of mycobacteria. Polyketide synthases similar to those that make mycobacterial wall lipids are abundant in oocysts of Toxoplasma and Eimeria and are predicted in Cryptosporidium. The outer layer of oocyst wall of Eimeria and the entire oocyst wall of Cryptosporidium are dissolved by organic solvents. Oocyst wall lipids are complex mixtures of triglycerides, some of which contain polyhydroxy fatty acyl chains like those present in plant cutin or elongated fatty acyl chains like mycolic acids. We propose a two-layered model of the oocyst wall (glucan and acid-fast lipids) that resembles the two-layered walls of mycobacteria (peptidoglycan and acid-fast lipids) and plants (cellulose and cutin). PMID:24003177

Corrected and Republished from: Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

PubMed Central

Verbree, Carolin T.; Dätwyler, Steven M.; Meile, Susanne; Eichenseher, Fritz; Donovan, David M.; Loessner, Martin J.

2017-01-01

ABSTRACT Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have used a microtiter plate-based protocol to screen a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk. Eight suitable PGH constructs were identified by this approach, and their efficacies against S. aureus in heat-treated milk were compared by time-kill assays. The two most active enzymes (lysostaphin and CHAPK_CWT-LST) reduced S. aureus numbers in milk to undetectable levels within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The PGH combination completely eradicated S. aureus from milk: no more bacteria were detected within 24 h after the addition of the enzymes, corresponding to a reduction of >9 log units from the level in the control. Efficacy was also retained at different inoculum levels (3 log versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas lysostaphin retained its activity, reducing bacterial numbers by >3.5 log units within 3 h. IMPORTANCE Staphylococci, and S. aureus in particular, are a major cause of bovine mastitis, an inflammation of the mammary gland in cows that is associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by

Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan

PubMed Central

Vieira, Diana; Figueiredo, Teresa A.; Verma, Anil; Sobral, Rita G.; Ludovice, Ana M.; de Lencastre, Hermínia; Trincao, Jose

2014-01-01

Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases. PMID:24817726

Engineering membrane and cell-wall programs for tolerance to toxic chemicals: Beyond solo genes.

PubMed

Sandoval, Nicholas R; Papoutsakis, Eleftherios T

2016-10-01

Metabolite toxicity in microbes, particularly at the membrane, remains a bottleneck in the production of fuels and chemicals. Under chemical stress, native adaptation mechanisms combat hyper-fluidization by modifying the phospholipids in the membrane. Recent work in fluxomics reveals the mechanism of how membrane damage negatively affects energy metabolism while lipidomic and transcriptomic analyses show that strains evolved to be tolerant maintain membrane fluidity under stress through a variety of mechanisms such as incorporation of cyclopropanated fatty acids, trans-unsaturated fatty acids, and upregulation of cell wall biosynthesis genes. Engineered strains with modifications made in the biosynthesis of fatty acids, peptidoglycan, and lipopolysaccharide have shown increased tolerance to exogenous stress as well as increased production of desired metabolites of industrial importance. We review recent advances in elucidation of mechanisms or toxicity and tolerance as well as efforts to engineer the bacterial membrane and cell wall. Copyright © 2016 Elsevier Ltd. All rights reserved.

DFT study of adsorption of picric acid molecule on the surface of single-walled ZnO nanotube; as potential new chemical sensor

NASA Astrophysics Data System (ADS)

Farmanzadeh, Davood; Tabari, Leila

2015-01-01

Using density functional theory (DFT), we have investigated the adsorption of picric acid (PA) molecule on the surface of (8,0) single-walled ZnO nanotube (ZnONT). The results show that the PA molecule can be chemisorbed on the surface of ZnONT with adsorption energies of -82.01 and -75.26 kJ/mol in gas and aqueous phase, respectively. Frontier molecular orbital analysis show that HOMO/LUMO gap of ZnONT reduces from 1.66 and 1.75 eV in the pristine nanotube to 0.83 and 0.72 eV in PA-adsorbed form in gas and aqueous phase, respectively. It suggests that the process can affect the electronic properties of the studied nanotube which would lead to its conductance change upon the adsorption of PA molecule. The modifying effect on the electrical conductance of ZnONT underlies the working mechanism of gas sensors for detecting the PA molecules. Analyses of the adsorption behavior of the electrically charged ZnONT toward PA molecule in the gas phase show that the PA molecule can be strongly adsorbed on the negatively charged ZnONT surface with significant adsorption energy (-135.1 kJ/mol). However, from the HOMO/LUMO gap changes, it can be concluded that the positive ZnONT might sensitively detect the PA molecule in comparison to the negative tube. These results can provide helpful information for experimental investigation to develop novel nanotube-based sensors.

Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus

PubMed Central

Foster, Timothy J.; Geoghegan, Joan A.; Ganesh, Vannakambadi K.; Höök, Magnus

2014-01-01

Staphylococcus aureus is an important opportunistic pathogen and persistently colonizes about 20% of the human population. Its surface is ‘decorated’ with proteins that are covalently anchored to the cell wall peptidoglycan. Structural and functional analysis has identified four distinct classes of surface proteins, of which microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) are the largest class. These surface proteins have numerous functions, including adhesion to and invasion of host cells and tissues, evasion of immune responses and biofilm formation. Thus, cell wall-anchored proteins are essential virulence factors for the survival of S. aureus in the commensal state and during invasive infections, and targeting them with vaccines could combat S. aureus infections. PMID:24336184

Potential of the virion-associated peptidoglycan hydrolase HydH5 and its derivative fusion proteins in milk biopreservation

USDA-ARS?s Scientific Manuscript database

Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [(a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-ph...

High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography*

PubMed Central

Desmarais, Samantha M.; Tropini, Carolina; Miguel, Amanda; Cava, Felipe; Monds, Russell D.; de Pedro, Miguel A.; Huang, Kerwyn Casey

2015-01-01

The bacterial cell wall is a network of glycan strands cross-linked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination. PMID:26468288

Diffusion of microspheres in shear flow near a wall: use to measure binding rates between attached molecules.

PubMed Central

Pierres, A; Benoliel, A M; Zhu, C; Bongrand, P

2001-01-01

The rate and distance-dependence of association between surface-attached molecules may be determined by monitoring the motion of receptor-bearing spheres along ligand-coated surfaces in a flow chamber (Pierres et al., Proc. Natl. Acad. Sci. U.S.A. 95:9256-9261, 1998). Particle arrests reveal bond formation, and the particle-to-surface distance may be estimated from the ratio between the velocity and the wall shear rate. However, several problems are raised. First, data interpretation requires extensive computer simulations. Second, the relevance of standard results from fluid mechanics to micrometer-size particles separated from surfaces by nanometer distances is not fully demonstrated. Third, the wall shear rate must be known with high accuracy. Here we present a simple derivation of an algorithm permitting one to simulate the motion of spheres near a plane in shear flow. We check that theoretical predictions are consistent with the experimental dependence of motion on medium viscosity or particle size, and the requirement for equilibrium particle height distribution to follow Boltzman's law. The determination of the statistical relationship between particle velocity and acceleration allows one to derive the wall shear rate with 1-s(-1) accuracy and the Hamaker constant of interaction between the particle and the wall with a sensitivity better than 10(-21) J. It is demonstrated that the correlation between particle height and mean velocity during a time interval Deltat is maximal when Deltat is about 0.1-0.2 s for a particle of 1.4-microm radius. When the particle-to-surface distance ranges between 10 and 40 nm, the particle height distribution may be obtained with a standard deviation ranging between 8 and 25 nm, provided the average velocity during a 160-ms period of time is determined with 10% accuracy. It is concluded that the flow chamber allows one to detect the formation of individual bonds with a minimal lifetime of 40 ms in presence of a disruptive force

MreB: pilot or passenger of cell wall synthesis?

PubMed

White, Courtney L; Gober, James W

2012-02-01

The discovery that the bacterial cell shape determinant MreB is related to actin spurred new insights into bacterial morphogenesis and development. The trafficking and mechanical roles of the eukaryotic cytoskeleton were hypothesized to have a functional ancestor in MreB based on evidence implicating MreB as an organizer of cell wall synthesis. Genetic, biochemical and cytological studies implicate MreB as a coordinator of a large multi-protein peptidoglycan (PG) synthesizing holoenzyme. Recent advances in microscopy and new biochemical evidence, however, suggest that MreB may function differently than previously envisioned. This review summarizes our evolving knowledge of MreB and attempts to refine the generalized model of the proteins organizing PG synthesis in bacteria. This is generally thought to be conserved among eubacteria and the majority of the discussion will focus on studies from a few well-studied model organisms. Copyright © 2011 Elsevier Ltd. All rights reserved.

Detection of single-molecule H2O2 signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Jin, Hong; Heller, Daniel A.; Kalbacova, Marie; Kim, Jong-Ho; Zhang, Jingqing; Boghossian, Ardemis A.; Maheshri, Narendra; Strano, Michael S.

2010-04-01

An emerging concept in cell signalling is the natural role of reactive oxygen species such as hydrogen peroxide (H2O2) as beneficial messengers in redox signalling pathways. The nature of H2O2 signalling is confounded, however, by difficulties in tracking it in living systems, both spatially and temporally, at low concentrations. Here, we develop an array of fluorescent single-walled carbon nanotubes that can selectively record, in real time, the discrete, stochastic quenching events that occur as H2O2 molecules are emitted from individual human epidermal carcinoma cells stimulated by epidermal growth factor. We show mathematically that such arrays can distinguish between molecules originating locally on the cell membrane from other contributions. We find that epidermal growth factor induces 2 nmol H2O2 locally over a period of 50 min. This platform promises a new approach to understanding the signalling of reactive oxygen species at the cellular level.

Phage lytic proteins: biotechnological applications beyond clinical antimicrobials

USDA-ARS?s Scientific Manuscript database

Most bacteriophages encode two types of cell wall lytic proteins: Endolysins (lysins) and virion-associated peptidoglycan hydrolases. Both enzymes have the ability to degrade the peptidoglycan of Gram positive bacteria resulting in cell lysis when they are applied externally. Bacteriophage lytic p...

Deciphering the mode of action of cell wall-inhibiting antibiotics using metabolic labeling of growing peptidoglycan in Streptococcus pyogenes.

PubMed

Sugimoto, Atsushi; Maeda, Asuka; Itto, Kaori; Arimoto, Hirokazu

2017-04-25

Because of the scanty pipeline of antibiotics newly obtained from nature, chemical modification of established drugs is one of the major streams of current antibacterial research. Intuitive and easy-to-use assays are critical for identifying drug candidates with novel modes of action. In this study, we demonstrated that metabolic fluorescent staining of growing cell walls is a powerful tool for mode-of-action analyses of antibiotics using Streptococcus pyogenes. A set of major cell-wall-inhibiting antibiotics (bacitracin, D-cycloserine, flavomycin, oxacillin, ramoplanin, and vancomycin) was employed to validate the potential of the assay. The mechanistic differences of these antibiotics were successfully observed. For instance, D-cycloserine treatment induced fluorescently stained, excessive peripheral cell wall growth. This may indicate that the switch from the peripheral growth stage to the succeeding septal growth was disturbed by the treatment. We then applied this assay to analyze a series of vancomycin derivatives. The assay was sufficiently sensitive to detect the effects of single-site chemical modification of vancomycin on its modes of action. This metabolic fluorescent labeling method is easy to perform, especially because it does not require radiolabeled substrates. Thus, it is suitable for the preliminary evaluation of antibacterial mechanisms during antibacterial research.

Phosphorylation of the Streptococcus pneumoniae cell wall biosynthesis enzyme MurC by a eukaryotic-like Ser/Thr kinase.

PubMed

Falk, Shaun P; Weisblum, Bernard

2013-03-01

Streptococcus pneumoniae contains a single Ser/Thr kinase-phosphatase pair known as StkP-PhpP. Here, we report the interaction of StkP-PhpP with S. pneumoniae UDP-N-acetylmuramoyl:L-alanine ligase, MurC, an enzyme that synthesizes an essential intermediate of the cell wall peptidoglycan pathway. Combinatorial phage display using StkP as target selected the peptide sequence YEVCGSDTVGC as an interacting partner and subsequently confirmed by ELISA. The phage peptide sequence YEVCGSDTVGC aligns closely with the MurC motif spanning S. pneumoniae amino acid coordinates 31-37. We show that MurC is phosphorylated by StkP and that phosphoMurC is dephosphorylated by PhpP. These data suggest a link between StkP-PhpP with the coordinated regulation of cell wall biosynthesis via MurC. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Desleucyl-Oritavancin with a Damaged d-Ala-d-Ala Binding Site Inhibits the Transpeptidation Step of Cell-Wall Biosynthesis in Whole Cells of Staphylococcus aureus.

PubMed

Kim, Sung Joon; Singh, Manmilan; Sharif, Shasad; Schaefer, Jacob

2017-03-14

We have used solid-state nuclear magnetic resonance to characterize the exact nature of the dual mode of action of oritavancin in preventing cell-wall assembly in Staphylococcus aureus. Measurements performed on whole cells labeled selectively in vivo have established that des-N-methylleucyl-N-4-(4-fluorophenyl)benzyl-chloroeremomycin, an Edman degradation product of [ 19 F]oritavancin, which has a damaged d-Ala-d-Ala binding aglycon, is a potent inhibitor of the transpeptidase activity of cell-wall biosynthesis. The desleucyl drug binds to partially cross-linked peptidoglycan by a cleft formed between the drug aglycon and its biphenyl hydrophobic side chain. This type of binding site is present in other oritavancin-like glycopeptides, which suggests that for these drugs a similar transpeptidase inhibition occurs.

β-1,3-Glucan, Which Can Be Targeted by Drugs, Forms a Trabecular Scaffold in the Oocyst Walls of Toxoplasma and Eimeria

PubMed Central

Bushkin, G. Guy; Motari, Edwin; Magnelli, Paula; Gubbels, Marc-Jan; Dubey, Jitender P.; Miska, Katarzyna B.; Bullitt, Esther; Costello, Catherine E.; Robbins, Phillips W.; Samuelson, John

2012-01-01

ABSTRACT The walls of infectious pathogens, which are essential for transmission, pathogenesis, and diagnosis, contain sugar polymers that are defining structural features, e.g., β-1,3-glucan and chitin in fungi, chitin in Entamoeba cysts, β-1,3-GalNAc in Giardia cysts, and peptidoglycans in bacteria. The goal here was to determine in which of three walled forms of Toxoplasma gondii (oocyst, sporocyst, or tissue cyst) is β-1,3-glucan, the product of glucan synthases and glucan hydrolases predicted by whole-genome sequences of the parasite. The three most important discoveries were as follows. (i) β-1,3-glucan is present in oocyst walls of Toxoplasma and Eimeria (a chicken parasite that is a model for intestinal stages of Toxoplasma) but is absent from sporocyst and tissue cyst walls. (ii) Fibrils of β-1,3-glucan are part of a trabecular scaffold in the inner layer of the oocyst wall, which also includes a glucan hydrolase that has a novel glucan-binding domain. (iii) Echinocandins, which target the glucan synthase and kill fungi, arrest development of the Eimeria oocyst wall and prevent release of the parasites into the intestinal lumen. In summary, β-1,3-glucan, which can be targeted by drugs, is an important component of oocyst walls of Toxoplasma but is not a component of sporocyst and tissue cyst walls. PMID:23015739

Role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins in AmpC expression, β-lactam resistance, and peptidoglycan structure.

PubMed

Ropy, Alaa; Cabot, Gabriel; Sánchez-Diener, Irina; Aguilera, Cristian; Moya, Bartolome; Ayala, Juan A; Oliver, Antonio

2015-07-01

This study aimed to characterize the role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins (LMM PBPs), namely, PBP4 (DacB), PBP5 (DacC), and PBP7 (PbpG), in peptidoglycan composition, β-lactam resistance, and ampC regulation. For this purpose, we constructed all single and multiple mutants of dacB, dacC, pbpG, and ampC from the wild-type P. aeruginosa PAO1 strain. Peptidoglycan composition was determined by high-performance liquid chromatography (HPLC), ampC expression by reverse transcription-PCR (RT-PCR), PBP patterns by a Bocillin FL-binding test, and antimicrobial susceptibility by MIC testing for a panel of β-lactams. Microscopy and growth rate analyses revealed no apparent major morphological changes for any of the mutants compared to the wild-type PAO1 strain. Of the single mutants, only dacC mutation led to significantly increased pentapeptide levels, showing that PBP5 is the major dd-carboxypeptidase in P. aeruginosa. Moreover, our results indicate that PBP4 and PBP7 play a significant role as dd-carboxypeptidase only if PBP5 is absent, and their dd-endopeptidase activity is also inferred. As expected, the inactivation of PBP4 led to a significant increase in ampC expression (around 50-fold), but, remarkably, the sequential inactivation of the three LMM PBPs produced a much greater increase (1,000-fold), which correlated with peptidoglycan pentapeptide levels. Finally, the β-lactam susceptibility profiles of the LMM PBP mutants correlated well with the ampC expression data. However, the inactivation of ampC in these mutants also evidenced a role of LMM PBPs, especially PBP5, in intrinsic β-lactam resistance. In summary, in addition to assessing the effect of P. aeruginosa LMM PBPs on peptidoglycan structure for the first time, we obtained results that represent a step forward in understanding the impact of these PBPs on β-lactam resistance, apparently driven by the interplay between their roles in AmpC induction, �

Otitis media induced by peptidoglycan-polysaccharide (PGPS) in TLR2-deficient (Tlr2(-/-)) mice for developing drug therapy.

PubMed

Zhang, Xiaolin; Zheng, Tihua; Sang, Lu; Apisa, Luke; Zhao, Hongchun; Fu, Fenghua; Wang, Qingzhu; Wang, Yanfei; Zheng, Qingyin

2015-10-01

Toll like receptor 2 (TLR2) signaling can regulate the pathogenesis of otitis media (OM). However, the precise role of TLR2 signaling in OM has not been clarified due to the lack of an optimal animal model. Peptidoglycan-polysaccharide (PGPS) of the bacterial cell wall can induce inflammation by activating the TLR2 signaling. This study aimed at examining the pathogenic characteristics of OM induced by PGPS in Tlr2(-/-) mice, and the potential therapeutic effect of sodium aescinate (SA) in this model. Wild-type (WT) and Tlr2(-/-) mice were inoculated with streptococcal PGPS into their middle ears (MEs) and treated intravenously with vehicle or SA daily beginning at 3days prior to PGPS for 6 consecutive days. The pathologic changes of individual mice were evaluated longitudinally. In comparison with WT mice, Tlr2(-/-) mice were susceptible to PGPS-induced OM. Tlr2(-/-) mice displayed greater hearing loss, tympanic membrane damage, ME mucosal thickening, longer inflammation state, cilia and goblet cell loss. SA-treatment decreased neutrophil infiltration, modulated TLR2-related gene expression and improved ciliary organization. PGPS induced a relatively stable OM in Tlr2(-/-) mice, providing a new model for OM research. Treatment with SA mitigated the pathogenic damage in the ME and may be valuable for intervention of OM. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison of the Immunostimulatory and Proinflammatory Activities of Candidate Gram-Positive Endotoxins, Lipoteichoic Acid, Peptidoglycan, and Lipopeptides in Murine and Human Cells

PubMed Central

Kimbrell, Matthew R.; Warshakoon, Hemamali; Cromer, Jens R.; Malladi, Subbalakshmi; Hood, Jennifer D.; Balakrishna, Rajalakshmi; Scholdberg, Tandace A.; David, Sunil A.

2008-01-01

1. Summary The role of lipopolysaccharide (LPS) in the pathogenesis of Gram-negative septic shock is well established. The corresponding proinflammatory and immunostimulatory molecule(s) on the Gram-positive bacteria is less well understood, and their identification and characterization would be a key prerequisite in designing specific sequestrants of the Gram-positive endotoxin(s). We report in this paper the comparison of NF-κB-, cytokine- and chemokine-inducing activities of the TLR2 ligands, lipoteichoic acid (LTA), peptidoglycan (PGN), and lipopeptides, to LPS, a prototype TLR4 agonist, in murine macrophage cell-lines as well as in human blood. In murine cells, di- and triacyl liopopeptides are equipotent in their NF-κB inducing activity relative to LPS, but elicit much lower proinflammatory cytokines. However, both LPS and the lipopeptides potently induce the secretion of a pattern of chemokines that is suggestive of the engagement of a TLR4-independent TRIF pathway. In human blood, although the lipopeptides induce p38 MAP kinase phosphorylation and CD11b upregulation in granulocytes at ng/ml concentrations, they do not elicit proinflammatory cytokine production even at very high doses; LTA, however, activates neutrophils and induces cytokine secretion, although its potency is considerably less than that of LPS, presumably due to its binding to plasma proteins. We conclude that, in human blood, the pattern of immunostimulation and proinflammatory mediator production elicited by LTA parallels that of LPS. PMID:18468694

In Situ Oxidation Synthesis of p-Type Composite with Narrow-Bandgap Small Organic Molecule Coating on Single-Walled Carbon Nanotube: Flexible Film and Thermoelectric Performance.

PubMed

Gao, Caiyan; Chen, Guangming

2018-03-01

Although composites of organic polymers or n-type small molecule/carbon nanotube (CNT) have achieved significant advances in thermoelectric (TE) applications, p-type TE composites of small organic molecules as thick surface coating layers on the surfaces of inorganic nanoparticles still remain a great challenge. Taking advantage of in situ oxidation reaction of thieno[3,4-b]pyrazine (TP) into TP di-N-oxide (TPNO) on single-walled CNT (SWCNT) surface, a novel synthesis strategy is proposed to achieve flexible films of TE composites with narrow-bandgap (1.19 eV) small molecule coating on SWCNT surface. The TE performance can be effectively enhanced and conveniently tuned by poly(sodium-p-styrenesulfonate) content, TPNO/SWCNT mass ratio, and posttreatment by various polar solvents. The maximum of the composite power factor at room temperature is 29.4 ± 1.0 µW m -1 K -2 . The work presents a way to achieve flexible films of p-type small organic molecule/inorganic composites with clear surface coating morphology for TE application. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Redundancy of Peptidoglycan Carboxypeptidases Ensures Robust Cell Shape Maintenance in Escherichia coli

PubMed Central

Peters, Katharina; Kannan, Suresh; Rao, Vincenzo A.; Biboy, Jacob; Vollmer, Daniela; Erickson, Stephen W.; Lewis, Richard J.

2016-01-01

ABSTRACT Peptidoglycan (PG) is an essential structural component of the bacterial cell wall and maintains the integrity and shape of the cell by forming a continuous layer around the cytoplasmic membrane. The thin PG layer of Escherichia coli resides in the periplasm, a unique compartment whose composition and pH can vary depending on the local environment of the cell. Hence, the growth of the PG layer must be sufficiently robust to allow cell growth and division under different conditions. We have analyzed the PG composition of 28 mutants lacking multiple PG enzymes (penicillin-binding proteins [PBPs]) after growth in acidic or near-neutral-pH media. Statistical analysis of the muropeptide profiles identified dd-carboxypeptidases (DD-CPases) that were more active in cells grown at acidic pH. In particular, the absence of the DD-CPase PBP6b caused a significant increase in the pentapeptide content of PG as well as morphological defects when the cells were grown at acidic pH. Other DD-CPases (PBP4, PBP4b, PBP5, PBP6a, PBP7, and AmpH) and the PG synthase PBP1B made a smaller or null contribution to the pentapeptide-trimming activity at acidic pH. We solved the crystal structure of PBP6b and also demonstrated that the enzyme is more stable and has a lower Km at acidic pH, explaining why PBP6b is more active at low pH. Hence, PBP6b is a specialized DD-CPase that contributes to cell shape maintenance at low pH, and E. coli appears to utilize redundant DD-CPases for normal growth under different conditions. PMID:27329754

Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a Uropathogenic Escherichia coli Isolate

PubMed Central

Diao, Jingyu; Bouwman, Catrien; Yan, Donghong; Kang, Jing; Katakam, Anand K.; Liu, Peter; Pantua, Homer; Abbas, Alexander R.; Nickerson, Nicholas N.; Austin, Cary; Reichelt, Mike; Sandoval, Wendy; Xu, Min

2017-01-01

ABSTRACT Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli. Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro, the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro. The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo. Compared to the wild-type strain, the lpp mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for “group 2” capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic E. coli isolates. PMID:28536290

Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem.

PubMed

Patin, Delphine; Bostock, Julieanne; Chopra, Ian; Mengin-Lecreulx, Dominique; Blanot, Didier

2012-06-01

Chlamydiaceae are obligate intracellular bacteria that do not synthesise detectable peptidoglycan although they possess an almost complete arsenal of genes encoding peptidoglycan biosynthetic activities. In this paper, the murF gene from Chlamydia trachomatis was shown to be capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase activity. Recombinant MurF from C. trachomatis was overproduced and purified from E. coli. It exhibited ATP-dependent UDP-MurNAc-X-γ-D-Glu-meso-A(2)pm:D-Ala-D-Ala ligase activity in vitro. No significant difference of kinetic parameters was seen when X was L-Ala, L-Ser or Gly. The L-Lys-containing UDP-MurNAc-tripeptide was a poorer substrate as compared to the meso-A(2)pm-containing one. Based on the respective substrate specificities of the chlamydial MurC, MurE, MurF and Ddl enzymes, a sequence L-Ala/L-Ser/Gly-γ-D-Glu-meso-A(2)pm-D-Ala-D-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.

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

PubMed

Primo, Emiliano D; Otero, Lisandro H; Ruiz, Francisco; Klinke, Sebastián; Giordano, Walter

2018-01-01

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. © 2017 The International Union of Biochemistry and Molecular Biology.

Cell shape acquisition and maintenance in rodlike bacteria

NASA Astrophysics Data System (ADS)

van Teeffelen, Sven; Wingreen, Ned; Gitai, Zemer

2010-03-01

The shape of rodlike bacteria such as Escherichia coli is mainly governed by the expansion and reorganization of the peptidoglycan cell wall. The cell wall is a huge, mostly single-layered molecule of stiff glycan strands that typically run perpendicular to the long axis and are crosslinked by short peptides. The wall resists the excess pressure from inside the cell. Although much is known about the enzymes that synthesize the wall, the mechanisms by which the cell maintains a constant rod diameter and uniform glycan strand orientation during growth remain unknown. Here we present quantitative results on the structure and dynamics of two essential proteins, which are believed to play an important role in cell wall synthesis. In particular, we have focused on the filament-forming protein MreB, an actin homolog that forms a long helical bundle along the inner membrane of the cell, and penicillin-binding protein 2, an essential protein for peptide bond formation in the periplasm. Based on their interplay we discuss the possibility of MreB serving as a guide and ruler for cell wall synthesis.

Building and Breaking the Cell Wall in Four Acts: A Kinesthetic and Tactile Role-Playing Exercise for Teaching Beta-Lactam Antibiotic Mechanism of Action and Resistance †

PubMed Central

Popovich, John; Stephens, Michelle; Celaya, Holly; Suwarno, Serena; Barclay, Shizuka; Yee, Emily; Dean, David A.; Farris, Megan; Haydel, Shelley E.

2018-01-01

“Building and breaking the cell wall” is designed to review the bacterial cell envelope, previously learned in lower-division biology classes, while introducing new topics such as antibiotics and bacterial antibiotic resistance mechanisms. We developed a kinesthetic and tactile modeling activity where students act as cellular components and construct the cell wall. In the first two acts, students model a portion of the gram-positive bacterial cell envelope and then demonstrate in detail how the peptidoglycan is formed. Act III involves student demonstration of the addition of β-lactam antibiotics to the environment and how they inhibit the formation of peptidoglycan, thereby preventing bacterial replication. Using Staphylococcus aureus as a model for gram-positive bacteria, students finish the activity (Act IV) by acting out how S. aureus often becomes resistant to β-lactam antibiotics. A high level of student engagement was observed, and the activity received positive feedback. In an assessment administered prior to and two months after the activity, significant improvements in scores were observed (p < 0.0001), demonstrating increased understanding and retention. This activity allows students to (i) visualize, role play, and kinesthetically “build” the cell envelope and form the peptidoglycan layer, (ii) understand the mechanism of action for β-lactam antibiotics, as well as how gene acquisition and protein changes result in resistance, and (iii) work cooperatively and actively to promote long-term retention of the subject material. PMID:29904519

Cell wall amidase AmiC1 is required for cellular communication and heterocyst development in the cyanobacterium Anabaena PCC 7120 but not for filament integrity.

PubMed

Berendt, Susanne; Lehner, Josef; Zhang, Yao Vincent; Rasse, Tobias M; Forchhammer, Karl; Maldener, Iris

2012-10-01

Filamentous cyanobacteria of the order Nostocales display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N(2) takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue amiC2 gene in Nostoc punctiforme ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two amiC paralogues from Anabaena sp. strain PCC 7120. The amiC1 (alr0092) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the amiC2 (alr0093) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.

Cloning and analysis of peptidoglycan recognition protein-LC and immune deficiency from the diamondback moth, Plutella xylostella.

PubMed

Zhan, Ming-Yue; Yang, Pei-Jin; Rao, Xiang-Jun

2018-02-01

Peptidoglycan (PGN) exists in both Gram-negative and Gram-positive bacteria as a component of the cell wall. PGN is an important target to be recognized by the innate immune system of animals. PGN recognition proteins (PGRP) are responsible for recognizing PGNs. In Drosophila melanogaster, PGRP-LC and IMD (immune deficiency) are critical for activating the Imd pathway. Here, we report the cloning and analysis of PGRP-LC and IMD (PxPGRP-LC and PxIMD) from diamondback moth, Plutella xylostella (L.), the insect pest of cruciferous vegetables. PxPGRP-LC gene consists of six exons encoding a polypeptide of 308 amino acid residues with a transmembrane region and a PGRP domain. PxIMD cDNA encodes a polypeptide of 251 amino acid residues with a death domain. Sequence comparisons indicate that they are characteristic of Drosophila PGRP-LC and IMD homologs. PxPGRP-LC and PxIMD were expressed in various tissues and developmental stages. Their mRNA levels were affected by bacterial challenges. The PGRP domain of PxPGRP-LC lacks key residues for the amidase activity, but it can recognize two types of PGNs. Overexpression of full-length and deletion mutants in Drosophila S2 cells induced expression of some antimicrobial peptide genes. These results indicate that PxPGRP-LC and PxIMD may be involved in the immune signaling of P. xylostella. This study provides a foundation for further studies of the immune system of P. xylostella. © 2017 Wiley Periodicals, Inc.

An Iron-Regulated Autolysin Remodels the Cell Wall To Facilitate Heme Acquisition in Staphylococcus lugdunensis

PubMed Central

Farrand, Allison J.; Haley, Kathryn P.; Lareau, Nichole M.; Heilbronner, Simon; McLean, John A.; Foster, Timothy

2015-01-01

Bacteria alter their cell surface in response to changing environments, including those encountered upon invasion of a host during infection. One alteration that occurs in several Gram-positive pathogens is the presentation of cell wall-anchored components of the iron-regulated surface determinant (Isd) system, which extracts heme from host hemoglobin to fulfill the bacterial requirement for iron. Staphylococcus lugdunensis, an opportunistic pathogen that causes infective endocarditis, encodes an Isd system. Unique among the known Isd systems, S. lugdunensis contains a gene encoding a putative autolysin located adjacent to the Isd operon. To elucidate the function of this putative autolysin, here named IsdP, we investigated its contribution to Isd protein localization and hemoglobin-dependent iron acquisition. S. lugdunensis IsdP was found to be iron regulated and cotranscribed with the Isd operon. IsdP is a specialized peptidoglycan hydrolase that cleaves the stem peptide and pentaglycine crossbridge of the cell wall and alters processing and anchoring of a major Isd system component, IsdC. Perturbation of IsdC localization due to isdP inactivation results in a hemoglobin utilization growth defect. These studies establish IsdP as an autolysin that functions in heme acquisition and describe a role for IsdP in cell wall reorganization to accommodate nutrient uptake systems during infection. PMID:26123800

Peptidoglycan Recognition Protein S2 From Silkworm Integument: Characterization, Microbe-Induced Expression, and Involvement in the Immune-Deficiency Pathway

PubMed Central

Yang, Jie; Wang, Xiaonan; Tang, Shunming; Shen, Zhongyuan; Wu, Jinmei

2015-01-01

Peptidoglycan recognition protein (PGRP) binds specifically to peptidoglycan and plays an important role as a pattern recognition receptor in the innate immunity of insects. The cDNA of a short-type PGRP, an open reading frame of 588 bp encoding a polypeptide of 196 amino acids, was cloned from Bombyx mori. A phylogenetic tree was constructed, and the results showed that BmPGRP-S2 was most similar to Drosophila melanogaster PGRP (DmPGRP-SA). The induced expression profile of BmPGRP-S2 in healthy Escherichia coli- and Bacillus subtilis-challenged B. mori was measured using semiquantitative reverse transcriptase polymerase chain reaction analysis. The expression of BmPGRP-S2 was upregulated at 24 h by E. coli and Ba. subtilis challenge. In addition, in the integument of B. mori, RNAi knockdown of BmPGRP-S2 caused an obvious reduction in the transcription expression of the transcription factor Relish and in antibacterial effector genes Attacin, Gloverin, and Moricin. The results indicated that BmPGRP-S2 participates in the signal transduction pathway of B. mori. PMID:25797797

Bacterial Cell Mechanics.

PubMed

Auer, George K; Weibel, Douglas B

2017-07-25

Cellular mechanical properties play an integral role in bacterial survival and adaptation. Historically, the bacterial cell wall and, in particular, the layer of polymeric material called the peptidoglycan were the elements to which cell mechanics could be primarily attributed. Disrupting the biochemical machinery that assembles the peptidoglycan (e.g., using the β-lactam family of antibiotics) alters the structure of this material, leads to mechanical defects, and results in cell lysis. Decades after the discovery of peptidoglycan-synthesizing enzymes, the mechanisms that underlie their positioning and regulation are still not entirely understood. In addition, recent evidence suggests a diverse group of other biochemical elements influence bacterial cell mechanics, may be regulated by new cellular mechanisms, and may be triggered in different environmental contexts to enable cell adaptation and survival. This review summarizes the contributions that different biomolecular components of the cell wall (e.g., lipopolysaccharides, wall and lipoteichoic acids, lipid bilayers, peptidoglycan, and proteins) make to Gram-negative and Gram-positive bacterial cell mechanics. We discuss the contribution of individual proteins and macromolecular complexes in cell mechanics and the tools that make it possible to quantitatively decipher the biochemical machinery that contributes to bacterial cell mechanics. Advances in this area may provide insight into new biology and influence the development of antibacterial chemotherapies.

Single molecule detection of nitric oxide enabled by d(AT)15 DNA adsorbed to near infrared fluorescent single-walled carbon nanotubes.

PubMed

Zhang, Jingqing; Boghossian, Ardemis A; Barone, Paul W; Rwei, Alina; Kim, Jong-Ho; Lin, Dahua; Heller, Daniel A; Hilmer, Andrew J; Nair, Nitish; Reuel, Nigel F; Strano, Michael S

2011-01-26

We report the selective detection of single nitric oxide (NO) molecules using a specific DNA sequence of d(AT)(15) oligonucleotides, adsorbed to an array of near-infrared fluorescent semiconducting single-walled carbon nanotubes (AT(15)-SWNT). While SWNT suspended with eight other variant DNA sequences show fluorescence quenching or enhancement from analytes such as dopamine, NADH, L-ascorbic acid, and riboflavin, d(AT)(15) imparts SWNT with a distinct selectivity toward NO. In contrast, the electrostatically neutral polyvinyl alcohol enables no response to nitric oxide, but exhibits fluorescent enhancement to other molecules in the tested library. For AT(15)-SWNT, a stepwise fluorescence decrease is observed when the nanotubes are exposed to NO, reporting the dynamics of single-molecule NO adsorption via SWNT exciton quenching. We describe these quenching traces using a birth-and-death Markov model, and the maximum likelihood estimator of adsorption and desorption rates of NO is derived. Applying the method to simulated traces indicates that the resulting error in the estimated rate constants is less than 5% under our experimental conditions, allowing for calibration using a series of NO concentrations. As expected, the adsorption rate is found to be linearly proportional to NO concentration, and the intrinsic single-site NO adsorption rate constant is 0.001 s(-1) μM NO(-1). The ability to detect nitric oxide quantitatively at the single-molecule level may find applications in new cellular assays for the study of nitric oxide carcinogenesis and chemical signaling, as well as medical diagnostics for inflammation.

Inhomogeneous hard homonuclear molecules

NASA Astrophysics Data System (ADS)

Quintana, Jacqueline

A review is given of some features of theories for inhomogeneous fluids of nonspherical molecules that take as input the direct correlation function of the corresponding homogeneous system. Two different methods are described for defining the structure of hard homonuclear molecules close to a hard planar wall. A spherical harmonics expanison (SHE) within the integral equation (IE) method is presented and, for comparison, a version of density functional theory for orientable hard bodies. In both cases the Pynn-Lado model is employed and a comparison is made with Monte Carlo data. The results indicate that for hard molecules the IE approach does not always capture the effects of orientation due to the characteristics of the SHE for the step function. This disadvantage is particularly true in the case of the orientationally averaged density profile.

DNA Molecules in Microfluidic Oscillatory Flow

PubMed Central

Chen, Y.-L.; Graham, M.D.; de Pablo, J.J.; Jo, K.; Schwartz, D.C.

