scholarly journals MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling

2021 ◽  
Author(s):  
Sabrina Wamp ◽  
Patricia Rothe ◽  
Gudrun Holland ◽  
Sven Halbedel
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Protein Kinase ◽  
Bacterial Cell ◽  
Bacterial Cell Wall ◽  
Escape Mutation ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Main Component ◽  
Biosynthesis Regulation

AbstractGram-positive bacteria are protected by a thick mesh of peptidoglycan (PG) completely engulfing their cells. This PG network is the main component of the bacterial cell wall, it provides rigidity and acts as foundation for the attachment of other surface molecules. Biosynthesis of PG consumes a high amount of cellular resources and therefore requires careful adjustments to environmental conditions.An important switch in the control of PG biosynthesis of Listeria monocytogenes, a Gram-positive pathogen with a high infection fatality rate, is the serine/threonine protein kinase PrkA. A key substrate of this kinase is the small cytosolic protein ReoM. We have shown previously that ReoM phosphorylation regulates PG formation through control of MurA stability. MurA catalyzes the first step in PG biosynthesis and the current model suggests that phosphorylated ReoM prevents MurA degradation by the ClpCP protease. In contrast, conditions leading to ReoM dephosphorylation stimulate MurA degradation. How ReoM controls degradation of MurA and potential other substrates is not understood. Also, the individual contribution of the ∼20 other known PrkA targets to PG biosynthesis regulation is unknown.We here present murA mutants which escape proteolytic degradation. The release of MurA from ClpCP-dependent proteolysis was able to constitutively activate PG biosynthesis and further enhances the intrinsic cephalosporin resistance of L. monocytogenes. This activation required the RodA3/PBP B3 transglycosylase/transpeptidase pair as additional effectors of the PrkA signaling route. One murA escape mutation not only fully rescued an otherwise non-viable prkA mutant during growth in batch culture and inside macrophages but also overcompensated cephalosporin hypersensitivity. Our data collectively indicate that the main purpose of PrkA-mediated signaling in L. monocytogenes is control of MurA stability during extra- and intracellular growth. These findings have important implications for the understanding of PG biosynthesis regulation and β-lactam resistance of L. monocytogenes and related Gram-positive bacteria.Author SummaryPeptidoglycan (PG) is the main component of the bacterial cell wall and many of the PG synthesizing enzymes are antibiotic targets. We previously have discovered a new signaling route controlling PG production in the human pathogen Listeria monocytogenes. This route also determines the intrinsic resistance of L. monocytogenes against cephalosporins, a group of β-lactam antibiotics. Signaling involves PrkA, a membrane-embedded protein kinase, that is activated during cell wall stress to phosphorylate its target ReoM. Depending on its phosphorylation, ReoM activates or inactivates PG production by controlling the proteolytic stability of MurA, which catalyzes the first step in PG biosynthesis. MurA degradation depends on the ClpCP protease and we here have isolated murA mutations that escape this degradation. Using these mutants, we could show that regulation of PG biosynthesis through control of MurA stability is the primary purpose of PrkA-mediated signaling in L. monocytogenes. Further experiments identified the transglycosylase RodA and the transpeptidase PBP B3 as additional effectors of PrkA signaling. Our results suggest that both proteins act together to translate the signals received by PrkA into intensification of PG biosynthesis. These findings shed new light on the regulation of PG biosynthesis in Gram-positive bacteria with intrinsic β-lactam resistance.

Infectious disease

2019 ◽  
Author(s):  
Stevan R. Emmett ◽  
Nicola Hill ◽  
Federico Dajas-Bailador
Keyword(s):  
Cell Wall ◽  
Protein Synthesis ◽  
Nucleic Acid ◽  
Outer Membrane ◽  
Bacterial Cell ◽  
Bacterial Cell Wall ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

