scholarly journals Remodeling of Crossbridges Controls Peptidoglycan Cross-linking Levels in Bacterial Cell Walls

2019 ◽  
Author(s):  
Sean E. Pidgeon ◽  
Alexis J. Apostolos ◽  
Marcos M. Pires
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Bacterial Species ◽  
Physiological Role ◽  
Primary Component ◽  
Acyl Donor ◽  
Bacterial Cells ◽  
Structural Variations ◽  
Acyl Acceptor ◽  
Donor And Acceptor

ABSTRACTCell walls are barriers found in almost all known bacterial cells. These structures establish a controlled interface between the external environment and vital cellular components. A primary component of cell wall is a highly crosslinked matrix called peptidoglycan (PG). PG crosslinking, carried out by transglycosylases and transpeptidases, is necessary for proper cell wall assembly. Transpeptidases, targets of β-lactam antibiotics, stitch together two neighboring PG stem peptides (acyl-donor and acyl-acceptor strands). We recently described a novel class of cellular PG probes that were processed exclusively as acyl-donor strands. Herein, we have accessed the other half of the transpeptidase reaction by developing probes that are processed exclusively as acyl-acceptor strands. The critical nature of the crossbridge on the PG peptide was demonstrated in live bacterial cells and surprising promiscuity in crossbridge primary sequence was found in various bacterial species. Additionally, acyl-acceptor probes provided insight into how chemical remodeling of the PG crossbridge (e.g., amidation) can modulate crosslinking levels, thus establishing a physiological role of PG structural variations. Together, the acyl-donor and -acceptor probes will provide a versatile platform to interrogate PG crosslinking in physiologically relevant settings.SYNOPSIS TOC

scholarly journals Facile Synthesis and Metabolic Incorporation of m-DAP Bioisosteres Into Cell Walls of Live Bacteria

2020 ◽  
Author(s):  
Alexis J. Apostolos ◽  
Julia M. Nelson ◽  
Marcos M. Pires
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Cell Walls ◽  
Drug Targets ◽  
Solid Phase ◽  
Bacterial Species ◽  
Building Blocks ◽  
Structural Features ◽  
Diaminopimelic Acid ◽  
Bacterial Cells

AbstractBacterial cell walls contain peptidoglycan (PG), a scaffold that provides proper rigidity to resist lysis from internal osmotic pressure and a barrier to protect cells against external stressors. It consists of repeating sugar units with a linkage to a stem peptide that becomes highly crosslinked by cell wall transpeptidases (TP). Because it is an essential component of the bacterial cell, the PG biosynthetic machinery is often the target of antibiotics. For this reason, cellular probes that advance our understanding of PG biosynthesis and its maintenance can be powerful tools to reveal novel drug targets. While synthetic PG fragments containing L-Lysine in the 3rd position on the stem peptide are easier to access, those with meso-diaminopimelic acid (m-DAP) pose a severe synthetic challenge. Herein, we describe a solid phase synthetic scheme based on the widely available Fmoc-protected L-Cysteine building block to assemble meso-cystine (m-CYT), which mimics key structural features of m-DAP. To demonstrate proper mimicry of m-DAP, cell wall probes were synthesized with m-CYT in place of m-DAP and evaluated for their metabolic processing in live bacterial cells. We found that m-CYT-based cell wall probes were properly processed by TPs in various bacterial species that endogenously contain m-DAP in their PG. We anticipate that this strategy, which is based on the use of inexpensive and commercially available building blocks, can be widely adopted to provide greater accessibility of PG mimics for m-DAP containing organisms.