2008-01-01

The conformation and dynamics of a single DNA molecule undergoing oscillatory pressure-driven flow in microfluidic channels is studied using Brownian dynamics simulations, accounting for hydrodynamic interactions between segments in the bulk and between the chain and the walls. Oscillatory flow provides a scenario under which the polymers may remain in the channel for an indefinite amount of time as they are stretched and migrate away from the channel walls. We show that by controlling the chain length, flow rate and oscillatory flow frequency, we are able to manipulate the chain extension and the chain migration from the channel walls. The chain stretch and the chain depletion layer thickness near the wall are found to increase as the Weissenberg number increases and as the oscillatory frequency decreases. PMID:19057656

Influence of Wall Material on VUV Emission from Hydrogen Plasma in H- Source

NASA Astrophysics Data System (ADS)

Bacal, M.; Glass-Maujean, M.; Ivanov, A. A., Jr; Nishiura, M.; Sasao, M.; Wada, M.

2002-11-01

The study of VUV emission from a hydrogen plasma produced in a filament discharge in a magnetic multicusp device showed that the use of tantalum and tungsten filaments leads to significant differences in the spectra. The effect of the filament material is interpreted in terms of the fresh film of this material, deposited on the wall. The synthetic spectrum convoluted with our apparatus function for the conditions of this experiment (gas temperature 500 K, electron energy 100 eV) agrees roughly well with the spectrum obtained with tungsten covered walls, but not with the spectrum obtained with tantalum covered walls. We show that in the case of tungsten covered walls the E-V singlet excitation is indeed a two-step Franck-Condon transition, going through either B or C state from an initial H2 molecule with v"=0, added to a Franck-Condon transition to highly excited states cascading to the B or C states. The excitation process to high v" states in the case of tantalum covered walls is a three step process, in which the first step is the formation by recombinative desorption on the wall of a vibrationally excited molecule with v"=1 or 2, which serves as the initial molecule in the subsequent E-V excitation through the B state. The results indicate a larger recombination coefficient of atoms on the tantalum covered wall.

Amino acids in cell wall of Gram-positive bacterium Micrococcus sp. hsn08 with flocculation activity on Chlorella vulgaris biomass.

PubMed

Li, Yi; Xu, Yanting; Zheng, Tianling; Wang, Hailei

2018-02-01

The aim of this work was to investigate the flocculation mechanism by Gram-positive bacterium, Micrococcus sp. hsn08 as a means for harvesting Chlorella vulgaris biomass. Bacterial cells of Micrococcus sp. hsn08 were added into algal culture to harvest algal cells through direct contacting with algae to form flocs. Viability dependence test confirmed that flocculation activity does not depend on live bacteria, but on part of the peptidoglycan. The further investigation has determined that amino acids in cell wall play an important role to flocculate algal cells. Positively charged calcium can combine bacterial and algal cells together, and form a bridge between them, thereby forming the flocs, suggesting that ions bridging is the main flocculation mechanism. These results suggest that bacterial cells of Micrococcus sp. hsn08 can be applied to harvest microalgae biomass with the help of amino acids in cell wall. Copyright © 2017 Elsevier Ltd. All rights reserved.

Porins in the Cell Wall of Mycobacteria

NASA Astrophysics Data System (ADS)

Trias, Joaquim; Jarlier, Vincent; Benz, Roland

1992-11-01

The cell wall of mycobacteria is an efficient permeability barrier that makes mycobacteria naturally resistant to most antibiotics. Liposome swelling assays and planar bilayer experiments were used to investigate the diffusion process of hydrophilic molecules through the cell wall of Mycobacterium chelonae and identify the main hydrophilic pathway. A 59-kilodalton cell wall protein formed a water-filled channel with a diameter of 2.2 nanometers and an average single-channel conductance equal to 2.7 nanosiemens in 1 M potassium chloride. These results suggest that porins can be found in the cell wall of a Gram-positive bacterium. A better knowledge of the hydrophilic pathways should help in the design of more effective antimycobacterial agents.

Detection of wood cell wall porosity using small carbohydrate molecules and confocal fluorescence microscopy.

PubMed

Donaldson, L A; Kroese, H W; Hill, S J; Franich, R A

2015-09-01

A novel approach to nanoscale detection of cell wall porosity using confocal fluorescence microscopy is described. Infiltration of cell walls with a range of nitrophenyl-substituted carbohydrates of different molecular weights was assessed by measuring changes in the intensity of lignin fluorescence, in response to the quenching effect of the 4-nitrophenyl group. The following carbohydrates were used in order of increasing molecular weight; 4-nitrophenyl β-D-glucopyrano-side (monosaccharide), 4-nitrophenyl β-D-lactopyranoside (disaccharide), 2-chloro-4-nitrophenyl β-D-maltotrioside (trisaccharide), and 4-nitrophenyl α-D-maltopentaoside (pentasaccharide). This technique was used to compare cell wall porosity in wood which had been dewatered to 40% moisture content using supercritical CO2, where cell walls remain fully hydrated, with kiln dried wood equilibrated to 12% moisture content. Infiltration of cell walls as measured by fluorescence quenching, was found to decrease with increasing molecular weight, with the pentasaccharide being significantly excluded compared to the monosaccharide. Porosity experiments were performed on blocks and sections to assess differences in cell wall accessibility. Dewatered and kiln dried wood infiltrated as blocks showed similar results, but greater infiltration was achieved by using sections, indicating that not all pores were easily accessible by infiltration from the lumen surface. In wood blocks infiltrated with 4-nitrophenyl α-D-maltopentaoside, quenching of the secondary wall was quite variable, especially in kiln dried wood, indicating limited connectivity of pores accessible from the lumen surface. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Lytic activity of the virion-associated peptidoglycan hydrolase HydH5 of staphylococcus aureus bacteriophage vB_SauS-phiIPLA88

USDA-ARS?s Scientific Manuscript database

Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 (phiIPLA88) contains a virion-associated muralytic enzyme (HydH5) encoded by orf58, which is located in the morphogenetic module. Comparative bioinformatic analysis revealed that HydH5 significantly resembled other peptidoglycan hydrolases encode...

The Effect of Water Molecules on Mechanical Properties of Cell Walls

NASA Astrophysics Data System (ADS)

Rahbar, Nima; Youssefian, Sina

The unique properties of bamboo fibers come from their natural composite structures that comprise mainly cellulose nanofibrils in a matrix of intertwined hemicellulose and lignin called lignin-carbohydrate complex (LCC). Here, we have utilized atomistic simulations to investigate the mechanical properties and mechanisms of interactions between these materials, in the presence of water molecules. The role of hemicellulose found to be enhancing the mechanical properties and lignin found to be providing the strength of bamboo fibers. The abundance of Hbonds in hemicellulose chains is responsible for improving the mechanical behavior of LCC. The strong van der Waals forces between lignin molecules and cellulose nanofibrils are responsible for higher adhesion energy between LCC/cellulose nanofibrils. We also found out that the amorphous regions of cellulose nanofibrils is the weakest interface in bamboo Microfibrils. In presence of water, the elastic modulus of lignin increases at low water content and decreases in higher water content, whereas the hemicellulose elastic modulus constantly decreases. The variations of Radial Distribution Function and Free Fractional Volume of these materials with water suggest that water molecules enhance the mechanical properties of lignin by filling voids in the system and creating Hbond bridges between polymer chains. For hemicellulose, however, the effect is always regressive due to the destructive effect of water molecules on the Hbond of its dense structure.

Critical cell wall hole size for lysis in Gram-positive bacteria

NASA Astrophysics Data System (ADS)

Mitchell, Gabriel; Wiesenfeld, Kurt; Nelson, Daniel; Weitz, Joshua

2013-03-01

Gram-positive bacteria transport molecules necessary for their survival through holes in their cell wall. The holes in cell walls need to be large enough to let critical nutrients pass through. However, the cell wall must also function to prevent the bacteria's membrane from protruding through a large hole into the environment and lysing the cell. As such, we hypothesize that there exists a range of cell wall hole sizes that allow for molecule transport but prevent membrane protrusion. Here we develop and analyze a biophysical theory of the response of a Gram-positive cell's membrane to the formation of a hole in the cell wall. We predict a critical hole size in the range 15-24nm beyond which lysis occurs. To test our theory, we measured hole sizes in Streptococcus pyogenes cells undergoing enzymatic lysis via transmission electron microscopy. The measured hole sizes are in strong agreement with our theoretical prediction. Together, the theory and experiments provide a means to quantify the mechanisms of death of Gram-positive cells via enzymatically mediated lysis and provides insight into the range of cell wall hole sizes compatible with bacterial homeostasis.

Ion penetration depth in the plant cell wall

NASA Astrophysics Data System (ADS)

Yu, L. D.; Vilaithong, T.; Phanchaisri, B.; Apavatjrut, P.; Anuntalabhochai, S.; Evans, P.; Brown, I. G.

2003-05-01

This study investigates the depth of ion penetration in plant cell wall material. Based on the biological structure of the plant cell wall, a physical model is proposed which assumes that the wall is composed of randomly orientated layers of cylindrical microfibrils made from cellulose molecules of C 6H 12O 6. With this model, we have determined numerical factors for ion implantation in the plant cell wall to correct values calculated from conventional ion implantation programs. Using these correction factors, it is possible to apply common ion implantation programs to estimate the ion penetration depth in the cell for bioengineering purposes. These estimates are compared with measured data from experiments and good agreement is achieved.

Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis

PubMed Central

Liu, Xiaokun; Grabherr, Heini M; Willmann, Roland; Kolb, Dagmar; Brunner, Frédéric; Bertsche, Ute; Kühner, Daniel; Franz-Wachtel, Mirita; Amin, Bushra; Felix, Georg; Ongena, Marc; Nürnberger, Thorsten; Gust, Andrea A

2014-01-01

Peptidoglycans (PGNs) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants. It is generally unknown how complex bacterial structures such as PGNs are perceived by plant pattern recognition receptors (PRRs) and whether host hydrolytic activities facilitate decomposition of bacterial matrices and generation of soluble PRR ligands. Here we show that Arabidopsis thaliana, upon bacterial infection or exposure to microbial patterns, produces a metazoan lysozyme-like hydrolase (lysozyme 1, LYS1). LYS1 activity releases soluble PGN fragments from insoluble bacterial cell walls and cleavage products are able to trigger responses typically associated with plant immunity. Importantly, LYS1 mutant genotypes exhibit super-susceptibility to bacterial infections similar to that observed on PGN receptor mutants. We propose that plants employ hydrolytic activities for the decomposition of complex bacterial structures, and that soluble pattern generation might aid PRR-mediated immune activation in cell layers adjacent to infection sites. DOI: http://dx.doi.org/10.7554/eLife.01990.001 PMID:24957336

2-Fluoro-L-Fucose Is a Metabolically Incorporated Inhibitor of Plant Cell Wall Polysaccharide Fucosylation

PubMed Central

Wallace, Ian S.

2015-01-01

The monosaccharide L-fucose (L-Fuc) is a common component of plant cell wall polysaccharides and other plant glycans, including the hemicellulose xyloglucan, pectic rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II), arabinogalactan proteins, and N-linked glycans. Mutations compromising the biosynthesis of many plant cell wall polysaccharides are lethal, and as a result, small molecule inhibitors of plant cell wall polysaccharide biosynthesis have been developed because these molecules can be applied at defined concentrations and developmental stages. In this study, we characterize novel small molecule inhibitors of plant fucosylation. 2-fluoro-L-fucose (2F-Fuc) analogs caused severe growth phenotypes when applied to Arabidopsis seedlings, including reduced root growth and altered root morphology. These phenotypic defects were dependent upon the L-Fuc salvage pathway enzyme L-Fucose Kinase/ GDP-L-Fucose Pyrophosphorylase (FKGP), suggesting that 2F-Fuc is metabolically converted to the sugar nucleotide GDP-2F-Fuc, which serves as the active inhibitory molecule. The L-Fuc content of cell wall matrix polysaccharides was reduced in plants treated with 2F-Fuc, suggesting that this molecule inhibits the incorporation of L-Fuc into these polysaccharides. Additionally, phenotypic defects induced by 2F-Fuc treatment could be partially relieved by the exogenous application of boric acid, suggesting that 2F-Fuc inhibits RG-II biosynthesis. Overall, the results presented here suggest that 2F-Fuc is a metabolically incorporated inhibitor of plant cellular fucosylation events, and potentially suggest that other 2-fluorinated monosaccharides could serve as useful chemical probes for the inhibition of cell wall polysaccharide biosynthesis. PMID:26414071

Molecular Sieving by the Bacillus megaterium Cell Wall and Protoplast

PubMed Central

Scherrer, Rene; Gerhardt, Philipp

1971-01-01

Passive permeabilities of the cell wall and protoplast of Bacillus megaterium strain KM were characterized by use of 50 hydrophilic probing molecules (tritiated water, sugars, dextrans, glycols, and polyglycols) which varied widely in size. Weight per cent uptake values (Rw) were measured at diffusional equilibrium under conditions that negated the influences of adsorption or active transport. Plots of Rw for intact cells as a function of number-average molecular weight (¯Mn) or Einstein-Stokes hydrodynamic radius (¯rES) of the solutes showed three phases: a protoplast uptake phase with a polydisperse exclusion threshold of ¯Mn = 0.6 × 103 to 1.1 × 103, ¯rES = 0.6 to 1.1 nm; a cell wall uptake phase with a polydisperse exclusion threshold of ¯Mn = 0.7 × 105 to 1.2 × 105, ¯rES ≅ 8.3 nm; and a total exclusion phase. Isolated cell walls showed only the latter two phases. However, it became evident that the cell wall selectively passed only the smallest molecules in a heterodisperse polymer sample. When the molecular-weight distributions of polyglycol samples (¯Mn = 1,000, 1,450, and 3,350) were determined by analytical gel chromatography before and after uptake by intact cells or isolated cell walls, a quasi-monodisperse exclusion threshold was obtained corresponding to Mn = 1,200, rES = 1.1 nm. The permeability of isolated protoplasts was assessed by the relative ability of solutes to effect osmotic stabilization. An indefinite exclusion threshold, evident even with monodisperse sugars, was attributed to lengthwise orientation of the penetrating rod-shaped molecules. Altogether, the best estimate of the limiting equivalent porosity of the protoplast was 0.4 to 0.6 nm in radius and of the cell wall, 1.1 nm. PMID:4999413

Studies on the genetic control of murine humoral response to immunization with a peptidoglycan-containing fraction extracted from Brucella melitensis.

PubMed

Cannat, A; Feingold, N; Caffin, J C; Serre, A

1979-01-01

A peptidoglycan containing fraction (fraction "5") extracted from Brucella melitensis has been injected in low infra-vaccinating doses into inbred mice. The genetic control of the resulting anti-Brucella humoral response has been studied in the C57BL/6 "good responder" X DBA2 "low responder" model. The results observed in F1, F2 and reciprocal backcrosses show that the "good responder" character, although transmitted as a dominant trait, is under polygenic control and independent of H2 haplotype, Ig allotype, sexual chromosoms or the "d" coat color gene. On the other hand, the phenotypic expression of at least one of the genes involved is sex-limited and influenced by hormonal environmental factors. Moreover the expression in females of one of these sex-dependent genes is associated with the "b" coat color gene. These results are discussed in terms of their possible relevance in spontaneous or vaccinal resistance to experimental brucellosis, of the relative role of the peptidoglycan and lipoprotein moieties in fraction "5" and of the possible importance of sex-dependent and chromosome 4-linked genetic factors for B-cell functions.

Phenotype-Based Screening of Small Molecules to Modify Plant Cell Walls Using BY-2 Cells.

PubMed

Okubo-Kurihara, Emiko; Matsui, Minami

2018-01-01

The plant cell wall is an important and abundant biomass with great potential for use as a modern recyclable resource. For effective utilization of this cellulosic biomass, its ability to degrade efficiently is key point. With the aim of modifying the cell wall to allow easy decomposition, we used chemical biological technology to alter its structure. As a first step toward evaluating the chemicals in the cell wall we employed a phenotype-based approach using high-throughput screening. As the plant cell wall is essential in determining cell morphology, phenotype-based screening is particularly effective in identifying compounds that bring about alterations in the cell wall. For rapid and reproducible screening, tobacco BY-2 cell is an excellent system in which to observe cell morphology. In this chapter, we provide a detailed chemical biological methodology for studying cell morphology using tobacco BY-2 cells.

Target molecules detection by waveguiding in a photonic silicon membrane

DOEpatents

Letant, Sonia E [Livermore, CA; Van Buuren, Anthony [Livermore, CA; Terminello, Louis [Danville, CA; Hart, Bradley R [Brentwood, CA

2006-12-26

Disclosed herein is a porous silicon filter capable of binding and detecting biological and chemical target molecules in liquid or gas samples. A photonic waveguiding silicon filter with chemical and/or biological anchors covalently attached to the pore walls bind target molecules. The system uses transmission curve engineering principles to allow measurements to be made in situ and in real time to detect the presence of various target molecules and calculate the concentration of bound target.

Target molecules detection by waveguiding in a photonic silicon membrane

DOEpatents

Letant, Sonia; Van Buuren, Anthony; Terminello, Louis

2004-08-31

Disclosed herein is a photonic silicon filter capable of binding and detecting biological and chemical target molecules in liquid or gas samples. A photonic waveguiding silicon filter with chemical and/or biological anchors covalently attached to the pore walls selectively bind target molecules. The system uses transmission curve engineering principles to allow measurements to be made in situ and in real time to detect the presence of various target molecules and determine the concentration of bound target.

Molecular deformation mechanisms of the wood cell wall material.

PubMed

Jin, Kai; Qin, Zhao; Buehler, Markus J

2015-02-01

Wood is a biological material with outstanding mechanical properties resulting from its hierarchical structure across different scales. Although earlier work has shown that the cellular structure of wood is a key factor that renders it excellent mechanical properties at light weight, the mechanical properties of the wood cell wall material itself still needs to be understood comprehensively. The wood cell wall material features a fiber reinforced composite structure, where cellulose fibrils act as stiff fibers, and hemicellulose and lignin molecules act as soft matrix. The angle between the fiber direction and the loading direction has been found to be the key factor controlling the mechanical properties. However, how the interactions between theses constitutive molecules contribute to the overall properties is still unclear, although the shearing between fibers has been proposed as a primary deformation mechanism. Here we report a molecular model of the wood cell wall material with atomistic resolution, used to assess the mechanical behavior under shear loading in order to understand the deformation mechanisms at the molecular level. The model includes an explicit description of cellulose crystals, hemicellulose, as well as lignin molecules arranged in a layered nanocomposite. The results obtained using this model show that the wood cell wall material under shear loading deforms in an elastic and then plastic manner. The plastic regime can be divided into two parts according to the different deformation mechanisms: yielding of the matrix and sliding of matrix along the cellulose surface. Our molecular dynamics study provides insights of the mechanical behavior of wood cell wall material at the molecular level, and paves a way for the multi-scale understanding of the mechanical properties of wood. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evidence for a structural role for acid-fast lipids in oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria.

PubMed

Bushkin, G Guy; Motari, Edwin; Carpentieri, Andrea; Dubey, Jitender P; Costello, Catherine E; Robbins, Phillips W; Samuelson, John

2013-09-03

Coccidia are protozoan parasites that cause significant human disease and are of major agricultural importance. Cryptosporidium spp. cause diarrhea in humans and animals, while Toxoplasma causes disseminated infections in fetuses and untreated AIDS patients. Eimeria is a major pathogen of commercial chickens. Oocysts, which are the infectious form of Cryptosporidium and Eimeria and one of two infectious forms of Toxoplasma (the other is tissue cysts in undercooked meat), have a multilayered wall. Recently we showed that the inner layer of the oocyst walls of Toxoplasma and Eimeria is a porous scaffold of fibers of β-1,3-glucan, which are also present in fungal walls but are absent from Cryptosporidium oocyst walls. Here we present evidence for a structural role for lipids in the oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. Briefly, oocyst walls of each organism label with acid-fast stains that bind to lipids in the walls of mycobacteria. Polyketide synthases similar to those that make mycobacterial wall lipids are abundant in oocysts of Toxoplasma and Eimeria and are predicted in Cryptosporidium. The outer layer of oocyst wall of Eimeria and the entire oocyst wall of Cryptosporidium are dissolved by organic solvents. Oocyst wall lipids are complex mixtures of triglycerides, some of which contain polyhydroxy fatty acyl chains like those present in plant cutin or elongated fatty acyl chains like mycolic acids. We propose a two-layered model of the oocyst wall (glucan and acid-fast lipids) that resembles the two-layered walls of mycobacteria (peptidoglycan and acid-fast lipids) and plants (cellulose and cutin). Oocysts, which are essential for the fecal-oral spread of coccidia, have a wall that is thought responsible for their survival in the environment and for their transit through the stomach and small intestine. While oocyst walls of Toxoplasma and Eimeria are strengthened by a porous scaffold of fibrils of β-1,3-glucan and by proteins cross

Molecular discriminators using single wall carbon nanotubes

NASA Astrophysics Data System (ADS)

Bhattacharyya, Tamoghna; Dasgupta, Anjan Kr; Ranjan Ray, Nihar; Sarkar, Sabyasachi

2012-09-01

The interaction between single wall carbon nanotubes (SWNTs) and amphiphilic molecules has been studied in a solid phase. SWNTs are allowed to interact with different amphiphilic probes (e.g. lipids) in a narrow capillary interface. Contact between strong hydrophobic and amphiphilic interfaces leads to a molecular restructuring of the lipids at the interface. The geometry of the diffusion front and the rate and the extent of diffusion of the interface are dependent on the structure of the lipid at the interface. Lecithin having a linear tail showed greater mobility of the interface as compared to a branched tail lipid like dipalmitoyl phosphatidylcholine, indicating the hydrophobic interaction between single wall carbon nanotube core and the hydrophobic tail of the lipid. Solid phase interactions between SWNT and lipids can thus become a very simple but efficient means of discriminating amphiphilic molecules in general and lipids in particular.

Modification of Helicobacter pylori peptidoglycan enhances NOD1 activation and promotes cancer of the stomach

DOE PAGES

Suarez, Giovanni; Romero-Gallo, Judith; Piazuelo, M. Blanca; ...

2015-03-02

Helicobacter pylori is the strongest known risk factor for gastric carcinogenesis. One cancer-linked locus is the cag pathogenicity island, which translocates components of peptidoglycan (PGN) into host cells. NOD1 is an intracellular immune receptor that senses PGN from Gram-negative bacteria and responds by inducing autophagy and activating NF-κB, leading to inflammation-mediated bacterial clearance; however chronic pathogens can evade NOD1-mediated clearance by altering PGN structure. We previously demonstrated that the H. pylori cag+ strain 7.13 rapidly induces gastric cancer in Mongolian gerbils. Using 2D-DIGE and mass spectrometry, we identified a novel mutation within the gene encoding the peptidoglycan deacetylase PgdA; therefore,more » we sought to define the role of H. pylori PgdA in NOD1-dependent activation of NF-κB, inflammation, and cancer. Co-culture of H. pylori strain 7.13 or its pgdA$-$ isogenic mutant with AGS gastric epithelial cells or HEK293 epithelial cells expressing a NF-κB reporter revealed that pgdA inactivation significantly decreased NOD1-dependent NF-κB activation and autophagy. Infection of Mongolian gerbils with an H. pylori pgdA$-$ mutant strain led to significantly decreased levels of inflammation and malignant lesions in the stomach; however, pre-activation of NOD1 prior to bacterial challenge reciprocally suppressed inflammation and cancer in response to wild-type H. pylori. Expression of NOD1 differs in human gastric cancer specimens compared to non-cancer samples harvested from the same patients. In conclusion, these results indicate that PGN deacetylation plays an important role in modulating host inflammatory responses to H. pylori, allowing the bacteria to persist and induce carcinogenic consequences in the gastric niche.« less

The Cell Wall Integrity Signaling Pathway and Its Involvement in Secondary Metabolite Production.

PubMed

Valiante, Vito

2017-12-06

The fungal cell wall is the external and first layer that fungi use to interact with the environment. Every stress signal, before being translated into an appropriate stress response, needs to overtake this layer. Many signaling pathways are involved in translating stress signals, but the cell wall integrity (CWI) signaling pathway is the one responsible for the maintenance and biosynthesis of the fungal cell wall. In fungi, the CWI signal is composed of a mitogen-activated protein kinase (MAPK) module. After the start of the phosphorylation cascade, the CWI signal induces the expression of cell-wall-related genes. However, the function of the CWI signal is not merely the activation of cell wall biosynthesis, but also the regulation of expression and production of specific molecules that are used by fungi to better compete in the environment. These molecules are normally defined as secondary metabolites or natural products. This review is focused on secondary metabolites affected by the CWI signal pathway with a special focus on relevant natural products such as melanins, mycotoxins, and antibacterial compounds.

Noise characteristics of single-walled carbon nanotube network transistors.

PubMed

Kim, Un Jeong; Kim, Kang Hyun; Kim, Kyu Tae; Min, Yo-Sep; Park, Wanjun

2008-07-16

The noise characteristics of randomly networked single-walled carbon nanotubes grown directly by plasma enhanced chemical vapor deposition (PECVD) are studied with field effect transistors (FETs). Due to the geometrical complexity of nanotube networks in the channel area and the large number of tube-tube/tube-metal junctions, the inverse frequency, 1/f, dependence of the noise shows a similar level to that of a single single-walled carbon nanotube transistor. Detailed analysis is performed with the parameters of number of mobile carriers and mobility in the different environment. This shows that the change in the number of mobile carriers resulting in the mobility change due to adsorption and desorption of gas molecules (mostly oxygen molecules) to the tube surface is a key factor in the 1/f noise level for carbon nanotube network transistors.

MreB filaments align along greatest principal membrane curvature to orient cell wall synthesis

PubMed Central

Szwedziak, Piotr; Wong, Felix; Schaefer, Kaitlin; Izoré, Thierry; Renner, Lars D; Holmes, Matthew J; Sun, Yingjie; Bisson-Filho, Alexandre W; Walker, Suzanne; Amir, Ariel; Löwe, Jan

2018-01-01

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape. PMID:29469806

Single wall carbon nanotubes dispersion study of different dye molecules and chitosan

NASA Astrophysics Data System (ADS)

Ramli, Muhammad M.; Isa, Siti S. Mat; Abdullah, M. M. A. B.; Murad, S. A. Z.

2017-09-01

Carbon Nanotubes (CNTs) is known for their hydrophobicity ability. However, this ability can become the bottleneck for the application of CNTs where a highly dispersion of materials are needed. In this project, different dispersing agents were investigated namely dye molecules and chitosan. Three different dyes are studied with different concentration, including 0.05 % of chitosan. The dispersion quality is determined by examining through UV-Vis-NIR. The best dispersion quality investigated here is when the concentration of dye molecules is higher, which is around 2.5 mM.

Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins).

PubMed

Pratt, R F

2008-07-01

The DD-peptidase enzymes (penicillin-binding proteins) catalyze the final transpeptidation reaction of bacterial cell wall (peptidoglycan) biosynthesis. Although there is now much structural information available about these enzymes, studies of their activity as enzymes lag. It is now established that representatives of two low-molecular-mass classes of DD-peptidases recognize elements of peptidoglycan structure and rapidly react with substrates and inhibitors incorporating these elements. No members of other DD-peptidase classes, including the high-molecular-mass enzymes, essential for bacterial growth, appear to interact strongly with any particular elements of peptidoglycan structure. Rational design of inhibitors for these enzymes is therefore challenging.

Vapor Wall Deposition in Chambers: Theoretical Considerations

NASA Astrophysics Data System (ADS)

McVay, R.; Cappa, C. D.; Seinfeld, J.

2014-12-01

In order to constrain the effects of vapor wall deposition on measured secondary organic aerosol (SOA) yields in laboratory chambers, Zhang et al. (2014) varied the seed aerosol surface area in toluene oxidation and observed a clear increase in the SOA yield with increasing seed surface area. Using a coupled vapor-particle dynamics model, we examine the extent to which this increase is the result of vapor wall deposition versus kinetic limitations arising from imperfect accommodation of organic species into the particle phase. We show that a seed surface area dependence of the SOA yield is present only when condensation of vapors onto particles is kinetically limited. The existence of kinetic limitation can be predicted by comparing the characteristic timescales of gas-phase reaction, vapor wall deposition, and gas-particle equilibration. The gas-particle equilibration timescale depends on the gas-particle accommodation coefficient αp. Regardless of the extent of kinetic limitation, vapor wall deposition depresses the SOA yield from that in its absence since vapor molecules that might otherwise condense on particles deposit on the walls. To accurately extrapolate chamber-derived yields to atmospheric conditions, both vapor wall deposition and kinetic limitations must be taken into account.

Positioning cell wall synthetic complexes by the bacterial morphogenetic proteins MreB and MreD.

PubMed

White, Courtney L; Kitich, Aleksandar; Gober, James W

2010-05-01

In Caulobacter crescentus, intact cables of the actin homologue, MreB, are required for the proper spatial positioning of MurG which catalyses the final step in peptidoglycan precursor synthesis. Similarly, in the periplasm, MreC controls the spatial orientation of the penicillin binding proteins and a lytic transglycosylase. We have now found that MreB cables are required for the organization of several other cytosolic murein biosynthetic enzymes such as MraY, MurB, MurC, MurE and MurF. We also show these proteins adopt a subcellular pattern of localization comparable to MurG, suggesting the existence of cytoskeletal-dependent interactions. Through extensive two-hybrid analyses, we have now generated a comprehensive interaction map of components of the bacterial morphogenetic complex. In the cytosol, this complex contains both murein biosynthetic enzymes and morphogenetic proteins, including RodA, RodZ and MreD. We show that the integral membrane protein, MreD, is essential for lateral peptidoglycan synthesis, interacts with the precursor synthesizing enzymes MurG and MraY, and additionally, determines MreB localization. Our results suggest that the interdependent localization of MreB and MreD functions to spatially organize a complex of peptidoglycan precursor synthesis proteins, which is required for propagation of a uniform cell shape and catalytically efficient peptidoglycan synthesis.

Use of the gram stain in microbiology.

PubMed

Beveridge, T J

2001-05-01

The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be "gram-positive," whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be "gram-negative." This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.

Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC).

PubMed

Pazos, Manuel; Otten, Christian; Vollmer, Waldemar

2018-03-20

Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by 'attacking' enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius . The TLC method can also monitor the digestion of undecaprenyl (pyro)phosphate, whereas the HPLC method allows to separate the di-, mono- or unphosphorylated disaccharide pentapeptide products of lipid II.

Cytoplasmic peptidoglycan intermediate levels in Staphylococcus aureus.

PubMed

Vemula, Harika; Ayon, Navid J; Gutheil, William G

2016-02-01

Intracellular cytoplasmic peptidoglycan (PG) intermediate levels were determined in Staphylococcus aureus during log-phase growth in enriched media. Levels of UDP-linked intermediates were quantitatively determined using ion pairing LC-MS/MS in negative mode, and amine intermediates were quantitatively determined stereospecifically as their Marfey's reagent derivatives in positive mode. Levels of UDP-linked intermediates in S. aureus varied from 1.4 μM for UDP-GlcNAc-Enolpyruvyate to 1200 μM for UDP-MurNAc. Levels of amine intermediates (L-Ala, D-Ala, D-Ala-D-Ala, L-Glu, D-Glu, and L-Lys) varied over a range of from 860 μM for D-Ala-D-Ala to 30-260 mM for the others. Total PG was determined from the D-Glu content of isolated PG, and used to estimate the rate of PG synthesis (in terms of cytoplasmic metabolite flux) as 690 μM/min. The total UDP-linked intermediates pool (2490 μM) is therefore sufficient to sustain growth for 3.6 min. Comparison of UDP-linked metabolite levels with published pathway enzyme characteristics demonstrates that enzymes on the UDP-branch range from >80% saturation for MurA, Z, and C, to <5% saturation for MurB. Metabolite levels were compared with literature values for Escherichia coli, with the major difference in UDP-intermediates being the level of UDP-MurNAc, which was high in S. aureus (1200 μM) and low in E. coli (45 μM). Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Theoretical Investigation of Single-Molecule Sensing Using Nanotube-Enhanced Circular Dichroism.

PubMed

Silva, Jaime; Milne, Bruce F; Nogueira, Fernando

2018-06-19

First-principles calculations have been used to investigate the potential use of circular dichroism (CD) spectroscopy in single-molecule sensing. Using a real-space implementation of time-dependent density functional theory (TDDFT), several systems involving single-walled carbon nanotubes (SWCNT) and small molecules have been studied to evaluate their CD response. Large induced CD (ICD) effects, differing for each test molecule, were observed in all SWCNT-molecule complexes. As the SWCNT used in this study shows no intrinsic CD response, the ICD spectra are the result of interaction with the small molecules. This finding is general and independent of the (a)chiral nature of the adsorbed molecule. Our results indicate that it is possible to design a system that uses SWCNT for detection of molecules using the change in CD spectrum of the system induced by adsorption of the molecule onto the SWCNT surface.

Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC)

PubMed Central

Pazos, Manuel; Otten, Christian; Vollmer, Waldemar

2018-01-01

Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by ‘attacking’ enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius. The TLC method can also monitor the digestion of undecaprenyl (pyro)phosphate, whereas the HPLC method allows to separate the di-, mono- or unphosphorylated disaccharide pentapeptide products of lipid II. PMID:29651453

Anti-staphylococcal activities of lysostaphin and LytM catalytic domain

PubMed Central

2012-01-01

Background Lysostaphin and the catalytic domain of LytM cleave pentaglycine crossbridges of Staphylococcus aureus peptidoglycan. The bacteriocin lysostaphin is secreted by Staphylococcus simulans biovar staphylolyticus and directed against the cell walls of competing S. aureus. LytM is produced by S. aureus as a latent autolysin and can be activated in vitro by the removal of an N-terminal domain and occluding region. Results We compared the efficacies of the lysostaphin and LytM catalytic domains using a newly developed model of chronic S. aureus infected eczema. Lysostaphin was effective, like in other models. In contrast, LytM was not significantly better than control. The different treatment outcomes could be correlated with in vitro properties of the proteins, including proteolytic stability, affinity to cell wall components other than peptidoglycan, and sensitivity to the ionic milieu. Conclusions Although lysostaphin and LytM cleave the same peptide bond in the peptidoglycan, the two enzymes have very different environmental requirements what is reflected in their contrasting performance in mouse eczema model. PMID:22672475

Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome.

PubMed

Faheem, Muhammad; Martins-de-Sa, Diogo; Vidal, Julia F D; Álvares, Alice C M; Brandão-Neto, José; Bird, Louise E; Tully, Mark D; von Delft, Frank; Souto, Betulia M; Quirino, Betania F; Freitas, Sonia M; Barbosa, João Alexandre R G

2016-12-09

A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a K b of 1.8 × 10 5  M -1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space R g of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features.

Quantum Behavior of Water Molecules Confined to Nanocavities in Gemstones.

PubMed

Gorshunov, Boris P; Zhukova, Elena S; Torgashev, Victor I; Lebedev, Vladimir V; Shakurov, Gil'man S; Kremer, Reinhard K; Pestrjakov, Efim V; Thomas, Victor G; Fursenko, Dimitry A; Dressel, Martin

2013-06-20

When water is confined to nanocavities, its quantum mechanical behavior can be revealed by terahertz spectroscopy. We place H2O molecules in the nanopores of a beryl crystal lattice and observe a rich and highly anisotropic set of absorption lines in the terahertz spectral range. Two bands can be identified, which originate from translational and librational motions of the water molecule isolated within the cage; they correspond to the analogous broad bands in liquid water and ice. In the present case of well-defined and highly symmetric nanocavities, the observed fine structure can be explained by macroscopic tunneling of the H2O molecules within a six-fold potential caused by the interaction of the molecule with the cavity walls.

