Staphylococcus aureusPenicillin-Binding Protein 2 Can Use Depsi-Lipid II Derived from Vancomycin-Resistant Strains for Cell Wall Synthesis

ABSTRACT Several non-β-lactam compounds were active against various gram-positive and gram-negative bacterial strains. The MICs of arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones were lower than those of ampicillin and cefotaxime for methicillin-resistant Staphylococcus aureus MI339 and vancomycin-resistant Enterococcus faecium EF12. Several compounds were found to inhibit the cell wall synthesis of S. aureus and the last two steps of peptidoglycan biosynthesis catalyzed by ether-treated cells of Escherichia coli or cell wall membrane preparations of Bacillus megaterium. The effects of the arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-one derivatives on E. coli PBP 3 and PBP 5, Streptococcus pneumoniae PBP 2xS (PBP 2x from a penicillin-sensitive strain) and PBP 2xR (PBP 2x from a penicillin-resistant strain), low-affinity PBP 2a of S. aureus, and the Actinomadura sp. strain R39 and Streptomyces sp. strain R61 dd-peptidases were studied. Some of the compounds exhibited inhibitory activities in the 10 to 100 μM concentration range. The inhibition of PBP 2xS by several of them appeared to be noncompetitive. The dissociation constant for the best inhibitor (Ki = 10 μM) was not influenced by the presence of the substrate.

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Penicillin-Binding Protein 2 Is Essential for Expression of High-Level Vancomycin Resistance and Cell Wall Synthesis in Vancomycin- Resistant Staphylococcus aureus Carrying the Enterococcal vanA Gene Complex

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.12.4566-4573.2004 ◽

2004 ◽

Vol 48 (12) ◽

pp. 4566-4573 ◽

Cited By ~ 31

Author(s):

Anatoly Severin ◽

Shang Wei Wu ◽

Keiko Tabei ◽

Alexander Tomasz

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Binding Protein ◽

Inducible Promoter ◽

Protein Product ◽

Vancomycin Resistance ◽

Penicillin Binding Protein ◽

Oxacillin Resistance ◽

Vancomycin Resistant ◽

High Level

ABSTRACT A combination of biochemical and genetic experiments were performed in order to better understand the mechanism of expression of high-level vancomycin resistance in Staphylococcus aureus. The transcription of pbp2 of the highly vancomycin- and oxacillin-resistant strain COLVA200 and its mutant derivative with inactivated mecA were put under the control of an inducible promoter, and the dependence of oxacillin and vancomycin resistance and cell wall composition on the concentration of the isopropyl-β-d-thiogalactopyranoside inducer was determined. The results indicate that mecA—the genetic determinant of oxacillin resistance—while essential for oxacillin resistance, is not involved with the expression of vancomycin resistance. Penicillin binding protein 2A, the protein product of mecA, appears to be unable to utilize the depsipeptide cell wall precursor produced in the vancomycin-resistant cells for transpeptidation. The key penicillin binding protein essential for vancomycin resistance and for the synthesis of the abnormally structured cell walls characteristic of vancomycin-resistant S. aureus (A. Severin, K. Tabei, F. Tenover, M. Chung, N. Clarke, and A. Tomasz, J. Biol. Chem. 279:3398-3407, 2004) is penicillin binding protein 2.

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THE EFFECT OF PENICILLIN ON THE STRUCTURE OF STAPHYLOCOCCAL CELL WALLS

Canadian Journal of Microbiology ◽

10.1139/m59-078 ◽

1959 ◽

Vol 5 (6) ◽

pp. 641-648 ◽

Cited By ~ 24

Author(s):

R. G. E. Murray ◽

W. H. Francombe ◽

B. H. Mayall

Keyword(s):

