d‑Amino Acid Derivatives as in Situ
Probes for Visualizing Bacterial Peptidoglycan Biosynthesis
Yen-Pang Hsu
Garrett Booher
Alexander Egan
Waldemar Vollmer
Michael S. VanNieuwenhze
10.1021/acs.accounts.9b00311.s001
https://acs.figshare.com/articles/journal_contribution/d_Amino_Acid_Derivatives_as_in_Situ_Probes_for_Visualizing_Bacterial_Peptidoglycan_Biosynthesis/9639296
ConspectusThe 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.
2019-08-16 17:05:22
PG synthase activity assays
antibiotic targets
imaging techniques
knowledge gaps
PG synthases
PG biosynthesis pathway
PG biosynthesis
enzyme-mediated incorporation
membrane permeability
cell morphology
antibiotic target
d-amino acids
peptidoglycan biosynthesis
PG-targeting antibiotics
Peptidoglycan Biosynthesis ConspectusThe
peptidoglycan synthesis
Situ Probes
PG-specific probes
PG formation
study PG synthesis
morphology analyses
antibiotic resistance
membrane layers
wheat germ agglutinin
PG specificity
cell wall
health threat
antibiotic inhibition tests
PG growth model studies
enzyme-mediated incorporation pathway
FDAA incorporation
PG synthesis.The emergence
novel enzymes
life cycle.In
PG synthesis