Environmental and Biofilm-dependent Changes in a Bacillus cereus Secondary Cell Wall Polysaccharide*

PubMed Central

Candela, Thomas; Maes, Emmanuel; Garénaux, Estelle; Rombouts, Yoann; Krzewinski, Frédéric; Gohar, Michel; Guérardel, Yann

2011-01-01

Bacterial species from the Bacillus genus, including Bacillus cereus and Bacillus anthracis, synthesize secondary cell wall polymers (SCWP) covalently associated to the peptidoglycan through a phospho-diester linkage. Although such components were observed in a wide panel of B. cereus and B. anthracis strains, the effect of culture conditions or of bacterial growth state on their synthesis has never been addressed. Herein we show that B. cereus ATCC 14579 can synthesize not only one, as previously reported, but two structurally unrelated secondary cell wall polymers (SCWP) polysaccharides. The first of these SCWP, →4)[GlcNAc(β1–3)]GlcNAc(β1–6)[Glc(β1-3)][ManNAc(α1–4)]GalNAc(α1–4)ManNAc(β1→, although presenting an original sequence, fits to the already described the canonical sequence motif of SCWP. In contrast, the second polysaccharide was made up by a totally original sequence, →6)Gal(α1–2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(β1→, which no equivalent has ever been identified in the Bacillus genus. In addition, we established that the syntheses of these two polysaccharides were differently regulated. The first one is constantly expressed at the surface of the bacteria, whereas the expression of the second is tightly regulated by culture conditions and growth states, planktonic, or biofilm. PMID:21784857

The secondary cell wall polysaccharide of Bacillus anthracis provides the specific binding ligand for the C-terminal cell wall-binding domain of two phage endolysins, PlyL and PlyG

PubMed Central

Ganguly, Jhuma; Low, Lieh Y; Kamal, Nazia; Saile, Elke; Forsberg, L Scott; Gutierrez-Sanchez, Gerardo; Hoffmaster, Alex R; Liddington, Robert; Quinn, Conrad P; Carlson, Russell W; Kannenberg, Elmar L

2013-01-01

Endolysins are bacteriophage enzymes that lyse their bacterial host for phage progeny release. They commonly contain an N-terminal catalytic domain that hydrolyzes bacterial peptidoglycan (PG) and a C-terminal cell wall-binding domain (CBD) that confers enzyme localization to the PG substrate. Two endolysins, phage lysin L (PlyL) and phage lysin G (PlyG), are specific for Bacillus anthracis. To date, the cell wall ligands for their C-terminal CBD have not been identified. We recently described structures for a number of secondary cell wall polysaccharides (SCWPs) from B. anthracis and B. cereus strains. They are covalently bound to the PG and are comprised of a -ManNAc-GlcNAc-HexNAc- backbone with various galactosyl or glucosyl substitutions. Surface plasmon resonance (SPR) showed that the endolysins PlyL and PlyG bind to the SCWP from B. anthracis (SCWPBa) with high affinity (i.e. in the μM range with dissociation constants ranging from 0.81 × 10−6 to 7.51 × 10−6 M). In addition, the PlyL and PlyG SCWPBa binding sites reside with their C-terminal domains. The dissociation constants for the interactions of these endolysins and their derived C-terminal domains with the SCWPBa were in the range reported for other protein–carbohydrate interactions. Our findings show that the SCWPBa is the ligand that confers PlyL and PlyG lysin binding and localization to the PG. PlyL and PlyG also bound the SCWP from B. cereus G9241 with comparable affinities to SCWPBa. No detectable binding was found to the SCWPs from B. cereus ATCC (American Type Culture Collection) 10987 and ATCC 14579, thus demonstrating specificity of lysin binding to SCWPs. PMID:23493680

Kinetic description of finite-wall catalysis for monatomic molecular recombination

NASA Astrophysics Data System (ADS)

Yano, Ryosuke; Suzuki, Kojiro

2011-11-01

In our previous study on hypothetical diatomic molecular dissociation and monatomic molecular recombination, A2 + M ↔ A + A + M [Yano et al., Phys. Fluids 21, 127101 (2009)], the interaction between the wall and A2* intermediates was not formulated. In this paper, we consider the effect of finite-wall catalysis on recombination of a monatomic molecule A via the interaction between the wall and A2*. According to the proposed Boltzmann model equation, the catalytic recombination rate depends on two quantities; the vibrational temperature and the translational temperature of A2* intermediates that are emitted from the wall. In particular, the translational temperature of A2* is related to its lifetime. In this paper, we investigate the change in the catalytic recombination rate of A upon changing the vibrational temperature of A2* intermediates that are emitted from the wall. As an object of analysis, the rarefied hypersonic flow around a cylinder with a finite wall-catalysis is considered using the proposed Boltzmann model equation. Numerical results confirm that a decrease in the vibrational temperature of A2* intermediates that are emitted from the wall results in an increase in recombination of A near the wall.

From cells to muropeptide structures in 24 h: Peptidoglycan mapping by UPLC-MS

PubMed Central

Kühner, Daniel; Stahl, Mark; Demircioglu, Dogan D.; Bertsche, Ute

2014-01-01

Peptidoglycan (PGN) is ubiquitous in nearly all bacterial species. The PGN sacculus protects the cells against their own internal turgor making PGN one of the most important targets for antibacterial treatment. Within the last sixty years PGN composition has been intensively studied by various methods. The breakthrough was the application of HPLC technology on the analysis of muropeptides. However, preparation of pure PGN relied on a very time consuming method of about one week. We established a purification protocol for both Gram-positive and Gram-negative bacteria which can be completely performed in plastic reaction tubes yielding pure muropeptides within 24 hours. The muropeptides can be analyzed by UPLC-MS, allowing their immediate determination. This new rapid method provides the feasibility to screen PGN composition even in high throughput, making it a highly useful tool for basic research as well as for the pharmaceutical industry. PMID:25510564

Adsorption of insulin peptide on charged single-walled carbon nanotubes: significant role of ordered water molecules.

PubMed

Shen, Jia-Wei; Wu, Tao; Wang, Qi; Kang, Yu; Chen, Xin

2009-06-02

Ordered hydration shells: The more ordered hydration shells outside the charged CNT surfaces prevent more compact adsorption of the peptide in the charged CNT systems [picture: see text], but peptide binding strengths on the charged CNT surfaces are stronger due to the electrostatic interaction.Studies of adsorption dynamics and stability for peptides/proteins on single-walled carbon nanotubes (SWNTs) are of great importance for a better understanding of the properties and nature of nanotube-based biosystems. Herein, the dynamics and mechanism of the adsorption of the insulin chain B peptide on different charged SWNTs are investigated by explicit solvent molecular dynamics simulations. The results show that all types of surfaces effectively attract the model peptide. Water molecules play a significant role in peptide adsorption on the surfaces of charged carbon nanotubes (CNTs). Compared to peptide adsorption on neutral CNT surfaces, the more ordered hydration shells outside the tube prevent more compact adsorption of the peptide in charged CNT systems. This shield effect leads to a smaller conformational change and van der Waals interaction between the peptide and surfaces, but peptide binding strengths on charged CNT surfaces are stronger than those on the neutral CNT surface due to the strong electrostatic interaction. The result of these simulations implies the possibility of improving the binding strength of peptides/proteins on CNT surfaces, as well as keeping the integrity of the peptide/protein conformation in peptide/protein-CNT complexes by charging the CNTs.

Extracellular Vesicle-Associated Transitory Cell Wall Components and Their Impact on the Interaction of Fungi with Host Cells.

PubMed

Nimrichter, Leonardo; de Souza, Marcio M; Del Poeta, Maurizio; Nosanchuk, Joshua D; Joffe, Luna; Tavares, Patricia de M; Rodrigues, Marcio L

2016-01-01

Classic cell wall components of fungi comprise the polysaccharides glucans and chitin, in association with glycoproteins and pigments. During the last decade, however, system biology approaches clearly demonstrated that the composition of fungal cell walls include atypical molecules historically associated with intracellular or membrane locations. Elucidation of mechanisms by which many fungal molecules are exported to the extracellular space suggested that these atypical components are transitorily located to the cell wall. The presence of extracellular vesicles (EVs) at the fungal cell wall and in culture supernatants of distinct pathogenic species suggested a highly functional mechanism of molecular export in these organisms. Thus, the passage of EVs through fungal cell walls suggests remarkable molecular diversity and, consequently, a potentially variable influence on the host antifungal response. On the basis of information derived from the proteomic characterization of fungal EVs from the yeasts Cryptoccocus neoformans and Candida albicans and the dimorphic fungi Histoplasma capsulatum and Paracoccidioides brasiliensis, our manuscript is focused on the clear view that the fungal cell wall is much more complex than previously thought.

Simulation of diatomic gas-wall interaction and accommodation coefficients for negative ion sources and accelerators.

PubMed

Sartori, E; Brescaccin, L; Serianni, G

2016-02-01

Particle-wall interactions determine in different ways the operating conditions of plasma sources, ion accelerators, and beams operating in vacuum. For instance, a contribution to gas heating is given by ion neutralization at walls; beam losses and stray particle production-detrimental for high current negative ion systems such as beam sources for fusion-are caused by collisional processes with residual gas, with the gas density profile that is determined by the scattering of neutral particles at the walls. This paper shows that Molecular Dynamics (MD) studies at the nano-scale can provide accommodation parameters for gas-wall interactions, such as the momentum accommodation coefficient and energy accommodation coefficient: in non-isothermal flows (such as the neutral gas in the accelerator, coming from the plasma source), these affect the gas density gradients and influence efficiency and losses in particular of negative ion accelerators. For ideal surfaces, the computation also provides the angular distribution of scattered particles. Classical MD method has been applied to the case of diatomic hydrogen molecules. Single collision events, against a frozen wall or a fully thermal lattice, have been simulated by using probe molecules. Different modelling approximations are compared.

Simulation of diatomic gas-wall interaction and accommodation coefficients for negative ion sources and accelerators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sartori, E., E-mail: emanuele.sartori@igi.cnr.it; Serianni, G.; Brescaccin, L.

2016-02-15

Particle-wall interactions determine in different ways the operating conditions of plasma sources, ion accelerators, and beams operating in vacuum. For instance, a contribution to gas heating is given by ion neutralization at walls; beam losses and stray particle production—detrimental for high current negative ion systems such as beam sources for fusion—are caused by collisional processes with residual gas, with the gas density profile that is determined by the scattering of neutral particles at the walls. This paper shows that Molecular Dynamics (MD) studies at the nano-scale can provide accommodation parameters for gas-wall interactions, such as the momentum accommodation coefficient andmore » energy accommodation coefficient: in non-isothermal flows (such as the neutral gas in the accelerator, coming from the plasma source), these affect the gas density gradients and influence efficiency and losses in particular of negative ion accelerators. For ideal surfaces, the computation also provides the angular distribution of scattered particles. Classical MD method has been applied to the case of diatomic hydrogen molecules. Single collision events, against a frozen wall or a fully thermal lattice, have been simulated by using probe molecules. Different modelling approximations are compared.« less

Antibiotic Effects on Methicillin-Resistant Staphylococcus aureus Cytoplasmic Peptidoglycan Intermediate Levels and Evidence for Potential Metabolite Level Regulatory Loops.

PubMed

Vemula, Harika; Ayon, Navid J; Burton, Alloch; Gutheil, William G

2017-06-01

Cytoplasmic peptidoglycan (PG) precursor levels were determined in methicillin-resistant Staphylococcus aureus (MRSA) after exposure to several cell wall-targeting antibiotics. Three experiments were performed: (i) exposure to 4× MIC levels (acute); (ii) exposure to sub-MIC levels (subacute); (iii) a time course experiment of the effect of vancomycin. In acute exposure experiments, fosfomycin increased UDP-GlcNAc, as expected, and resulted in substantially lower levels of total UDP-linked metabolite accumulation relative to other pathway inhibitors, indicating reduced entry into this pathway. Upstream inhibitors (fosfomycin, d-cycloserine, or d-boroalanine) reduced UDP-MurNAc-pentapeptide levels by more than fourfold. Alanine branch inhibitors (d-cycloserine and d-boroalanine) reduced d-Ala-d-Ala levels only modestly (up to 4-fold) but increased UDP-MurNAc-tripeptide levels up to 3,000-fold. Downstream pathway inhibitors (vancomycin, bacitracin, moenomycin, and oxacillin) increased UDP-MurNAc-pentapeptide levels up to 350-fold and UDP-MurNAc-l-Ala levels up to 80-fold, suggesting reduced MurD activity by downstream inhibitor action. Sub-MIC exposures demonstrated effects even at 1/8× MIC which strongly paralleled acute exposure changes. Time course data demonstrated that UDP-linked intermediate levels respond rapidly to vancomycin exposure, with several intermediates increasing three- to sixfold within minutes. UDP-linked intermediate level changes were also multiphasic, with some increasing, some decreasing, and some increasing and then decreasing. The total (summed) UDP-linked intermediate pool increased by 1,475 μM/min during the first 10 min after vancomycin exposure, providing a revised estimate of flux in this pathway during logarithmic growth. These observations outline the complexity of PG precursor response to antibiotic exposure in MRSA and indicate likely sites of regulation (entry and MurD). Copyright © 2017 American Society for Microbiology.

Phosphorylated hydroxyethylamines as novel inhibitors of the bacterial cell wall biosynthesis enzymes MurC to MurF.

PubMed

Sova, Matej; Kovac, Andreja; Turk, Samo; Hrast, Martina; Blanot, Didier; Gobec, Stanislav

2009-12-01

Enzymes involved in the biosynthesis of bacterial peptidoglycan represent important targets for development of new antibacterial drugs. Among them, Mur ligases (MurC to MurF) catalyze the formation of the final cytoplasmic precursor UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid. We present the design, synthesis and biological evaluation of a series of phosphorylated hydroxyethylamines as new type of small-molecule inhibitors of Mur ligases. We show that the phosphate group attached to the hydroxyl moiety of the hydroxyethylamine core is essential for good inhibitory activity. The IC(50) values of these inhibitors were in the micromolar range, which makes them a promising starting point for the development of multiple inhibitors of Mur ligases as potential antibacterial agents. In addition, 1-(4-methoxyphenylsulfonamido)-3-morpholinopropan-2-yl dihydrogen phosphate 7a was discovered as one of the best inhibitors of MurE described so far.

Proteolytic Cascade for the Activation of the Insect Toll Pathway Induced by the Fungal Cell Wall Component

PubMed Central

Roh, Kyung-Baeg; Kim, Chan-Hee; Lee, Hanna; Kwon, Hyun-Mi; Park, Ji-Won; Ryu, Ji-Hwan; Kurokawa, Kenji; Ha, Nam-Chul; Lee, Won-Jae; Lemaitre, Bruno; Söderhäll, Kenneth; Lee, Bok-Luel

2009-01-01

The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-κB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Recently, we identified three serine proteases mediating Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative-binding protein 3 (GNBP3), a receptor for a major cell wall component β-1,3-glucan of fungi, and their order of activation have not been characterized yet. Here, we identified three serine proteases that are required for Toll activation by β-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, we show that injection of β-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal β-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation. PMID:19473968

Dynamic behaviors and transport properties of ethanol molecules in transmembrane cyclic peptide nanotubes.

PubMed

Li, Rui; Fan, Jianfen; Li, Hui; Yan, Xiliang; Yu, Yi

2015-07-07

Classical molecular dynamics simulations have been performed to investigate the dynamic behaviors and transport properties of ethanol molecules in transmembrane cyclic peptide nanotubes (CPNTs) with various radii, i.e., 8×(WL¯)n=3,4,5/POPE. The results show that ethanol molecules spontaneously fill the octa- and deca-CPNTs, but not the hexa-CPNT. In the octa-CPNT, ethanol molecules are trapped at individual gaps with their carbon skeletons perpendicular to the tube axis and hydroxyl groups towards the tube wall, forming a broken single-file chain. As the channel radius increases, ethanol molecules inside the deca-CPNT tend to form a tubular layer and the hydroxyl groups mainly stretch towards the tube axis. Computations of diffusion coefficients indicate that ethanol molecules in the octa-CPNT nearly lost their diffusion abilities, while those in the deca-CPNT diffuse as 4.5 times as in a (8, 8) carbon nanotube with a similar tube diameter. The osmotic and diffusion permeabilities (pf and pd, respectively) of the octa- and deca-CPNTs transporting ethanol were deduced for the first time. The distributions of the gauche and trans conformers of ethanol molecules in two CPNTs are quite similar, both with approximately 57% gauche conformers. The non-bonded interactions of channel ethanol with a CPNT wall and surrounding ethanol were explored. The potential of mean force elucidates the mechanism underlying the transporting characteristics of channel ethanol in a transmembrane CPNT.

Wall Conditioning Characterization in NSTX-U

NASA Astrophysics Data System (ADS)

Caron, D.; Soukhanovskii, V.; Scotti, F.; Weller, M.

2016-10-01

Impurities in tokamak plasmas can lead to disruptive instabilities due to radiative energy loss which impede access to high-confinements mode. One source of impurities in NSTX-U are water molecules trapped in graphite plasma facing components (PFCs), which make up the walls and divertors. Hydrogen and oxygen impurities are released into the plasma due to plasma surface interactions. Extreme ultraviolet (EUV) and visible spectrometers are used in conjunction with a residual gas analyzer (RGA) to characterize the source and amount of released impurities. A high resolution visible spectrometer measured H/D Balmer- α intensity ratio on the inner wall, the upper and lower divertors, and provided a hydrogen time history for shot-to-shot trends. The RGA provided partial pressure trends of masses 2 (H2) , 16 (O2) , and 18 (H2O). Trends of O VIII and C VI spectral line intensities from the core plasma were obtained from the EUV spectrometer. The trends are correlated with wall conditioning, namely helium glow discharge cleaning and boronization. Using these trends, impurity content monitoring and recommendations for wall conditioning can be implemented. Work supported by DOE under Contracts DE-AC52-07NA27344 and DE-AC02-09CH11466.

Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan.

PubMed

Sundarrajan, Sudarson; Raghupatil, Junjappa; Vipra, Aradhana; Narasimhaswamy, Nagalakshmi; Saravanan, Sanjeev; Appaiah, Chemira; Poonacha, Nethravathi; Desai, Srividya; Nair, Sandhya; Bhatt, Rajagopala Narayana; Roy, Panchali; Chikkamadaiah, Ravisha; Durgaiah, Murali; Sriram, Bharathi; Padmanabhan, Sriram; Sharma, Umender

2014-10-01

P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other β-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains. © 2014 The Authors.

Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a Uropathogenic Escherichia coli Isolate.

PubMed

Diao, Jingyu; Bouwman, Catrien; Yan, Donghong; Kang, Jing; Katakam, Anand K; Liu, Peter; Pantua, Homer; Abbas, Alexander R; Nickerson, Nicholas N; Austin, Cary; Reichelt, Mike; Sandoval, Wendy; Xu, Min; Whitfield, Chris; Kapadia, Sharookh B

2017-05-23

Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro , the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo Compared to the wild-type strain, the lpp mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for "group 2" capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic E. coli isolates. IMPORTANCE Uropathogenic E. coli (UPEC) isolates represent a significant cause of nosocomial urinary tract and bloodstream infections. Many UPEC isolates are resistant to serum killing. Here, we show that a major cell-envelope lipoprotein (murein lipoprotein) is required for serum resistance in vitro and for complement-mediated bacterial clearance in vivo This is mediated, in part, through a novel mechanism by which murein lipoprotein affects the proper assembly of a key component of the machinery involved in production of "group 2" capsules. The absence of murein lipoprotein results in impaired production of the capsule layer, a known participant in complement resistance. These results demonstrate an important role for

Peptidoglycan-associated lipoprotein (Pal) of Gram-negative bacteria: function, structure, role in pathogenesis and potential application in immunoprophylaxis.

PubMed

Godlewska, Renata; Wiśniewska, Katarzyna; Pietras, Zbigniew; Jagusztyn-Krynicka, Elzbieta Katarzyna

2009-09-01

The protein Pal (peptidoglycan-associated lipoprotein) is anchored in the outer membrane (OM) of Gram-negative bacteria and interacts with Tol proteins. Tol-Pal proteins form two complexes: the first is composed of three inner membrane Tol proteins (TolA, TolQ and TolR); the second consists of the TolB and Pal proteins linked to the cell's OM. These complexes interact with one another forming a multiprotein membrane-spanning system. It has recently been demonstrated that Pal is essential for bacterial survival and pathogenesis, although its role in virulence has not been clearly defined. This review summarizes the available data concerning the structure and function of Pal and its role in pathogenesis.

Characterization of a Glucosamine/Glucosaminide N-Acetyltransferase of Clostridium acetobutylicum▿†

PubMed Central

Reith, Jan; Mayer, Christoph

2011-01-01

Many bacteria, in particular Gram-positive bacteria, contain high proportions of non-N-acetylated amino sugars, i.e., glucosamine (GlcN) and/or muramic acid, in the peptidoglycan of their cell wall, thereby acquiring resistance to lysozyme. However, muramidases with specificity for non-N-acetylated peptidoglycan have been characterized as part of autolytic systems such as of Clostridium acetobutylicum. We aim to elucidate the recovery pathway for non-N-acetylated peptidoglycan fragments and present here the identification and characterization of an acetyltransferase of novel specificity from C. acetobutylicum, named GlmA (for glucosamine/glucosaminide N-acetyltransferase). The enzyme catalyzes the specific transfer of an acetyl group from acetyl coenzyme A to the primary amino group of GlcN, thereby generating N-acetylglucosamine. GlmA is also able to N-acetylate GlcN residues at the nonreducing end of glycosides such as (partially) non-N-acetylated peptidoglycan fragments and β-1,4-glycosidically linked chitosan oligomers. Km values of 114, 64, and 39 μM were determined for GlcN, (GlcN)2, and (GlcN)3, respectively, and a 3- to 4-fold higher catalytic efficiency was determined for the di- and trisaccharides. GlmA is the first cloned and biochemically characterized glucosamine/glucosaminide N-acetyltransferase and a member of the large GCN5-related N-acetyltransferases (GNAT) superfamily of acetyltransferases. We suggest that GlmA is required for the recovery of non-N-acetylated muropeptides during cell wall rescue in C. acetobutylicum. PMID:21784938

Reactive oxygen species, essential molecules, during plant-pathogen interactions.

PubMed

Camejo, Daymi; Guzmán-Cedeño, Ángel; Moreno, Alexander

2016-06-01

Reactive oxygen species (ROS) are continually generated as a consequence of the normal metabolism in aerobic organisms. Accumulation and release of ROS into cell take place in response to a wide variety of adverse environmental conditions including salt, temperature, cold stresses and pathogen attack, among others. In plants, peroxidases class III, NADPH oxidase (NOX) locates in cell wall and plasma membrane, respectively, may be mainly enzymatic systems involving ROS generation. It is well documented that ROS play a dual role into cells, acting as important signal transduction molecules and as toxic molecules with strong oxidant power, however some aspects related to its function during plant-pathogen interactions remain unclear. This review focuses on the principal enzymatic systems involving ROS generation addressing the role of ROS as signal molecules during plant-pathogen interactions. We described how the chloroplasts, mitochondria and peroxisomes perceive the external stimuli as pathogen invasion, and trigger resistance response using ROS as signal molecule. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Selective degradation of the recalcitrant cell wall of Scenedesmus quadricauda CASA CC202.

PubMed

Reshma, Ragini; Arumugam, Muthu

2017-10-01

An eco-friendly cell wall digestion strategy was developed to enhance the availability of nutritionally important bio molecules of edible microalgae and exploit them for cloning, transformation, and expression of therapeutic proteins. Microalgae are the source for many nutritionally important bioactive compounds and potential drugs. Even though edible microalgae are rich in nutraceutical, bioavailability of all these molecules is very less due to their rigid recalcitrant cell wall. For example, the cell wall of Scenedesmus quadricauda CASA CC202 is made up of three layers comprising of rigid outer pectin and inner cellulosic layer separated by a thin middle layer. In the present investigation, a comprehensive method has been developed for the selective degradation of S. quadricauda CASA CC202 cell wall, by employing both mechanical and enzymatic treatments. The efficiency of cell wall removal was evaluated by measuring total reducing sugar (TRS), tannic acid-ferric chloride staining, calcoflour white staining, scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR) analysis. It was confirmed that the yield of TRS increased from 129.82 mg/g in 14 h from pectinase treatment alone to 352.44 mg/g by combined sonication and enzymatic treatment within 12 h. As a result, the combination method was found to be effective for the selective degradation of S. quadricauda CASA CC202 cell wall. This study will form a base for our future works, where this will help to enhance the digestibility and availability of nutraceutically important proteins.

Teaching Introductory Mineralogy With the GeoWall

NASA Astrophysics Data System (ADS)

Anderson, C. D.; Haymon, R. M.

2003-12-01

Mineralogy, like many topics in Earth Sciences, contains inherently three-dimensional topics that are difficult to teach. Concepts such as crystal symmetry and forms, Miller indices, the polymerization of silica tetrahedra and resulting structures of silicate mineral groups, and the interaction of light and minerals are particularly difficult. Two-dimensional diagrams are limited in their effectiveness, and physical models, while effective, are expensive and do not work as well in large class settings. The GeoWall system brings the effectiveness of physical models to the large classroom. In Fall 2003, we will integrate the GeoWall into our introductory mineralogy classes at UCSB using a combination of commercial software, atomic structure models available on the web, and custom content created in-house. The commercial software SHAPE (www.shapesoftware.com) allows users to build and display crystal shapes and their symmetry. Though not designed for the GeoWall, the software's stereopair display mode works perfectly on the system. Using the Chime web browser plug-in (www.mdl.com), computer models of silicate minerals available from the Virtual Museum of Minerals and Molecules (www.soils.umn.eduvirtual_museum) provide an interactive display of silicate mineral structure that illustrates the tetrahedral framework. Again, while not developed for the GeoWall, the Chime plug-in works seamlessly with the GeoWall hardware. 3-D GeoWall images that display light paths through minerals, and reveal relationships between crystal symmetry and optical indicatrix properties, have been developed in-house using a combination of SHAPE and 3D modeling software. The 3-D GeoWall images should convey in an instant these difficult concepts that students historically have struggled to visualize. Initial assessment of the GeoWall's effectiveness as a mineralogy teaching aid at UCSB in Fall 2003 will come from the instructor's impressions and by comparing test scores with classes from

Contactless experiments on individual DNA molecules show no evidence for molecular wire behavior.

PubMed

Gómez-Navarro, C; Moreno-Herrero, F; de Pablo, P J; Colchero, J; Gómez-Herrero, J; Baró, A M

2002-06-25

A fundamental requirement for a molecule to be considered a molecular wire (MW) is the ability to transport electrical charge with a reasonably low resistance. We have carried out two experiments that measure first, the charge transfer from an electrode to the molecule, and second, the dielectric response of the MW. The latter experiment requires no contacts to either end of the molecule. From our experiments we conclude that adsorbed individual DNA molecules have a resistivity similar to mica, glass, and silicon oxide substrates. Therefore adsorbed DNA is not a conductor, and it should not be considered as a viable candidate for MW applications. Parallel studies on other nanowires, including single-walled carbon nanotubes, showed conductivity as expected.

Genes Sufficient for Synthesizing Peptidoglycan are Retained in Gymnosperm Genomes, and MurE from Larix gmelinii can Rescue the Albino Phenotype of Arabidopsis MurE Mutation.

PubMed

Lin, Xiaofei; Li, Ningning; Kudo, Hiromi; Zhang, Zhe; Li, Jinyu; Wang, Li; Zhang, Wenbo; Takechi, Katsuaki; Takano, Hiroyoshi

2017-03-01

The endosymbiotic theory states that plastids are derived from a single cyanobacterial ancestor that possessed a cell wall. Peptidoglycan (PG), the main component of the bacteria cell wall, gradually degraded during plastid evolution. PG-synthesizing Mur genes have been found to be retained in the genomes of basal streptophyte plants, although many of them have been lost from the genomes of angiosperms. The enzyme encoded by bacterial MurE genes catalyzes the formation of the UDP-N-acetylmuramic acid (UDP-MurNAc) tripeptide in bacterial PG biosynthesis. Knockout of the MurE gene in the moss Physcomitrella patens resulted in defects of chloroplast division, whereas T-DNA-tagged mutants of Arabidopsis thaliana for MurE revealed inhibition of chloroplast development but not of plastid division, suggesting that AtMurE is functionally divergent from the bacterial and moss MurE proteins. Here, we could identify 10 homologs of bacterial Mur genes, including MurE, in the recently sequenced genomes of Picea abies and Pinus taeda, suggesting the retention of the plastid PG system in gymnosperms. To investigate the function of gymnosperm MurE, we isolated an ortholog of MurE from the larch, Larix gmelinii (LgMurE) and confirmed its presence as a single copy per genome, as well as its abundant expression in the leaves of larch seedlings. Analysis with a fusion protein combining green fluorescent protein and LgMurE suggested that it localizes in chloroplasts. Cross-species complementation assay with MurE mutants of A. thaliana and P. patens showed that the expression of LgMurE cDNA completely rescued the albefaction defects in A. thaliana but did not rescue the macrochloroplast phenotype in P. patens. The evolution of plastid PG and the mechanism behind the functional divergence of MurE genes are discussed in the context of information about plant genomes at different evolutionary stages. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of

Discovery of Novel Cell Wall-Active Compounds Using PywaC, a Sensitive Reporter of Cell Wall Stress, in the Model Gram-Positive Bacterium Bacillus subtilis

PubMed Central

Czarny, T. L.; Perri, A. L.; French, S.

2014-01-01

The emergence of antibiotic resistance in recent years has radically reduced the clinical efficacy of many antibacterial treatments and now poses a significant threat to public health. One of the earliest studied well-validated targets for antimicrobial discovery is the bacterial cell wall. The essential nature of this pathway, its conservation among bacterial pathogens, and its absence in human biology have made cell wall synthesis an attractive pathway for new antibiotic drug discovery. Herein, we describe a highly sensitive screening methodology for identifying chemical agents that perturb cell wall synthesis, using the model of the Gram-positive bacterium Bacillus subtilis. We report on a cell-based pilot screen of 26,000 small molecules to look for cell wall-active chemicals in real time using an autonomous luminescence gene cluster driven by the promoter of ywaC, which encodes a guanosine tetra(penta)phosphate synthetase that is expressed under cell wall stress. The promoter-reporter system was generally much more sensitive than growth inhibition testing and responded almost exclusively to cell wall-active antibiotics. Follow-up testing of the compounds from the pilot screen with secondary assays to verify the mechanism of action led to the discovery of 9 novel cell wall-active compounds. PMID:24687489

Measurements of Gas-Wall Partitioning of Oxidized Species in Environmental Smog Chambers and Teflon Sampling Lines

NASA Astrophysics Data System (ADS)

Krechmer, J.; Pagonis, D.; Ziemann, P. J.; Jimenez, J. L.

2015-12-01

Environmental "smog" chambers have played an integral role in atmospheric aerosol research for decades. Recently, many works have demonstrated that the loss of gas-phase material to fluorinated ethylene propylene (FEP) chamber walls can have significant effects on secondary organic aerosol (SOA) yield results. The effects of gas-wall partitioning on highly oxidized species is still controversial, however. In this work we performed a series of experiments examining the losses of oxidized gas-phase compounds that were generated in-situ­ in an environmental chamber. The loss of species to the walls was measured using three chemical ionization mass spectrometry techniques: proton-transfer-reaction (PTR), nitrate (NO3-) ion, and iodide (I-). Many oxidized species have wall loss timescales ranging between 15 to 45 minutes and scale according to the molecule's estimated saturation concentration c* and functional groups. By comparing results of the different techniques, and in particular by the use of the "wall-less" NO3- source, we find that measuring species with high chamber wall-loss rates is complicated by the use of a standard ion-molecule reaction (IMR) region, as well as long Teflon sampling lines, which can be important sinks for gas-phase species. This effect is observed even for semi-volatile species and could have significant effects on ambient sampling techniques that make highly time-resolved measurements using long sampling lines, such as eddy covariance measurements.

Process for separating metallic from semiconducting single-walled carbon nanotubes

NASA Technical Reports Server (NTRS)

Sun, Ya-Ping (Inventor)

2008-01-01

A method for separating semiconducting single-walled carbon nanotubes from metallic single-walled carbon nanotubes is disclosed. The method utilizes separation agents that preferentially associate with semiconducting nanotubes due to the electrical nature of the nanotubes. The separation agents are those that have a planar orientation, .pi.-electrons available for association with the surface of the nanotubes, and also include a soluble portion of the molecule. Following preferential association of the separation agent with the semiconducting nanotubes, the agent/nanotubes complex is soluble and can be solubilized with the solution enriched in semiconducting nanotubes while the residual solid is enriched in metallic nanotubes.

Role of Two Cell Wall Amidases in Septal Junction and Nanopore Formation in the Multicellular Cyanobacterium Anabaena sp. PCC 7120

PubMed Central

Bornikoel, Jan; Carrión, Alejandro; Fan, Qing; Flores, Enrique; Forchhammer, Karl; Mariscal, Vicente; Mullineaux, Conrad W.; Perez, Rebeca; Silber, Nadine; Wolk, C. Peter; Maldener, Iris

2017-01-01

Filamentous cyanobacteria have developed a strategy to perform incompatible processes in one filament by differentiating specialized cell types, N2-fixing heterocysts and CO2-fixing, photosynthetic, vegetative cells. These bacteria can be considered true multicellular organisms with cells exchanging metabolites and signaling molecules via septal junctions, involving the SepJ and FraCD proteins. Previously, it was shown that the cell wall lytic N-acetylmuramyl-L-alanine amidase, AmiC2, is essential for cell–cell communication in Nostoc punctiforme. This enzyme perforates the septal peptidoglycan creating an array of nanopores, which may be the framework for septal junction complexes. In Anabaena sp. PCC 7120, two homologs of AmiC2, encoded by amiC1 and amiC2, were identified and investigated in two different studies. Here, we compare the function of both AmiC proteins by characterizing different Anabaena amiC mutants, which was not possible in N. punctiforme, because there the amiC1 gene could not be inactivated. This study shows the different impact of each protein on nanopore array formation, the process of cell–cell communication, septal protein localization, and heterocyst differentiation. Inactivation of either amidase resulted in significant reduction in nanopore count and in the rate of fluorescent tracer exchange between neighboring cells measured by FRAP analysis. In an amiC1 amiC2 double mutant, filament morphology was affected and heterocyst differentiation was abolished. Furthermore, the inactivation of amiC1 influenced SepJ localization and prevented the filament-fragmentation phenotype that is characteristic of sepJ or fraC fraD mutants. Our findings suggest that both amidases are to some extent redundant in their function, and describe a functional relationship of AmiC1 and septal proteins SepJ and FraCD. PMID:28929086

Role of Two Cell Wall Amidases in Septal Junction and Nanopore Formation in the Multicellular Cyanobacterium Anabaena sp. PCC 7120.

PubMed

Bornikoel, Jan; Carrión, Alejandro; Fan, Qing; Flores, Enrique; Forchhammer, Karl; Mariscal, Vicente; Mullineaux, Conrad W; Perez, Rebeca; Silber, Nadine; Wolk, C Peter; Maldener, Iris

2017-01-01

Filamentous cyanobacteria have developed a strategy to perform incompatible processes in one filament by differentiating specialized cell types, N 2 -fixing heterocysts and CO 2 -fixing, photosynthetic, vegetative cells. These bacteria can be considered true multicellular organisms with cells exchanging metabolites and signaling molecules via septal junctions, involving the SepJ and FraCD proteins. Previously, it was shown that the cell wall lytic N -acetylmuramyl-L-alanine amidase, AmiC2, is essential for cell-cell communication in Nostoc punctiforme . This enzyme perforates the septal peptidoglycan creating an array of nanopores, which may be the framework for septal junction complexes. In Anabaena sp. PCC 7120, two homologs of AmiC2, encoded by amiC1 and amiC2 , were identified and investigated in two different studies. Here, we compare the function of both AmiC proteins by characterizing different Anabaena amiC mutants, which was not possible in N. punctiforme , because there the amiC1 gene could not be inactivated. This study shows the different impact of each protein on nanopore array formation, the process of cell-cell communication, septal protein localization, and heterocyst differentiation. Inactivation of either amidase resulted in significant reduction in nanopore count and in the rate of fluorescent tracer exchange between neighboring cells measured by FRAP analysis. In an amiC1 amiC2 double mutant, filament morphology was affected and heterocyst differentiation was abolished. Furthermore, the inactivation of amiC1 influenced SepJ localization and prevented the filament-fragmentation phenotype that is characteristic of sepJ or fraC fraD mutants. Our findings suggest that both amidases are to some extent redundant in their function, and describe a functional relationship of AmiC1 and septal proteins SepJ and FraCD.

Peptidoglycan inhibits progesterone and androstenedione production in bovine ovarian theca cells.

PubMed

Magata, F; Horiuchi, M; Miyamoto, A; Shimizu, T

2014-08-01

Uterine bacterial infection perturbs uterine and ovarian functions in postpartum dairy cows. Peptidoglycan (PGN) produced by gram-positive bacteria has been shown to disrupt the ovarian function in ewes. The aim of this study was to determine the effect of PGN on steroid production in bovine theca cells at different stages of follicular development. Bovine theca cells isolated from pre- and post-selection ovarian follicles (<8.5mm and >8.5mm in diameter, respectively) were cultured in vitro and challenged with PGN. Steroid production was evaluated by measuring progesterone (P4) and androstenedione (A4) concentration in culture media after 48 h or 96 h of culture. Bovine theca cells expressed PGN receptors including Toll-like receptor 2 and nucleotide-binding oligomerization domain 1 and 2. Treatment with PGN (1, 10, or 50 μg/ml) led to a decrease in P4 and A4 production by theca cells in both pre- and post-selection follicles. The mRNA expression of steroidogenic enzymes were decreased by PGN treatment. Moreover, A4 production was further suppressed when theca cells of post-selection follicles were simultaneously treated by PGN and lipopolysaccharide (0.1, 1, or 10 μg/ml). These findings indicate that bacterial toxins may act locally on ovarian steroidogenic cells and compromise follicular development in postpartum dairy cows. Copyright © 2014 Elsevier Ltd. All rights reserved.

Investigation of the mechanism for penetration of low density lipoprotein into the arterial wall

NASA Astrophysics Data System (ADS)

Glukhova, O. E.; Zyktin, A. A.; Slepchenkov, M. M.

2018-02-01

Currently, the pathology of the cardiovascular system is an extremely urgent problem of fundamental and clinical medicine. These diseases are caused, mainly, by atherosclerotic changes in the wall of blood vessels. The predominant role in the development of atherosclerosis is attributed to the penetration of various kinds of lipoproteins into the arterial intima. In this paper, we in silico investigated the dynamics of the penetration of low density lipoprotein (LDL) through the intercellular gap using molecular modeling methods. The simulation was carried out in the GROMACS software package using a coarse-grained MARTINI model. During investigation we carried out the LDL self-assembly for the first time. The coarse-grained model of LDL was collected from the following molecules: POPC (phosphatidylcholine) - 630 molecules, LPC (lysophosphatidylcholine) - 80 molecules CHOL (cholesterol) - 600 molecules CHYO (cholesteryl oleate) - 1600 molecules TOG (glycerol trioleate) 180 Molecules. The coarse-grained model of the intercellular endothelial gap was based on a model of lipid bilayer consisting of DPPC phospholipids and cholesterol in a percentage ratio of 70% and 30%, respectively. Based on the obtained results, we can predict the mechanism of LDL diffusion. Lipoproteins can be deformed so as to pass through narrow gaps. Our investigations open the way for the research of the behavior dynamics of LDL moving with the blood flow rate when interacting with the intercellular gaps of the endothelial layer of the vessel inner wall.