Cell Wall ◽

Cell Walls ◽

Untreated Control ◽

Osmium Tetroxide ◽

Wall Material ◽

Cell Wall Material ◽

Cell Wall Synthesis ◽

Cell Wall Component ◽

A Cell ◽

Resistant Strains

Cultures of sensitive stains of Staphylococcus aureus were fixed with osmium tetroxide after 1–5 hours' exposure to various does of pencillin and were embedded in methacrylate for sectioning and electron microscopy. They were compared with untreated, control cultures. The contrast of the cell wall material was untreated, control cultures. The contrast of the cell wall material was increased, by cutting the section of lanthanum nitrate.The cells increased in size and the surrounding cell wall was thinner than normal. The main lesions appeared in the developing cell wall septa, which showed a loss in density and gross irregularity of shape. Some questionable inclusions were seen in the cytoplasm. Lysis was prevented in a medium containing 0.3 M sucrose and the stable spheroplasts retained a recognizable cell wall after 24 hours' exposure to penicillin. However, the septa could not be demonstrated in the cells treated in sucrose medium.Two resistant strains were exposed to penicillin. In one, the cells showed no morphological effects; in the other, there was temporary damage to the cell septa with complete recovery.The observations support the hypothesis that penicillin interferes with the synthesis of a cell wall component and indicate that the main point of cell wall synthesis is at the site of septum formation.

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ChemInform Abstract: Lipid II: Total Synthesis of the Bacterial Cell Wall Precursor and Utilization as a Substrate for Glycosyltransfer and Transpeptidation by Penicillin Binding Protein (PBP) 1b of Eschericia coli.

ChemInform ◽

10.1002/chin.200216208 ◽

2010 ◽

Vol 33 (16) ◽

pp. no-no

Author(s):

Benjamin Schwartz ◽

Jay A. Markwalder ◽

Yi Wang

Keyword(s):

Cell Wall ◽

Total Synthesis ◽

Bacterial Cell ◽

Binding Protein ◽

Bacterial Cell Wall ◽

Penicillin Binding Protein ◽

Lipid Ii

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Substrate Inhibition of VanA by d-Alanine Reduces Vancomycin Resistance in a VanX-Dependent Manner

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00276-16 ◽

2016 ◽

Vol 60 (8) ◽

pp. 4930-4939 ◽

Cited By ~ 6

Author(s):

Lizah T. van der Aart ◽

Nicole Lemmens ◽

Willem J. van Wamel ◽

Gilles P. van Wezel

Keyword(s):

Cell Wall ◽

Streptomyces Coelicolor ◽

Substrate Inhibition ◽

Model Organism ◽

Vancomycin Resistance ◽

Dependent Manner ◽

Wild Type ◽

Content Type ◽

Lipid Ii ◽

Vancomycin Resistant

ABSTRACTThe increasing resistance of clinical pathogens against the glycopeptide antibiotic vancomycin, a last-resort drug against infections with Gram-positive pathogens, is a major problem in the nosocomial environment. Vancomycin inhibits peptidoglycan synthesis by binding to thed-Ala–d-Ala terminal dipeptide moiety of the cell wall precursor lipid II. Plasmid-transferable resistance is conferred by modification of the terminal dipeptide into the vancomycin-insensitive variantd-Ala–d-Lac, which is produced by VanA. Here we show that exogenousd-Ala competes withd-Lac as a substrate for VanA, increasing the ratio of wild-type to mutant dipeptide, an effect that was augmented by several orders of magnitude in the absence of thed-Ala–d-Ala peptidase VanX. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that high concentrations ofd-Ala led to the production of a significant amount of wild-type cell wall precursors, whilevanX-null mutants produced primarily wild-type precursors. This enhanced the efficacy of vancomycin in the vancomycin-resistant model organismStreptomyces coelicolor, and the susceptibility of vancomycin-resistant clinical isolates ofEnterococcus faecium(VRE) increased by up to 100-fold. The enhanced vancomycin sensitivity ofS. coelicolorcells correlated directly to increased binding of the antibiotic to the cell wall. Our work offers new perspectives for the treatment of diseases associated with vancomycin-resistant pathogens and for the development of drugs that target vancomycin resistance.

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BceAB-type antibiotic resistance transporters appear to act by target protection of cell wall synthesis

10.1101/835702 ◽

2019 ◽

Cited By ~ 1

Author(s):

Carolin M Kobras ◽

Hannah Piepenbreier ◽

Jennifer Emenegger ◽

Andre Sim ◽

Georg Fritz ◽

...