Antibiotics include an extensive range of agents able to kill or prevent reproduction of bacteria in the body, without being overly toxic to the patient. Traditionally derived from living organisms, most are now chemically synthesized and act to disrupt the integrity of the bacterial cell wall, or penetrate the cell and disrupt protein synthesis or nucleic acid replication. Typically, bacteria are identified according to their ap­pearance under the microscope depending on shape and response to the Gram stain test. Further identification is obtained by growth characteristics on various types of culture media, based on broth or agar, biochemical and immunological profiles. Further testing on broth or agar determines antibiotic sensitivity to guide on anti­biotic therapy in individual patients. This process can take 24– 48 hours to culture and a further 24– 48 hours to measure sensitivities. Increasingly, new technology, e.g. Matrix Assisted Laser Desorption Ionization— Time of Flight (MALDI- TOF) and nucleic acid amplification as­says, are being used to provide more rapid identification. The Gram classification, however, is still widely referred to as it differentiates bacteria by the presence or absence of the outer lipid membrane (see Figure 11.1), a fundamental characteristic that influences antibiotic management. Antimicrobial agents rely on selective action exploiting genetic differences between bacterial and eukaryotic cells. They target bacterial cell wall synthesis, bacterial protein synthesis, microbial DNA or RNA synthesis, by acting on bacterial cell metabolic pathways or by inhibiting the ac­tion of a bacterial toxin (see Table 11.1). Both Gram- positive and Gram- negative bacteria possess a rigid cell wall able to protect the bacteria from varying osmotic pressures (Figure 11.1). Peptidoglycan gives the cell wall its rigidity and is composed of a glycan chain of complex alternating carbohydrates, N- acetylglucosamide (N- ATG), and N- acetylmurcarinic acid (N- ATM), that are cross- linked by peptide (or glycine) chains. In Gram-positive bacteria, the cell wall contains multiple peptido­glycan layers, interspersed with teichoic acids, whereas Gram- negative bacteria contain only one or two peptido­glycan layers that are surrounded by an outer membrane attached by lipoproteins. The outer membrane contains porins (which regulate transport of substances into and out of the cell), lipopolysaccharides, and outer proteins in a phospholipid bilayer. For both Gram- negative and Gram-positive bacteria, peptidoglycan synthesis involves about 30 bacterial enzymes acting over three stages. Since the cell wall is unique to bacteria, it makes a suitable target for antibiotic therapy.

scholarly journals Lysozyme from Chicken Egg White as an Antibacterial Agent

2019 ◽  
Vol 28 (4) ◽  
pp. 175
Author(s):  
Harsi Dewantari Kusumaningrum ◽  
Syahrizal Nasution ◽  
E Kusumaningtyas ◽  
D N Faridah
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Antibacterial Activity ◽  
Bacterial Cell ◽  
Egg White ◽  
Bacterial Cell Wall ◽  
Enzymatic Treatment ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Chicken Egg

<p class="00-6Abstrak2Wtz">Lysozyme is one of the constituent proteins of chicken egg white that plays an important role in a protection system during the embryo growing process. Lysozyme protection systems can be applied in food and for health. This paper aims to describe the role of egg white lysozyme which has the antibacterial activity to improve food safety and health. Mechanism and activity of lysozyme protection can be explored by understanding the structure of proteins, type of amino acids, and the sequence of amino acids. The mechanism of lysozyme antibacterial activity against Gram positive bacteria occurs through its ability to breakdown the peptidoglycan glycosidic bonds in the bacterial cell wall membrane. The antibacterial activity of lysozyme can be increased when its normal lysozyme form was denatured/hydrolyzed through modifying the lysozyme structure by heat and enzymatic treatment. Heat treatment will reveal the active site of lysozyme after denaturation, leading to the destruction of the bacterial cell wall membrane. Enzymatic treatment through hydrolysis process of hydrolysis by protease enzymes will generate antibacterial peptides, which have the ability to inhibit the growth of Gram positive bacteria and Gram negative bacteria. Antibacterial lysozyme peptides and heat treated lysozyme are dominated by hydrophobic amino acids and positively charged to facilitate interaction between lysozyme and lipopolysaccharide which coats peptidoglycan. Antibacterial lysozyme peptide can be used as an antibiotic and a safe natural preservative compared to synthetic material and can reduce the risk of destructive bacteria and pathogenic bacteria. Lysozyme peptide is potential to protect human health since it has also other activities such as inhibition of angiotensin-converting enzyme, antihypertensive, antitumor, antioxidant, and antiviral agents.</p>

scholarly journals Rhodomyrtone Accumulates in Bacterial Cell Wall and Cell Membrane and Inhibits the Synthesis of Multiple Cellular Macromolecules in Epidemic Methicillin-Resistant Staphylococcus aureus

Antibiotics ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 543
Author(s):  
Ozioma F. Nwabor ◽  
Sukanlaya Leejae ◽  
Supayang P. Voravuthikunchai
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Membrane ◽  
Bacterial Cell ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Treatment Options ◽  
Bacterial Cell Wall ◽  
Methicillin Resistant ◽  
Gram Positive Bacteria ◽  
Inhibitor Compounds