Influence of the Cell Wall on Intracellular Delivery to Algal Cells by Electroporation and Sonication

PubMed Central

Azencott, Harold R.; Peter, Gary F.; Prausnitz, Mark R.

2007-01-01

To assess the cell wall’s role as a barrier to intracellular delivery, wild-type Chlamydomonas reinhardtii algal cells and mutant cells lacking a cell wall were exposed to electroporation or sonication. Flow cytometry determined intracellular uptake of calcein and bovine serum albumin (BSA) and loss of cell viability as functions of electroporation transmembrane potential and acoustic energy. Electroporation of wild-type cells increased calcein uptake with increasing transmembrane potential, but delivered much less BSA. Electroporation of wall-deficient cells had similar effects on calcein uptake, but increased BSA uptake as much as 7.5-fold relative to wild-type cells, which indicated that the cell wall was a significant barrier to BSA delivery during electroporation. Sonication of wild-type cells caused calcein and BSA uptake at similar levels. This suggests that the cell wall barrier to BSA delivery can be overcome by sonication. Increased electroporation transmembrane potential or acoustic energy also caused increased loss of cell viability, where wall-deficient cells were especially susceptible to lysis. Overall, we believe this is the first study to compare the effects of electroporation and sonication in a direct fashion in any cell type. Specifically, these findings suggest that electroporation primarily transports molecules across the plasma membrane, because its mechanism is specific to lipid bilayer disruption, whereas sonication transports molecules across both the plasma membrane and cell wall, because it non-specifically disrupts cell-surface barriers. PMID:17602827

Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane.

PubMed

Puech, V; Chami, M; Lemassu, A; Lanéelle, M A; Schiffler, B; Gounon, P; Bayan, N; Benz, R; Daffé, M

2001-05-01

With the recent success of the heterologous expression of mycobacterial antigens in corynebacteria, in addition to the importance of these bacteria in biotechnology and medicine, a better understanding of the structure of their cell envelopes was needed. A combination of molecular compositional analysis, ultrastructural appearance and freeze-etch electron microscopy study was used to arrive at a chemical model, unique to corynebacteria but consistent with their phylogenetic relatedness to mycobacteria and other members of the distinctive suprageneric actinomycete taxon. Transmission electron microscopy and chemical analyses showed that the cell envelopes of the representative strains of corynebacteria examined consisted of (i) an outer layer composed of polysaccharides (primarily a high-molecular-mass glucan and arabinomannans), proteins, which include the mycoloyltransferase PS1, and lipids; (ii) a cell wall glycan core of peptidoglycan-arabinogalactan which may contain other sugar residues and was usually esterified by corynomycolic acids; and (iii) a typical plasma membrane bilayer. Freeze-etch electron microscopy showed that most corynomycolate-containing strains exhibited a main fracture plane in their cell wall and contained low-molecular-mass porins, while the fracture occurred within the plasma membrane of strains devoid of both corynomycolate and pore-forming proteins. Importantly, in most strains, the amount of cell wall-linked corynomycolates was not sufficient to cover the bacterial surface; interestingly, the occurrence of a cell wall fracture plane correlated with the amount of non-covalently bound lipids of the strains. Furthermore, these lipids were shown to spontaneously form liposomes, indicating that they may participate in a bilayer structure. Altogether, the data suggested that the cell wall permeability barrier in corynebacteria involved both covalently linked corynomycolates and non-covalently bound lipids of their cell envelopes.

Synthesis of avibactam derivatives and activity on β-lactamases and peptidoglycan biosynthesis enzymes of mycobacteria.

PubMed

Edoo, Zainab; Iannazzo, Laura; Compain, Fabrice; Li de la Sierra Gallay, Inès; van Tilbeurgh, Herman; Fonvielle, Matthieu; Bouchet, Flavie; Le Run, Eva; Mainardi, Jean-Luc; Arthur, Michel; Ethève-Quelquejeu, Mélanie; Hugonnet, Jean-Emmanuel

2018-03-30

There is a renewed interest for β-lactams for treating infections due to Mycobacterium tuberculosis and M. abscessus since their β-lactamases are inhibited by classical (clavulanate) or new generation (avibactam) inhibitors, respectively. Here, we report access to an azido derivative of the diazabicyclooctane (DBO) scaffold of avibactam for functionalization by the Huisgen-Sharpless cycloaddition reaction. The amoxicillin-DBO combinations were active indicating that the triazole ring is compatible with drug penetration (minimal inhibitory concentration of 16 µg/ml for both species). Mechanistically, β-lactamase inhibition was not sufficient to account for the potentiation of amoxicillin by DBOs. Thus, we investigated the latter compounds as inhibitors of L,D-transpeptidases (LDTs), which are the main peptidoglycan polymerases in mycobacteria. The DBOs acted as slow-binding inhibitors of LDTs by S-carbamoylation indicating that optimization of DBOs for LDT inhibition is an attractive strategy to obtain drugs selectively active on mycobacteria. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibitors of the bacterial cell wall biosynthesis enzyme MurC.

PubMed

Reck, F; Marmor, S; Fisher, S; Wuonola, M A

2001-06-04

A series of phosphinate transition-state analogues of the L-alanine adding enzyme (MurC) of bacterial peptidoglycan biosynthesis was prepared and tested as inhibitors of the Escherichia coli enzyme. Compound 4 was identified as a potent inhibitor of MurC from Escherichia coli with an IC(50) of 49nM.

Discovery of novel cell wall-active compounds using P ywaC, a sensitive reporter of cell wall stress, in the model gram-positive bacterium Bacillus subtilis.

PubMed

Czarny, T L; Perri, A L; French, S; Brown, E D

2014-06-01

The emergence of antibiotic resistance in recent years has radically reduced the clinical efficacy of many antibacterial treatments and now poses a significant threat to public health. One of the earliest studied well-validated targets for antimicrobial discovery is the bacterial cell wall. The essential nature of this pathway, its conservation among bacterial pathogens, and its absence in human biology have made cell wall synthesis an attractive pathway for new antibiotic drug discovery. Herein, we describe a highly sensitive screening methodology for identifying chemical agents that perturb cell wall synthesis, using the model of the Gram-positive bacterium Bacillus subtilis. We report on a cell-based pilot screen of 26,000 small molecules to look for cell wall-active chemicals in real time using an autonomous luminescence gene cluster driven by the promoter of ywaC, which encodes a guanosine tetra(penta)phosphate synthetase that is expressed under cell wall stress. The promoter-reporter system was generally much more sensitive than growth inhibition testing and responded almost exclusively to cell wall-active antibiotics. Follow-up testing of the compounds from the pilot screen with secondary assays to verify the mechanism of action led to the discovery of 9 novel cell wall-active compounds. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division

PubMed Central

Gray, Andrew N; Egan, Alexander JF; van't Veer, Inge L; Verheul, Jolanda; Colavin, Alexandre; Koumoutsi, Alexandra; Biboy, Jacob; Altelaar, A F Maarten; Damen, Mirjam J; Huang, Kerwyn Casey; Simorre, Jean-Pierre; Breukink, Eefjan; den Blaauwen, Tanneke; Typas, Athanasios; Gross, Carol A; Vollmer, Waldemar

2015-01-01

To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope machines facilitating septal PG synthesis (PBP1B-LpoB complex) and OM constriction (Tol system) are physically and functionally coordinated via YbgF, renamed CpoB (Coordinator of PG synthesis and OM constriction, associated with PBP1B). CpoB localizes to the septum concurrent with PBP1B-LpoB and Tol at the onset of constriction, interacts with both complexes, and regulates PBP1B activity in response to Tol energy state. This coordination links PG synthesis with OM invagination and imparts a unique mode of bifunctional PG synthase regulation by selectively modulating PBP1B cross-linking activity. Coordination of the PBP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division. DOI: http://dx.doi.org/10.7554/eLife.07118.001 PMID:25951518

Molecular imprinting at walls of silica nanotubes for TNT recognition.

PubMed

Xie, Chenggen; Liu, Bianhua; Wang, Zhenyang; Gao, Daming; Guan, Guijian; Zhang, Zhongping

2008-01-15

This paper reports the molecular imprinting at the walls of highly uniform silica nanotubes for the recognition of 2,4,6-trinitrotoluene (TNT). It has been demonstrated that TNT templates were efficiently imprinted into the matrix of silica through the strong acid-base pairing interaction between TNT and 3-aminopropyltriethoxysilane (APTS). TNT-imprinted silica nanotubes were synthesized by the gelation reaction between APTS and tetraethylorthosilicate (TEOS), selectively occurring at the porous walls of APTS-modified alumina membranes. The removal of the original TNT templates leaves the imprinted cavities with covalently anchored amine groups at the cavity walls. A high density of recognition sites with molecular selectivity to the TNT analyte was created at the wall of silica nanotubes. Furthermore, most of these recognition sites are situated at the inside and outside surfaces of tubular walls and in the proximity of the two surfaces due to the ultrathin wall thickness of only 15 nm, providing a better site accessibility and lower mass-transfer resistance. Therefore, greater capacity and faster kinetics of uptaking target species were achieved. The silica nanotube reported herein is an ideal form of material for imprinting various organic or biological molecules toward applications in chemical/biological sensors and bioassay.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Longenecker, Kenton L.; Stamper, Geoffrey F.; Hajduk, Philip J.

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differsmore » dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.« less

Phototransformation-Induced Aggregation of Functionalized Single-Walled Carbon Nanotubes: The Importance of Amorphous Carbon

EPA Science Inventory

Single-walled carbon nanotubes (SWCNTs) with proper functionalization are desirable for applications that require dispersion in aqueous and biological environments, and functionalized SWCNTs also serve as building blocks for conjugation with specific molecules in these applicatio...

Chimeric Ply187 endolysin kills Staphylococcus aureus more effectively than the parental enzyme.

USDA-ARS?s Scientific Manuscript database

Peptidoglycan hydrolases are an effective new source of antimicrobials. A chimeric fusion protein of the Ply187 endopeptidase domain and LysK SH3b cell wall binding domain is a potent agent against Staphylococcus aureus in three functional assays....

Morphological Changes and Antibiotic-Induced Thermal Resistance in Vegetative Cells of Bacillus subtilis

PubMed Central

Dul, Michael J.; McDonald, William C.

1971-01-01

The morphology and thermal resistance of vegetative cells of Bacillus subtilis W168 were examined after growth at 37 and 53 C. Vegetative cells grown at 37 C exhibited a typical trilaminar morphology, whereas cells grown at 53 C exhibited a cell wall which was apparently thicker and more loosely organized and had a poorly defined periphery. A concurrent increase in thermal resistance to a heat shock of 60 C occurs with the change in cell wall morphology. The change to the aberrant cell wall form, or its reversal to the normal form, is always accompanied by the gain or the loss of thermal resistance, respectively. The inhibition of protein synthesis by chloramphenicol has little effect upon the acquisition of thermal resistance at 53 C. Addition of the disaccharide pentapeptide subunit to the cell wall peptidoglycan is apparently essential to growth at 53 C and the acquisition of thermal resistance, since both growth and thermal resistance are inhibited by bacitracin. Two antibiotics, penicillin and cycloserine, which inhibit the final cross-linking of the cell wall peptidoglycan at two separate points, do not affect the acquisition of thermal resistance at 53 C. These same antibiotics induce a high degree of thermal resistance at 37 C. It is proposed that a change in the cell wall structure is related to an increased thermal resistance. Images PMID:4995654

Protein secretion and surface display in Gram-positive bacteria

PubMed Central

Schneewind, Olaf; Missiakas, Dominique M.

2012-01-01

The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions. PMID:22411983

Roles of Cells from the Arterial Vessel Wall in Atherosclerosis.

PubMed

Wang, Di; Wang, Zhiyan; Zhang, Lili; Wang, Yi

2017-01-01

Atherosclerosis has been identified as a chronic inflammatory disease of the arterial vessel wall. Accumulating evidence indicates that different cells from the tunica intima, media, adventitia, and perivascular adipose tissue not only comprise the intact and normal arterial vessel wall but also participate all in the inflammatory response of atherosclerosis via multiple intricate pathways. For instance, endothelial dysfunction has historically been considered to be the initiator of the development of atherosclerosis. The migration and proliferation of smooth muscle cells also play a pivotal role in the progression of atherosclerosis. Additionally, the fibroblasts from the adventitia and adipocytes from perivascular adipose tissue have received considerable attention given their special functions that contribute to atherosclerosis. In addition, numerous types of cytokines produced by different cells from the arterial vessel wall, including endothelium-derived relaxing factors, endothelium-derived contracting factors, tumor necrosis factors, interleukin, adhesion molecules, interferon, and adventitium-derived relaxing factors, have been implicated in atherosclerosis. Herein, we summarize the possible roles of different cells from the entire arterial vessel wall in the pathogenesis of atherosclerosis.

Biophysical dynamics in disorderly environments.

PubMed

Nelson, David R

2012-01-01

Three areas where time-independent disorder plays a key role in biological dynamics far from equilibrium are reviewed. We first discuss the anomalous localization dynamics that arises when a single species spreads in space and time via diffusion and fluid advection in the presence of frozen heterogeneities in the growth rate. Next we treat the unzipping of double-stranded DNA as a function of force and temperature, a challenge that must be surmounted every time a cell divides. Heterogeneity in the DNA sequence dominates the physics of single-molecule force-extension curves for a broad range of forces upon approaching a sharp unzipping transition. The dynamics of the unzipping fork exhibits anomalous drift and diffusion in a similar range above this transition, with energy barriers that scale as the square root of the genome size. Finally, we describe how activated peptidoglycan strand extension sites, called dislocations in materials science, can mediate the growth of bacterial cell walls. Enzymatically driven circumferential motions of a few dozen of these defects are sufficient to describe the exponential elongation rates observed in experiments on Escherichia coli in a nutrient-rich environment. However, long-range elastic forces transmitted by the peptidoglycan meshwork cause the moving dislocations to interact not only with each other, but also with a disorderly array of frozen, inactivated strand ends.

Observation of the time-course for peptidoglycan lipid intermediate II polymerization by Staphylococcus aureus monofunctional transglycosylase.

PubMed

Braddick, Darren; Sandhu, Sandeep; Roper, David I; Chappell, Michael J; Bugg, Timothy D H

2014-08-01

The polymerization of lipid intermediate II by the transglycosylase activity of penicillin-binding proteins (PBPs) represents an important target for antibacterial action, but limited methods are available for quantitative assay of this reaction, or screening potential inhibitors. A new labelling method for lipid II polymerization products using Sanger's reagent (fluoro-2,4-dinitrobenzene), followed by gel permeation HPLC analysis, has permitted the observation of intermediate polymerization products for Staphylococcus aureus monofunctional transglycosylase MGT. Peak formation is inhibited by 6 µM ramoplanin or enduracidin. Characterization by mass spectrometry indicates the formation of tetrasaccharide and octasaccharide intermediates, but not a hexasaccharide intermediate, suggesting a dimerization of a lipid-linked tetrasaccharide. Numerical modelling of the time-course data supports a kinetic model involving addition to lipid-linked tetrasaccharide of either lipid II or lipid-linked tetrasaccharide. Observation of free octasaccharide suggests that hydrolysis of the undecaprenyl diphosphate lipid carrier occurs at this stage in peptidoglycan transglycosylation. © 2014 The Authors.

Peptidoglycan Synthesis Machinery in Agrobacterium tumefaciens During Unipolar Growth and Cell Division

PubMed Central

Cameron, Todd A.; Anderson-Furgeson, James; Zupan, John R.; Zik, Justin J.

2014-01-01

ABSTRACT The synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the Rhizobiales. To better understand polar growth in the Rhizobiales Agrobacterium tumefaciens, we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from A. tumefaciens, we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a Rhizobiales- and Rhodobacterales-specific l,d-transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous d-amino acids. Thus, unipolar growth in the Rhizobiales is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli. PMID:24865559

Controlling the crystalline three-dimensional order in bulk materials by single-wall carbon nanotubes.

PubMed

López-Andarias, Javier; López, Juan Luis; Atienza, Carmen; Brunetti, Fulvio G; Romero-Nieto, Carlos; Guldi, Dirk M; Martín, Nazario

2014-04-29

The construction of ordered single-wall carbon nanotube soft-materials at the nanoscale is currently an important challenge in science. Here we use single-wall carbon nanotubes as a tool to gain control over the crystalline ordering of three-dimensional bulk materials composed of suitably functionalized molecular building blocks. We prepare p-type nanofibres from tripeptide and pentapeptide-containing small molecules, which are covalently connected to both carboxylic and electron-donating 9,10-di(1,3-dithiol-2-ylidene)-9,10-dihydroanthracene termini. Adding small amounts of single-wall carbon nanotubes to the so-prepared p-nanofibres together with the externally controlled self assembly by charge screening by means of Ca(2+) results in new and stable single-wall carbon nanotube-based supramolecular gels featuring remarkably long-range internal order.

Biochemical and immunocytological characterizations of Arabidopsis pollen tube cell wall.

PubMed

Dardelle, Flavien; Lehner, Arnaud; Ramdani, Yasmina; Bardor, Muriel; Lerouge, Patrice; Driouich, Azeddine; Mollet, Jean-Claude

2010-08-01

During plant sexual reproduction, pollen germination and tube growth require development under tight spatial and temporal control for the proper delivery of the sperm cells to the ovules. Pollen tubes are fast growing tip-polarized cells able to perceive multiple guiding signals emitted by the female organ. Adhesion of pollen tubes via cell wall molecules may be part of the battery of signals. In order to study these processes, we investigated the cell wall characteristics of in vitro-grown Arabidopsis (Arabidopsis thaliana) pollen tubes using a combination of immunocytochemical and biochemical techniques. Results showed a well-defined localization of cell wall epitopes. Low esterified homogalacturonan epitopes were found mostly in the pollen tube wall back from the tip. Xyloglucan and arabinan from rhamnogalacturonan I epitopes were detected along the entire tube within the two wall layers and the outer wall layer, respectively. In contrast, highly esterified homogalacturonan and arabinogalactan protein epitopes were found associated predominantly with the tip region. Chemical analysis of the pollen tube cell wall revealed an important content of arabinosyl residues (43%) originating mostly from (1-->5)-alpha-L-arabinan, the side chains of rhamnogalacturonan I. Finally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of endo-glucanase-sensitive xyloglucan showed mass spectra with two dominant oligosaccharides (XLXG/XXLG and XXFG), both being mono O-acetylated, and accounting for over 68% of the total ion signals. These findings demonstrate that the Arabidopsis pollen tube wall has its own characteristics compared with other cell types in the Arabidopsis sporophyte. These structural features are discussed in terms of pollen tube cell wall biosynthesis and growth dynamics.

Single-walled carbon nanotubes as near-infrared optical biosensors for life sciences and biomedicine.

PubMed

Jain, Astha; Homayoun, Aida; Bannister, Christopher W; Yum, Kyungsuk

2015-03-01

Single-walled carbon nanotubes that emit photostable near-infrared fluorescence have emerged as near-infrared optical biosensors for life sciences and biomedicine. Since the discovery of their near-infrared fluorescence, researchers have engineered single-walled carbon nanotubes to function as an optical biosensor that selectively modulates its fluorescence upon binding of target molecules. Here we review the recent advances in the single-walled carbon nanotube-based optical sensing technology for life sciences and biomedicine. We discuss the structure and optical properties of single-walled carbon nanotubes, the mechanisms for molecular recognition and signal transduction in single-walled carbon nanotube complexes, and the recent development of various single-walled carbon nanotube-based optical biosensors. We also discuss the opportunities and challenges to translate this emerging technology into biomedical research and clinical use, including the biological safety of single-walled carbon nanotubes. The advances in single-walled carbon nanotube-based near-infrared optical sensing technology open up a new avenue for in vitro and in vivo biosensing with high sensitivity and high spatial resolution, beneficial for many areas of life sciences and biomedicine. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Near-Wall Reynolds-Stress Closure Without Wall Normals

NASA Technical Reports Server (NTRS)

Yuan, S. P.; So, R. M. C.

1997-01-01

Turbulent wall-bounded complex flows are commonly encountered in engineering practice and are of considerable interest in a variety of industrial applications. The presence of a wall significantly affects turbulence characteristics. In addition to the wall effects, turbulent wall-bounded flows become more complicated by the presence of additional body forces (e.g. centrifugal force and Coriolis force) and complex geometry. Most near-wall Reynolds stress models are developed from a high-Reynolds-number model which assumes turbulence is homogenous (or quasi-homogenous). Near-wall modifications are proposed to include wall effects in near-wall regions. In this process, wall normals are introduced. Good predictions could be obtained by Reynolds stress models with wall normals. However, ambiguity arises when the models are applied in flows with multiple walls. Many models have been proposed to model turbulent flows. Among them, Reynolds stress models, in which turbulent stresses are obtained by solving the Reynolds stress transport equations, have been proved to be the most successful ones. To apply the Reynolds stress models to wall-bounded flows, near-wall corrections accounting for the wall effects are needed, and the resulting models are called near-wall Reynolds stress models. In most of the existing near-wall models, the near-wall corrections invoke wall normals. These wall-dependent near-wall models are difficult to implement for turbulent flows with complex geometry and may give inaccurate predictions due to the ambiguity of wall normals at corners connecting multiple walls. The objective of this study is to develop a more general and flexible near-wall Reynolds stress model without using any wall-dependent variable for wall-bounded turbulent flows. With the aid of near-wall asymptotic analysis and results of direct numerical simulation, a new near-wall Reynolds stress model (NNWRS) is formulated based on Speziale et al.'s high-Reynolds-stress model with wall

Peptidoglycan from the gut microbiota governs the lifespan of circulating phagocytes at homeostasis.

PubMed

Hergott, Christopher B; Roche, Aoife M; Tamashiro, Edwin; Clarke, Thomas B; Bailey, Aubrey G; Laughlin, Alice; Bushman, Frederic D; Weiser, Jeffrey N

2016-05-19

Maintenance of myeloid cell homeostasis requires continuous turnover of phagocytes from the bloodstream, yet whether environmental signals influence phagocyte longevity in the absence of inflammation remains unknown. Here, we show that the gut microbiota regulates the steady-state cellular lifespan of neutrophils and inflammatory monocytes, the 2 most abundant circulating myeloid cells and key contributors to inflammatory responses. Treatment of mice with broad-spectrum antibiotics, or with the gut-restricted aminoglycoside neomycin alone, accelerated phagocyte turnover and increased the rates of their spontaneous apoptosis. Metagenomic analyses revealed that neomycin altered the abundance of intestinal bacteria bearing γ-d-glutamyl-meso-diaminopimelic acid, a ligand for the intracellular peptidoglycan sensor Nod1. Accordingly, signaling through Nod1 was both necessary and sufficient to mediate the stimulatory influence of the flora on myeloid cell longevity. Stimulation of Nod1 signaling increased the frequency of lymphocytes in the murine intestine producing the proinflammatory cytokine interleukin 17A (IL-17A), and liberation of IL-17A was required for transmission of Nod1-dependent signals to circulating phagocytes. Together, these results define a mechanism through which intestinal microbes govern a central component of myeloid homeostasis and suggest perturbations of commensal communities can influence steady-state regulation of cell fate. © 2016 by The American Society of Hematology.

Peptidoglycan from the gut microbiota governs the lifespan of circulating phagocytes at homeostasis

PubMed Central

Hergott, Christopher B.; Roche, Aoife M.; Tamashiro, Edwin; Clarke, Thomas B.; Bailey, Aubrey G.; Laughlin, Alice; Bushman, Frederic D.

2016-01-01

Maintenance of myeloid cell homeostasis requires continuous turnover of phagocytes from the bloodstream, yet whether environmental signals influence phagocyte longevity in the absence of inflammation remains unknown. Here, we show that the gut microbiota regulates the steady-state cellular lifespan of neutrophils and inflammatory monocytes, the 2 most abundant circulating myeloid cells and key contributors to inflammatory responses. Treatment of mice with broad-spectrum antibiotics, or with the gut-restricted aminoglycoside neomycin alone, accelerated phagocyte turnover and increased the rates of their spontaneous apoptosis. Metagenomic analyses revealed that neomycin altered the abundance of intestinal bacteria bearing γ-d-glutamyl-meso-diaminopimelic acid, a ligand for the intracellular peptidoglycan sensor Nod1. Accordingly, signaling through Nod1 was both necessary and sufficient to mediate the stimulatory influence of the flora on myeloid cell longevity. Stimulation of Nod1 signaling increased the frequency of lymphocytes in the murine intestine producing the proinflammatory cytokine interleukin 17A (IL-17A), and liberation of IL-17A was required for transmission of Nod1-dependent signals to circulating phagocytes. Together, these results define a mechanism through which intestinal microbes govern a central component of myeloid homeostasis and suggest perturbations of commensal communities can influence steady-state regulation of cell fate. PMID:26989200

Recognition, survival and persistence of Staphylococcus aureus in the model host Tenebrio molitor.

PubMed

Dorling, Jack; Moraes, Caroline; Rolff, Jens

2015-02-01

The degree of specificity of any given immune response to a parasite is governed by the complexity and variation of interactions between host and pathogen derived molecules. Here, we assess the extent to which recognition and immuno-resistance of cell wall mutants of the pathogen Staphylococcus aureus may contribute to establishment and maintenance of persistent infection in the model insect host, Tenebrio molitor. The cell surface of S. aureus is decorated with various molecules, including glycopolymers such as wall teichoic acid (WTA). WTA is covalently bound to peptidoglycan (PGN) and its absence has been associated with increased recognition of PGN by host receptors (PGRPs). WTA is also further modified by other molecules such as D-alanine (D-alanylation). Both the level of WTA expression and its D-alanylation were found to be important in the mediation of the host-parasite interaction in this model system. Specifically, WTA itself was seen to influence immune recognition, while D-alanylation of WTA was found to increase immuno-resistance and was associated with prolonged persistence of S. aureus in T. molitor. These results implicate WTA and its D-alanylation as important factors in the establishment and maintenance of persistent infection, affecting different critical junctions in the immune response; through potential evasion of recognition by PGRPs and resistance to humoral immune effectors during prolonged exposure to the immune system. This highlights a mechanism by which specificity in this host-parasite interaction may arise. Copyright © 2014 Elsevier Ltd. All rights reserved.

Optical properties of graphene nanoribbons encapsulated in single-walled carbon nanotubes.

PubMed

Chernov, Alexander I; Fedotov, Pavel V; Talyzin, Alexandr V; Suarez Lopez, Inma; Anoshkin, Ilya V; Nasibulin, Albert G; Kauppinen, Esko I; Obraztsova, Elena D

2013-07-23

We report the photoluminescence (PL) from graphene nanoribbons (GNRs) encapsulated in single-walled carbon nanotubes (SWCNTs). New PL spectral features originating from GNRs have been detected in the visible spectral range. PL peaks from GNRs have resonant character, and their positions depend on the ribbon geometrical structure in accordance with the theoretical predictions. GNRs were synthesized using confined polymerization and fusion of coronene molecules. GNR@SWCNTs material demonstrates a bright photoluminescence both in infrared (IR) and visible regions. The photoluminescence excitation mapping in the near-IR spectral range has revealed the geometry-dependent shifts of the SWCNT peaks (up to 11 meV in excitation and emission) after the process of polymerization of coronene molecules inside the nanotubes. This behavior has been attributed to the strain of SWCNTs induced by insertion of the coronene molecules.

Theoretical study on adsorption and dissociation of NO2 molecules on BNNT surface

NASA Astrophysics Data System (ADS)

Singla, Preeti; Singhal, Sonal; Goel, Neetu

2013-10-01

The adsorption of NO2 molecules on (8,0) zigzag single-walled boron nitride nanotube surface is investigated using density functional theory calculations. Two interaction modes, nitro (interacting atom is N) and nitrite (O interacts with BNNT) have been studied with increase in number of NO2 molecules. The adsorption of single NO2 molecule in both configurations is observed to be exothermic and physical in nature. However, in nitrite configuration, NO2 molecules are chemisorbed on the surface leading to the dissociation of NO2 molecules into NO and O. The density of states, natural bond orbital analysis and frontier orbital pictures provide rational understanding of the charge transfer involved in the process and predict significant enhancement in the conductivity of the BNNT after NO2 adsorption. The DFT calculations show that NO2 adsorption introduces new impurity states in the band gap of bare BNNT and expand their applications as NO2 molecule gas sensor and catalytic surface for Nsbnd O dissociation depending upon the mode of adsorption.

Invariant amino acids in the Mur peptide synthetases of bacterial peptidoglycan synthesis and their modification by site-directed mutagenesis in the UDP-MurNAc:L-alanine ligase from Escherichia coli.

PubMed

Bouhss, A; Mengin-Lecreulx, D; Blanot, D; van Heijenoort, J; Parquet, C

1997-09-30

The comparison of the amino acid sequences of 20 cytoplasmic peptidoglycan synthetases (MurC, MurD, MurE, MurF, and Mpl) from various bacterial organisms has allowed us to detect common invariants: seven amino acids and the ATP-binding consensus sequence GXXGKT/S all at the same position in the alignment. The Mur synthetases thus appeared as a well-defined class of closely functionally related proteins. The conservation of a constant backbone length between certain invariants suggested common structural motifs. Among the other enzymes catalyzing a peptide bond formation driven by ATP hydrolysis to ADP and Pi, only folylpoly-gamma-l-glutamate synthetases presented the same common conserved amino acid residues, except for the most N-terminal invariant D50. Site-directed mutageneses were carried out to replace the K130, E174, H199, N293, N296, R327, and D351 residues by alanine in the MurC protein from Escherichia coli taken as model. For this purpose, plasmid pAM1005 was used as template, MurC being highly overproduced in this genetic setting. Analysis of the Vmax values of the mutated proteins suggested that residues K130, E174, and D351 are essential for the catalytic process whereas residues H199, N293, N296, and R327 were not. Mutations K130A, H199A, N293A, N296A, and R327A led to important variations of the Km values for one or more substrates, thereby indicating that these residues are involved in the structure of the active site and suggesting that the binding order of the substrates could be ATP, UDP-MurNAc, and alanine. The various mutated murC plasmids were tested for their effects on the growth, cell morphology, and peptidoglycan cell content of a murC thermosensitive strain at 42 degrees C. The observed effects (complementation, altered morphology, and reduced peptidoglycan content) paralleled more or less the decreased values of the MurC activity of each mutant.

Plant cell wall sugars: sweeteners for a bio-based economy.

PubMed

Van de Wouwer, Dorien; Boerjan, Wout; Vanholme, Bartel

2018-02-12

Global warming and the consequent climate change is one of the major environmental challenges we are facing today. The driving force behind the rise in temperature is our fossil-based economy, which releases massive amounts of the greenhouse gas carbon dioxide into the atmosphere. In order to reduce greenhouse gas emission, we need to scale down our dependency on fossil resources, implying that we need other sources for energy and chemicals to feed our economy. Here, plants have an important role to play; by means of photosynthesis, plants capture solar energy to split water and fix carbon derived from atmospheric carbon dioxide. A significant fraction of the fixed carbon ends up as polysaccharides in the plant cell wall. Fermentable sugars derived from cell wall polysaccharides form an ideal carbon source for the production of bio-platform molecules. However, a major limiting factor in the use of plant biomass as feedstock for the bio-based economy is the complexity of the plant cell wall and its recalcitrance towards deconstruction. To facilitate the release of fermentable sugars during downstream biomass processing, the composition and structure of the cell wall can be engineered. Different strategies to reduce cell wall recalcitrance will be described in this review. The ultimate goal is to obtain a tailor-made biomass, derived from plants with a cell wall optimized for particular industrial or agricultural applications, without affecting plant growth and development. This article is protected by copyright. All rights reserved.

Thioridazine Induces Major Changes in Global Gene Expression and Cell Wall Composition in Methicillin-Resistant Staphylococcus aureus USA300

PubMed Central

Thorsing, Mette; Klitgaard, Janne K.; Atilano, Magda L.; Skov, Marianne N.; Kolmos, Hans Jørn; Filipe, Sérgio R.; Kallipolitis, Birgitte H.

2013-01-01

Subinhibitory concentrations of the neuroleptic drug thioridazine (TDZ) are well-known to enhance the killing of methicillin-resistant Staphylococcus aureus (MRSA) by β-lactam antibiotics, however, the mechanism underlying the synergy between TDZ and β-lactams is not fully understood. In the present study, we have examined the effect of a subinhibitory concentration of TDZ on antimicrobial resistance, the global transcriptome, and the cell wall composition of MRSA USA300. We show that TDZ is able to sensitize the bacteria to several classes of antimicrobials targeting the late stages of peptidoglycan (PGN) synthesis. Furthermore, our microarray analysis demonstrates that TDZ modulates the expression of genes encoding membrane and surface proteins, transporters, and enzymes involved in amino acid biosynthesis. Interestingly, resemblance between the transcriptional profile of TDZ treatment and the transcriptomic response of S. aureus to known inhibitors of cell wall synthesis suggests that TDZ disturbs PGN biosynthesis at a stage that precedes transpeptidation by penicillin-binding proteins (PBPs). In support of this notion, dramatic changes in the muropeptide profile of USA300 were observed following growth in the presence of TDZ, indicating that TDZ can interfere with the formation of the pentaglycine branches. Strikingly, the addition of glycine to the growth medium relieved the effect of TDZ on the muropeptide profile. Furthermore, exogenous glycine offered a modest protective effect against TDZ-induced β-lactam sensitivity. We propose that TDZ exposure leads to a shortage of intracellular amino acids, including glycine, which is required for the production of normal PGN precursors with pentaglycine branches, the correct substrate of S. aureus PBPs. Collectively, this work demonstrates that TDZ has a major impact on the cell wall biosynthesis pathway in S. aureus and provides new insights into how MRSA may be sensitized towards β-lactam antibiotics. PMID

Monitoring Single-Molecule Protein Dynamics with a Carbon Nanotube Transistor

NASA Astrophysics Data System (ADS)

Collins, Philip G.

2014-03-01

Nanoscale electronic devices like field-effect transistors have long promised to provide sensitive, label-free detection of biomolecules. Single-walled carbon nanotubes press this concept further by not just detecting molecules but also monitoring their dynamics in real time. Recent measurements have demonstrated this premise by monitoring the single-molecule processivity of three different enzymes: lysozyme, protein Kinase A, and the Klenow fragment of DNA polymerase I. With all three enzymes, single molecules tethered to nanotube transistors were electronically monitored for 10 or more minutes, allowing us to directly observe a range of activity including rare transitions to chemically inactive and hyperactive conformations. The high bandwidth of the nanotube transistors further allow every individual chemical event to be clearly resolved, providing excellent statistics from tens of thousands of turnovers by a single enzyme. Initial success with three different enzymes indicates the generality and attractiveness of the nanotube devices as a new tool to complement other single-molecule techniques. Research on transduction mechanisms provides the design rules necessary to further generalize this architecture and apply it to other proteins. The purposeful incorporation of just one amino acid is sufficient to fabricate effective, single molecule sensors from a wide range of enzymes or proteins.

Lysozyme as an alternative to growth promoting antibiotics in swine production

USDA-ARS?s Scientific Manuscript database

Lysozyme is a naturally occurring enzyme found in bodily secretions such as tears, saliva, and milk. It functions as an antimicrobial agent by cleaving the peptidoglycan component of bacterial cell walls, which leads to cell death. Antibiotics are also antimicrobials and have been fed at subtherape...

Single Molecule Detection in Living Biological Cells using Carbon Nanotube Optical Probes

NASA Astrophysics Data System (ADS)

Strano, Michael

2009-03-01

Nanoscale sensing elements offer promise for single molecule analyte detection in physically or biologically constrained environments. Molecular adsorption can be amplified via modulation of sharp singularities in the electronic density of states that arise from 1D quantum confinement [1]. Single-walled carbon nanotubes (SWNT), as single molecule optical sensors [2-3], offer unique advantages such as photostable near-infrared (n-IR) emission for prolonged detection through biological media, single-molecule sensitivity and, nearly orthogonal optical modes for signal transduction that can be used to identify distinct classes of analytes. Selective binding to the SWNT surface is difficult to engineer [4]. In this lecture, we will briefly review the immerging field of fluorescent diagnostics using band gap emission from SWNT. In recent work, we demonstrate that even a single pair of SWNT provides at least four optical modes that can be modulated to uniquely fingerprint chemical agents by the degree to which they alter either the emission band intensity or wavelength. We validate this identification method in vitro by demonstrating detection and identification of six genotoxic analytes, including chemotherapeutic drugs and reactive oxygen species (ROS), which are spectroscopically differentiated into four distinct classes. We also demonstrate single-molecule sensitivity in detecting hydrogen peroxide, one of the most common genotoxins and an important cellular signal. Finally, we employ our sensing and fingerprinting method of these analytes in real time within live 3T3 cells, demonstrating the first multiplexed optical detection from a nanoscale biosensor and the first label-free tool to optically discriminate between genotoxins. We will also discuss our recent efforts to fabricate biomedical sensors for real time detection of glucose and other important physiologically relevant analytes in-vivo. The response of embedded SWNT in a swellable hydrogel construct to

Employing proteomic analysis to compare Paracoccidioides lutzii yeast and mycelium cell wall proteins.