Controlled lysis of bacterial cells utilizing mutants with defective synthesis of D-alanine

1988 ◽  
Vol 34 (3) ◽  
pp. 256-261 ◽  
Author(s):  
Michael P. Heaton ◽  
Robert B. Johnston ◽  
Thomas L. Thompson
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Walls ◽  
Alanine Racemase ◽  
Growth Media ◽  
Bacterial Cells ◽  
Cell Wall Formation ◽  
Fermentation Processes ◽  
Intracellular Enzymes ◽  
Dense Cell

An alanine racemase (EC 5.1.1.1) mutant (Dal−) of Bacillus subtilis required small amounts of D-alanine to synthesize an osmotically stable cell wall in certain growth media. Investigation of the conditions which caused lysis in hypotonic media revealed that in addition to complex media, such as nutrient broth and acid-hydrolyzed casein, glycine inhibited stable cell wall formation. D-Alanine prevented the glycine inhibition. Up to 99% lysis occurred in both dilute and dense cell suspensions (optical densities up to 110) within 2.5 h after adding 1% glycine to late log phase cultures. Intracellular enzymes recovered from the lysate were as active as those from lysozyme-disrupted cells. No amino acid tested other than glycine induced lysis. Dal− mutants can be used for controlled lysis of bacterial cells to facilitate the isolation of normal intracellular constituents and bioengineered products from fermentation processes. Cell walls of most bacteria contain D-alanine; thus, this strategy should be applicable to a wide variety of microorganisms.

scholarly journals Factors That Affect the Enlargement of Bacterial Protoplasts and Spheroplasts

2020 ◽  
Vol 21 (19) ◽  
pp. 7131
Author(s):  
Hiromi Nishida
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Bacterial Cell ◽  
Metal Ion ◽  
Bacterial Species ◽  
Bacterial Cells ◽  
Cell Enlargement ◽  
Ion Composition ◽  
Gene Manipulation ◽  
Peptidoglycan Biosynthesis

Cell enlargement is essential for the microinjection of various substances into bacterial cells. The cell wall (peptidoglycan) inhibits cell enlargement. Thus, bacterial protoplasts/spheroplasts are used for enlargement because they lack cell wall. Though bacterial species that are capable of gene manipulation are limited, procedure for bacterial cell enlargement does not involve any gene manipulation technique. In order to prevent cell wall resynthesis during enlargement of protoplasts/spheroplasts, incubation media are supplemented with inhibitors of peptidoglycan biosynthesis such as penicillin. Moreover, metal ion composition in the incubation medium affects the properties of the plasma membrane. Therefore, in order to generate enlarged cells that are suitable for microinjection, metal ion composition in the medium should be considered. Experiment of bacterial protoplast or spheroplast enlargement is useful for studies on bacterial plasma membrane biosynthesis. In this paper, we have summarized the factors that influence bacterial cell enlargement.

scholarly journals Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

2021 ◽  
Vol 17 (3) ◽  
pp. e1009468
Author(s):  
Joshua A. F. Sutton ◽  
Oliver T. Carnell ◽  
Lucia Lafage ◽  
Joe Gray ◽  
Jacob Biboy ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Walls ◽  
Ex Vivo ◽  
Cell Wall Structure ◽  
Wall Structure ◽  
Bacterial Cells ◽  
Human Macrophages ◽  
Structure And Dynamics

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which correlated with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.

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

2019 ◽  
Vol 26 (10) ◽  
pp. 1950079 ◽  
Author(s):  
WALEED K. ABDUL KADHIM ◽  
UDAY M. NAYEF ◽  
MAJID S. JABIR
Keyword(s):  
Cell Wall ◽  
Antibacterial Activity ◽  
Polyethylene Glycol ◽  
Nucleic Acid ◽  
Antibacterial Agent ◽  
Bacterial Species ◽  
Good Method ◽  
Diffusion Method ◽  
Bacterial Cells ◽  
Average Size

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

scholarly journals Discovery of a Diverse Set of Bacteria That Build Their Cell Walls without the Canonical Peptidoglycan Polymerase aPBP

mBio ◽  
2021 ◽  
Author(s):  
Sharanjeet Atwal ◽  
Suthida Chuenklin ◽  
Edward M. Bonder ◽  
Juan Flores ◽  
Joseph J. Gillespie ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Wall ◽  
Cell Walls ◽  
Osmotic Shock ◽  
Bacterial Species ◽  
Potent Stimulator

Peptidoglycan (PG) is a large, cross-linked polymer that forms the cell wall of most bacterial species and confers shape, rigidity, and protection from osmotic shock. It is also a potent stimulator of the immune response in animals.

