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d‑Amino Acid Derivatives as in Situ Probes for Visualizing Bacterial Peptidoglycan Biosynthesis
journal contribution
posted on 2019-08-16, 17:05 authored by Yen-Pang Hsu, Garrett Booher, Alexander Egan, Waldemar Vollmer, Michael S. VanNieuwenhzeConspectusThe bacterial cell wall is composed of membrane
layers and a rigid
yet flexible scaffold called peptidoglycan (PG). PG provides mechanical
strength to enable bacteria to resist damage from the environment
and lysis due to high internal turgor. PG also has a critical role
in dictating bacterial cell morphology. The essential nature of PG
for bacterial propagation, as well as its value as an antibiotic target,
has led to renewed interest in the study of peptidoglycan biosynthesis.
However, significant knowledge gaps remain that must be addressed
before a clear understanding of peptidoglycan synthesis and dynamics
is realized. For example, the enzymes involved in the PG biosynthesis
pathway have not been fully characterized. Our understanding of PG
biosynthesis has been frequently revamped by the discovery of novel
enzymes or newly characterized functions of known enzymes. In addition,
we do not clearly know how the respective activities of these enzymes
are coordinated with each other and how they control the spatial and
temporal dynamics of PG synthesis.The emergence of molecular
probes and imaging techniques has significantly
advanced the study PG synthesis and modification. Prior efforts utilized
the specificity of PG-targeting antibiotics and proteins to develop
PG-specific probes, such as fluorescent vancomycin and fluorescent
wheat germ agglutinin. However, these probes suffer from limitations
due to toxic effects toward bacterial cells and poor membrane permeability.
To address these issues, we designed and introduced a family of novel
molecular probes, fluorescent d-amino acids (FDAAs), which
are covalently incorporated into PG through the activities of endogenous
bacterial transpeptidases. Their high biocompatibility and PG specificity
have made them powerful tools for labeling peptidoglycan. In addition,
their enzyme-mediated incorporation faithfully reflects the activity
of PG synthases, providing a direct in situ method for studying PG
formation during the bacterial life cycle.In this Account,
we describe our efforts directed at the development
of FDAAs and their derivatives. These probes have enabled for the
first time the ability to visualize PG synthesis in live bacterial
cells and in real time. We summarize experimental evidence for FDAA
incorporation into PG and the enzyme-mediated incorporation pathway.
We demonstrate various applications of FDAAs, including bacterial
morphology analyses, PG growth model studies, investigation of PG–enzyme
correlation, in vitro PG synthase activity assays, and antibiotic
inhibition tests. Finally, we discuss the current limitations of the
probes and our ongoing efforts to improve them. We are confident that
these probes will prove to be valuable tools that will enable the
discovery of new antibiotic targets and expand the available arsenal
directed at the public health threat posed by antibiotic resistance.
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PG synthase activity assaysantibiotic targetsimaging techniquesknowledge gapsPG synthasesPG biosynthesis pathwayPG biosynthesisenzyme-mediated incorporationmembrane permeabilitycell morphologyantibiotic targetd-amino acidspeptidoglycan biosynthesisPG-targeting antibioticsPeptidoglycan Biosynthesis ConspectusThepeptidoglycan synthesisSitu ProbesPG-specific probesPG formationstudy PG synthesismorphology analysesantibiotic resistancemembrane layerswheat germ agglutininPG specificitycell wallhealth threatantibiotic inhibition testsPG growth model studiesenzyme-mediated incorporation pathwayFDAA incorporationPG synthesis.The emergencenovel enzymeslife cycle.InPG synthesis
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