PubMed

Araújo, Danielle Silva; de Sousa Lima, Patrícia; Baeza, Lilian Cristiane; Parente, Ana Flávia Alves; Melo Bailão, Alexandre; Borges, Clayton Luiz; de Almeida Soares, Célia Maria

2017-11-01

Paracoccidioidomycosis is an important systemic mycosis caused by thermodimorphic fungi of the Paracoccidioides genus. During the infective process, the cell wall acts at the interface between the fungus and the host. In this way, the cell wall has a key role in growth, environment sensing and interaction, as well as morphogenesis of the fungus. Since the cell wall is absent in mammals, it may present molecules that are described as target sites for new antifungal drugs. Despite its importance, up to now few studies have been conducted employing proteomics in for the identification of cell wall proteins in Paracoccidioides spp. Here, a detailed proteomic approach, including cell wall-fractionation coupled to NanoUPLC-MS E , was used to study and compare the cell wall fractions from Paracoccidioides lutzii mycelia and yeast cells. The analyzed samples consisted of cell wall proteins extracted by hot SDS followed by extraction by mild alkali. In summary, 512 proteins constituting different cell wall fractions were identified, including 7 predicted GPI-dependent cell wall proteins that are potentially involved in cell wall metabolism. Adhesins previously described in Paracoccidioides spp. such as enolase, glyceraldehyde-3-phosphate dehydrogenase were identified. Comparing the proteins in mycelium and yeast cells, we detected some that are common to both fungal phases, such as Ecm33, and some specific proteins, as glucanase Crf1. All of those proteins were described in the metabolism of cell wall. Our study provides an important elucidation of cell wall composition of fractions in Paracoccidioides, opening a way to understand the fungus cell wall architecture. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular coordination of Staphylococcus aureus cell division

PubMed Central

Cotterell, Bryony E; Walther, Christa G; Fenn, Samuel J; Grein, Fabian; Wollman, Adam JM; Leake, Mark C; Olivier, Nicolas; Cadby, Ashley; Mesnage, Stéphane; Jones, Simon

2018-01-01

The bacterial cell wall is essential for viability, but despite its ability to withstand internal turgor must remain dynamic to permit growth and division. Peptidoglycan is the major cell wall structural polymer, whose synthesis requires multiple interacting components. The human pathogen Staphylococcus aureus is a prolate spheroid that divides in three orthogonal planes. Here, we have integrated cellular morphology during division with molecular level resolution imaging of peptidoglycan synthesis and the components responsible. Synthesis occurs across the developing septal surface in a diffuse pattern, a necessity of the observed septal geometry, that is matched by variegated division component distribution. Synthesis continues after septal annulus completion, where the core division component FtsZ remains. The novel molecular level information requires re-evaluation of the growth and division processes leading to a new conceptual model, whereby the cell cycle is expedited by a set of functionally connected but not regularly distributed components. PMID:29465397

Bacteriophage endolysins as novel antimicrobials

PubMed Central

Schmelcher, Mathias; Donovan, David M; Loessner, Martin J

2013-01-01

Endolysins are enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. Due to the absence of an outer membrane in the Gram-positive bacterial cell wall, endolysins can access the peptidoglycan and destroy these organisms when applied externally, making them interesting antimicrobial candidates, particularly in light of increasing bacterial drug resistance. This article reviews the modular structure of these enzymes, in which cell wall binding and catalytic functions are separated, as well as their mechanism of action, lytic activity and potential as antimicrobials. It particularly focuses on molecular engineering as a means of optimizing endolysins for specific applications, highlights new developments that may render these proteins active against Gram-negative and intracellular pathogens and summarizes the most recent applications of endolysins in the fields of medicine, food safety, agriculture and biotechnology. PMID:23030422

Potential of the virion-associated peptidoglycan hydrolase HydH5 and its derivative fusion proteins in milk biopreservation.

PubMed

Rodríguez-Rubio, Lorena; Martínez, Beatriz; Donovan, David M; García, Pilar; Rodríguez, Ana

2013-01-01

Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88], and three different fusion proteins created between HydH5 and lysostaphin has been assessed. The lytic activity of the five proteins (HydH5, HydH5Lyso, HydH5SH3b, CHAPSH3b and lysostaphin) was confirmed using commercial whole extended shelf-life milk (ESL) in challenge assays with 10(4) CFU/mL of the strain S. aureus Sa9. HydH5, HydH5Lyso and HydH5SH3b (3.5 µM) kept the staphylococcal viable counts below the control cultures for 6 h at 37°C. The effect is apparent just 15 minutes after the addition of the lytic enzyme. Of note, lysostaphin and CHAPSH3b showed the highest staphylolytic protection as they were able to eradicate the initial staphylococcal challenge immediately or 15 min after addition, respectively, at lower concentration (1 µM) at 37°C. CHAPSH3b showed the same antistaphyloccal effect at room temperature (1.65 µM). No re-growth was observed for the remainder of the experiment (up to 6 h). CHAPSH3b activity (1.65 µM) was also assayed in raw (whole and skim) and pasteurized (whole and skim) milk. Pasteurization of milk clearly enhanced CHAPSH3b staphylolytic activity in both whole and skim milk at both temperatures. This effect was most dramatic at room temperature as this protein was able to reduce S. aureus viable counts to undetectable levels immediately after addition with no re-growth detected for the duration of the experiment (360 min). Furthermore, CHAPSH3b protein is known to be heat tolerant and retained some lytic activity after pasteurization treatment and after storage at 4°C for 3 days. These results might facilitate the use of the peptidoglycan hydrolase HydH5 and its derivative fusions, particularly CHAPSH3b, as biocontrol

Structure-based design of diverse inhibitors of Mycobacterium tuberculosis N-acetylglucosamine-1-phosphate uridyltransferase: combined molecular docking, dynamic simulation, and biological activity.

PubMed

Soni, Vijay; Suryadevara, Priyanka; Sriram, Dharmarajan; Kumar, Santhosh; Nandicoori, Vinay Kumar; Yogeeswari, Perumal

2015-07-01

Persistent nature of Mycobacterium tuberculosis is one of the major factors which make the drug development process monotonous against this organism. The highly lipophilic cell wall, which constituting outer mycolic acid and inner peptidoglycan layers, acts as a barrier for the drugs to enter the bacteria. The rigidity of the cell wall is imparted by the peptidoglycan layer, which is covalently linked to mycolic acid by arabinogalactan. Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) serves as the starting material in the biosynthesis of this peptidoglycan layers. This UDP-GlcNAc is synthesized by N-acetylglucosamine-1-phosphate uridyltransferase (GlmU(Mtb)), a bi-functional enzyme with two functional sites, acetyltransferase site and uridyltransferase site. Here, we report design and screening of nine inhibitors against UTP and NAcGlc-1-P of uridyltransferase active site of glmU(Mtb). Compound 4 was showing good inhibition and was selected for further analysis. The isothermal titration calorimetry (ITC) experiments showed the binding energy pattern of compound 4 to the uridyltransferase active site is similar to that of substrate UTP. In silico molecular dynamics (MD) simulation studies, for compound 4, carried out for 10 ns showed the protein-compound complex to be stable throughout the simulation with relative rmsd in acceptable range. Hence, these compounds can serve as a starting point in the drug discovery processes against Mycobacterium tuberculosis.

Wall characterization for through-the-wall radar applications

NASA Astrophysics Data System (ADS)

Greneker, Gene; Rausch, E. O.

2008-04-01

There has been continuing interest in the penetration of multilayer building materials, such as wood walls with air gaps and concrete hollow core block, using through-the-wall (TTW) radar systems. TTW operational techniques and signal propagation paths vary depending on how the TTW system is intended to be operated. For example, the operator of a TTW radar may be required to place the radar against the intervening wall of interest while collecting data. Other operational doctrines allow the radar to be operated in a stand-off mode from the wall. The stand-off distances can vary from feet to hundreds of feet, depending on the type of radar being used. When a signal is propagated through a multilayer wall with air gaps between the material and the wall construction uses materials of radically different dielectric constants, attenuation may not be the only effect that the probing signal experiences passing through the wall. This paper presents measurements of a hollow core concrete block wall and the measurement of a standard wall constructed of siding and wallboard. Both types of walls are typically found in most U.S. homes. These limited measurements demonstrate that the type of wall being penetrated by a wideband signal can modify the probing signal.

A Near-Wall Reynolds-Stress Closure without Wall Normals

NASA Technical Reports Server (NTRS)

Yuan, S. P.; So, R. M. C.

1997-01-01

With the aid of near-wall asymptotic analysis and results of direct numerical simulation, a new near-wall Reynolds stress model (NNWRS) is formulated based on the SSG high-Reynolds-stress model with wall-independent near-wall corrections. Only one damping function is used for flows with a wide range of Reynolds numbers to ensure that the near-wall modifications diminish away from the walls. The model is able to reproduce complicated flow phenomena induced by complex geometry, such as flow recirculation, reattachment and boundary-layer redevelopment in backward-facing step flow and secondary flow in three-dimensional square duct flow. In simple flows, including fully developed channel/pipe flow, Couette flow and boundary-layer flow, the wall effects are dominant, and the NNWRS model predicts less degree of turbulent anisotropy in the near-wall region compared with a wall-dependent near-wall Reynolds Stress model (NWRS) developed by So and colleagues. The comparison of the predictions given by the two models rectifies the misconception that the overshooting of skin friction coefficient in backward-facing step flow prevalent in those near-wall, models with wall normal is caused by he use of wall normal.

The MreB-like protein Mbl of Streptomyces coelicolor A3(2) depends on MreB for proper localization and contributes to spore wall synthesis.

PubMed

Heichlinger, Andrea; Ammelburg, Moritz; Kleinschnitz, Eva-Maria; Latus, Annette; Maldener, Iris; Flärdh, Klas; Wohlleben, Wolfgang; Muth, Günther

2011-04-01

Most bacteria with a rod-shaped morphology contain an actin-like cytoskeleton consisting of MreB polymers, which form helical spirals underneath the cytoplasmic membrane to direct peptidoglycan synthesis for the elongation of the cell wall. In contrast, MreB of Streptomyces coelicolor is not required for vegetative growth but has a role in sporulation. Besides MreB, S. coelicolor encodes two further MreB-like proteins, Mbl and SCO6166, whose function is unknown. Whereas MreB and Mbl are highly similar, SCO6166 is shorter, lacking the subdomains IB and IIB of actin-like proteins. Here, we showed that MreB and Mbl are not functionally redundant but cooperate in spore wall synthesis. Expression analysis by semiquantitative reverse transcription-PCR revealed distinct expression patterns. mreB and mbl are induced predominantly during morphological differentiation. In contrast, sco6166 is strongly expressed during vegetative growth but switched off during sporulation. All genes could be deleted without affecting viability. Even a ΔmreB Δmbl double mutant was viable. Δsco6166 had a wild-type phenotype. ΔmreB, Δmbl, and ΔmreB Δmbl produced swollen, prematurely germinating spores that were sensitive to various kinds of stress, suggesting a defect in spore wall integrity. During aerial mycelium formation, an Mbl-mCherry fusion protein colocalized with an MreB-enhanced green fluorescent protein (MreB-eGFP) fusion protein at the sporulation septa. Whereas MreB-eGFP localized properly in the Δmbl mutant, Mbl-mCherry localization depended on the presence of a functional MreB protein. Our results revealed that MreB and Mbl cooperate in the synthesis of the thickened spore wall, while SCO6166 has a nonessential function during vegetative growth.

Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock.

PubMed

Van Amersfoort, Edwin S; Van Berkel, Theo J C; Kuiper, Johan

2003-07-01

Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins.

Accessibility and contribution to glucan masking of natural and genetically tagged versions of yeast wall protein 1 of Candida albicans

PubMed Central

2018-01-01

-anchored Ywp1 were previously created by others, and were further explored here. As above, rare cells with much greater accessibility of the HA epitopes were isolated, and also found to exhibit greater exposure of Ywp1 and β-1,3-glucan. The placement of the HA cassette inhibited the normal N-glycosylation and propeptide cleavage of Ywp1, but the wall-anchored Ywp1-HA-Ywp1 still accumulated in the cell wall of yeast forms. Bifunctional transformation cassettes were used to additionally tag these molecules with Gfp, generating soluble Ywp1-HA-Gfp and wall-anchored Ywp1-HA-Gfp-Ywp1 molecules. The former revealed unexpected electrophoretic properties caused by the HA insertion, while the latter further highlighted differences between the presence of a tagged Ywp1 molecule (as revealed by Gfp fluorescence) and its accessibility in the cell wall to externally applied antibodies specific for HA, Gfp and Ywp1, with accessibility being greatest in the rapidly expanding walls of budding daughter cells. These strains and results increase our understanding of cell wall properties and how C. albicans masks itself from recognition by the human immune system. PMID:29329339

Accessibility and contribution to glucan masking of natural and genetically tagged versions of yeast wall protein 1 of Candida albicans.

PubMed

Granger, Bruce L

2018-01-01

-anchored Ywp1 were previously created by others, and were further explored here. As above, rare cells with much greater accessibility of the HA epitopes were isolated, and also found to exhibit greater exposure of Ywp1 and β-1,3-glucan. The placement of the HA cassette inhibited the normal N-glycosylation and propeptide cleavage of Ywp1, but the wall-anchored Ywp1-HA-Ywp1 still accumulated in the cell wall of yeast forms. Bifunctional transformation cassettes were used to additionally tag these molecules with Gfp, generating soluble Ywp1-HA-Gfp and wall-anchored Ywp1-HA-Gfp-Ywp1 molecules. The former revealed unexpected electrophoretic properties caused by the HA insertion, while the latter further highlighted differences between the presence of a tagged Ywp1 molecule (as revealed by Gfp fluorescence) and its accessibility in the cell wall to externally applied antibodies specific for HA, Gfp and Ywp1, with accessibility being greatest in the rapidly expanding walls of budding daughter cells. These strains and results increase our understanding of cell wall properties and how C. albicans masks itself from recognition by the human immune system.

Therapeutic use of chimeric bacteriophage (phage) lysins in staphylococcal endophthalmitis

USDA-ARS?s Scientific Manuscript database

Purpose: Phage endolysins are peptidoglycan hydrolases that are produced at the end of the phage lytic cycle to digest the host bacterial cell wall, facilitating the release of mature phage progeny. The aim of this study is to determine the antimicrobial activity of chimeric phage lysins against cli...

The role of endoxyloglucan transferase in the organization of plant cell walls.

PubMed

Nishitani, K

1997-01-01

The plant cell wall plays a central role in morphogenesis as well as responsiveness to environmental signals. Xyloglucans are the principal component of the plant cell wall matrix and serve as cross-links between cellulose microfibrils to form the cellulose-xyloglucan framework. Endoxyloglucan transferase (EXGT), which was isolated and characterized in 1992, is an enzyme that mediates molecular grafting reaction between xyloglucan molecules. Structural studies on cDNAs encoding EXGT and its related proteins have disclosed the ubiquitous presence in the plant kingdom of a large multigene family of xyloglucan-related proteins (XRPs). Each XRP functions as either hydrolase or transferase acting on xyloglucans and is considered to be responsible for rearrangement of the cellulose-xyloglucan framework, the processes essential for the construction, modification, and degradation of plant cell walls. Different XRP genes exhibit potentially different expression profiles with respect to tissue specificity and responsiveness to hormonal and mechanical signals. The molecular approach to individual XRP genes will open a new path for exploring the controlling mechanisms by which the plant cell wall is constructed and reformed during plant growth and development.

Structure of the Bacteriophage [phi]KZ Lytic Transglycosylase gp144

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fokine, Andrei; Miroshnikov, Konstantin A.; Shneider, Mikhail M.

2008-04-02

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage {phi}KZ has been determined to 2.5-{angstrom} resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. {phi}KZ gp144 is a 260-residue {alpha}-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The foldmore » of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G d-Ala-d-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu{sup 115} in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the {phi}KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine){sub 4} has been determined to 2.6-{angstrom} resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.« less

Detection of muramic acid in a carbohydrate fraction of human spleen.

PubMed Central

Hoijer, M A; Melief, M J; van Helden-Meeuwsen, C G; Eulderink, F; Hazenberg, M P

1995-01-01

In previous studies, we showed that peptidoglycan polysaccharides from anaerobic bacteria normally present in the human gut induced severe chronic joint inflammation in rats. Our hypothesis is that peptidoglycan from the gut flora is involved in perpetuation of idiopathic inflammation. However, in the literature, the presence of peptidoglycan or subunits like muramyl peptides in blood or tissues is still a matter of debate. We were able to stain red pulp macrophages in all six available human spleens by immunohistochemical techniques using a monoclonal antibody against gut flora-derived antigens. Therefore, these human spleens were extracted, and after removal of most of the protein, the carbohydrate fraction was investigated for the presence of muramic acid, an amino sugar characteristic for peptidoglycan. Using three different methods for detection of muramic acid, we found a mean of 3.3 mumol of muramic acid with high-pressure liquid chromatography, 1.9 mumol with a colorimetric method for detection of lactate, and 0.8 mumol with an enzymatic method for detection of D-lactate per spleen (D-lactate is a specific group of the muramic acid molecule). It is concluded that peptidoglycan is present in human spleen not as small muramyl peptides as were previously searched for by other investigators but as larger macromolecules probably stored in spleen macrophages. PMID:7729869

Shearing of nanoscopic bridges in two-component thin liquid layers between chemically patterned walls.

PubMed

Hemming, C J; Patey, G N

2004-10-01

Bridge phases associated with a phase transition between two liquid phases occur when a two-component liquid mixture is confined between chemically patterned walls. In the bulk the liquid mixture with components A, B undergoes phase separation into an A-rich phase and a B-rich phase. The walls bear stripes attractive to A. In the bridge phase A-rich and B-rich regions alternate. Grand canonical Monte Carlo studies are performed with the alignment between stripes on opposite walls varied. Misalignment of the stripes places the nanoscopic liquid bridges under shear strain. The bridges exert a Hookean restoring force on the walls for small displacements from equilibrium. As the strain increases there are deviations from Hooke's law. Eventually there is an abrupt yielding of the bridges. Molecular dynamics simulations show the bridges form or disintegrate on time scales which are fast compared to wall motion and transport of molecules into or from the confined space. Some interesting possible applications of the phenomena are discussed. (c) 2004 American Institute of Physics

Roles of the bacterial cell wall and capsule in induction of tumor necrosis factor alpha by type III group B streptococci.

PubMed Central

Vallejo, J G; Baker, C J; Edwards, M S

1996-01-01

Group B streptococci (GBS) are the major cause of sepsis and fatal shock in neonates in the United States. The precise role of tumor necrosis factor alpha (TNF-alpha) in the development of human GBS sepsis has not been defined; however, whole GBS have been shown to induce the production of this inflammatory cytokine. We sought to determine which bacterial cell wall components of GBS are responsible for triggering TNF-alpha production. Human cord blood monocytes were stimulated with encapsulated (COH1) or unencapsulated (COH1-13) whole type III GBS or with purified bacterial components, including type III capsular polysaccharide (III-PS), group B polysaccharide (GB-PS), lipoteichoic acid (LTA), or peptidoglycan (PG). Lipopolysaccharide from Escherichia coli served as a control. Supernatants were harvested at specific timed intervals, and TNF-alpha levels were measured by enzyme-linked immunosorbent assay. Monocytes exposed to COH1 and COH1-13 induced similar amounts of TNF-alpha. III-PS, GB-PS, LTA, and PG each induced TNF-alpha in a time- and concentration-dependent manner. However, TNF-alpha release was significantly greater after stimulation by the GB-PS or PG than after stimulation by III-PS or LTA (P < 0.05). Our findings indicate that GB-PS and PG are the bacterial cell wall components primarily evoking TNF-alpha release. These, alone or in concert with other factors, may be responsible for septic shock accompanying GBS sepsis. PMID:8945544

Modeling Radiation Effectiveness for Inactivation of Bacillus Spores

DTIC Science & Technology

2015-09-17

are the exosporium, the spore coat, the outer membrane, the cortex, the germ cell wall, the inner membrane, and the core. These are illustrated in...small amounts of carbohydrates and lipids. The 6 coat acts as the spore’s first line of defense against some chemical infiltration such as lytic enzymes...the spore as water makes up 48-57 percent of the cortex [2]. Immediately interior to the cortex is the germ cell wall which is also a peptidoglycan

The MreB-Like Protein Mbl of Streptomyces coelicolor A3(2) Depends on MreB for Proper Localization and Contributes to Spore Wall Synthesis▿ †

PubMed Central

Heichlinger, Andrea; Ammelburg, Moritz; Kleinschnitz, Eva-Maria; Latus, Annette; Maldener, Iris; Flärdh, Klas; Wohlleben, Wolfgang; Muth, Günther

2011-01-01

Most bacteria with a rod-shaped morphology contain an actin-like cytoskeleton consisting of MreB polymers, which form helical spirals underneath the cytoplasmic membrane to direct peptidoglycan synthesis for the elongation of the cell wall. In contrast, MreB of Streptomyces coelicolor is not required for vegetative growth but has a role in sporulation. Besides MreB, S. coelicolor encodes two further MreB-like proteins, Mbl and SCO6166, whose function is unknown. Whereas MreB and Mbl are highly similar, SCO6166 is shorter, lacking the subdomains IB and IIB of actin-like proteins. Here, we showed that MreB and Mbl are not functionally redundant but cooperate in spore wall synthesis. Expression analysis by semiquantitative reverse transcription-PCR revealed distinct expression patterns. mreB and mbl are induced predominantly during morphological differentiation. In contrast, sco6166 is strongly expressed during vegetative growth but switched off during sporulation. All genes could be deleted without affecting viability. Even a ΔmreB Δmbl double mutant was viable. Δsco6166 had a wild-type phenotype. ΔmreB, Δmbl, and ΔmreB Δmbl produced swollen, prematurely germinating spores that were sensitive to various kinds of stress, suggesting a defect in spore wall integrity. During aerial mycelium formation, an Mbl-mCherry fusion protein colocalized with an MreB-enhanced green fluorescent protein (MreB-eGFP) fusion protein at the sporulation septa. Whereas MreB-eGFP localized properly in the Δmbl mutant, Mbl-mCherry localization depended on the presence of a functional MreB protein. Our results revealed that MreB and Mbl cooperate in the synthesis of the thickened spore wall, while SCO6166 has a nonessential function during vegetative growth. PMID:21257777

Density functional theory (DFT) study of a new novel bionanosensor hybrid; tryptophan/Pd doped single walled carbon nanotube

NASA Astrophysics Data System (ADS)

Yoosefian, Mehdi; Etminan, Nazanin

2016-07-01

In order to explore a new novel L-amino acid/transition metal doped single walled carbon nanotube based biosensor, density functional theory calculations were studied. These hybrid structures of organic-inorganic nanobiosensors are able to detect the smallest amino acid building block of proteins. The configurations of amine and carbonyl group coordination of tryptophan aromatic amino acid adsorbed on Pd/doped single walled carbon nanotube were compared. The frontier molecular orbital theory, quantum theory atom in molecule and natural bond orbital analysis were performed. The molecular electrostatic potential and the electron density surfaces were constructed. The calculations indicated that the Pd/SWCNT was sensitive to tryptophan suggesting the importance of interaction with biological molecule and potential detecting application. The proposed nanobiosensor represents a highly sensitive detection of protein at ultra-low concentration in diagnosis applications.

Chemical Organization of the Cell Wall Polysaccharide Core of Malassezia restricta

PubMed Central

Stalhberger, Thomas; Simenel, Catherine; Clavaud, Cécile; Eijsink, Vincent G. H.; Jourdain, Roland; Delepierre, Muriel; Latgé, Jean-Paul; Breton, Lionel; Fontaine, Thierry

2014-01-01

Malassezia species are ubiquitous residents of human skin and are associated with several diseases such as seborrheic dermatitis, tinea versicolor, folliculitis, atopic dermatitis, and scalp conditions such as dandruff. Host-Malassezia interactions and mechanisms to evade local immune responses remain largely unknown. Malassezia restricta is one of the most predominant yeasts of the healthy human skin, its cell wall has been investigated in this paper. Polysaccharides in the M. restricta cell wall are almost exclusively alkali-insoluble, showing that they play an essential role in the organization and rigidity of the M. restricta cell wall. Fractionation of cell wall polymers and carbohydrate analyses showed that the polysaccharide core of the cell wall of M. restricta contained an average of 5% chitin, 20% chitosan, 5% β-(1,3)-glucan, and 70% β-(1,6)-glucan. In contrast to other yeasts, chitin and chitosan are relatively abundant, and β-(1,3)-glucans constitute a minor cell wall component. The most abundant polymer is β-(1,6)-glucans, which are large molecules composed of a linear β-(1,6)-glucan chains with β-(1,3)-glucosyl side chain with an average of 1 branch point every 3.8 glucose unit. Both β-glucans are cross-linked, forming a huge alkali-insoluble complex with chitin and chitosan polymers. Data presented here show that M. restricta has a polysaccharide organization very different of all fungal species analyzed to date. PMID:24627479

Chemical organization of the cell wall polysaccharide core of Malassezia restricta.

PubMed

Stalhberger, Thomas; Simenel, Catherine; Clavaud, Cécile; Eijsink, Vincent G H; Jourdain, Roland; Delepierre, Muriel; Latgé, Jean-Paul; Breton, Lionel; Fontaine, Thierry

2014-05-02

Malassezia species are ubiquitous residents of human skin and are associated with several diseases such as seborrheic dermatitis, tinea versicolor, folliculitis, atopic dermatitis, and scalp conditions such as dandruff. Host-Malassezia interactions and mechanisms to evade local immune responses remain largely unknown. Malassezia restricta is one of the most predominant yeasts of the healthy human skin, its cell wall has been investigated in this paper. Polysaccharides in the M. restricta cell wall are almost exclusively alkali-insoluble, showing that they play an essential role in the organization and rigidity of the M. restricta cell wall. Fractionation of cell wall polymers and carbohydrate analyses showed that the polysaccharide core of the cell wall of M. restricta contained an average of 5% chitin, 20% chitosan, 5% β-(1,3)-glucan, and 70% β-(1,6)-glucan. In contrast to other yeasts, chitin and chitosan are relatively abundant, and β-(1,3)-glucans constitute a minor cell wall component. The most abundant polymer is β-(1,6)-glucans, which are large molecules composed of a linear β-(1,6)-glucan chains with β-(1,3)-glucosyl side chain with an average of 1 branch point every 3.8 glucose unit. Both β-glucans are cross-linked, forming a huge alkali-insoluble complex with chitin and chitosan polymers. Data presented here show that M. restricta has a polysaccharide organization very different of all fungal species analyzed to date.

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

DOE PAGES

Hasim, Sahar; Allison, David P.; Retterer, Scott T.; ...

2016-11-14

Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is alteredmore » or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in this paper in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. Finally, AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system.« less

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

PubMed Central

Hasim, Sahar; Allison, David P.; Retterer, Scott T.; Hopke, Alex; Wheeler, Robert T.; Doktycz, Mitchel J.

2016-01-01

ABSTRACT Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is altered or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system. PMID:27849179

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hasim, Sahar; Allison, David P.; Retterer, Scott T.

Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is alteredmore » or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in this paper in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. Finally, AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system.« less

Measuring Conformational Dynamics of Single Biomolecules Using Nanoscale Electronic Devices

NASA Astrophysics Data System (ADS)

Akhterov, Maxim V.; Choi, Yongki; Sims, Patrick C.; Olsen, Tivoli J.; Gul, O. Tolga; Corso, Brad L.; Weiss, Gregory A.; Collins, Philip G.

2014-03-01

Molecular motion can be a rate-limiting step of enzyme catalysis, but motions are typically too quick to resolve with fluorescent single molecule techniques. Recently, we demonstrated a label-free technique that replaced fluorophores with nano-electronic circuits to monitor protein motions. The solid-state electronic technique used single-walled carbon nanotube (SWNT) transistors to monitor conformational motions of a single molecule of T4 lysozyme while processing its substrate, peptidoglycan. As lysozyme catalyzes the hydrolysis of glycosidic bonds, two protein domains undergo 8 Å hinge bending motion that generates an electronic signal in the SWNT transistor. We describe improvements to the system that have extended our temporal resolution to 2 μs . Electronic recordings at this level of detail directly resolve not just transitions between open and closed conformations but also the durations for those transition events. Statistical analysis of many events determines transition timescales characteristic of enzyme activity and shows a high degree of variability within nominally identical chemical events. The high resolution technique can be readily applied to other complex biomolecules to gain insights into their kinetic parameters and catalytic function.

Bio-inspired synthetic receptor molecules towards mimicry of vancomycin.

PubMed

Monnee, M C; Brouwer, A J; Verbeek, L M; van Wageningen, A M; Liskamp, R M

2001-06-18

A 512-member library of bio-inspired synthetic receptor molecules was prepared featuring a triazacyclophane scaffold. The purpose of this scaffold was to orient three (identical) peptide 'binding arms' in order to mimic an antibiotic binding cavity as is present in the vancomycin antibiotics. The library was screened with D-Ala-D-Ala and D-Ala-D-Lac containing ligands, which are present in the cell wall precursors of pathogenic bacteria. Screening and validation led to identification of a synthetic receptor capable of binding these ligands.

Dissecting Bacterial Cell Wall Entry and Signaling in Eukaryotic Cells: an Actin-Dependent Pathway Parallels Platelet-Activating Factor Receptor-Mediated Endocytosis.

PubMed

Loh, Lip Nam; Gao, Geli; Tuomanen, Elaine I

2017-01-03

The Gram-positive bacterial cell wall (CW) peptidoglycan-teichoic acid complex is released into the host environment during bacterial metabolism or death. It is a highly inflammatory Toll-like receptor 2 (TLR2) ligand, and previous in vivo studies have demonstrated its ability to recapitulate pathological features of pneumonia and meningitis. We report that an actin-dependent pathway is involved in the internalization of the CW by epithelial and endothelial cells, in addition to the previously described platelet-activating factor receptor (PAFr)-dependent uptake pathway. Unlike the PAFr-dependent pathway, which is mediated by clathrin and dynamin and does not lead to signaling, the alternative pathway is sensitive to 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and engenders Rac1, Cdc42, and phosphatidylinositol 3-kinase (PI3K) signaling. Upon internalization by this macropinocytosis-like pathway, CW is trafficked to lysosomes. Intracellular CW trafficking is more complex than previously recognized and suggests multiple points of interaction with and without innate immune signaling. Streptococcus pneumoniae is a major human pathogen infecting the respiratory tract and brain. It is an established model organism for understanding how infection injures the host. During infection or bacterial growth, bacteria shed their cell wall (CW) into the host environment and trigger inflammation. A previous study has shown that CW enters and crosses cell barriers by interacting with a receptor on the surfaces of host cells, termed platelet-activating factor receptor (PAFr). In the present study, by using cells that are depleted of PAFr, we identified a second pathway with features of macropinocytosis, which is a receptor-independent fluid uptake mechanism by cells. Each pathway contributes approximately the same amount of cell wall trafficking, but the PAFr pathway is silent, while the new pathway appears to contribute to the host inflammatory response to CW insult. Copyright © 2017

Small molecules targeting LapB protein prevent Listeria attachment to catfish muscle

PubMed Central

Das, Bhaskar; Lawrence, Mark

2017-01-01

Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listeriosis. L. monocytogenes lapB gene encodes a cell wall surface anchor protein, and mutation of this gene causes Listeria attenuation in mice. In this work, the potential role of Listeria LapB protein in catfish fillet attachment was investigated. To achieve this, boron-based small molecules designed to interfere with the active site of the L. monocytogenes LapB protein were developed, and their ability to prevent L. monocytogenes attachment to fish fillet was tested. Results indicated that seven out of nine different small molecules were effective in reducing the Listeria attachment to catfish fillets. Of these, three small molecules (SM3, SM5, and SM7) were highly effective in blocking Listeria attachment to catfish fillets. This study suggests an alternative strategy for reduction of L. monocytogenes contamination in fresh and frozen fish products. PMID:29253892

A Versatile Click-Compatible Monolignol Probe to Study Lignin Deposition in Plant Cell Walls

PubMed Central

Pandey, Jyotsna L.; Wang, Bo; Diehl, Brett G.; Richard, Tom L.; Chen, Gong; Anderson, Charles T.

2015-01-01

Lignin plays important structural and functional roles in plants by forming a hydrophobic matrix in secondary cell walls that enhances mechanical strength and resists microbial decay. While the importance of the lignin matrix is well documented and the biosynthetic pathways for monolignols are known, the process by which lignin precursors or monolignols are transported and polymerized to form this matrix remains a subject of considerable debate. In this study, we have synthesized and tested an analog of coniferyl alcohol that has been modified to contain an ethynyl group at the C-3 position. This modification enables fluorescent tagging and imaging of this molecule after its incorporation into plant tissue by click chemistry-assisted covalent labeling with a fluorescent azide dye, and confers a distinct Raman signature that could be used for Raman imaging. We found that this monolignol analog is incorporated into in vitro-polymerized dehydrogenation polymer (DHP) lignin and into root epidermal cell walls of 4-day-old Arabidopsis seedlings. Incorporation of the analog in stem sections of 6-week-old Arabidopsis thaliana plants and labeling with an Alexa-594 azide dye revealed the precise locations of new lignin polymerization. Results from this study indicate that this molecule can provide high-resolution localization of lignification during plant cell wall maturation and lignin matrix assembly. PMID:25884205

Observation of anisotropic interactions between metastable atoms and target molecules by two-dimensional collisional ionization electron spectroscopy

NASA Astrophysics Data System (ADS)

Kishimoto, Naoki; Ohno, Koichi

Excited metastable atoms colliding with target molecules can sensitively probe outer properties of molecules by chemi-ionization (Penning ionization) from molecular orbitals in the outer region, since metastable atoms cannot penetrate into the repulsive interaction wall around the molecules. By means of two-dimensional measurements using kinetic energy analysis of electrons combined with a velocity-resolved metastable beam, one can obtain information on the anisotropic interaction between the colliding particles without any control of orientation or alignment of target molecules. We have developed a classical trajectory method to calculate the collision energy dependence of partial ionization cross-sections (CEDPICS) on the anisotropic interaction potential energy surface, which has enabled us to study stereodynamics between metastable atoms and target molecules as well as the spatial distribution of molecular orbitals and electron ejection functions which have a relation with entrance and exit channels of the reaction. Based on the individual CEDPICS, the electronic structure of molecules can also be elucidated.

Long-Range Activation of Systemic Immunity through Peptidoglycan Diffusion in Drosophila

PubMed Central

Gendrin, Mathilde; Welchman, David P.; Poidevin, Mickael; Hervé, Mireille; Lemaitre, Bruno

2009-01-01

The systemic immune response of Drosophila is known to be induced both by septic injury and by oral infection with certain bacteria, and is characterized by the secretion of antimicrobial peptides (AMPs) into the haemolymph. To investigate other possible routes of bacterial infection, we deposited Erwinia carotovora (Ecc15) on various sites of the cuticle and monitored the immune response via expression of the AMP gene Diptericin. A strong response was observed to deposition on the genital plate of males (up to 20% of a septic injury response), but not females. We show that the principal response to genital infection is systemic, but that some AMPs, particularly Defensin, are induced locally in the genital tract. At late time points we detected bacteria in the haemolymph of immune deficient RelishE20 flies, indicating that the genital plate can be a route of entry for pathogens, and that the immune response protects flies against the progression of genital infection. The protective role of the immune response is further illustrated by our observation that RelishE20 flies exhibit significant lethality in response to genital Ecc15 infections. We next show that a systemic immune response can be induced by deposition of the bacterial elicitor peptidoglycan (PGN), or its terminal monomer tracheal cytotoxin (TCT), on the genital plate. This immune response is downregulated by PGRP-LB and Pirk, known regulators of the Imd pathway, and can be suppressed by the overexpression of PGRP-LB in the haemolymph compartment. Finally, we provide strong evidence that TCT can activate a systemic response by crossing epithelia, by showing that radiolabelled TCT deposited on the genital plate can subsequently be detected in the haemolymph. Genital infection is thus an intriguing new model for studying the systemic immune response to local epithelial infections and a potential route of entry for naturally occurring pathogens of Drosophila. PMID:20019799

Long-range activation of systemic immunity through peptidoglycan diffusion in Drosophila.

PubMed

Gendrin, Mathilde; Welchman, David P; Poidevin, Mickael; Hervé, Mireille; Lemaitre, Bruno

2009-12-01

The systemic immune response of Drosophila is known to be induced both by septic injury and by oral infection with certain bacteria, and is characterized by the secretion of antimicrobial peptides (AMPs) into the haemolymph. To investigate other possible routes of bacterial infection, we deposited Erwinia carotovora (Ecc15) on various sites of the cuticle and monitored the immune response via expression of the AMP gene Diptericin. A strong response was observed to deposition on the genital plate of males (up to 20% of a septic injury response), but not females. We show that the principal response to genital infection is systemic, but that some AMPs, particularly Defensin, are induced locally in the genital tract. At late time points we detected bacteria in the haemolymph of immune deficient Relish(E20) flies, indicating that the genital plate can be a route of entry for pathogens, and that the immune response protects flies against the progression of genital infection. The protective role of the immune response is further illustrated by our observation that Relish(E20) flies exhibit significant lethality in response to genital Ecc15 infections. We next show that a systemic immune response can be induced by deposition of the bacterial elicitor peptidoglycan (PGN), or its terminal monomer tracheal cytotoxin (TCT), on the genital plate. This immune response is downregulated by PGRP-LB and Pirk, known regulators of the Imd pathway, and can be suppressed by the overexpression of PGRP-LB in the haemolymph compartment. Finally, we provide strong evidence that TCT can activate a systemic response by crossing epithelia, by showing that radiolabelled TCT deposited on the genital plate can subsequently be detected in the haemolymph. Genital infection is thus an intriguing new model for studying the systemic immune response to local epithelial infections and a potential route of entry for naturally occurring pathogens of Drosophila.