scholarly journals SaccuFlow: High-throughput Analysis Platform to Investigate Bacterial Cell Wall Interactions

2021 ◽  
Author(s):  
Alexis J Apostolos ◽  
Noel J Ferraro ◽  
Brianna E Dalesandro ◽  
Marcos Pires
Keyword(s):  
Cell Wall ◽  
High Throughput ◽  
Bacterial Cell ◽  
Cell Walls ◽  
Turgor Pressure ◽  
Principal Component ◽  
Bacterial Cells ◽  
High Throughput Analysis ◽  
Peptidoglycan Biosynthesis ◽  
Analysis Platform

Bacterial cell walls are formidable barriers that protect bacterial cells against external insults and oppose internal turgor pressure. While cell wall composition is variable across species, peptidoglycan is the principal component of all cell walls. Peptidoglycan is a mesh-like scaffold composed of crosslinked strands that can be heavily decorated with anchored proteins. The biosynthesis and remodeling of peptidoglycan must be tightly regulated by cells because disruption to this biomacromolecule is lethal. This essentiality is exploited by the human innate immune system in resisting colonization and by a number of clinically relevant antibiotics that target peptidoglycan biosynthesis. Evaluation of molecules or proteins that interact with peptidoglycan can be a complicated and, typically, qualitative effort. We have developed a novel assay platform (SaccuFlow) that preserves the native structure of bacterial peptidoglycan and is compatible with high-throughput flow cytometry analysis. We show that the assay is facile and versatile as demonstrated by its compatibility with sacculi from Gram-positive bacteria, Gram-negative bacteria, and mycobacteria. Finally, we highlight the utility of this assay to assess the activity of sortase from Staphylococcus aureus against potential anti-virulence agents.

scholarly journals Immobilization of Phosphatidylserine by Ethanol and Lysozyme on the Cell Surface for Evaluation of Apoptosis-Like Decay in Activated-Sludge Bacteria

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Ben Chen ◽  
Yasi Zhao ◽  
Zemin Li ◽  
Jianxin Pan ◽  
Haizhen Wu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Activated Sludge ◽  
Bacterial Cell ◽  
Cell Membranes ◽  
Bacterial Species ◽  
Accurate Determination ◽  
Bacterial Cells ◽  
Soil Matrix ◽  
Common Component ◽  
Bacterial Decay

ABSTRACT Accurate determination of microbial viability can be crucial in microbe-dominated biosystems. However, the identification of metabolic decay in bacterial cells can be elaborate and difficult. We sought to identify apoptosis-like bacterial processes by using annexin V-fluorescein isothiocyanate (FITC) (AVF), a probe typically used to stain phosphatidylserine (PS) on exposed cell membranes. The bacterial cell wall provides a barrier that is responsible for low efficiency of direct PS staining of decayed bacterial cells. This can be overcome by pretreatment of the bacteria with 70% ethanol, which fixates the bacteria and preserves the PS status, combined with lysozyme treatment to hydrolyze the cell wall. That treatment improved the efficiency of AVF staining considerably, as shown for pure strains of an Ochrobactrum sp. and a Micrococcus sp. Using this method, decayed bacterial cells (induced by starvation) were more strongly stained, indicating externalization of PS to a greater extent than seen for cells harvested at logarithmic growth. A multispecies microbial sludge was artificially decayed by heat treatment or alternating anoxic-oxic treatment, which also induced increased AVF staining, again presumably via decay-related PS externalization. The method developed proved to be efficient for identification of bacterial decay and has potential for the evaluation of multispecies bacterial samples from sources like soil matrix, bioaerosol, and activated sludge. IMPORTANCE Since the externalization of phosphatidylserine (PS) is considered a crucial characteristic of apoptosis, we sought to identify apoptosis-like decay in bacterial cells by PS staining using AVF. We show that this is possible, provided the bacteria are pretreated with ethanol plus lysozyme to remove a physical staining barrier and preserve the original, decay-related externalization of PS. Our work suggests that PS externalization occurs in starved bacteria and this can be quantified with AVF staining, providing a measure of bacterial decay. Since PS is the common component of the lipid bilayer in bacterial cell membranes, this approach also has potential for evaluation of cell decay of other bacterial species.

