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Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis

[Image: see text] Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphologica...

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Autores principales: Kuru, Erkin, Radkov, Atanas, Meng, Xin, Egan, Alexander, Alvarez, Laura, Dowson, Amanda, Booher, Garrett, Breukink, Eefjan, Roper, David I., Cava, Felipe, Vollmer, Waldemar, Brun, Yves, VanNieuwenhze, Michael S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2019
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929685/
https://www.ncbi.nlm.nih.gov/pubmed/31743648
http://dx.doi.org/10.1021/acschembio.9b00664
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author Kuru, Erkin
Radkov, Atanas
Meng, Xin
Egan, Alexander
Alvarez, Laura
Dowson, Amanda
Booher, Garrett
Breukink, Eefjan
Roper, David I.
Cava, Felipe
Vollmer, Waldemar
Brun, Yves
VanNieuwenhze, Michael S.
author_facet Kuru, Erkin
Radkov, Atanas
Meng, Xin
Egan, Alexander
Alvarez, Laura
Dowson, Amanda
Booher, Garrett
Breukink, Eefjan
Roper, David I.
Cava, Felipe
Vollmer, Waldemar
Brun, Yves
VanNieuwenhze, Michael S.
author_sort Kuru, Erkin
collection PubMed
description [Image: see text] Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.
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spelling pubmed-69296852019-12-26 Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis Kuru, Erkin Radkov, Atanas Meng, Xin Egan, Alexander Alvarez, Laura Dowson, Amanda Booher, Garrett Breukink, Eefjan Roper, David I. Cava, Felipe Vollmer, Waldemar Brun, Yves VanNieuwenhze, Michael S. ACS Chem Biol [Image: see text] Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs. American Chemical Society 2019-11-19 2019-12-20 /pmc/articles/PMC6929685/ /pubmed/31743648 http://dx.doi.org/10.1021/acschembio.9b00664 Text en Copyright © 2019 American Chemical Society This is an open access article published under a Creative Commons Attribution (CC-BY) License (http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html) , which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
spellingShingle Kuru, Erkin
Radkov, Atanas
Meng, Xin
Egan, Alexander
Alvarez, Laura
Dowson, Amanda
Booher, Garrett
Breukink, Eefjan
Roper, David I.
Cava, Felipe
Vollmer, Waldemar
Brun, Yves
VanNieuwenhze, Michael S.
Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis
title Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis
title_full Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis
title_fullStr Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis
title_full_unstemmed Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis
title_short Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis
title_sort mechanisms of incorporation for d-amino acid probes that target peptidoglycan biosynthesis
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929685/
https://www.ncbi.nlm.nih.gov/pubmed/31743648
http://dx.doi.org/10.1021/acschembio.9b00664
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