Keyword(s):

Cell Wall ◽

Antimicrobial Peptides ◽

Gc Content ◽

Critical Factor ◽

Peptide Antibiotics ◽

Cell Wall Synthesis ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Lipid Ii ◽

High Level

ABSTRACTResistance against cell wall-active antimicrobial peptides in bacteria is often mediated by transporters. In low GC-content Gram-positive bacteria, a wide-spread type of such transporters are the BceAB-like systems, which frequently provide a high level of resistance against peptide antibiotics that target intermediates of the lipid II cycle of cell wall synthesis. How a transporter can offer protection from drugs that are active on the cell surface, however, has presented researchers with a conundrum. Multiple theories have been discussed, ranging from removal of the peptides from the membrane, internalisation of the drug for degradation, to removal of the cellular target rather than the drug itself. To resolve this much-debated question, we here investigated the mode of action of the transporter BceAB of Bacillus subtilis. We show that it does not inactivate or import its substrate antibiotic bacitracin. Moreover, we present evidence that the critical factor driving transport activity is not the drug itself, but instead the concentration of drug-target complexes in the cell. Our results, together with previously reported findings, lead us to propose that BceAB-type transporters act by transiently freeing lipid II cycle intermediates from the inhibitory grip of antimicrobial peptides, and thus provide resistance through target protection of cell wall synthesis. Target protection has so far only been reported for resistance against antibiotics with intracellular targets, such as the ribosome. However, this mechanism offers a plausible explanation for the use of transporters as resistance determinants against cell wall-active antibiotics in Gram-positive bacteria where cell wall synthesis lacks the additional protection of an outer membrane.

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An Alternative and Conserved Cell Wall Enzyme That Can Substitute for the Lipid II Synthase MurG

mBio ◽

10.1128/mbio.03381-20 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

L. Zhang ◽

K. Ramijan ◽

V. J. Carrión ◽

L. T. van der Aart ◽

J. Willemse ◽

...

Keyword(s):

Cell Wall ◽

Sequence Similarity ◽

Streptomyces Coelicolor ◽

Genomic Analysis ◽

Environmental Harm ◽

Cell Wall Synthesis ◽

Precursor Molecule ◽

Content Type ◽

Lipid Ii ◽

A Cell

ABSTRACT The cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here, we present the discovery of a cell wall enzyme that can substitute for MurG. A mutant of Kitasatospora viridifaciens lacking a significant part of the dcw cluster, including murG, surprisingly produced lipid II and wild-type peptidoglycan. Genomic analysis identified a distant murG homologue, which encodes a putative enzyme that shares only around 31% amino acid sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MglA. Orthologues of mglA are present in 38% of all genomes of Kitasatospora and members of the sister genus Streptomyces. CRISPR interference experiments showed that K. viridifaciens mglA can also functionally replace murG in Streptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. All together, these results identify MglA as a bona fide lipid II synthase, thus demonstrating plasticity in cell wall synthesis. IMPORTANCE Almost all bacteria are surrounded by a cell wall, which protects cells from environmental harm. Formation of the cell wall requires the precursor molecule lipid II, which in bacteria is universally synthesized by the conserved and essential lipid II synthase MurG. We here exploit the unique ability of an actinobacterial strain capable of growing with or without its cell wall to discover an alternative lipid II synthase, MglA. Although this enzyme bears only weak sequence similarity to MurG, it can functionally replace MurG and can even do so in organisms that naturally have only a canonical MurG. The observation that MglA proteins are found in many actinobacteria highlights the plasticity in cell wall synthesis in these bacteria and demonstrates that important new cell wall biosynthetic enzymes remain to be discovered.