Phenotypic and genotypic characterization of peptidoglycan hydrolases of Lactobacillus sakei

PubMed Central

Najjari, Afef; Amairi, Houda; Chaillou, Stéphane; Mora, Diego; Boudabous, Abdellatif; Zagorec, Monique; Ouzari, Hadda

2015-01-01

Lactobacillus sakei, a lactic acid bacterium naturally found in fresh meat and sea products, is considered to be one of the most important bacterial species involved in meat fermentation and bio-preservation. Several enzymes of Lb. sakei species contributing to microbial safeguarding and organoleptic properties of fermented-meat were studied. However, the specific autolytic mechanisms and associated enzymes involved in Lb. sakei are not well understood. The autolytic phenotype of 22 Lb. sakei strains isolated from Tunisian meat and seafood products was evaluated under starvation conditions, at pH 6.5 and 8.5, and in the presence of different carbon sources. A higher autolytic rate was observed when cells were grown in the presence of glucose and incubated at pH 6.5. Almost all strains showed high resistance to mutanolysin, indicating a minor role of muramidases in Lb. sakei cell lysis. Using Micrococcus lysodeikticus cells as a substrate in activity gels zymogram, peptidoglycan hydrolase (PGH) patterns for all strains was characterized by two lytic bands of ∼80 (B1) and ∼70 kDa (B2), except for strain BMG.167 which harbored two activity signals at a lower MW. Lytic activity was retained in high salt and in acid/basic conditions and was active toward cells of Lb. sakei, Listeria monocytogenes, Listeria ivanovii and Listeria innocua. Analysis of five putative PGH genes found in the Lb. sakei 23 K model strain genome, indicated that one gene, lsa1437, could encode a PGH (N-acetylmuramoyl-L-alanine amidase) containing B1 and B2 as isoforms. According to this hypothesis, strain BMG.167 showed an allelic version of lsa1437 gene deleted of one of the five LysM domains, leading to a reduction in the MW of lytic bands and the high autolytic rate of this strain. Characterization of autolytic phenotype of Lb. sakei should expand the knowledge of their role in fermentation processes where they represent the dominant species. PMID:26843981

Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum

PubMed Central

2014-01-01

Background Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production. Results The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion. Conclusion There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system. PMID:24731213

Two DD-carboxypeptidases from Mycobacterium smegmatis affect cell surface properties through regulation of peptidoglycan cross-linking and glycopeptidolipids.

PubMed

Pandey, Satya Deo; Pal, Shilpa; Kumar N, Ganesh; Bansal, Ankita; Mallick, Sathi; Ghosh, Anindya S

2018-05-07

During the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between two meso-DAP) and 4-3 cross-links (between D-ala and meso-DAP), though there is a predominance (60-80%) of 3-3 cross-links. The DD-CPases act on pentapeptides to generate tetrapeptides that are used by LD-transpeptidases as substrates to form 3-3 cross-links. Therefore, DD-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology of DD-CPases in mycobacteria is relatively unexplored. Here, we deleted two DD-CPase genes, msmeg_2433 , and msmeg_2432 , both individually and in combination, from Mycobacterium smegmatis mc 2 155. Though the single DD-CPase deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double deletion mutant, viz. , a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced and susceptibility towards β-lactams and anti-tubercular agents was enhanced. Moreover, the existence of the double mutant within murine macrophages was better as compared to the parent. Interestingly, the complementation with any one of the DD-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3: 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation,, drug susceptibility and subsistence of the cells within macrophages. Importance The glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. The DD-CPases generate tetrapeptides by acting on the pentapeptides, and LD-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. Here, we showed that simultaneous deletions of two DD-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression

Identification and Deletion of Tft1, a Predicted Glycosyltransferase Necessary for Cell Wall β-1,3;1,4-Glucan Synthesis in Aspergillus fumigatus

PubMed Central

Samar, Danial; Kieler, Joshua B.; Klutts, J. Stacey

2015-01-01

Aspergillus fumigatus is an environmental mold that causes severe, often fatal invasive infections in immunocompromised patients. The search for new antifungal drug targets is critical, and the synthesis of the cell wall represents a potential area to find such a target. Embedded within the main β-1,3-glucan core of the A. fumigatus cell wall is a mixed linkage, β-D-(1,3;1,4)-glucan. The role of this molecule or how it is synthesized is unknown, though it comprises 10% of the glucans within the wall. While this is not a well-studied molecule in fungi, it has been studied in plants. Using the sequences of two plant mixed linkage glucan synthases, a single ortholog was identified in A. fumigatus (Tft1). A strain lacking this enzyme (tft1Δ) was generated along with revertant strains containing the native gene under the control of either the native or a strongly expressing promoter. Immunofluorescence staining with an antibody against β-(1,3;1,4)-glucan and biochemical quantification of this polysaccharide in the tft1Δ strain demonstrated complete loss of this molecule. Reintroduction of the gene into the knockout strain yielded reappearance in amounts that correlated with expected expression of the gene. The loss of Tft1 and mixed linkage glucan yielded no in vitro growth phenotype. However, there was a modest increase in virulence for the tft1Δ strain in a wax worm model. While the precise roles for β-(1,3;1,4)-glucan within A. fumigatus cell wall are still uncertain, it is clear that Tft1 plays a pivotal role in the biosynthesis of this cell wall polysaccharide. PMID:25723175

Surface enhanced Raman scattering analyses of individual silver nanoaggregates on living single yeast cell wall

NASA Astrophysics Data System (ADS)

Sujith, Athiyanathil; Itoh, Tamitake; Abe, Hiroko; Anas, Abdul Aziz; Yoshida, Kenichi; Biju, Vasudevanpillai; Ishikawa, Mitsuru

2008-03-01

We labeled the living yeast cell surface (Saccharomyces cerevisiae strain W303-1A) by silver nanoparticles which can form nanoaggregates and found to show surface enhanced Raman scattering (SERS) activity. Blinking of SERS and its polarization dependence reveal that SERS signals are from amplified electromagnetic field at nanometric Ag nanoparticles gaps with single or a few molecules sensitivity. We tentatively assigned SERS spectra from a yeast cell wall to mannoproteins. Nanoaggregate-by-nanoaggregate variations and temporal fluctuations of SERS spectra are discussed in terms of inhomogeneous mannoprotein distribution on a cell wall and possible ways of Ag nanoaggregate adsorption, respectively.

Dissecting single-molecule signal transduction in carbon nanotube circuits with protein engineering

PubMed Central

Choi, Yongki; Olsen, Tivoli J.; Sims, Patrick C.; Moody, Issa S.; Corso, Brad L.; Dang, Mytrang N.; Weiss, Gregory A.; Collins, Philip G.

2013-01-01

Single molecule experimental methods have provided new insights into biomolecular function, dynamic disorder, and transient states that are all invisible to conventional measurements. A novel, non-fluorescent single molecule technique involves attaching single molecules to single-walled carbon nanotube field-effective transistors (SWNT FETs). These ultrasensitive electronic devices provide long-duration, label-free monitoring of biomolecules and their dynamic motions. However, generalization of the SWNT FET technique first requires design rules that can predict the success and applicability of these devices. Here, we report on the transduction mechanism linking enzymatic processivity to electrical signal generation by a SWNT FET. The interaction between SWNT FETs and the enzyme lysozyme was systematically dissected using eight different lysozyme variants synthesized by protein engineering. The data prove that effective signal generation can be accomplished using a single charged amino acid, when appropriately located, providing a foundation to widely apply SWNT FET sensitivity to other biomolecular systems. PMID:23323846

Reinforcement mechanism of multi-anchor wall with double wall facing

NASA Astrophysics Data System (ADS)

Suzuki, Kouta; Kobayashi, Makoto; Miura, Kinya; Konami, Takeharu; Hayashi, Taketo

2017-10-01

The reinforced soil wall has high seismic performance as generally known. However, the seismic behavior has not been clarified accurately yet, especially on multi-anchor wall with double wall facing. Indefinite behavior of reinforced soil wall during earthquake make us complicated in case with adopting to the abutment, because of arrangement of anchor plate as reinforcement often different according to the width of roads. In this study, a series of centrifuge model tests were carried out to investigate the reinforcement mechanism of multi anchor wall with double wall facing from the perspective of the vertical earth pressure. Several types of reinforce arrangement and rigid wall were applied in order to verify the arch function in the reinforced regions. The test results show unique behavior of vertical earth pressure, which was affected by arch action. All the vertical earth pressure placed behind facing panel, are larger than that of middle part between facing panel despite of friction between backfill and facing panel. Similar results were obtained in case using rigid wall. On the other hands, the vertical earth pressure, which were measured at the 3cm high from bottom of model container, shows larger than that of bottom. This results show the existence of arch action between double walls. In addition, it implies that the wall facing of such soil structure confined the backfill as pseudo wall, which is very reason that the multi anchor wall with double wall facing has high seismic performance.

Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamada, Mototsugu, E-mail: mototsugu-yamada@meiji.co.jp; Watanabe, Takashi; Baba, Nobuyoshi

The selenomethionyl-substituted transpeptidase domain of penicillin-binding protein (PBP) 2B from S. pneumoniae was isolated from a limited proteolysis digest of the soluble form of recombinant PBP 2B and then crystallized. MAD data were collected to 2.4 Å resolution. Penicillin-binding protein (PBP) 2B from Streptococcus pneumoniae catalyzes the cross-linking of peptidoglycan precursors that occurs during bacterial cell-wall biosynthesis. A selenomethionyl (SeMet) substituted PBP 2B transpeptidase domain was isolated from a limited proteolysis digest of a soluble form of recombinant PBP 2B and then crystallized. The crystals belonged to space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 86.39,more » c = 143.27 Å. Diffraction data were collected to 2.4 Å resolution using the BL32B2 beamline at SPring-8. The asymmetric unit contains one protein molecule and 63.7% solvent.« less

Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins.

PubMed

Peaucelle, Alexis; Louvet, Romain; Johansen, Jorunn N; Höfte, Herman; Laufs, Patrick; Pelloux, Jérome; Mouille, Grégory

2008-12-23

Plant organs are produced from meristems in a characteristic pattern. This pattern, referred to as phyllotaxis, is thought to be generated by local gradients of an information molecule, auxin. Some studies propose a key role for the mechanical properties of the cell walls in the control of organ outgrowth. A major cell-wall component is the linear alpha-1-4-linked D-GalAp pectic polysaccharide homogalacturonan (HG), which plays a key role in cell-to-cell cohesion. HG is deposited in the cell wall in a highly (70%-80%) methyl-esterified form and is subsequently de-methyl-esterified by pectin methyl-esterases (PME, EC 3.1.1.11). PME activity is itself regulated by endogenous PME inhibitor (PMEI) proteins. PME action modulates cell-wall-matrix properties and plays a role in the control of cell growth. Here, we show that the formation of flower primordia in the Arabidopsis shoot apical meristem is accompanied by the de-methyl-esterification of pectic polysaccharides in the cell walls. In addition, experimental perturbation of the methyl-esterification status of pectins within the meristem dramatically alters the phyllotactic pattern. These results demonstrate that regulated de-methyl-esterification of pectins is a key event in the outgrowth of primordia and possibly also in phyllotactic patterning.

Electromagnetic approaches to wall characterization, wall mitigation, and antenna design for through-the-wall radar systems

NASA Astrophysics Data System (ADS)

Thajudeen, Christopher

Through-the-wall imaging (TWI) is a topic of current interest due to its wide range of public safety, law enforcement, and defense applications. Among the various available technologies such as, acoustic, thermal, and optical imaging, which can be employed to sense and image targets of interest, electromagnetic (EM) imaging, in the microwave frequency bands, is the most widely utilized technology and has been at the forefront of research in recent years. The primary objectives for any Through-the-Wall Radar Imaging (TWRI) system are to obtain a layout of the building and/or inner rooms, detect if there are targets of interest including humans or weapons, determine if there are countermeasures being employed to further obscure the contents of a building or room of interest, and finally to classify the detected targets. Unlike conventional radar scenarios, the presence of walls, made of common construction materials such as brick, drywall, plywood, cinder block, and solid concrete, adversely affects the ability of any conventional imaging technique to properly image targets enclosed within building structures as the propagation through the wall can induce shadowing effects on targets of interest which may result in image degradation, errors in target localization, and even complete target masking. For many applications of TWR systems, the wall ringing signals are strong enough to mask the returns from targets not located a sufficient distance behind the wall, beyond the distance of the wall ringing, and thus without proper wall mitigation, target detection becomes extremely difficult. The results presented in this thesis focus on the development of wall parameter estimation, and intra-wall and wall-type characterization techniques for use in both the time and frequency domains as well as analysis of these techniques under various real world scenarios such as reduced system bandwidth scenarios, various wall backing scenarios, the case of inhomogeneous walls, presence

Differentiated roles for MreB-actin isologues and autolytic enzymes in Bacillus subtilis morphogenesis.

PubMed

Domínguez-Cuevas, Patricia; Porcelli, Ida; Daniel, Richard A; Errington, Jeff

2013-09-01

Cell morphogenesis in most bacteria is governed by spatiotemporal growth regulation of the peptidoglycan cell wall layer. Much is known about peptidoglycan synthesis but regulation of its turnover by hydrolytic enzymes is much less well understood. Bacillus subtilis has a multitude of such enzymes. Two of the best characterized are CwlO and LytE: cells lacking both enzymes have a lethal block in cell elongation. Here we show that activity of CwlO is regulated by an ABC transporter, FtsEX, which is required for cell elongation, unlike cell division as in Escherichia coli. Actin-like MreB proteins are thought to play a key role in orchestrating cell wall morphogenesis. B. subtilis has three MreB isologues with partially differentiated functions. We now show that the three MreB isologues have differential roles in regulation of the CwlO and LytE systems and that autolysins control different aspects of cell morphogenesis. The results add major autolytic activities to the growing list of functions controlled by MreB isologues in bacteria and provide new insights into the different specialized functions of essential cell wall autolysins. © 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

A sweet new role for LCP enzymes in protein glycosylation

DOE PAGES

Amer, Brendan R.; Clubb, Robert T.

2014-11-21

The peptidoglycan that surrounds Gram-positive bacteria is affixed with a range of macromolecules that enable the microbe to effectively interact with its environment. Distinct enzymes decorate the cell wall with proteins and glycopolymers. Sortase enzymes covalently attach proteins to the peptidoglycan, while LytRCpsA-Psr (LCP) proteins are thought to attach teichoic acid polymers and capsular polysaccharides. Ton-That and colleagues have discovered a new glycosylation pathway in the oral bacterium Actinomyces oris in which sortase and LCP enzymes operate on the same protein substrate. The A. oris LCP protein has a novel function, acting on the cell surface to transfer glycan macromoleculesmore » to a protein, which is then attached to the cell wall by a sortase. The reactions are tightly coupled, as elimination of the sortase causes the lethal accumulation of glycosylated protein in the membrane. Furthermore, since sortase enzymes are attractive drug targets, this novel finding may provide a convenient cell-based tool to discover inhibitors of this important enzyme family.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amer, Brendan R.; Clubb, Robert T.

The peptidoglycan that surrounds Gram-positive bacteria is affixed with a range of macromolecules that enable the microbe to effectively interact with its environment. Distinct enzymes decorate the cell wall with proteins and glycopolymers. Sortase enzymes covalently attach proteins to the peptidoglycan, while LytRCpsA-Psr (LCP) proteins are thought to attach teichoic acid polymers and capsular polysaccharides. Ton-That and colleagues have discovered a new glycosylation pathway in the oral bacterium Actinomyces oris in which sortase and LCP enzymes operate on the same protein substrate. The A. oris LCP protein has a novel function, acting on the cell surface to transfer glycan macromoleculesmore » to a protein, which is then attached to the cell wall by a sortase. The reactions are tightly coupled, as elimination of the sortase causes the lethal accumulation of glycosylated protein in the membrane. Furthermore, since sortase enzymes are attractive drug targets, this novel finding may provide a convenient cell-based tool to discover inhibitors of this important enzyme family.« less

Localization and structural analysis of a conserved pyruvylated epitope in Bacillus anthracis secondary cell wall polysaccharides and characterization of the galactose-deficient wall polysaccharide from avirulent B. anthracis CDC 684.

PubMed

Forsberg, L Scott; Abshire, Teresa G; Friedlander, Arthur; Quinn, Conrad P; Kannenberg, Elmar L; Carlson, Russell W

2012-08-01

Bacillus anthracis CDC 684 is a naturally occurring, avirulent variant and close relative of the highly pathogenic B. anthracis Vollum. Bacillus anthracis CDC 684 contains both virulence plasmids, pXO1 and pXO2, yet is non-pathogenic in animal models, prompting closer scrutiny of the molecular basis of attenuation. We structurally characterized the secondary cell wall polysaccharide (SCWP) of B. anthracis CDC 684 (Ba684) using chemical and NMR spectroscopy analysis. The SCWP consists of a HexNAc trisaccharide backbone having identical structure as that of B. anthracis Pasteur, Sterne and Ames, →4)-β-d-ManpNAc-(1 → 4)-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNAc-(1→. Remarkably, although the backbone is fully polymerized, the SCWP is the devoid of all galactosyl side residues, a feature which normally comprises 50% of the glycosyl residues on the highly galactosylated SCWPs from pathogenic strains. This observation highlights the role of defective wall assembly in virulence and indicates that polymerization occurs independently of galactose side residue attachment. Of particular interest, the polymerized Ba684 backbone retains the substoichiometric pyruvate acetal, O-acetate and amino group modifications found on SCWPs from normal B. anthracis strains, and immunofluorescence analysis confirms that SCWP expression coincides with the ability to bind the surface layer homology (SLH) domain containing S-layer protein extractable antigen-1. Pyruvate was previously demonstrated as part of a conserved epitope, mediating SLH-domain protein attachment to the underlying peptidoglycan layer. We find that a single repeating unit, located at the distal (non-reducing) end of the Ba684 SCWP, is structurally modified and that this modification is present in identical manner in the SCWPs of normal B. anthracis strains. These polysaccharides terminate in the sequence: (S)-4,6-O-(1-carboxyethylidene)-β-d-ManpNAc-(1 → 4)-[3-O-acetyl]-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNH(2)-(1→.

Molecular adsorption study of nicotine and caffeine on single-walled carbon nanotubes from first principles

NASA Astrophysics Data System (ADS)

Lee, Hyung-June; Kim, Gunn; Kwon, Young-Kyun

2013-08-01

Using first-principles calculations, we investigate the electronic structures and binding properties of nicotine and caffeine adsorbed on single-walled carbon nanotubes to determine whether CNTs are appropriate for filtering or sensing nicotine and caffeine molecules. We find that caffeine adsorbs more strongly than nicotine. The different binding characteristics are discussed by analyzing the modification of the electronic structure of the molecule-adsorbed CNTs. We also calculate the quantum conductance of the CNTs in the presence of nicotine or caffeine adsorbates and demonstrate that the influence of caffeine is stronger than nicotine on the conductance of the host CNT.

Cell Wall Structure of Coccoid Green Algae as an Important Trade-Off Between Biotic Interference Mechanisms and Multidimensional Cell Growth.

PubMed

Dunker, Susanne; Wilhelm, Christian

2018-01-01

Coccoid green algae can be divided in two groups based on their cell wall structure. One group has a highly chemical resistant cell wall (HR-cell wall) containing algaenan. The other group is more susceptible to chemicals (LR-cell wall - Low resistant cell wall). Algaenan is considered as important molecule to explain cell wall resistance. Interestingly, cell wall types (LR- and HR-cell wall) are not in accordance with the taxonomic classes Chlorophyceae and Trebouxiophyceae, which makes it even more interesting to consider the ecological function. It was already shown that algaenan helps to protect against virus, bacterial and fungal attack, but in this study we show for the first time that green algae with different cell wall properties show different sensitivity against interference competition with the cyanobacterium Microcystis aeruginosa . Based on previous work with co-cultures of M. aeruginosa and two green algae ( Acutodesmus obliquus and Oocystis marssonii ) differing in their cell wall structure, it was shown that M. aeruginosa could impair only the growth of the green algae if they belong to the LR-cell wall type. In this study it was shown that the sensitivity to biotic interference mechanism shows a more general pattern within coccoid green algae species depending on cell wall structure.

Tunicamycins: translocase-I inhibitors that target bacterial cell wall and mammalian N-glycoproteins. The potential for selective inhibitors

USDA-ARS?s Scientific Manuscript database

Tunicamycins are a heterologous family of nucleoside antibiotics that target the biosynthesis of bacterial peptidoglycan and eukaryotic N-glycoproteins. The mechanism of action is known, with the tunicamycin-Mg2+ complex established as a transition state analog for hexosamine-1-phosphate: prenol pho...

Wind tunnels with adapted walls for reducing wall interference

NASA Technical Reports Server (NTRS)

Ganzer, U.

1979-01-01

The basic principle of adaptable wind tunnel walls is explained. First results of an investigation carried out at the Aero-Space Institute of Berlin Technical University are presented for two dimensional flexible walls and a NACA 0012 airfoil. With five examples exhibiting very different flow conditions it is demonstrated that it is possible to reduce wall interference and to avoid blockage at transonic speeds by wall adaptation.

Method for delivery of small molecules and proteins across the cell wall of algae using molecular transporters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geihe, Erika; Trantow, Brian; Wender, Paul

The introduction of tools to study, control or expand the inner-workings of algae has been slow to develop. Provided are embodiments of a molecular method based on guanidinium-rich molecular transporters (GR-MoTrs) for bringing molecular cargos into algal cells. The methods of the disclosure have been shown to work in wild-type algae that have an intact cell wall. Developed using Chlamydomonas reinhardtii, this method is also successful with less studied algae, including Neochloris oleoabundans and Scenedesmus dimorphus, thus providing a new and versatile tool for algal research and modification. The method of delivering a cargo compound to an algal cell comprisesmore » contacting an algal cell with a guanidinium-rich delivery vehicle comprising a guanidinium-rich molecular transporter (GR-MoTr) linked to a cargo compound desired to be delivered to the algal cell, whereby the guanidinium-rich molecular transporter can traverse the algal cell wall, thereby delivering the cargo compound to the algal cell.« less

Inverse measurement of wall pressure field in flexible-wall wind tunnels using global wall deformation data

NASA Astrophysics Data System (ADS)

Brown, Kenneth; Brown, Julian; Patil, Mayuresh; Devenport, William

2018-02-01

The Kevlar-wall anechoic wind tunnel offers great value to the aeroacoustics research community, affording the capability to make simultaneous aeroacoustic and aerodynamic measurements. While the aeroacoustic potential of the Kevlar-wall test section is already being leveraged, the aerodynamic capability of these test sections is still to be fully realized. The flexibility of the Kevlar walls suggests the possibility that the internal test section flow may be characterized by precisely measuring small deflections of the flexible walls. Treating the Kevlar fabric walls as tensioned membranes with known pre-tension and material properties, an inverse stress problem arises where the pressure distribution over the wall is sought as a function of the measured wall deflection. Experimental wall deformations produced by the wind loading of an airfoil model are measured using digital image correlation and subsequently projected onto polynomial basis functions which have been formulated to mitigate the impact of measurement noise based on a finite-element study. Inserting analytic derivatives of the basis functions into the equilibrium relations for a membrane, full-field pressure distributions across the Kevlar walls are computed. These inversely calculated pressures, after being validated against an independent measurement technique, can then be integrated along the length of the test section to give the sectional lift of the airfoil. Notably, these first-time results are achieved with a non-contact technique and in an anechoic environment.

ATP-binding cassette transporter 1 participates in LDL oxidation by artery wall cells.

PubMed

Reddy, Srinivasa T; Hama, Susan; Ng, Carey; Grijalva, Victor; Navab, Mohamad; Fogelman, Alan M

2002-11-01

We have previously reported that products of the lipoxygenase pathway, hydroperoxyoctadecadienoic acid and hydroperoxyeicosatetraenoic acid, as well as cholesterol linoleate hydroperoxides, collectively termed seeding molecules, are removed by apolipoprotein A-I (apoA-I) from the artery wall cells and render low density lipoprotein (LDL) resistant to oxidation by human artery wall cells. The mechanisms by which oxidized lipids are transported and/or transferred to lipoproteins and the pathways by which apoA-I facilitates their removal remain unclear. ATP-binding cassette transporter 1 (ABCA1) is known to facilitate the release of cellular phospholipids and cholesterol from the plasma membrane to apoA-I and high density lipoprotein. Therefore, we evaluated whether ABCA1 participates in LDL oxidation. In this report, we show that (1) chemical inhibitors of ABCA1 function, glyburide and DIDS, block artery wall cell-mediated oxidative modification of LDL, (2) inhibition of ABCA1 with the use of antisense (but not sense) oligonucleotides prevents LDL-induced lipid hydroperoxide formation and LDL-induced monocyte chemotactic activity by the artery wall cells, and (3) oxysterols that induce ABCA1 expression, such as 22(R)hydroxycholesterol, enhance cell-mediated LDL oxidation. Furthermore, we also show that 22(R)hydroxycholesterol induces the production of reactive oxygen species in the artery wall cells, which can be removed by incubating the artery wall cells with apoA-I. Our data suggest that ABCA1 plays an important role in artery wall cell-mediated modification/oxidation of LDL by modulating the release of reactive oxygen species from artery wall cells that are necessary for LDL oxidation.

Electronic structure of multi-walled carbon fullerenes

NASA Astrophysics Data System (ADS)

Doore, Keith; Cook, Matthew; Clausen, Eric; Lukashev, Pavel V.; Kidd, Tim E.; Stollenwerk, Andrew J.

2017-02-01

Despite an enormous amount of research on carbon based nanostructures, relatively little is known about the electronic structure of multi-walled carbon fullerenes, also known as carbon onions. In part, this is due to the very high computational expense involved in estimating electronic structure of large molecules. At the same time, experimentally, the exact crystal structure of the carbon onion is usually unknown, and therefore one relies on qualitative arguments only. In this work we present the results of a computational study on a series of multi-walled fullerenes and compare their electronic structures to experimental data. Experimentally, the carbon onions were fabricated using ultrasonic agitation of isopropanol alcohol and deposited onto the surface of highly ordered pyrolytic graphite using a drop cast method. Scanning tunneling microscopy images indicate that the carbon onions produced using this technique are ellipsoidal with dimensions on the order of 10 nm. The majority of differential tunneling spectra acquired on individual carbon onions are similar to that of graphite with the addition of molecular-like peaks, indicating that these particles span the transition between molecules and bulk crystals. A smaller, yet sizable number exhibited a semiconducting gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) levels. These results are compared with the electronic structure of different carbon onion configurations calculated using first-principles. Similar to the experimental results, the majority of these configurations are metallic with a minority behaving as semiconductors. Analysis of the configurations investigated here reveals that each carbon onion exhibiting an energy band gap consisted only of non-metallic fullerene layers, indicating that the interlayer interaction is not significant enough to affect the total density of states in these structures.

Reciprocal Interactions between Cadmium-Induced Cell Wall Responses and Oxidative Stress in Plants

PubMed Central

Loix, Christophe; Huybrechts, Michiel; Vangronsveld, Jaco; Gielen, Marijke; Keunen, Els; Cuypers, Ann

2017-01-01

Cadmium (Cd) pollution renders many soils across the world unsuited or unsafe for food- or feed-orientated agriculture. The main mechanism of Cd phytotoxicity is the induction of oxidative stress, amongst others through the depletion of glutathione. Oxidative stress can damage lipids, proteins, and nucleic acids, leading to growth inhibition or even cell death. The plant cell has a variety of tools to defend itself against Cd stress. First and foremost, cell walls might prevent Cd from entering and damaging the protoplast. Both the primary and secondary cell wall have an array of defensive mechanisms that can be adapted to cope with Cd. Pectin, which contains most of the negative charges within the primary cell wall, can sequester Cd very effectively. In the secondary cell wall, lignification can serve to immobilize Cd and create a tougher barrier for entry. Changes in cell wall composition are, however, dependent on nutrients and conversely might affect their uptake. Additionally, the role of ascorbate (AsA) as most important apoplastic antioxidant is of considerable interest, due to the fact that oxidative stress is a major mechanism underlying Cd toxicity, and that AsA biosynthesis shares several links with cell wall construction. In this review, modifications of the plant cell wall in response to Cd exposure are discussed. Focus lies on pectin in the primary cell wall, lignification in the secondary cell wall and the importance of AsA in the apoplast. Regarding lignification, we attempt to answer the question whether increased lignification is merely a consequence of Cd toxicity, or rather an elicited defense response. We propose a model for lignification as defense response, with a central role for hydrogen peroxide as substrate and signaling molecule. PMID:29163592

A post-labeling strategy based on dye-induced peeling of the aptamer off single-walled carbon nanotubes for electrochemical aptasensing.

PubMed

Fu, Yingchun; Wang, Ting; Bu, Lijuan; Xie, Qingji; Li, Penghao; Chen, Jinhua; Yao, Shouzhuo

2011-03-07

A simple and efficient post-labeling strategy based on dye-induced peeling of the aptamer molecules off single-walled carbon nanotubes was developed for electrochemical aptasensing of thrombin with a detection limit down to 3 pM.

Homology modeling and docking analyses of M. leprae Mur ligases reveals the common binding residues for structure based drug designing to eradicate leprosy.

PubMed

Shanmugam, Anusuya; Natarajan, Jeyakumar

2012-06-01

Multi drug resistance capacity for Mycobacterium leprae (MDR-Mle) demands the profound need for developing new anti-leprosy drugs. Since most of the drugs target a single enzyme, mutation in the active site renders the antibiotic ineffective. However, structural and mechanistic information on essential bacterial enzymes in a pathway could lead to the development of antibiotics that targets multiple enzymes. Peptidoglycan is an important component of the cell wall of M. leprae. The biosynthesis of bacterial peptidoglycan represents important targets for the development of new antibacterial drugs. Biosynthesis of peptidoglycan is a multi-step process that involves four key Mur ligase enzymes: MurC (EC:6.3.2.8), MurD (EC:6.3.2.9), MurE (EC:6.3.2.13) and MurF (EC:6.3.2.10). Hence in our work, we modeled the three-dimensional structure of the above Mur ligases using homology modeling method and analyzed its common binding features. The residues playing an important role in the catalytic activity of each of the Mur enzymes were predicted by docking these Mur ligases with their substrates and ATP. The conserved sequence motifs significant for ATP binding were predicted as the probable residues for structure based drug designing. Overall, the study was successful in listing significant and common binding residues of Mur enzymes in peptidoglycan pathway for multi targeted therapy.

Biochemical and Immunocytological Characterizations of Arabidopsis Pollen Tube Cell Wall1[C][W][OA

PubMed Central

Dardelle, Flavien; Lehner, Arnaud; Ramdani, Yasmina; Bardor, Muriel; Lerouge, Patrice; Driouich, Azeddine; Mollet, Jean-Claude

2010-01-01

During plant sexual reproduction, pollen germination and tube growth require development under tight spatial and temporal control for the proper delivery of the sperm cells to the ovules. Pollen tubes are fast growing tip-polarized cells able to perceive multiple guiding signals emitted by the female organ. Adhesion of pollen tubes via cell wall molecules may be part of the battery of signals. In order to study these processes, we investigated the cell wall characteristics of in vitro-grown Arabidopsis (Arabidopsis thaliana) pollen tubes using a combination of immunocytochemical and biochemical techniques. Results showed a well-defined localization of cell wall epitopes. Low esterified homogalacturonan epitopes were found mostly in the pollen tube wall back from the tip. Xyloglucan and arabinan from rhamnogalacturonan I epitopes were detected along the entire tube within the two wall layers and the outer wall layer, respectively. In contrast, highly esterified homogalacturonan and arabinogalactan protein epitopes were found associated predominantly with the tip region. Chemical analysis of the pollen tube cell wall revealed an important content of arabinosyl residues (43%) originating mostly from (1→5)-α-l-arabinan, the side chains of rhamnogalacturonan I. Finally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of endo-glucanase-sensitive xyloglucan showed mass spectra with two dominant oligosaccharides (XLXG/XXLG and XXFG), both being mono O-acetylated, and accounting for over 68% of the total ion signals. These findings demonstrate that the Arabidopsis pollen tube wall has its own characteristics compared with other cell types in the Arabidopsis sporophyte. These structural features are discussed in terms of pollen tube cell wall biosynthesis and growth dynamics. PMID:20547702

A glycosylphosphatidylinositol anchor is required for membrane localization but dispensable for cell wall association of chitin deacetylase 2 in Cryptococcus neoformans.

PubMed

Gilbert, Nicole M; Baker, Lorina G; Specht, Charles A; Lodge, Jennifer K

2012-01-01

Cell wall proteins (CWPs) mediate important cellular processes in fungi, including adhesion, invasion, biofilm formation, and flocculation. The current model of fungal cell wall organization includes a major class of CWPs covalently bound to β-1,6-glucan via a remnant of a glycosylphosphatidylinositol (GPI) anchor. This model was established by studies of ascomycetes more than a decade ago, and relatively little work has been done with other fungi, although the presumption has been that proteins identified in the cell wall which contain a predicted GPI anchor are covalently linked to cell wall glucans. The pathogenic basidiomycete Cryptococcus neoformans encodes >50 putatively GPI-anchored proteins, some of which have been identified in the cell wall. One of these proteins is chitin deacetylase 2 (Cda2), an enzyme responsible for converting chitin to chitosan, a cell wall polymer recently established as a virulence factor for C. neoformans infection of mammalian hosts. Using a combination of biochemistry, molecular biology, and genetics, we show that Cda2 is GPI anchored to membranes but noncovalently associated with the cell wall by means independent of both its GPI anchor and β-1,6-glucan. We also show that Cda2 produces chitosan when localized to the plasma membrane, but association with the cell wall is not essential for this process, thereby providing insight into the mechanism of chitosan biosynthesis. These results increase our understanding of the surface of C. neoformans and provide models of cell walls likely applicable to other undercharacterized basidiomycete pathogenic fungi. The surface of a pathogenic microbe is a major interface with its host. In fungi, the outer surface consists of a complex matrix known as the cell wall, which includes polysaccharides, proteins, and other molecules. The mammalian host recognizes many of these surface molecules and mounts appropriate responses to combat the microbial infection. Cryptococcus neoformans is a

Spontaneous Behaviors and Wall-Curvature Lead to Apparent Wall Preference in Planarian.

PubMed

Akiyama, Yoshitaro; Agata, Kiyokazu; Inoue, Takeshi

2015-01-01

The planarian Dugesia japonica tends to stay near the walls of its breeding containers and experimental dishes in the laboratory, a phenomenon called "wall preference". This behavior is thought to be important for environmental adaptation, such as hiding by planarians in nature. However, the mechanisms regulating wall-preference behavior are not well understood, since this behavior occurs in the absence of any particular stimulation. Here we show the mechanisms of wall-preference behavior. Surprisingly, planarian wall-preference behavior was also shown even by the head alone and by headless planarians. These results indicate that planarian "wall-preference" behavior only appears to be a "preference" behavior, and is actually an outcome of spontaneous behaviors, rather than of brain function. We found that in the absence of environmental cues planarians moved basically straight ahead until they reached a wall, and that after reaching a wall, they changed their direction of movement to one tangential to the wall, suggesting that this spontaneous behavior may play a critical role in the wall preference. When we tested another spontaneous behavior, the wigwag movement of the planarian head, using computer simulation with various wigwag angles and wigwag intervals, large wigwag angle and short wigwag interval reduced wall-preference behavior. This indicated that wigwag movement may determine the probability of staying near the wall or leaving the wall. Furthermore, in accord with this simulation, when we tested planarian wall-preference behavior using several assay fields with different curvature of the wall, we found that concavity and sharp curvature of walls negatively impacted wall preference by affecting the permissible angle of the wigwag movement. Together, these results indicate that planarian wall preference may be involuntarily caused by the combination of two spontaneous planarian behaviors: moving straight ahead until reaching a wall and then moving along it

Modification of near-wall coherent structures in polymer drag reduced flow: simulation

NASA Astrophysics Data System (ADS)

Dubief, Yves; White, Christopher; Shaqfeh, Eric; Moin, Parviz; Lele, Sanjiva

2002-11-01

Polymer drag reduced flows are investigated through direct numerical simulations of viscoelastic flows. The solver for the viscoelastic model (FENE-P) is based on higher-order finite difference schemes and a novel implicit time integration method. Its robustness allows the simulation of all drag reduction (DR) regimes from the onset to the maximum drag reduction (MDR). It also permits the use of realistic polymer length and concentration. The maximum polymer extension in our simulation matches that of a polystyrene molecule of 10^6 molecular weight. Two distinct regimes of polymer drag reduced flows are observed: at low drag reduction (LDR, DR< 40-50%), the near-wall structure is essentially similar to Newtonian wall turbulence whereas the high drag reduction regime (HDR, DR from 40-50% to MDR) shows significant differences in the organization of the coherent structures. The 3D information provided by numerical simulations allows the determination of the interaction of polymers and near-wall coherent structures. To isolate the contribution of polymers in the viscous sublayer, the buffer and the outer region of the flow, numerical experiments are performed where the polymer concentration is varied in the wall-normal direction. Finally a mechanism of polymer drag reduction derived from our results and PIV measurements is discussed.

Imaging fluorescence detected linear dichroism of plant cell walls in laser scanning confocal microscope.