Digestion of cell-wall monosaccharides of ryegrass and alfalfa hays by the ruminal bacteria Fibrobacter succinogenes and Butyrivibrio fibrisolvens

1993 ◽  
Vol 39 (8) ◽  
pp. 780-786 ◽  
Author(s):  
Joshua Miron ◽  
Daniel Ben-Ghedalia
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Cell Walls ◽  
Surface Structures ◽  
Fibrobacter Succinogenes ◽  
Bacterial Cells ◽  
Ruminal Bacteria ◽  
Butyrivibrio Fibrisolvens ◽  
Scanning Electron ◽  
Better Than

The ruminal bacteria Fibrobacter succinogenes strains S85 and BL2 were grown in monocultures or in coculture with strain D1 of Butyrivibrio fibrisolvens, and the solubilization of ryegrass and alfalfa cell walls (CW) and digestion of CW monosaccharides were measured. Fibrobacter succinogenes monocultures and cocultures with B. fibrisolvens D1 degraded 58–69% of ryegrass CW, solubilizing 67–78% of CW glucose, 65–71% of CW xylose, 69–75% of hemicellulose, and 68–77% of total CW monosaccharides. When grown on alfalfa CW, those cultures degraded 28–39% of alfalfa CW, solubilizing 42–58% of CW glucose, 30–36% of CW xylose, and 37–45% of hemicellulose. With respect to both substrates, F. succinogenes strains solubilized CW carbohydrates better than did B. fibrisolvens D1. Complementary interaction between B. fibrisolvens D1 and the F. succinogenes strains was identified with respect to the utilization of some solubilized carbohydrates, but not with respect to the extent of CW solubilization, which was determined mainly by the F. succinogenes strains. For both substrates, utilization of solubilized cellulose by F. succinogenes monocultures was high (96–98%), whereas that of hemicellulose was lower (24–26% in ryegrass and 49–50% in alfalfa). Under scanning electron microscopy, F. succinogenes bacterial cells attached to and colonized on CW particles were characterized by the appearance of protuberant surface structures that we have identified as "polycellulosome complexes." Key words: cell wall monosaccharides, ryegrass, alfalfa, ruminal bacteria, Fibrobacter succinogenes, Butyrivibrio fibrisolvens.

Factors affecting the susceptibility of bacterial cell walls to the action of lysozyme

1967 ◽  
Vol 167 (1009) ◽  
pp. 446-447 ◽  
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Mode Of Action ◽  
Cell Walls ◽  
Bacterial Species ◽  
Bacterial Cell Wall ◽  
Low Molecular Weight ◽  
Factors Affecting ◽  
Structural Differences ◽  
Bacterial Cell Walls

Although we have heard a lot about the mode of binding of low molecular weight, soluble, lysozyme substrates, we have heard little about the mode of action of lysozyme on its natural insoluble substrate, the bacterial cell wall; so I want to bring a biological flavour into this discussion. Lysozyme was the name given by Fleming (1922) to the powerful bacteriolytic agent found in various cells and secretions; it was particularly active against a new bacterial species which he named Micrococcus lysodeikticus . The walls of this species still provide us with one of the best substrates for the study of lysozyme action. Salton showed that there is a considerable spectrum of activity of lysozyme in solubilizing walls of other species of bacteria. For example, walls of M. lysodeikticus are attacked rapidly by a concentration of enzyme of 1 μg/ml., Bacillus megaterium walls need 50 μg/ml., while walls of B. cereus are hardly changed visibly by 50 μg/ml. Consideration of the structure of the basal mucopeptide unit of bacterial cell walls, illustrated by Dr Perkins, shows that there are many ways in which structural differences could be introduced. Knowledge of the effects of some of these differences on lysozyme sensitivity may help in elucidating the mode of action of lysozyme on the complete bacterial cell wall.

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