14 research outputs found
Bacterial Derived Carbohydrates Bind Cyr1 and Trigger Hyphal Growth in <i>Candida albicans</i>
The
dimorphic yeast <i>Candida albicans</i> is the most common
pathogenic fungus found in humans. While this species is normally
commensal, a morphological switch from budding yeast to filamentous
hyphae allows the fungi to invade epithelial cells and cause infections.
The phenotypic change is controlled by the adenylyl cyclase, Cyr1.
Interestingly, this protein contains a leucine-rich repeat (LRR) domain,
which is commonly found in innate immune receptors from plants and
animals. A functional and pure LRR domain was obtained in high yields
from <i>E. coli</i> expression. Utilizing a surface
plasmon resonance assay, the LRR was found to bind diverse bacterial
derived carbohydrates with high affinity. This domain is capable of
binding fragments of peptidoglycan, a carbohydrate polymer component
of the bacterial cell wall, as well as anthracyclines produced by <i>Streptomyces</i>, leading to hyphae formation. These findings
add another dimension to the human microbiome, taking into account
yeast–bacteria interactions that occur in the host
Postsynthetic Modification of Bacterial Peptidoglycan Using Bioorthogonal <i>N</i>‑Acetylcysteamine Analogs and Peptidoglycan <i>O</i>‑Acetyltransferase B
Bacteria have the natural ability
to install protective postsynthetic
modifications onto its bacterial peptidoglycan (PG), the coat woven
into bacterial cell wall. Peptidoglycan <i>O</i>-acetyltransferase
B (PatB) catalyzes the <i>O</i>-acetylation of PG in Gram
(−) bacteria, which aids in bacterial survival, as it prevents
autolysins such as lysozyme from cleaving the PG. We explored the
mechanistic details of PatB’s acetylation function and determined
that PatB has substrate specificity for bioorthgonal short <i>N</i>-acetyl cysteamine (SNAc) donors. A variety of functionality
including azides and alkynes were installed on tri-<i>N</i>-acetylglucosamine (NAG)<sub>3</sub>, a PG mimic, as well as PG isolated
from various Gram (+) and Gram (−) bacterial species. The bioorthogonal
modifications protect the isolated PG against lysozyme degradation <i>in vitro.</i> We further demonstrate that this postsynthetic
modification of PG can be extended to use click chemistry to fluorescently
label the mature PG in whole bacterial cells of Bacillus
subtilis. Modifying PG postsynthetically can aid in
the development of antibiotics and immune modulators by expanding
the understanding of how PG is processed by lytic enzymes
sj-pdf-1-ini-10.1177_17534259231207198 - Supplemental material for Synthesis and validation of click-modified NOD1/2 agonists
Supplemental material, sj-pdf-1-ini-10.1177_17534259231207198 for Synthesis and validation of click-modified NOD1/2 agonists by Ravi Bharadwaj, Madison V. Anonick, Swati Jaiswal, Siavash Mashayekh, Ashley Brown, Kimberly A. Wodzanowski, Kendi Okuda, Neal Silverman and Catherine L. Grimes in Innate Immunity</p
Synthesis of Functionalized <i>N</i>‑Acetyl Muramic Acids To Probe Bacterial Cell Wall Recycling and Biosynthesis
Uridine
diphosphate <i>N</i>-acetyl muramic acid (UDP
NAM) is a critical intermediate in bacterial peptidoglycan (PG) biosynthesis.
As the primary source of muramic acid that shapes the PG backbone,
modifications installed at the UDP NAM intermediate can be used to
selectively tag and manipulate this polymer via metabolic incorporation.
However, synthetic and purification strategies to access large quantities
of these PG building blocks, as well as their derivatives, are challenging.
A robust chemoenzymatic synthesis was developed using an expanded
NAM library to produce a variety of 2<i>-N</i>-functionalized
UDP NAMs. In addition, a synthetic strategy to access bio-orthogonal
3-lactic acid NAM derivatives was developed. The chemoenzymatic UDP
synthesis revealed that the bacterial cell wall recycling enzymes
MurNAc/GlcNAc anomeric kinase (AmgK) and NAM α-1 phosphate uridylyl
transferase (MurU) were permissive to permutations at the two and
three positions of the sugar donor. We further explored the utility
of these derivatives in the fluorescent labeling of both Gram (−)
and Gram (+) PG in whole cells using a variety of bio-orthogonal chemistries
including the tetrazine ligation. This report allows for rapid and
scalable access to a variety of functionalized NAMs and UDP NAMs,
which now can be used in tandem with other complementary bio-orthogonal
labeling strategies to address fundamental questions surrounding PG’s
role in immunology and microbiology
Crohn’s Disease Variants of Nod2 Are Stabilized by the Critical Contact Region of Hsp70
Nod2
is a cytosolic, innate immune receptor responsible for binding
to bacterial cell wall fragments such as muramyl dipeptide (MDP).