As the burden of antibacterial resistance worsens and treatment options become narrower, rhodomyrtone—a novel natural antibiotic agent with a new antibacterial mechanism—could replace existing antibiotics for the treatment of infections caused by multi-drug resistant Gram-positive bacteria. In this study, rhodomyrtone was detected within the cell by means of an easy an inexpensive method. The antibacterial effects of rhodomyrtone were investigated on epidemic methicillin-resistant Staphylococcus aureus. Thin-layer chromatography demonstrated the entrapment and accumulation of rhodomyrtone within the bacterial cell wall and cell membrane. The incorporation of radiolabelled precursors revealed that rhodomyrtone inhibited the synthesis of macromolecules including DNA, RNA, proteins, the cell wall, and lipids. Following the treatment with rhodomyrtone at MIC (0.5–1 µg/mL), the synthesis of all macromolecules was significantly inhibited (p ≤ 0.05) after 4 h. Inhibition of macromolecule synthesis was demonstrated after 30 min at a higher concentration of rhodomyrtone (4× MIC), comparable to standard inhibitor compounds. In contrast, rhodomyrtone did not affect lipase activity in staphylococci—both epidemic methicillin-resistant S. aureus and S. aureus ATCC 29213. Interfering with the synthesis of multiple macromolecules is thought to be one of the antibacterial mechanisms of rhodomyrtone.

scholarly journals Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues

BIO-PROTOCOL ◽  
2016 ◽  
Vol 6 (23) ◽  
Author(s):  
Beth Mann ◽  
Lip Loh ◽  
Geli Gao ◽  
Elaine Tuomanen
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Bacterial Cell Wall ◽  
Gram Positive ◽  
Fetal Tissues

scholarly journals The Sortase SrtA of Listeria monocytogenes Is Involved in Processing of Internalin and in Virulence

2002 ◽  
Vol 70 (3) ◽  
pp. 1382-1390 ◽  
Author(s):  
Caroline Garandeau ◽  
Hélène Réglier-Poupet ◽  
Iharilalao Dubail ◽  
Jean-Luc Beretti ◽  
Patrick Berche ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Protein A ◽  
Knockout Mutant ◽  
Reporter Protein ◽  
Gram Positive ◽  
Bacterial Surface ◽  
Gram Positive Bacteria ◽  
Covalently Linked

ABSTRACT Listeria monocytogenes is an intracellular gram-positive human pathogen that invades eucaryotic cells. Among the surface-exposed proteins playing a role in this invasive process, internalin belongs to the family of LPXTG proteins, which are known to be covalently linked to the bacterial cell wall in gram-positive bacteria. Recently, it has been shown in Staphylococcus aureus that the covalent anchoring of protein A, a typical LPXTG protein, is due to a cysteine protease, named sortase, required for bacterial virulence. Here, we identified in silico from the genome of L. monocytogenes a gene, designated srtA, encoding a sortase homologue. The role of this previously unknown sortase was studied by constructing a sortase knockout mutant. Internalin was used as a reporter protein to study the effects of the srtA mutation on cell wall anchoring of this LPXTG protein in L. monocytogenes. We show that the srtA mutant (i) is affected in the display of internalin at the bacterial surface, (ii) is significantly less invasive in vitro, and (iii) is attenuated in its virulence in the mouse. These results demonstrate that srtA of L. monocytogenes acts as a sortase and plays a role in the pathogenicity.

The structure of secondary cell wall polymers: how Gram-positive bacteria stick their cell walls together

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 643-651 ◽  
Author(s):  
Christina Schäffer ◽  
Paul Messner
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Bacterial Cell ◽  
Secondary Cell Wall ◽  
Chemical Study ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Bacterial Cell Surface ◽  
Cell Wall Polymers ◽  
Structural Principles

The cell wall of Gram-positive bacteria has been a subject of detailed chemical study over the past five decades. Outside the cytoplasmic membrane of these organisms the fundamental polymer is peptidoglycan (PG), which is responsible for the maintenance of cell shape and osmotic stability. In addition, typical essential cell wall polymers such as teichoic or teichuronic acids are linked to some of the peptidoglycan chains. In this review these compounds are considered as ‘classical’ cell wall polymers. In the course of recent investigations of bacterial cell surface layers (S-layers) a different class of ‘non-classical’ secondary cell wall polymers (SCWPs) has been identified, which is involved in anchoring of S-layers to the bacterial cell surface. Comparative analyses have shown considerable differences in chemical composition, overall structure and charge behaviour of these SCWPs. This review discusses the progress that has been made in understanding the structural principles of SCWPs, which may have useful applications in S-layer-based ‘supramolecular construction kits' in nanobiotechnology.

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