PubMed

Steinbach, Gábor; Pomozi, István; Zsiros, Ottó; Páy, Anikó; Horváth, Gábor V; Garab, Gyozo

2008-03-01

Anisotropy carries important information on the molecular organization of biological samples. Its determination requires a combination of microscopy and polarization spectroscopy tools. The authors constructed differential polarization (DP) attachments to a laser scanning microscope in order to determine physical quantities related to the anisotropic distribution of molecules in microscopic samples; here the authors focus on fluorescence-detected linear dichroism (FDLD). By modulating the linear polarization of the laser beam between two orthogonally polarized states and by using a demodulation circuit, the authors determine the associated transmitted and fluorescence intensity-difference signals, which serve the basis for LD (linear dichroism) and FDLD, respectively. The authors demonstrate on sections of Convallaria majalis root tissue stained with Acridin Orange that while (nonconfocal) LD images remain smeared and weak, FDLD images recorded in confocal mode reveal strong anisotropy of the cell wall. FDLD imaging is suitable for mapping the anisotropic distribution of transition dipoles in 3 dimensions. A mathematical model is proposed to account for the fiber-laminate ultrastructure of the cell wall and for the intercalation of the dye molecules in complex, highly anisotropic architecture. Copyright 2007 International Society for Analytical Cytology.

Revealing of Saccharomyces cerevisiae yeast cell wall proteins capable of binding thioflavin T, a fluorescent dye specifically interacting with amyloid fibrils.

PubMed

Gorkovskii, A A; Bezsonov, E E; Plotnikova, T A; Kalebina, T S; Kulaev, I S

2009-11-01

Proteins binding thioflavin T leading to its specific fluorescence were discovered in a fraction of noncovalently bound Saccharomyces cerevisiae yeast cell wall mannoproteins. Thioflavin-binding proteins display high resistance to trypsin digestion in solution. These data are the first experimental evidence for the presence of proteins whose properties are characteristic of amyloids in yeast cell wall, except for data on glucanotransferase Bgl2p that has amyloid properties. Our data suggest the anchoring of these proteins in the cell wall by a trypsin-sensitive part of the protein molecule. Experiments with a mutant strain devoid of the BGL2 gene suggest the compensation of absent amyloid-like protein Bgl2p by increase in contents of thioflavin-binding proteins in the cell wall.

An assessment for the erosion rate of DEMO first wall

NASA Astrophysics Data System (ADS)

Tokar, M. Z.

2018-01-01

In a fusion reactor a significant fraction of plasma particles lost from the confined volume will reach the vessel wall. The recombination of these charged species, electrons and ions of hydrogen isotopes, is a source of neutral molecules and atoms, recycling back into the plasma. Here they participate, in particular, in charge-exchange (c-x) collisions with the plasma ions and, as a result, atoms of high energies with chaotically oriented velocities are generated. A significant fraction of these hot neutrals will hit the wall, leading, as well as the outflowing fuel and impurity ions, to its erosion, limiting the reactor operation time. The rate of the wall erosion in DEMO is assessed by applying a one-dimensional model which takes into account the transport of charged and neutral species across the flux surfaces in the main part of the scrape-off layer, beyond the X-point vicinity and divertor, and by considering the shift of the centers of flux surfaces, their elongation and triangularity. Atoms generated by c-x of recycling neutrals are modeled kinetically to define firmly their energy spectrum, being of particular importance for the erosion assessment. It is demonstrated the erosion rate of the DEMO wall armor of tungsten will have a pronounced ballooning character with a significant maximum of 0.3 mm per full power year at the low field side, decreasing with an increase in the anomalous perpendicular transport in the ‘far’ SOL or the plasma density at the separatrix.

Magnetic resonance tells microbiology where to go; bacterial teichoic acid protects liquid water at sub-zero temperatures

NASA Astrophysics Data System (ADS)

Rice, Charles V.; Wickham, Jason R.; Eastman, Margaret A.; Harrison, William; Pereira, Mark P.; Brown, Eric D.

2008-08-01

Numerous chemical additives lower the freezing point of water, but life at sub-zero temperatures is sustained by a limited number of biological cryoprotectants. Antifreeze proteins in fish, plants, and insects provide protection to a few degrees below freezing. Microbes have been found to survive at even lower temperatures, although, with a few exceptions, antifreeze proteins are missing. Survival has been attributed to external factors, such as high salt concentration (brine veins) and adhesion to particulates or ice crystal defects. Teichoic acid is a phosphodiester polymer ubiquitous in Gram positive bacteria, composing 50% of the mass of the bacterial cell wall and excreted into the extracellular space of biofilm communities. We have found that when bound to the peptidoglycan cell wall (wall teichoic acid) or as a free molecule (lipoteichoic acid), teichoic acid is surrounded by liquid water at temperatures significantly below freezing. Using solid-state NMR, we are unable to collect 31P CPMAS spectra for frozen solutions of lipoteichoic acid at temperatures above -60 °C. For wall teichoic acid in D2O, signals are not seen above -30 °C. These results can be explained by the presence of liquid water, which permits rapid molecular motion to remove 1H/31P dipolar coupling. 2H quadrupole echo NMR spectroscopy reveals that both liquid and solid water are present. We suggest that teichoic acids could provide a shell of liquid water around biofilms and planktonic bacteria, removing the need for brine veins to prevent bacterial freezing.

Wall shear stress measurement in blade end-wall corner region

NASA Technical Reports Server (NTRS)

Bhargava, R.; Raj, R.; Boldman, D. R.

1987-01-01

The magnitude and the direction of wall shear stress and surface pressure in the blade end-wall corner region were investigated. The measurements were obtained on a specially designed Preston tube, the tip of which could be concentrically rotated about its axis of rotation at the measurement location. The magnitude of wall shear stress in the vicinity of the corner was observed to increase significantly (170 percent) compared to its far-upstream value; the increase was consistently higher on the blade surface compared to the value on the plate surface of the blade end-wall corner. On both surfaces in the blade end-wall corner, the variation of the wall shear stress direction was found to be more predominant in the vicinity of the blade leading-edge location. The trend of the measured wall shear stress direction showed good agreement with the limiting streamline directions obtained from the flow visualization studies.

Segmentation and additive approach: A reliable technique to study noncovalent interactions of large molecules at the surface of single-wall carbon nanotubes.

PubMed

Torres, Ana M; Scheiner, Steve; Roy, Ajit K; Garay-Tapia, Andrés M; Bustamante, John; Kar, Tapas

2016-08-05

This investigation explores a new protocol, named Segmentation and Additive approach (SAA), to study exohedral noncovalent functionalization of single-walled carbon nanotubes with large molecules, such as polymers and biomolecules, by segmenting the entire system into smaller units to reduce computational cost. A key criterion of the segmentation process is the preservation of the molecular structure responsible for stabilization of the entire system in smaller segments. Noncovalent interaction of linoleic acid (LA, C18 H32 O2 ), a fatty acid, at the surface of a (10,0) zigzag nanotube is considered for test purposes. Three smaller segmented models have been created from the full (10,0)-LA system and interaction energies were calculated for these models and compared with the full system at different levels of theory, namely ωB97XD, LDA. The success of this SAA is confirmed as the sum of the interaction energies is in very good agreement with the total interaction energy. Besides reducing computational cost, another merit of SAA is an estimation of the contributions from different sections of the large system to the total interaction energy which can be studied in-depth using a higher level of theory to estimate several properties of each segment. On the negative side, bulk properties, such as HOMO-LUMO (highest occupied molecular orbital - lowest occupied molecular orbital) gap, of the entire system cannot be estimated by adding results from segment models. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Wall-based identification of coherent structures in wall-bounded turbulence

NASA Astrophysics Data System (ADS)

Sanmiguel Vila, C.; Flores, O.

2018-04-01

During the last decades, a number of reduced order models based on coherent structures have been proposed to describe wall-bounded turbulence. Many of these models emphasize the importance of coherent wall-normal velocity eddies (ν-eddies), which drive the generation of the very long streamwise velocity structures observed in the logarithmic and outer region. In order to use these models to improve our ability to control wall-bounded turbulence in realistic applications, these ν-eddies need to be identified from the wall in a non-intrusive way. In this paper, the possibility of using the pressure signal at the wall to identify these ν-eddies is explored, analyzing the cross-correlation between the wall-normal velocity component and the pressure fluctuations at the wall in a DNS of a turbulent channel flow at Reτ = 939. The results show that the cross-correlation has a region of negative correlation upstream, and a region of positive correlation backwards. In the spanwise direction the correlation decays monotonously, except very close to the wall where a change of sign of the correlation coefficient is observed. Moreover, filtering the pressure fluctuations at the wall in space results in an increase of the region where the cross-correlation is strong, both for the positively and the negatively correlated regions. The use of a time filter for the pressure fluctuations at the wall yields different results, displacing the regions of strong correlation without changing much their sizes. The results suggest that space-filtering the pressure at the wall is a feasible way to identify ν-eddies of different sizes, which could be used to trigger turbulent control strategies.

Spontaneous Behaviors and Wall-Curvature Lead to Apparent Wall Preference in Planarian

PubMed Central

Akiyama, Yoshitaro; Agata, Kiyokazu; Inoue, Takeshi

2015-01-01

The planarian Dugesia japonica tends to stay near the walls of its breeding containers and experimental dishes in the laboratory, a phenomenon called “wall preference”. This behavior is thought to be important for environmental adaptation, such as hiding by planarians in nature. However, the mechanisms regulating wall-preference behavior are not well understood, since this behavior occurs in the absence of any particular stimulation. Here we show the mechanisms of wall-preference behavior. Surprisingly, planarian wall-preference behavior was also shown even by the head alone and by headless planarians. These results indicate that planarian “wall-preference” behavior only appears to be a “preference” behavior, and is actually an outcome of spontaneous behaviors, rather than of brain function. We found that in the absence of environmental cues planarians moved basically straight ahead until they reached a wall, and that after reaching a wall, they changed their direction of movement to one tangential to the wall, suggesting that this spontaneous behavior may play a critical role in the wall preference. When we tested another spontaneous behavior, the wigwag movement of the planarian head, using computer simulation with various wigwag angles and wigwag intervals, large wigwag angle and short wigwag interval reduced wall-preference behavior. This indicated that wigwag movement may determine the probability of staying near the wall or leaving the wall. Furthermore, in accord with this simulation, when we tested planarian wall-preference behavior using several assay fields with different curvature of the wall, we found that concavity and sharp curvature of walls negatively impacted wall preference by affecting the permissible angle of the wigwag movement. Together, these results indicate that planarian wall preference may be involuntarily caused by the combination of two spontaneous planarian behaviors: moving straight ahead until reaching a wall and then

Domain wall nanoelectronics

NASA Astrophysics Data System (ADS)

Catalan, G.; Seidel, J.; Ramesh, R.; Scott, J. F.

2012-01-01

Domains in ferroelectrics were considered to be well understood by the middle of the last century: They were generally rectilinear, and their walls were Ising-like. Their simplicity stood in stark contrast to the more complex Bloch walls or Néel walls in magnets. Only within the past decade and with the introduction of atomic-resolution studies via transmission electron microscopy, electron holography, and atomic force microscopy with polarization sensitivity has their real complexity been revealed. Additional phenomena appear in recent studies, especially of magnetoelectric materials, where functional properties inside domain walls are being directly measured. In this paper these studies are reviewed, focusing attention on ferroelectrics and multiferroics but making comparisons where possible with magnetic domains and domain walls. An important part of this review will concern device applications, with the spotlight on a new paradigm of ferroic devices where the domain walls, rather than the domains, are the active element. Here magnetic wall microelectronics is already in full swing, owing largely to the work of Cowburn and of Parkin and their colleagues. These devices exploit the high domain wall mobilities in magnets and their resulting high velocities, which can be supersonic, as shown by Kreines’ and co-workers 30 years ago. By comparison, nanoelectronic devices employing ferroelectric domain walls often have slower domain wall speeds, but may exploit their smaller size as well as their different functional properties. These include domain wall conductivity (metallic or even superconducting in bulk insulating or semiconducting oxides) and the fact that domain walls can be ferromagnetic while the surrounding domains are not.

19. INTERIOR OF UTILITY ROOM SHOWING STUCCO WALL/DRYWALL WALL TRANSITION, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

19. INTERIOR OF UTILITY ROOM SHOWING STUCCO WALL/DRYWALL WALL TRANSITION, ELECTRICAL JUNCTION BOXES, BUILT-IN WALL CABINETRY, AND ELECTRICAL WALL HEATER. VIEW TO NORTHEAST. - Bishop Creek Hydroelectric System, Plant 4, Worker Cottage, Bishop Creek, Bishop, Inyo County, CA

22. SIDE WALL CONSTRUCTION, NORTH TRAINING WALL, LOOKING WEST FROM ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

22. SIDE WALL CONSTRUCTION, NORTH TRAINING WALL, LOOKING WEST FROM THE SAME POINT AS VIEW NO. 21. - Oakland Harbor Training Walls, Mouth of Federal Channel to Inner Harbor, Oakland, Alameda County, CA

Co-Inactivation of GlnR and CodY Regulators Impacts Pneumococcal Cell Wall Physiology.

PubMed

Johnston, Calum; Bootsma, Hester J; Aldridge, Christine; Manuse, Sylvie; Gisch, Nicolas; Schwudke, Dominik; Hermans, Peter W M; Grangeasse, Christophe; Polard, Patrice; Vollmer, Waldemar; Claverys, Jean-Pierre

2015-01-01

CodY, a nutritional regulator highly conserved in low G+C Gram-positive bacteria, is essential in Streptococcus pneumoniae (the pneumococcus). A published codY mutant possessed suppressing mutations inactivating the fatC and amiC genes, respectively belonging to iron (Fat/Fec) and oligopeptide (Ami) ABC permease operons, which are directly repressed by CodY. Here we analyzed two additional published codY mutants to further explore the essentiality of CodY. We show that one, in which the regulator of glutamine/glutamate metabolism glnR had been inactivated by design, had only a suppressor in fecE (a gene in the fat/fec operon), while the other possessed both fecE and amiC mutations. Independent isolation of three different fat/fec suppressors thus establishes that reduction of iron import is crucial for survival without CodY. We refer to these as primary suppressors, while inactivation of ami, which is not essential for survival of codY mutants and acquired after initial fat/fec inactivation, can be regarded as a secondary suppressor. The availability of codY- ami+ cells allowed us to establish that CodY activates competence for genetic transformation indirectly, presumably by repressing ami which is known to antagonize competence. The glnR codY fecE mutant was then found to be only partially viable on solid medium and hypersensitive to peptidoglycan (PG) targeting agents such as the antibiotic cefotaxime and the muramidase lysozyme. While analysis of PG and teichoic acid composition uncovered no alteration in the glnR codY fecE mutant compared to wildtype, electron microscopy revealed altered ultrastructure of the cell wall in the mutant, establishing that co-inactivation of GlnR and CodY regulators impacts pneumococcal cell wall physiology. In light of rising levels of resistance to PG-targeting antibiotics of natural pneumococcal isolates, GlnR and CodY constitute potential alternative therapeutic targets to combat this debilitating pathogen, as co

Wall Shear Stress, Wall Pressure and Near Wall Velocity Field Relationships in a Whirling Annular Seal

NASA Technical Reports Server (NTRS)

Morrison, Gerald L.; Winslow, Robert B.; Thames, H. Davis, III

1996-01-01

The mean and phase averaged pressure and wall shear stress distributions were measured on the stator wall of a 50% eccentric annular seal which was whirling in a circular orbit at the same speed as the shaft rotation. The shear stresses were measured using flush mounted hot-film probes. Four different operating conditions were considered consisting of Reynolds numbers of 12,000 and 24,000 and Taylor numbers of 3,300 and 6,600. At each of the operating conditions the axial distribution (from Z/L = -0.2 to 1.2) of the mean pressure, shear stress magnitude, and shear stress direction on the stator wall were measured. Also measured were the phase averaged pressure and shear stress. These data were combined to calculate the force distributions along the seal length. Integration of the force distributions result in the net forces and moments generated by the pressure and shear stresses. The flow field inside the seal operating at a Reynolds number of 24,000 and a Taylor number of 6,600 has been measured using a 3-D laser Doppler anemometer system. Phase averaged wall pressure and wall shear stress are presented along with phase averaged mean velocity and turbulence kinetic energy distributions located 0.16c from the stator wall where c is the seal clearance. The relationships between the velocity, turbulence, wall pressure and wall shear stress are very complex and do not follow simple bulk flow predictions.

Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release

PubMed Central

Greene, Neil G.; Narciso, Ana R.; Filipe, Sergio R.; Camilli, Andrew

2015-01-01

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens. PMID:26114646

Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release.

PubMed

Greene, Neil G; Narciso, Ana R; Filipe, Sergio R; Camilli, Andrew

2015-06-01

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens.

5. Detail of bin wall, showing the thinner exterior wall ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

5. Detail of bin wall, showing the thinner exterior wall next to the inner wall with its alternating courses of channel tile and hollow tile. - Saint Anthony Elevator No. 3, 620 Malcom Avenue, Southeast, Minneapolis, Hennepin County, MN

Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM

NASA Astrophysics Data System (ADS)

Alsteens, David; Dupres, Vincent; McEvoy, Kevin; Wildling, Linda; Gruber, Hermann J.; Dufrêne, Yves F.

2008-09-01

Although the chemical composition of yeast cell walls is known, the organization, assembly, and interactions of the various macromolecules remain poorly understood. Here, we used in situ atomic force microscopy (AFM) in three different modes to probe the ultrastructure, cell wall elasticity and polymer properties of two brewing yeast strains, i.e. Saccharomyces carlsbergensis and S. cerevisiae. Topographic images of the two strains revealed smooth and homogeneous cell surfaces, and the presence of circular bud scars on dividing cells. Nanomechanical measurements demonstrated that the cell wall elasticity of S. carlsbergensis is homogeneous. By contrast, the bud scar of S. cerevisiae was found to be stiffer than the cell wall, presumably due to the accumulation of chitin. Notably, single molecule force spectroscopy with lectin-modified tips revealed major differences in polysaccharide properties of the two strains. Polysaccharides were clearly more extended on S. cerevisiae, suggesting that not only oligosaccharides, but also polypeptide chains of the mannoproteins were stretched. Consistent with earlier cell surface analyses, these findings may explain the very different aggregation properties of the two organisms. This study demonstrates the power of using multiple complementary AFM modalities for probing the organization and interactions of the various macromolecules of microbial cell walls.

The Glycogen Synthase Kinase 3α and β Isoforms Differentially Regulates Interleukin-12p40 Expression in Endothelial Cells Stimulated with Peptidoglycan from Staphylococcus aureus.

PubMed

Cortés-Vieyra, Ricarda; Silva-García, Octavio; Oviedo-Boyso, Javier; Huante-Mendoza, Alejandro; Bravo-Patiño, Alejandro; Valdez-Alarcón, Juan J; Finlay, B Brett; Baizabal-Aguirre, Víctor M

2015-01-01

Glycogen synthase kinase 3 (GSK3) is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3β is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN), one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3β activity in bovine endothelial cells (BEC) regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3β at Ser9, and NF-κB p65 subunit (p65) at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3β. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor). Inhibition of GSK3α and GSK3β with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3β. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3β gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus.

Structure of a small-molecule inhibitor complexed with GlmU from Haemophilus influenzae reveals an allosteric binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mochalkin, Igor; Lightle, Sandra; Narasimhan, Lakshmi

2008-04-02

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and an attractive target for antibiotic drug discovery. GlmU catalyzes the formation of uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), an important precursor in the peptidoglycan and lipopolisaccharide biosynthesis in both Gram-negative and Gram-positive bacteria. Here we disclose a 1.9 {angstrom} resolution crystal structure of a synthetic small-molecule inhibitor of GlmU from Haemophilus influenzae (hiGlmU). The compound was identified through a high-throughput screening (HTS) configured to detect inhibitors that target the uridyltransferase active site of hiGlmU. The original HTS hit exhibited a modest micromolar potency (IC{sub 50} - 18 {mu}M in a racemic mixture) againstmore » hiGlmU and no activity against Staphylococcus aureus GlmU (saGlmU). The determined crystal structure indicated that the inhibitor occupies an allosteric site adjacent to the GlcNAc-1-P substrate-binding region. Analysis of the mechanistic model of the uridyltransferase reaction suggests that the binding of this allosteric inhibitor prevents structural rearrangements that are required for the enzymatic reaction, thus providing a basis for structure-guided design of a new class of mechanism-based inhibitors of GlmU.« less

Biomolecular Doping of Single-Walled Carbon Nanotubes by Thyroid Hormone

NASA Astrophysics Data System (ADS)

Rojas, Enrique; Paulson, Scott; Stern, Mike; Staii, Cristian; Dratman, Mary; Johnson, Alan

2004-03-01

Electron doping of semiconducting single-walled carbon nanotubes (SWNTs) by the thyroid hormone triiodothyronine (T3) is observed. T3 is applied locally, in solution, to SWNT field effect transistors (FETs) and binds along the length of the nanotube. T3 acts as an electron donor, shifting the I-V gate characteristics towards negative values of gate voltage. Shifts in the characteristics are measured as a function of the concentration of the solution. The effect is nearly reversible by rinsing the FETs with the solvent. Several days after application of T3, with no solvent rinsing, the gate characteristics are also nearly reversed. Experiments with a similar molecule for which the phenol ring is brominated as well as experiments with the de-iodinated molecule (T0) are performed to inform the effect of the iodine. The interaction of T3 with SWNTs may suggest a electronic interaction of T3 with other one-dimensional systems such as DNA.

AcmB Is an S-Layer-Associated β-N-Acetylglucosaminidase and Functional Autolysin in Lactobacillus acidophilus NCFM

PubMed Central

Johnson, Brant R.

2016-01-01

ABSTRACT Autolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome of Lactobacillus acidophilus NCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins in L. acidophilus NCFM are S-layer-associated proteins (SLAPs) noncovalently colocalized along with the surface (S)-layer at the cell surface. One of these SLAPs, AcmB, a β-N-acetylglucosaminidase encoded by the gene lba0176 (acmB), was selected for functional analysis. In silico analysis revealed that acmB orthologs are found exclusively in S-layer- forming species of Lactobacillus. Chromosomal deletion of acmB resulted in aberrant cell division, autolysis, and autoaggregation. Complementation of acmB in the ΔacmB mutant restored the wild-type phenotype, confirming the role of this SLAP in cell division. The absence of AcmB within the exoproteome had a pleiotropic effect on the extracellular proteins covalently and noncovalently bound to the peptidoglycan, which likely led to the observed decrease in the binding capacity of the ΔacmB strain for mucin and extracellular matrices fibronectin, laminin, and collagen in vitro. These data suggest a functional association between the S-layer and the multiple autolysins noncovalently colocalized at the cell surface of L. acidophilus NCFM and other S-layer-producing Lactobacillus species. IMPORTANCE Lactobacillus acidophilus is one of the most widely used probiotic microbes incorporated in many dairy foods and dietary supplements. This organism produces a surface (S)-layer, which is a self-assembling crystalline array found as the outermost layer of the cell wall. The S-layer, along with colocalized associated proteins, is an important mediator of probiotic activity through intestinal adhesion and modulation of

AcmB Is an S-Layer-Associated β-N-Acetylglucosaminidase and Functional Autolysin in Lactobacillus acidophilus NCFM.

PubMed

Johnson, Brant R; Klaenhammer, Todd R

2016-09-15

Autolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome of Lactobacillus acidophilus NCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins in L. acidophilus NCFM are S-layer-associated proteins (SLAPs) noncovalently colocalized along with the surface (S)-layer at the cell surface. One of these SLAPs, AcmB, a β-N-acetylglucosaminidase encoded by the gene lba0176 (acmB), was selected for functional analysis. In silico analysis revealed that acmB orthologs are found exclusively in S-layer- forming species of Lactobacillus Chromosomal deletion of acmB resulted in aberrant cell division, autolysis, and autoaggregation. Complementation of acmB in the ΔacmB mutant restored the wild-type phenotype, confirming the role of this SLAP in cell division. The absence of AcmB within the exoproteome had a pleiotropic effect on the extracellular proteins covalently and noncovalently bound to the peptidoglycan, which likely led to the observed decrease in the binding capacity of the ΔacmB strain for mucin and extracellular matrices fibronectin, laminin, and collagen in vitro These data suggest a functional association between the S-layer and the multiple autolysins noncovalently colocalized at the cell surface of L. acidophilus NCFM and other S-layer-producing Lactobacillus species. Lactobacillus acidophilus is one of the most widely used probiotic microbes incorporated in many dairy foods and dietary supplements. This organism produces a surface (S)-layer, which is a self-assembling crystalline array found as the outermost layer of the cell wall. The S-layer, along with colocalized associated proteins, is an important mediator of probiotic activity through intestinal adhesion and modulation of the mucosal immune

Regulation of Cell Wall Plasticity by Nucleotide Metabolism in Lactococcus lactis*

PubMed Central

Solopova, Ana; Formosa-Dague, Cécile; Courtin, Pascal; Furlan, Sylviane; Veiga, Patrick; Péchoux, Christine; Armalyte, Julija; Sadauskas, Mikas; Kok, Jan; Hols, Pascal; Dufrêne, Yves F.; Kuipers, Oscar P.; Chapot-Chartier, Marie-Pierre; Kulakauskas, Saulius

2016-01-01

To ensure optimal cell growth and separation and to adapt to environmental parameters, bacteria have to maintain a balance between cell wall (CW) rigidity and flexibility. This can be achieved by a concerted action of peptidoglycan (PG) hydrolases and PG-synthesizing/modifying enzymes. In a search for new regulatory mechanisms responsible for the maintenance of this equilibrium in Lactococcus lactis, we isolated mutants that are resistant to the PG hydrolase lysozyme. We found that 14% of the causative mutations were mapped in the guaA gene, the product of which is involved in purine metabolism. Genetic and transcriptional analyses combined with PG structure determination of the guaA mutant enabled us to reveal the pivotal role of the pyrB gene in the regulation of CW rigidity. Our results indicate that conversion of l-aspartate (l-Asp) to N-carbamoyl-l-aspartate by PyrB may reduce the amount of l-Asp available for PG synthesis and thus cause the appearance of Asp/Asn-less stem peptides in PG. Such stem peptides do not form PG cross-bridges, resulting in a decrease in PG cross-linking and, consequently, reduced PG thickness and rigidity. We hypothesize that the concurrent utilization of l-Asp for pyrimidine and PG synthesis may be part of the regulatory scheme, ensuring CW flexibility during exponential growth and rigidity in stationary phase. The fact that l-Asp availability is dependent on nucleotide metabolism, which is tightly regulated in accordance with the growth rate, provides L. lactis cells the means to ensure optimal CW plasticity without the need to control the expression of PG synthesis genes. PMID:27022026

25. NORTH TRAINING WALL, EAST SECTION, SIDE WALL CONSTRUCTION, LOOKING ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

25. NORTH TRAINING WALL, EAST SECTION, SIDE WALL CONSTRUCTION, LOOKING WEST FROM A POINT ABOUT 500 FEET FROM THE MIDDLE HARBOR PARK FISHING PIER. (Panoramic view 1 of 2). - Oakland Harbor Training Walls, Mouth of Federal Channel to Inner Harbor, Oakland, Alameda County, CA

EDITORIAL: Focus on Cold and Ultracold Molecules FOCUS ON COLD AND ULTRACOLD MOLECULES

NASA Astrophysics Data System (ADS)

Carr, Lincoln D.; Ye, Jun

2009-05-01

öhlich, A Griesmaier, T Pfau, H Saito, Y Kawaguchi and M Ueda High-energy-resolution molecular beams for cold collision studies L P Parazzoli, N Fitch, D S Lobser and H J Lewandowski Collisional effects in the formation of cold guided beams of polar molecules M Motsch, C Sommer, M Zeppenfeld, L D van Buuren, P W H Pinkse and G Rempe Towards sympathetic cooling of large molecules: cold collisions between benzene and rare gas atoms P Barletta, J Tennyson and P F Barker Efficient formation of ground-state ultracold molecules via STIRAP from the continuum at a Feshbach resonance Elena Kuznetsova, Marko Gacesa, Philippe Pellegrini, Susanne F Yelin and Robin Côté Emergent timescales in entangled quantum dynamics of ultracold molecules in optical lattices M L Wall and L D Carr Rotational state resolved photodissociation spectroscopy of translationally and vibrationally cold MgH+ ions: toward rotational cooling of molecular ions K Højbjerre, A K Hansen, P S Skyt, P F Staanum and M Drewsen Collective transverse cavity cooling of a dense molecular beam Thomas Salzburger and Helmut Ritsch A Stark decelerator on a chip Samuel A Meek, Horst Conrad and Gerard Meijer Deceleration of molecules by dipole force potential: a numerical simulation Susumu Kuma and Takamasa Momose Ultracold molecules: vehicles to scalable quantum information processing Kathy-Anne Brickman Soderberg, Nathan Gemelke and Cheng Chin Magnetic field modification of ultracold molecule-molecule collisions T V Tscherbul, Yu V Suleimanov, V Aquilanti and R V Krems Spectroscopy of 39K85Rb triplet excited states using ultracold a 3Σ+ state molecules formed by photoassociation J T Kim, D Wang, E E Eyler, P L Gould and W C Stwalley Pumping vortex into a Bose-Einstein condensate of heteronuclear molecules Z F Xu, R Q Wang and L You Intense atomic and molecular beams via neon buffer-gas cooling David Patterson, Julia Rasmussen and John M Doyle Dynamical properties of dipolar Fermi gases T Sogo, L He, T Miyakawa, S Yi, H Lu

6-Mercaptopurine attenuates adhesive molecules in experimental vasospasm.

PubMed

Chang, Chih-Zen; Lin, Chih-Lung; Kassel, Neal F; Kwan, Aij-Lie; Howng, Shen-Long

2010-05-01

Adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, are important inflammatory mediators which are elevated in the serum of patients following aneurysmal subarachnoid hemorrhage (SAH). The authors previously found that 6-mercaptopurine (6-mp) was effective in preventing and reversing arterial narrowing in a rodent SAH model. The present study was to examine whether levels of adhesion molecules were altered after treatment with 6-mp in this animal model. Animals were each injected with autologous blood into the cisterna magna, and intraperitoneal treatment with 6-mp (2 mg/kg) was initiated 1 h before (prevention) or later (treatment). The compound was subsequently administered at 24 and 48 h post-SAH. Blood samples were collected at 72 h post-SAH to measure ICAM-1, VCAM-1, and E-selectin levels. The basilar arteries were harvested and sliced, and their cross-sectional areas were measured. Morphologically, convolution of the internal elastic lamina, distorted endothelial wall, and myonecrosis of the smooth muscle were prominently observed in the SAH only and vehicle-treated SAH groups, but not in the 6-mp-treated SAH group or in healthy controls. No significant differences were found in the levels of VCAM-1 among all groups. However, the levels of E-selectin were increased in all animals subjected to SAH (SAH only and SAH plus vehicle groups) compared with healthy controls (no SAH), but not in the 6-mp group (SAH plus 6-mp treatment and preventive treatment with 6-mp).Likewise, the levels of ICAM-1 in the SAH only and SAH plus vehicle groups were significantly elevated (p < 0.001), and pretreatment and treatment with 6-mp reduced ICAM-1 to control levels. These results show that ICAM-1 and E-selectin may play a role in mediating SAH-induced vasospasm and that a reduction of both adhesive molecules after SAH may partly contribute to the antispastic effect of 6-mp.

Wind tunnel wall interference

NASA Technical Reports Server (NTRS)

Newman, Perry A.; Mineck, Raymond E.; Barnwell, Richard W.; Kemp, William B., Jr.

1986-01-01

About a decade ago, interest in alleviating wind tunnel wall interference was renewed by advances in computational aerodynamics, concepts of adaptive test section walls, and plans for high Reynolds number transonic test facilities. Selection of NASA Langley cryogenic concept for the National Transonic Facility (NTF) tended to focus the renewed wall interference efforts. A brief overview and current status of some Langley sponsored transonic wind tunnel wall interference research are presented. Included are continuing efforts in basic wall flow studies, wall interference assessment/correction procedures, and adaptive wall technology.

Molecules Without Atoms

NASA Astrophysics Data System (ADS)

Ruth, Anthony; Collins, Laura; Gomes, Kenjiro; Janko, Boldizsar

We present a real-space representation of molecules which results in the normal bonding rules and electronic structure of chemistry without atom-centered coulomb potentials. Using a simple mapping, we can generate atomless molecules from the structure of real molecules. Additionally, molecules without atoms show similar covalent bonding energies and transfer of charge in ionic bonds as real molecules. The atomless molecules contain only the valence and conduction electronic structure of the real molecule. Using the framework of the Atoms in Molecules (AIM) theory of Bader, we prove that the topological features of the valence charge distribution of molecules without atoms are identical to that of real molecules. In particular, the charge basins of atomless molecules show identical location and quantities of representative charge. We compare the accuracy, computational cost, and intuition gained from electronic structure calculations of molecules without atoms with the use of pseudopotentials to represent atomic cores in density functional theory. A. R. acknowledges support from a NASA Space Technology Research Fellowship.

Skyrmion domain wall collision and domain wall-gated skyrmion logic

NASA Astrophysics Data System (ADS)

Xing, Xiangjun; Pong, Philip W. T.; Zhou, Yan

2016-08-01

Skyrmions and domain walls are significant spin textures of great technological relevance to magnetic memory and logic applications, where they can be used as carriers of information. The unique topology of skyrmions makes them display emergent dynamical properties as compared with domain walls. Some studies have demonstrated that the two topologically inequivalent magnetic objects could be interconverted by using cleverly designed geometric structures. Here, we numerically address the skyrmion domain wall collision in a magnetic racetrack by introducing relative motion between the two objects based on a specially designed junction. An electric current serves as the driving force that moves a skyrmion toward a trapped domain wall pair. We see different types of collision dynamics depending on the driving parameters. Most importantly, the modulation of skyrmion transport using domain walls is realized in this system, allowing a set of domain wall-gated logical NOT, NAND, and NOR gates to be constructed. This work provides a skyrmion-based spin-logic architecture that is fully compatible with racetrack memories.

Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

PubMed Central

Billaud, Marie; Lohman, Alexander W.; Johnstone, Scott R.; Biwer, Lauren A.; Mutchler, Stephanie; Isakson, Brant E.

2014-01-01

It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endothelium-derived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function. PMID:24671377

Regulation of cellular communication by signaling microdomains in the blood vessel wall.

PubMed

Billaud, Marie; Lohman, Alexander W; Johnstone, Scott R; Biwer, Lauren A; Mutchler, Stephanie; Isakson, Brant E

2014-01-01

It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endothelium-derived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function.

Single-molecule analysis of the major glycopolymers of pathogenic and non-pathogenic yeast cells

NASA Astrophysics Data System (ADS)

El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Alsteens, David; Sarazin, Aurore; Jouault, Thierry; Dufrêne, Yves F.

2013-05-01

Most microbes are coated with carbohydrates that show remarkable structural variability and play a crucial role in mediating microbial-host interactions. Understanding the functions of cell wall glycoconjugates requires detailed knowledge of their molecular organization, diversity and heterogeneity. Here we use atomic force microscopy (AFM) with tips bearing specific probes (lectins, antibodies) to analyze the major glycopolymers of pathogenic and non-pathogenic yeast cells at molecular resolution. We show that non-ubiquitous β-1,2-mannans are largely exposed on the surface of native cells from pathogenic Candida albicans and C. glabrata, the former species displaying the highest glycopolymer density and extensions. We also find that chitin, a major component of the inner layer of the yeast cell wall, is much more abundant in C. albicans. These differences in molecular properties, further supported by flow cytometry measurements, may play an important role in strengthening cell wall mechanics and immune interactions. This study demonstrates that single-molecule AFM, combined with immunological and fluorescence methods, is a powerful platform in fungal glycobiology for probing the density, distribution and extension of specific cell wall glycoconjugates. In nanomedicine, we anticipate that this new form of AFM-based nanoglycobiology will contribute to the development of sugar-based drugs, immunotherapeutics, vaccines and diagnostics.

PREFACE: Light element atom, molecule and radical behaviour in the divertor and edge plasma regions

NASA Astrophysics Data System (ADS)

Braams, Bastiaan J.; Chung, Hyun-Kung

2015-01-01

This volume of Journal of Physics: Conference Series contains contributions by participants in an International Atomic Energy Agency (IAEA) Coordinated Research Project (CRP) on "Light element atom, molecule and radical behaviour in the divertor and edge plasma regions" (in magnetic fusion devices). Light elements are the dominant impurity species in fusion experiments and in the near-wall plasma they occur as atoms or ions and also as hydrides and other molecules and molecular ions. Hydrogen (H or D, and T in a reactor) is the dominant species in fusion experiments, but all light elements He - O and Ne are of interest for various reasons. Helium is a product of the D+T fusion reaction and is introduced in experiments for transport studies. Lithium is used for wall coating and also as a beam diagnostic material. Beryllium is foreseen as a wall material for the ITER experiment and is used on the Joint European Torus (JET) experiment. Boron may be used as a coating material for the vessel walls. Carbon (graphite or carbon-fiber composite) is often used as the target material for wall regions subject to high heat load. Nitrogen may be used as a buffer gas for edge plasma cooling. Oxygen is a common impurity in experiments due to residual water vapor. Finally, neon is another choice as a buffer gas. Data for collisional and radiative processes involving these species are important for plasma modelling and for diagnostics. The participants in the CRP met 3 times over the years 2009-2013 for a research coordination meeting. Reports and presentation materials for these meetings are available through the web page on coordinated research projects of the (IAEA) Atomic and Molecular Data Unit [1]. Some of the numerical data generated in the course of the CRP is available through the ALADDIN database [2]. The IAEA takes the opportunity to thank the participants in the CRP for their dedicated efforts in the course of the CRP and for their contributions to this volume. The IAEA

Adaptive wall technology for minimization of wall interferences in transonic wind tunnels

NASA Technical Reports Server (NTRS)

Wolf, Stephen W. D.