Upon binding, subsequent downstream activation of the NF-κB
pathway leads to an immune response. Nod2 mutations are correlated
with an increased susceptibility to Crohn’s disease (CD) and
ultimately result in a misregulated immune response. Previous work
had demonstrated that Nod2 interacts with and is stabilized by the
molecular chaperone Hsp70. In this work, it is shown using purified
protein and <i>in vitro</i> biochemical assays that the
critical Nod2 CD mutations (G908R, R702W, and 1007fs) preserve the
ability to bind bacterial ligands. A limited proteolysis assay and
luciferase reporter assay reveal regions of Hsp70 that are capable
of stabilizing Nod2 and rescuing CD mutant activity. A minimal 71-amino
acid subset of Hsp70 that stabilizes the CD-associated variants of
Nod2 and restores a proper immune response upon activation with MDP
was identified. This work suggests that CD-associated Nod2 variants
could be stabilized <i>in vivo</i> with a molecular chaperone
Minimalist Tetrazine <i>N</i>‑Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion
N-Acetyl muramic acid (NAM) probes containing
alkyne or azide groups are commonly used to investigate aspects of
cell wall synthesis because of their small size and ability to incorporate
into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne–azide
cycloaddition (CuAAC) reactions are not compatible with live cells,
and strain-promoted alkyne–azide cycloaddition (SPAAC) reaction
rates are modest and, therefore, not as desirable for tracking the
temporal alterations of bacterial cell growth, remodeling, and division.
Alternatively, the tetrazine-trans-cyclooctene ligation
(Tz-TCO), which is the fastest known bioorthogonal reaction and not
cytotoxic, allows for rapid live-cell labeling of PG at biologically
relevant time scales and concentrations. Previous work to increase
reaction kinetics on the PG surface by using tetrazine probes was
limited because of low incorporation of the probe. Described here
are new approaches to construct a minimalist tetrazine (Tz)-NAM probe
utilizing recent advancements in asymmetric tetrazine synthesis. This
minimalist Tz-NAM probe was successfully incorporated into pathogenic
and commensal bacterial PG where fixed and rapid live-cell, no-wash
labeling was successful in both free bacterial cultures and in coculture
with human macrophages. Overall, this probe allows for expeditious
labeling of bacterial PG, thereby making it an exceptional tool for
monitoring PG biosynthesis for the development of new antibiotic screens.
The versatility and selectivity of this probe will allow for real-time
interrogation of the interactions of bacterial pathogens in a human
host and will serve a broader utility for studying glycans in multiple
complex biological systems
Minimalist Tetrazine <i>N</i>‑Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion
N-Acetyl muramic acid (NAM) probes containing
alkyne or azide groups are commonly used to investigate aspects of
cell wall synthesis because of their small size and ability to incorporate
into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne–azide
cycloaddition (CuAAC) reactions are not compatible with live cells,
and strain-promoted alkyne–azide cycloaddition (SPAAC) reaction
rates are modest and, therefore, not as desirable for tracking the
temporal alterations of bacterial cell growth, remodeling, and division.
Alternatively, the tetrazine-trans-cyclooctene ligation
(Tz-TCO), which is the fastest known bioorthogonal reaction and not
cytotoxic, allows for rapid live-cell labeling of PG at biologically
relevant time scales and concentrations. Previous work to increase
reaction kinetics on the PG surface by using tetrazine probes was
limited because of low incorporation of the probe. Described here
are new approaches to construct a minimalist tetrazine (Tz)-NAM probe
utilizing recent advancements in asymmetric tetrazine synthesis. This
minimalist Tz-NAM probe was successfully incorporated into pathogenic
and commensal bacterial PG where fixed and rapid live-cell, no-wash
labeling was successful in both free bacterial cultures and in coculture
with human macrophages. Overall, this probe allows for expeditious
labeling of bacterial PG, thereby making it an exceptional tool for
monitoring PG biosynthesis for the development of new antibiotic screens.