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An Association Between Peptidoglycan Synthesis and Organization of the Streptococcus pyogenes ExPortal

mBio ◽

10.1128/mbio.00485-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 12

Author(s):

Luis Alberto Vega ◽

Gary C. Port ◽

Michael G. Caparon

Keyword(s):

Cell Wall ◽

Protein Secretion ◽

Streptococcus Pyogenes ◽

Secretory Pathway ◽

Cell Wall Synthesis ◽

Gram Positive ◽

Content Type ◽

Peptidoglycan Synthesis ◽

Lipid Ii ◽

Protein Components

ABSTRACTThe ExPortal ofStreptococcus pyogenesis a focal microdomain of the cytoplasmic membrane that clusters the translocons of the general secretory pathway with accessory factors to facilitate the maturation of secreted polypeptides. While it is known that the ExPortal is enriched in anionic lipids, the mechanisms that organize the ExPortal are poorly understood. In the present study, we examined the role of the cell wall in organizing and maintaining the ExPortal. Removal of the cell wall resulted in a loss of ExPortal focal integrity accompanied by the circumferential redistribution of ExPortal lipid and protein components. A similar loss occurred upon treatment with gallidermin, a nonpermeabilizing lantibiotic that targets the lipid II precursor of peptidoglycan synthesis, and this treatment disrupted the secretion of several ExPortal substrates. Furthermore, several enzymes involved in the membrane-associated steps of lipid II synthesis, including MraY and MurN, were found to localize to a single discrete focus in the membrane that was coincident with the focal location of the secretory translocons and the anionic lipid microdomain. These data suggest that the ExPortal is associated with the site of peptidoglycan precursor synthesis and that peptidoglycan biogenesis influences ExPortal organization. These data add to an emerging literature indicating that cell wall biogenesis, cell division, and protein secretion are spatially coorganized processes.IMPORTANCESince Gram-positive bacteria lack a periplasmic space, they lack a protected compartment to spatially coordinate interaction between newly secreted proteins and the factors required to process them. This represents a significant problem for pathogens that depend on the secretion of toxins and cell wall-associated adhesins to cause disease. Streptococci solve this dilemma by restricting secretion and processing factors to a defined region of the membrane. However, the mechanisms that promote restriction are not understood. In this study, we show that restriction of these factors in the pathogenStreptococcus pyogenesis intimately linked with the presence of the cell wall and its synthesis. Furthermore, several cell wall synthesis proteins are also restricted to the site of protein secretion. This study contributes to our understanding of how the Gram-positive cell is organized to coordinate protein secretion and biogenesis with cell wall synthesis and to the ongoing development of antibiotics that target these processes.

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Lipid II: A central component in bacterial cell wall synthesis and a target for antibiotics

Prostaglandins Leukotrienes and Essential Fatty Acids ◽

10.1016/j.plefa.2008.09.020 ◽

2008 ◽

Vol 79 (3-5) ◽

pp. 117-121 ◽

Cited By ~ 68

Author(s):

Ben de Kruijff ◽

Vincent van Dam ◽

Eefjan Breukink

Keyword(s):

Cell Wall ◽

Bacterial Cell ◽

Bacterial Cell Wall ◽

Central Component ◽

Cell Wall Synthesis ◽

Lipid Ii

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Visualizing Conformation Transitions of the Lipid Flippase MurJ

10.1101/552745 ◽

2019 ◽

Author(s):

Alvin C. Y. Kuk ◽

Aili Hao ◽

Ziqiang Guan ◽

Seok-Yong Lee

Keyword(s):

Mass Spectrometry ◽

Cell Wall ◽

Bacterial Cell ◽

Structural Information ◽

Conformational Transitions ◽

Cell Wall Synthesis ◽

Lipid Ii ◽

Bacterial Peptidoglycan ◽

Lipid Flippase ◽

Conformation Transitions

AbstractThe biosynthesis of many polysaccharides, including bacterial peptidoglycan and eukaryotic N-linked glycans, requires transport of lipid-linked oligosaccharide (LLO) precursors across the membrane by specialized flippases. MurJ is the flippase for the lipid-linked peptidoglycan precursor Lipid II, a key player in bacterial cell wall synthesis, and a target of recently discovered antibacterials. However, the flipping mechanism of LLOs including Lipid II remains poorly understood due to a dearth of structural information. Here we report crystal structures of MurJ captured in inward-closed, inward-open, inward-occluded and outward-facing conformations. Together with cysteine accessibility, mass spectrometry, and complementation studies, we elucidate the conformational transitions in MurJ that mediate lipid flipping, identified the key ion for function, and provide a framework for the development of inhibitors.

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