1988-01-01

Modern experimental techniques to improve free air simulations in transonic wind tunnels by use of adaptive wall technology are reviewed. Considered are the significant advantages of adaptive wall testing techniques with respect to wall interferences, Reynolds number, tunnel drive power, and flow quality. The application of these testing techniques relies on making the test section boundaries adjustable and using a rapid wall adjustment procedure. A historical overview shows how the disjointed development of these testing techniques, since 1938, is closely linked to available computer support. An overview of Adaptive Wall Test Section (AWTS) designs shows a preference for use of relatively simple designs with solid adaptive walls in 2- and 3-D testing. Operational aspects of AWTS's are discussed with regard to production type operation where adaptive wall adjustments need to be quick. Both 2- and 3-D data are presented to illustrate the quality of AWTS data over the transonic speed range. Adaptive wall technology is available for general use in 2-D testing, even in cryogenic wind tunnels. In 3-D testing, more refinement of the adaptive wall testing techniques is required before more widespread use can be planned.

Response of Escherichia coli growth rate to osmotic shock.

PubMed

Rojas, Enrique; Theriot, Julie A; Huang, Kerwyn Casey

2014-05-27

It has long been proposed that turgor pressure plays an essential role during bacterial growth by driving mechanical expansion of the cell wall. This hypothesis is based on analogy to plant cells, for which this mechanism has been established, and on experiments in which the growth rate of bacterial cultures was observed to decrease as the osmolarity of the growth medium was increased. To distinguish the effect of turgor pressure from pressure-independent effects that osmolarity might have on cell growth, we monitored the elongation of single Escherichia coli cells while rapidly changing the osmolarity of their media. By plasmolyzing cells, we found that cell-wall elastic strain did not scale with growth rate, suggesting that pressure does not drive cell-wall expansion. Furthermore, in response to hyper- and hypoosmotic shock, E. coli cells resumed their preshock growth rate and relaxed to their steady-state rate after several minutes, demonstrating that osmolarity modulates growth rate slowly, independently of pressure. Oscillatory hyperosmotic shock revealed that although plasmolysis slowed cell elongation, the cells nevertheless "stored" growth such that once turgor was reestablished the cells elongated to the length that they would have attained had they never been plasmolyzed. Finally, MreB dynamics were unaffected by osmotic shock. These results reveal the simple nature of E. coli cell-wall expansion: that the rate of expansion is determined by the rate of peptidoglycan insertion and insertion is not directly dependent on turgor pressure, but that pressure does play a basic role whereby it enables full extension of recently inserted peptidoglycan.

Response of Escherichia coli growth rate to osmotic shock

PubMed Central

Rojas, Enrique; Theriot, Julie A.; Huang, Kerwyn Casey

2014-01-01

It has long been proposed that turgor pressure plays an essential role during bacterial growth by driving mechanical expansion of the cell wall. This hypothesis is based on analogy to plant cells, for which this mechanism has been established, and on experiments in which the growth rate of bacterial cultures was observed to decrease as the osmolarity of the growth medium was increased. To distinguish the effect of turgor pressure from pressure-independent effects that osmolarity might have on cell growth, we monitored the elongation of single Escherichia coli cells while rapidly changing the osmolarity of their media. By plasmolyzing cells, we found that cell-wall elastic strain did not scale with growth rate, suggesting that pressure does not drive cell-wall expansion. Furthermore, in response to hyper- and hypoosmotic shock, E. coli cells resumed their preshock growth rate and relaxed to their steady-state rate after several minutes, demonstrating that osmolarity modulates growth rate slowly, independently of pressure. Oscillatory hyperosmotic shock revealed that although plasmolysis slowed cell elongation, the cells nevertheless “stored” growth such that once turgor was reestablished the cells elongated to the length that they would have attained had they never been plasmolyzed. Finally, MreB dynamics were unaffected by osmotic shock. These results reveal the simple nature of E. coli cell-wall expansion: that the rate of expansion is determined by the rate of peptidoglycan insertion and insertion is not directly dependent on turgor pressure, but that pressure does play a basic role whereby it enables full extension of recently inserted peptidoglycan. PMID:24821776

Investigation of the ellipsoidal-statistical Bhatnagar-Gross-Krook kinetic model applied to gas-phase transport of heat and tangential momentum between parallel walls

NASA Astrophysics Data System (ADS)

Gallis, M. A.; Torczynski, J. R.

2011-03-01

The ellipsoidal-statistical Bhatnagar-Gross-Krook (ES-BGK) kinetic model is investigated for steady gas-phase transport of heat, tangential momentum, and mass between parallel walls (i.e., Fourier, Couette, and Fickian flows). This investigation extends the original study of Cercignani and Tironi, who first applied the ES-BGK model to heat transport (i.e., Fourier flow) shortly after this model was proposed by Holway. The ES-BGK model is implemented in a molecular-gas-dynamics code so that results from this model can be compared directly to results from the full Boltzmann collision term, as computed by the same code with the direct simulation Monte Carlo (DSMC) algorithm of Bird. A gas of monatomic molecules is considered. These molecules collide in a pairwise fashion according to either the Maxwell or the hard-sphere interaction and reflect from the walls according to the Cercignani-Lampis-Lord model with unity accommodation coefficients. Simulations are performed at pressures from near-free-molecular to near-continuum. Unlike the BGK model, the ES-BGK model produces heat-flux and shear-stress values that both agree closely with the DSMC values at all pressures. However, for both interactions, the ES-BGK model produces molecular-velocity-distribution functions that are qualitatively similar to those determined for the Maxwell interaction from Chapman-Enskog theory for small wall temperature differences and moment-hierarchy theory for large wall temperature differences. Moreover, the ES-BGK model does not produce accurate values of the mass self-diffusion coefficient for either interaction. Nevertheless, given its reasonable accuracy for heat and tangential-momentum transport, its sound theoretical foundation (it obeys the H-theorem), and its available extension to polyatomic molecules, the ES-BGK model may be a useful method for simulating certain classes of single-species noncontinuum gas flows, as Cercignani suggested.

Novel strategy for diameter-selective separation and functionalization of single-wall carbon nanotubes.

PubMed

Tromp, R M; Afzali, A; Freitag, M; Mitzi, D B; Chen, Zh

2008-02-01

The problem of separating single-wall carbon nanotubes (CNTs) by diameter and/or chirality is one of the greatest impediments toward the widespread application of these promising materials in nanoelectronics. In this paper, we describe a novel physical-chemical method for diameter-selective CNT separation that is both simple and effective and that allows up-scaling to large volumes at modest cost. Separation is based on size-selective noncovalent matching of an appropriate anchor molecule to the wall of the CNT, enabling suspension of the CNTs in solvents in which they would otherwise not be soluble. We demonstrate size-selective separation in the 1-2 nm diameter range using easily synthesized oligo-acene adducts as a diameter-selective molecular anchor. CNT field effect transistors fabricated from diameter-selected CNTs show markedly improved electrical properties as compared to nonselected CNTs.

Structural Aspects for Evolution of [beta]-Lactamases from Penicillin-Binding Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meroueh, Samy O.; Minasov, George; Lee, Wenlin

Penicillin-binding proteins (PBPs), biosynthetic enzymes of bacterial cell wall assembly, and {beta}-lactamases, resistance enzymes to {beta}-lactam antibiotics, are related to each other from an evolutionary point of view. Massova and Mobashery (Antimicrob. Agents Chemother. 1998, 42, 1-17) have proposed that for {beta}-lactamases to have become effective at their function as antibiotic resistance enzymes, they would have had to undergo structure alterations such that they would not interact with the peptidoglycan, which is the substrate for PBPs. A cephalosporin analogue, 7{beta}-[N-Acetyl-L-alanyl-{gamma}-D-glutamyl-L-lysine]-3-acetoxymethyl-3-cephem-carboxylic acid (compound 6), was conceived and synthesized to test this notion. The X-ray structure of the complex of this cephalosporinmore » bound to the active site of the deacylation-deficient Q120L/Y150E variant of the class C AmpC {beta}-lactamase from Escherichia coli was solved at 1.71 {angstrom} resolution. This complex revealed that the surface for interaction with the strand of peptidoglycan that acylates the active site, which is present in PBPs, is absent in the {beta}-lactamase active site. Furthermore, insertion of a peptide in the {beta}-lactamase active site at a location where the second strand of peptidoglycan in some PBPs binds has effectively abolished the possibility for such interaction with the {beta}-lactamase. A 2.6 ns dynamics simulation was carried out for the complex, which revealed that the peptidoglycan surrogate (i.e., the active-site-bound ligand) undergoes substantial motion and is not stabilized for binding within the active site. These factors taken together disclose the set of structure modifications in the antibiotic resistance enzyme that prevent it from interacting with the peptidoglycan, en route to achieving catalytic proficiency for their intended function.« less

Research on wall shear stress considering wall roughness when shear swirling flow vibration cementing

NASA Astrophysics Data System (ADS)

Cui, Zhihua; Ai, Chi; Feng, Fuping

2017-01-01

When shear swirling flow vibration cementing, the casing is revolving periodically and eccentrically, which leads to the annulus fluid in turbulent swirling flow state. The wall shear stress is more than that in laminar flow field when conventional cementing. The paper mainly studied the wall shear stress distribution on the borehole wall when shear swirling flow vibration cementing based on the finite volume method. At the same time, the wall roughness affected and changed the turbulent flow near the borehole wall and the wall shear stress. Based on the wall function method, the paper established boundary conditions considering the wall roughness and derived the formula of the wall shear stress. The results showed that the wall roughness significantly increases the wall shear stress. However, the larger the wall roughness, the greater the thickness of mud cake, which weakening the cementing strength. Considering the effects in a comprehensive way, it is discovered that the particle size of solid phase in drilling fluid is about 0.1 mm to get better cementing quality.

The Pseudomonas putida peptidoglycan-associated outer membrane lipoprotein is involved in maintenance of the integrity of the cell cell envelope.

PubMed Central

Rodríguez-Herva, J J; Ramos-Gonzalez, M I; Ramos, J L

1996-01-01

Pseudomonas putida 14G-3, a derivative of the natural soil inhabitant P. putida KT2440, exhibited a chromosomal insertion of a mini-Tn5/'phoA transposon that resulted in reduced ability to colonize soil. In vitro characterization of P. putida 14G-3 revealed that it exhibited an altered cell morphology and envelope, as revealed by electron microscopy. The derived strain was sensitive to sodium dodecyl sulfate, deoxycholate, and EDTA, produced clumps when it reached high cell densities in the late logarithmic growth phase, and did not grow on low-osmolarity medium. The P. putida DNA surrounding the mini-Tn5/'phoA insertion was cloned and used as a probe to rescue the wild-type gene, which was sequenced. Comparison of the deduced peptide sequence with sequences in the Swiss-Prot database allowed the knocked-out gene to be identified as that encoding the peptidoglycan-associated lipoprotein (Pal or OprL) of P. putida. The protein was identified in coupled transcription and translation assays in vitro. PMID:8626299

Molecule nanoweaver

DOEpatents

Gerald, II; Rex, E [Brookfield, IL; Klingler, Robert J [Glenview, IL; Rathke, Jerome W [Homer Glen, IL; Diaz, Rocio [Chicago, IL; Vukovic, Lela [Westchester, IL

2009-03-10

A method, apparatus, and system for constructing uniform macroscopic films with tailored geometric assemblies of molecules on the nanometer scale. The method, apparatus, and system include providing starting molecules of selected character, applying one or more force fields to the molecules to cause them to order and condense with NMR spectra and images being used to monitor progress in creating the desired geometrical assembly and functionality of molecules that comprise the films.

On investigating wall shear stress in two-dimensional plane turbulent wall jets

NASA Astrophysics Data System (ADS)

Mehdi, Faraz; Johansson, Gunnar; White, Christopher; Naughton, Jonathan

2012-11-01

Mehdi & White [Exp Fluids 50:43-51(2011)] presented a full momentum integral based method for determining wall shear stress in zero pressure gradient turbulent boundary layers. They utilized the boundary conditions at the wall and at the outer edge of the boundary layer. A more generalized expression is presented here that uses just one boundary condition at the wall. The method is mathematically exact and has an advantage of having no explicit streamwise gradient terms. It is successfully applied to two different experimental plane turbulent wall jet datasets for which independent estimates of wall shear stress were known. Complications owing to experimental inaccuracies in determining wall shear stress from the proposed method are also discussed.

Vacuum template synthesis of multifunctional nanotubes with tailored nanostructured walls

NASA Astrophysics Data System (ADS)

Filippin, A. Nicolas; Macias-Montero, Manuel; Saghi, Zineb; Idígoras, Jesús; Burdet, Pierre; Barranco, Angel; Midgley, Paul; Anta, Juan A.; Borras, Ana

2016-02-01

A three-step vacuum procedure for the fabrication of vertical TiO2 and ZnO nanotubes with three dimensional walls is presented. The method combines physical vapor deposition of small-molecules, plasma enhanced chemical vapor deposition of inorganic functional thin films and layers and a post-annealing process in vacuum in order to remove the organic template. As a result, an ample variety of inorganic nanotubes are made with tunable length, hole dimensions and shapes and tailored wall composition, microstructure, porosity and structure. The fabrication of multishell nanotubes combining different semiconducting oxides and metal nanoparticles is as well explored. This method provides a feasible and reproducible route for the fabrication of high density arrays of vertically alligned nanotubes on processable substrates. The emptying mechanism and microstructure of the nanotubes have been elucidated through SEM, STEM, HAADF-STEM tomography and energy dispersive X-ray spectroscopy. In this article, as a proof of concept, it is presented the straightforward integration of ZnO nanotubes as photoanode in a photovoltaic cell and as a photonic oxygen gas sensor.

Vacuum template synthesis of multifunctional nanotubes with tailored nanostructured walls

PubMed Central

Filippin, A. Nicolas; Macias-Montero, Manuel; Saghi, Zineb; Idígoras, Jesús; Burdet, Pierre; Barranco, Angel; Midgley, Paul; Anta, Juan A.; Borras, Ana

2016-01-01

A three-step vacuum procedure for the fabrication of vertical TiO2 and ZnO nanotubes with three dimensional walls is presented. The method combines physical vapor deposition of small-molecules, plasma enhanced chemical vapor deposition of inorganic functional thin films and layers and a post-annealing process in vacuum in order to remove the organic template. As a result, an ample variety of inorganic nanotubes are made with tunable length, hole dimensions and shapes and tailored wall composition, microstructure, porosity and structure. The fabrication of multishell nanotubes combining different semiconducting oxides and metal nanoparticles is as well explored. This method provides a feasible and reproducible route for the fabrication of high density arrays of vertically alligned nanotubes on processable substrates. The emptying mechanism and microstructure of the nanotubes have been elucidated through SEM, STEM, HAADF-STEM tomography and energy dispersive X-ray spectroscopy. In this article, as a proof of concept, it is presented the straightforward integration of ZnO nanotubes as photoanode in a photovoltaic cell and as a photonic oxygen gas sensor. PMID:26860367

Non-parametric wall model and methods of identifying boundary conditions for moments in gas flow equations

NASA Astrophysics Data System (ADS)

Liao, Meng; To, Quy-Dong; Léonard, Céline; Monchiet, Vincent

2018-03-01

In this paper, we use the molecular dynamics simulation method to study gas-wall boundary conditions. Discrete scattering information of gas molecules at the wall surface is obtained from collision simulations. The collision data can be used to identify the accommodation coefficients for parametric wall models such as Maxwell and Cercignani-Lampis scattering kernels. Since these scattering kernels are based on a limited number of accommodation coefficients, we adopt non-parametric statistical methods to construct the kernel to overcome these issues. Different from parametric kernels, the non-parametric kernels require no parameter (i.e. accommodation coefficients) and no predefined distribution. We also propose approaches to derive directly the Navier friction and Kapitza thermal resistance coefficients as well as other interface coefficients associated with moment equations from the non-parametric kernels. The methods are applied successfully to systems composed of CH4 or CO2 and graphite, which are of interest to the petroleum industry.

Identification of a novel gene cluster in the upstream region of the S-layer gene sbpA involved in cell wall metabolism of Lysinibacillus sphaericus CCM 2177 and characterization of the recombinantly produced autolysin and pyruvyl transferase.

PubMed

Pleschberger, Magdalena; Hildner, Florian; Rünzler, Dominik; Gelbmann, Nicola; Mayer, Harald F; Sleytr, Uwe B; Egelseer, Eva M

2013-05-01

The S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 assembles into a square (p4) lattice structure and recognizes a pyruvylated secondary cell wall polymer (SCWP) as the proper anchoring structure to the rigid cell wall layer. Sequencing of 8,004 bp in the 5'-upstream region of the S-layer gene sbpA led to five ORFs-encoding proteins involved in cell wall metabolism. After cloning and heterologous expression of ORF1 and ORF5 in Escherichia coli, the recombinant autolysin rAbpA and the recombinant pyruvyl transferase rCsaB were isolated, purified, and correct folding was confirmed by circular dichroism. Although rAbpA encoded by ORF1 showed amidase activity, it could attack whole cells of Ly. sphaericus CCM 2177 only after complete extraction of the S-layer lattice. Despite the presence of three S-layer-homology motifs on the N-terminal part, rAbpA did not show detectable affinity to peptidoglycan-containing sacculi, nor to isolated SCWP. As the molecular mass of the autolysin lies above the molecular exclusion limit of the S-layer, AbpA is obviously trapped within the rigid cell wall layer by the isoporous protein lattice. Immunogold-labeling of ultrathin-sectioned whole cells of Ly. sphaericus CCM 2177 with a polyclonal rabbit antiserum raised against rCsaB encoded by ORF5, and cell fractionation experiments demonstrated that the pyruvyl transferase was located in the cytoplasm, but not associated with cell envelope components including the plasma membrane. In enzymatic assays, rCsaB clearly showed pyruvyl transferase activity. By using RT-PCR, specific transcripts for each ORF could be detected. Cotranscription could be confirmed for ORF2 and ORF3.

Evidence for land plant cell wall biosynthetic mechanisms in charophyte green algae

PubMed Central

Mikkelsen, Maria D.; Harholt, Jesper; Ulvskov, Peter; Johansen, Ida E.; Fangel, Jonatan U.; Doblin, Monika S.; Bacic, Antony; Willats, William G. T.

2014-01-01

Background and Aims The charophyte green algae (CGA) are thought to be the closest living relatives to the land plants, and ancestral CGA were unique in giving rise to the land plant lineage. The cell wall has been suggested to be a defining structure that enabled the green algal ancestor to colonize land. These cell walls provide support and protection, are a source of signalling molecules, and provide developmental cues for cell differentiation and elongation. The cell wall of land plants is a highly complex fibre composite, characterized by cellulose cross-linked by non-cellulosic polysaccharides, such as xyloglucan, embedded in a matrix of pectic polysaccharides. How the land plant cell wall evolved is currently unknown: early-divergent chlorophyte and prasinophyte algae genomes contain a low number of glycosyl transferases (GTs), while land plants contain hundreds. The number of GTs in CGA is currently unknown, as no genomes are available, so this study sought to give insight into the evolution of the biosynthetic machinery of CGA through an analysis of available transcriptomes. Methods Available CGA transcriptomes were mined for cell wall biosynthesis GTs and compared with GTs characterized in land plants. In addition, gene cloning was employed in two cases to answer important evolutionary questions. Key Results Genetic evidence was obtained indicating that many of the most important core cell wall polysaccharides have their evolutionary origins in the CGA, including cellulose, mannan, xyloglucan, xylan and pectin, as well as arabino-galactan protein. Moreover, two putative cellulose synthase-like D family genes (CSLDs) from the CGA species Coleochaete orbicularis and a fragment of a putative CSLA/K-like sequence from a CGA Spirogyra species were cloned, providing the first evidence that all the cellulose synthase/-like genes present in early-divergent land plants were already present in CGA. Conclusions The results provide new insights into the evolution of

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins

PubMed Central

Ghamrawi, Sarah; Gastebois, Amandine; Zykwinska, Agata; Vandeputte, Patrick; Marot, Agnès; Mabilleau, Guillaume; Cuenot, Stéphane; Bouchara, Jean-Philippe

2015-01-01

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence. PMID:26038837

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins.

PubMed

Ghamrawi, Sarah; Gastebois, Amandine; Zykwinska, Agata; Vandeputte, Patrick; Marot, Agnès; Mabilleau, Guillaume; Cuenot, Stéphane; Bouchara, Jean-Philippe

2015-01-01

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence.

Biological Nanopores: Confined Spaces for Electrochemical Single-Molecule Analysis.

PubMed

Cao, Chan; Long, Yi-Tao

2018-02-20

, we develop an integrated current measurement system and an accurate data processing method for nanopore sensing. The unique geometric structure of a biological nanopore offers a distinct advantage as a nanosensor for single-molecule sensing. The construction of the pore entrance is responsible for capturing the target molecule, while the lumen region determines the translocation process of the single molecule. Since the capture of the target molecule is predominantly diffusion-limited, it is expected that the capture ability of the nanopore toward the target analyte could be effectively enhanced by site-directed mutations of key amino acids with desirable groups. Additionally, changing the side chains inside the wall of the biological nanopore could optimize the geometry of the pore and realize an optimal interaction between the single-molecule interface and the analyte. These improvements would allow for high spatial and current resolution of nanopore sensors, which would ensure the possibility of dynamic study of single biomolecules, including their metastable conformations, charge distributions, and interactions. In the future, data analysis with powerful algorithms will make it possible to automatically and statistically extract detailed information while an analyte translocates through the pore. We conclude that these improvements could have tremendous potential applications for nanopore sensing in the near future.

Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance.

PubMed

Ene, Iuliana V; Walker, Louise A; Schiavone, Marion; Lee, Keunsook K; Martin-Yken, Hélène; Dague, Etienne; Gow, Neil A R; Munro, Carol A; Brown, Alistair J P

2015-07-28

The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. The C. albicans cell wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antifungal therapy. Its tensile strength is conferred by

Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance

PubMed Central

Ene, Iuliana V.; Walker, Louise A.; Schiavone, Marion; Lee, Keunsook K.; Martin-Yken, Hélène; Dague, Etienne; Gow, Neil A. R.; Munro, Carol A.

2015-01-01

ABSTRACT The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. PMID:26220968

Direct calculation of wall interferences and wall adaptation for two-dimensional flow in wind tunnels with closed walls

NASA Technical Reports Server (NTRS)

Amecke, Juergen

1986-01-01

A method for the direct calculation of the wall induced interference velocity in two dimensional flow based on Cauchy's integral formula was derived. This one-step method allows the calculation of the residual corrections and the required wall adaptation for interference-free flow starting from the wall pressure distribution without any model representation. Demonstrated applications are given.

Experimental investigation of wall shock cancellation and reduction of wall interference in transonic testing

NASA Technical Reports Server (NTRS)

Ferri, A.; Roffe, G.

1975-01-01

A series of experiments were performed to evaluate the effectiveness of a three-dimensional land and groove wall geometry and a variable permeability distribution to reduce the interference produced by the porous walls of a supercritical transonic test section. The three-dimensional wall geometry was found to diffuse the pressure perturbations caused by small local mismatches in wall porosity permitting the use of a relatively coarse wall porosity control to reduce or eliminate wall interference effects. The wall porosity distribution required was found to be a sensitive function of Mach number requiring that the Mach number repeatability characteristics of the test apparatus be quite good. The effectiveness of a variable porosity wall is greatest in the upstream region of the test section where the pressure differences across the wall are largest. An effective variable porosity wall in the down stream region of the test section requires the use of a slightly convergent test section geometry.

Crystal structure of Bacillus anthracis transpeptidase enzyme CapD.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, R.; Richter, S.; Zhang, R.

2009-09-04

Bacillus anthracis elaborates a poly-{gamma}-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the {gamma}-glutamyltranspeptidase CapD with and without {alpha}-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-{gamma}-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-{gamma}-glutamatemore » binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro{sup 427}, Gly{sup 428}, and Gly{sup 429} activate the catalytic residue of the enzyme, Thr{sup 352}, and stabilize an oxyanion hole via main chain amide hydrogen bonds.« less

Tuberculosis: a balanced diet of lipids and carbohydrates.

PubMed

Bhowruth, Veemal; Alderwick, Luke J; Brown, Alistair K; Bhatt, Apoorva; Besra, Gurdyal S

2008-08-01

In spite of effective antibiotics to treat TB (tuberculosis) since the early 1960s, we enter the new millennium with TB currently the leading cause of death from a single infectious agent, killing more than 3 million people worldwide each year. Thus an understanding of drug-resistance mechanisms, the immunobiology of cell wall components to elucidate host-pathogen interactions and the discovery of new drug targets are now required for the treatment of TB. Above the plasma membrane is a classical chemotype IV peptidoglycan to which is attached the macromolecular structure, mycolyl-arabinogalactan via a unique diglycosylphosphoryl bridge. The present review discusses the assembly of the mAGP (mycolyl-arabinogalactan-peptidoglycan) complex and the site of action of EMB (ethambutol), bringing forward a new era in TB research and focus for new drugs to combat multidrug-resistant TB.

Peptidoglycan recognition protein genes and their roles in the innate immune pathways of the red flour beetle, Tribolium castaneum.

PubMed

Koyama, Hiroaki; Kato, Daiki; Minakuchi, Chieka; Tanaka, Toshiharu; Yokoi, Kakeru; Miura, Ken

2015-11-01

We have previously demonstrated that the functional Toll and IMD innate immune pathways indeed exist in the model beetle, Tribolium castaneum while the beetle's pathways have broader specificity in terms of microbial activation than that of Drosophila. To elucidate the molecular basis of this broad microbial activation, we here focused on potential upstream sensors of the T. castaneum innate immune pathways, peptidoglycan recognition proteins (PGRPs). Our phenotype analyses utilizing RNA interference-based comprehensive gene knockdown followed by bacterial challenge suggested: PGRP-LA functions as a pivotal sensor of the IMD pathway for both Gram-negative and Gram-positive bacteria; PGRP-LC acts as an IMD pathway-associated sensor mainly for Gram-negative bacteria; PGRP-LE also has some roles in Gram-negative bacterial recognition of the IMD pathway. On the other hand, we did not obtain clear phenotype changes by gene knockdown of short-type PGRP genes, probably because of highly inducible nature of these genes. Our results may collectively account for the promiscuous bacterial activation of the T. castaneum innate immune pathways at least in part. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization of Mutants Deficient in the l,d-Carboxypeptidase (DacB) and WalRK (VicRK) Regulon, Involved in Peptidoglycan Maturation of Streptococcus pneumoniae Serotype 2 Strain D39▿†

PubMed Central

Barendt, Skye M.; Sham, Lok-To; Winkler, Malcolm E.

2011-01-01

Peptidoglycan (PG) hydrolases play critical roles in the remodeling of bacterial cell walls during division. PG hydrolases have been studied extensively in several bacillus species, such as Escherichia coli and Bacillus subtilis, but remain relatively uncharacterized in ovococcus species, such as Streptococcus pneumoniae (pneumococcus). In this work, we identified genes that encode proteins with putative PG hydrolytic domains in the genome of S. pneumoniae strain D39. Knockout mutations in these genes were constructed, and the resulting mutants were characterized in comparison with the parent strain for growth, cell morphology, PG peptide incorporation, and in some cases, PG peptide composition. In addition, we characterized deletion mutations in nonessential genes of unknown function in the WalRKSpn two-component system regulon, which also contains the essential pcsB cell division gene. Several mutants did not show overt phenotypes, which is perhaps indicative of redundancy. In contrast, two new mutants showed distinct defects in PG biosynthesis. One mutation was in a gene designated dacB (spd_0549), which we showed encodes an l,d-carboxypeptidase involved in PG maturation. Notably, dacB mutants, similar to dacA (d,d-carboxypeptidase) mutants, exhibited defects in cell shape and septation, consistent with the idea that the availability of PG peptide precursors is important for proper PG biosynthesis. Epistasis analysis indicated that DacA functions before DacB in d-Ala removal, and immunofluorescence microscopy showed that DacA and DacB are located over the entire surface of pneumococcal cells. The other mutation was in WalRKSpn regulon gene spd_0703, which encodes a putative membrane protein that may function as a type of conserved streptococcal shape, elongation, division, and sporulation (SEDS) protein. PMID:21378199

A Generalized Wall Function

NASA Technical Reports Server (NTRS)

Shih, Tsan-Hsing; Povinelli, Louis A.; Liu, Nan-Suey; Potapczuk, Mark G.; Lumley, J. L.

1999-01-01

The asymptotic solutions, described by Tennekes and Lumley (1972), for surface flows in a channel, pipe or boundary layer at large Reynolds numbers are revisited. These solutions can be extended to more complex flows such as the flows with various pressure gradients, zero wall stress and rough surfaces, etc. In computational fluid dynamics (CFD), these solutions can be used as the boundary conditions to bridge the near-wall region of turbulent flows so that there is no need to have the fine grids near the wall unless the near-wall flow structures are required to resolve. These solutions are referred to as the wall functions. Furthermore, a generalized and unified law of the wall which is valid for whole surface layer (including viscous sublayer, buffer layer and inertial sublayer) is analytically constructed. The generalized law of the wall shows that the effect of both adverse and favorable pressure gradients on the surface flow is very significant. Such as unified wall function will be useful not only in deriving analytic expressions for surface flow properties but also bringing a great convenience for CFD methods to place accurate boundary conditions at any location away from the wall. The extended wall functions introduced in this paper can be used for complex flows with acceleration, deceleration, separation, recirculation and rough surfaces.

Inner- and outer-wall sorting of double-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Li, Han; Gordeev, Georgy; Wasserroth, Sören; Chakravadhanula, Venkata Sai Kiran; Neelakandhan, Shyam Kumar Chethala; Hennrich, Frank; Jorio, Ado; Reich, Stephanie; Krupke, Ralph; Flavel, Benjamin Scott

2017-12-01

Double-walled carbon nanotubes (DWCNTs) consist of two coaxially aligned single-walled carbon nanotubes (SWCNTs), and previous sorting methods only achieved outer-wall electronic-type selectivity. Here, a separation technique capable of sorting DWCNTs by semiconducting (S) or metallic (M) inner- and outer-wall electronic type is presented. Electronic coupling between the inner and outer wall is used to alter the surfactant coating around each of the DWCNT types, and aqueous gel permeation is used to separate them. Aqueous methods are used to remove SWCNT species from the raw material and prepare enriched DWCNT fractions. The enriched DWCNT fractions are then transferred into either chlorobenzene or toluene using the copolymer PFO-BPy to yield the four inner@outer combinations of M@M, M@S, S@M and S@S. The high purity of the resulting fractions is verified by absorption measurements, transmission electron microscopy, atomic force microscopy, resonance Raman mapping and high-density field-effect transistor devices.

Inner- and outer-wall sorting of double-walled carbon nanotubes.

PubMed

Li, Han; Gordeev, Georgy; Wasserroth, Sören; Chakravadhanula, Venkata Sai Kiran; Neelakandhan, Shyam Kumar Chethala; Hennrich, Frank; Jorio, Ado; Reich, Stephanie; Krupke, Ralph; Flavel, Benjamin Scott

2017-12-01

Double-walled carbon nanotubes (DWCNTs) consist of two coaxially aligned single-walled carbon nanotubes (SWCNTs), and previous sorting methods only achieved outer-wall electronic-type selectivity. Here, a separation technique capable of sorting DWCNTs by semiconducting (S) or metallic (M) inner- and outer-wall electronic type is presented. Electronic coupling between the inner and outer wall is used to alter the surfactant coating around each of the DWCNT types, and aqueous gel permeation is used to separate them. Aqueous methods are used to remove SWCNT species from the raw material and prepare enriched DWCNT fractions. The enriched DWCNT fractions are then transferred into either chlorobenzene or toluene using the copolymer PFO-BPy to yield the four inner@outer combinations of M@M, M@S, S@M and S@S. The high purity of the resulting fractions is verified by absorption measurements, transmission electron microscopy, atomic force microscopy, resonance Raman mapping and high-density field-effect transistor devices.

If walls could talk

NASA Technical Reports Server (NTRS)

Braam, J.; McIntire, L. V. (Principal Investigator)

1999-01-01

The plant cell wall is very complex, both in structure and function. The wall components and the mechanical properties of the wall have been implicated in conveying information that is important for morphogenesis. Proteoglycans, fragments of polysaccharides and the structural integrity of the wall may relay signals that influence cellular differentiation and growth control. Furthering our knowledge of cell wall structure and function is likely to have a profound impact on our understanding of how plant cells communicate with the extracellular environment.

Digestion of peptidoglycan near the cross-link is necessary for the growth of Bacillus subtilis.

PubMed

Hashimoto, Masayuki; Matsushima, Hiroaki; Suparthana, I Putu; Ogasawara, Hiroshi; Yamamoto, Hiroki; Teng, ChingHao; Sekiguchi, Junichi

2018-03-01

Bacterial cells are covered with peptidoglycan (PG) layer(s), serving as the cellular exoskeleton. The PG sacculus changes its shape during cell growth, and thus both the synthesis and disassembly of PG are important for cell proliferation. In Bacillus subtilis, four dl-endopeptidases (DLEPases; LytE, LytF, CwlO and CwlS) are involved in the maintenance of cell morphology. The lytE cwlO double mutant exhibits synthetic lethality and defective cell elongation, while the lytE lytF cwlS triple mutant exhibits defective cell separation, albeit with septum formation. LytE is involved in both cell separation and elongation. We propose that DLEPases have varied roles in cell separation and elongation. To determine these roles, the catalytic domain of LytE was substituted with another catalytic domain that digests the other bonds in PG. By using the chimeric enzymes, we assessed the suppression of the synthetic lethality by the cell elongation defect and the disruption of chain morphology by the cell separation defect. All the constructed chimeric enzymes suppressed the cell separation defect, restoring the chain morphology. Digestion at any position of PG broke the linkage between two daughter cells, releasing them from each other. However, only d,d-endopeptidases suppressed the lack of DLEPase in the lytE cwlO double mutant. This indicated that the release of tension on the expanding PG sacculus is not the sole essential function of DLEPases. Considering that the structure of the digested PG is important for cell elongation, the digested product might be reused in the growth process in some way.

Investigation of the mechanism of the cell wall DD-carboxypeptidase reaction of penicillin-binding protein 5 of Escherichia coli by quantum mechanics/molecular mechanics calculations.

PubMed

Shi, Qicun; Meroueh, Samy O; Fisher, Jed F; Mobashery, Shahriar

2008-07-23

Penicillin-binding protein 5 (PBP 5) of Escherichia coli hydrolyzes the terminal D-Ala-D-Ala peptide bond of the stem peptides of the cell wall peptidoglycan. The mechanism of PBP 5 catalysis of amide bond hydrolysis is initial acylation of an active site serine by the peptide substrate, followed by hydrolytic deacylation of this acyl-enzyme intermediate to complete the turnover. The microscopic events of both the acylation and deacylation half-reactions have not been studied. This absence is addressed here by the use of explicit-solvent molecular dynamics simulations and ONIOM quantum mechanics/molecular mechanics (QM/MM) calculations. The potential-energy surface for the acylation reaction, based on MP2/6-31+G(d) calculations, reveals that Lys47 acts as the general base for proton abstraction from Ser44 in the serine acylation step. A discrete potential-energy minimum for the tetrahedral species is not found. The absence of such a minimum implies a conformational change in the transition state, concomitant with serine addition to the amide carbonyl, so as to enable the nitrogen atom of the scissile bond to accept the proton that is necessary for progression to the acyl-enzyme intermediate. Molecular dynamics simulations indicate that transiently protonated Lys47 is the proton donor in tetrahedral intermediate collapse to the acyl-enzyme species. Two pathways for this proton transfer are observed. One is the direct migration of a proton from Lys47. The second pathway is proton transfer via an intermediary water molecule. Although the energy barriers for the two pathways are similar, more conformers sample the latter pathway. The same water molecule that mediates the Lys47 proton transfer to the nitrogen of the departing D-Ala is well positioned, with respect to the Lys47 amine, to act as the hydrolytic water in the deacylation step. Deacylation occurs with the formation of a tetrahedral intermediate over a 24 kcal x mol(-1) barrier. This barrier is approximately 2

Laser absorption spectroscopy of water vapor confined in nanoporous alumina: wall collision line broadening and gas diffusion dynamics.

PubMed

Svensson, Tomas; Lewander, Märta; Svanberg, Sune

2010-08-02

We demonstrate high-resolution tunable diode laser absorption spectroscopy (TDLAS) of water vapor confined in nanoporous alumina. Strong multiple light scattering results in long photon pathlengths (1 m through a 6 mm sample). We report on strong line broadening due to frequent wall collisions (gas-surface interactions). For the water vapor line at 935.685 nm, the HWHM of confined molecules are about 4.3 GHz as compared to 2.9 GHz for free molecules (atmospheric pressure). Gas diffusion is also investigated, and in contrast to molecular oxygen (that moves rapidly in and out of the alumina), the exchange of water vapor is found very slow.

Phosphatidyl-Inositol-3 Kinase Inhibitors Regulate Peptidoglycan-Induced Myeloid Leukocyte Recruitment, Inflammation, and Neurotoxicity in Mouse Brain.