The versatility and selectivity of this probe will allow for real-time
interrogation of the interactions of bacterial pathogens in a human
host and will serve a broader utility for studying glycans in multiple
complex biological systems
Minimalist Tetrazine <i>N</i>‑Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion
N-Acetyl muramic acid (NAM) probes containing
alkyne or azide groups are commonly used to investigate aspects of
cell wall synthesis because of their small size and ability to incorporate
into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne–azide
cycloaddition (CuAAC) reactions are not compatible with live cells,
and strain-promoted alkyne–azide cycloaddition (SPAAC) reaction
rates are modest and, therefore, not as desirable for tracking the
temporal alterations of bacterial cell growth, remodeling, and division.
Alternatively, the tetrazine-trans-cyclooctene ligation
(Tz-TCO), which is the fastest known bioorthogonal reaction and not
cytotoxic, allows for rapid live-cell labeling of PG at biologically
relevant time scales and concentrations. Previous work to increase
reaction kinetics on the PG surface by using tetrazine probes was
limited because of low incorporation of the probe. Described here
are new approaches to construct a minimalist tetrazine (Tz)-NAM probe
utilizing recent advancements in asymmetric tetrazine synthesis. This
minimalist Tz-NAM probe was successfully incorporated into pathogenic
and commensal bacterial PG where fixed and rapid live-cell, no-wash
labeling was successful in both free bacterial cultures and in coculture
with human macrophages. Overall, this probe allows for expeditious
labeling of bacterial PG, thereby making it an exceptional tool for
monitoring PG biosynthesis for the development of new antibiotic screens.
The versatility and selectivity of this probe will allow for real-time
interrogation of the interactions of bacterial pathogens in a human
host and will serve a broader utility for studying glycans in multiple
complex biological systems
Minimalist Tetrazine <i>N</i>‑Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion
N-Acetyl muramic acid (NAM) probes containing
alkyne or azide groups are commonly used to investigate aspects of
cell wall synthesis because of their small size and ability to incorporate
into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne–azide
cycloaddition (CuAAC) reactions are not compatible with live cells,
and strain-promoted alkyne–azide cycloaddition (SPAAC) reaction
rates are modest and, therefore, not as desirable for tracking the
temporal alterations of bacterial cell growth, remodeling, and division.
Alternatively, the tetrazine-trans-cyclooctene ligation
(Tz-TCO), which is the fastest known bioorthogonal reaction and not
cytotoxic, allows for rapid live-cell labeling of PG at biologically
relevant time scales and concentrations. Previous work to increase
reaction kinetics on the PG surface by using tetrazine probes was
limited because of low incorporation of the probe. Described here
are new approaches to construct a minimalist tetrazine (Tz)-NAM probe
utilizing recent advancements in asymmetric tetrazine synthesis. This
minimalist Tz-NAM probe was successfully incorporated into pathogenic
and commensal bacterial PG where fixed and rapid live-cell, no-wash
labeling was successful in both free bacterial cultures and in coculture
with human macrophages. Overall, this probe allows for expeditious
labeling of bacterial PG, thereby making it an exceptional tool for
monitoring PG biosynthesis for the development of new antibiotic screens.
The versatility and selectivity of this probe will allow for real-time
interrogation of the interactions of bacterial pathogens in a human
host and will serve a broader utility for studying glycans in multiple
complex biological systems
Minimalist Tetrazine <i>N</i>‑Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion
N-Acetyl muramic acid (NAM) probes containing
alkyne or azide groups are commonly used to investigate aspects of
cell wall synthesis because of their small size and ability to incorporate
into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne–azide
cycloaddition (CuAAC) reactions are not compatible with live cells,
and strain-promoted alkyne–azide cycloaddition (SPAAC) reaction
rates are modest and, therefore, not as desirable for tracking the
temporal alterations of bacterial cell growth, remodeling, and division.
Alternatively, the tetrazine-trans-cyclooctene ligation
(Tz-TCO), which is the fastest known bioorthogonal reaction and not
cytotoxic, allows for rapid live-cell labeling of PG at biologically
relevant time scales and concentrations. Previous work to increase
reaction kinetics on the PG surface by using tetrazine probes was
limited because of low incorporation of the probe. Described here
are new approaches to construct a minimalist tetrazine (Tz)-NAM probe
utilizing recent advancements in asymmetric tetrazine synthesis. This
minimalist Tz-NAM probe was successfully incorporated into pathogenic
and commensal bacterial PG where fixed and rapid live-cell, no-wash
labeling was successful in both free bacterial cultures and in coculture
with human macrophages. Overall, this probe allows for expeditious
labeling of bacterial PG, thereby making it an exceptional tool for
monitoring PG biosynthesis for the development of new antibiotic screens.
The versatility and selectivity of this probe will allow for real-time
interrogation of the interactions of bacterial pathogens in a human
host and will serve a broader utility for studying glycans in multiple
complex biological systems