The design, synthesis, and characterization
of enterobactin–antibiotic
conjugates, hereafter Ent-Amp/Amx, where the β-lactam antibiotics
ampicillin (Amp) and amoxicillin (Amx) are linked to a monofunctionalized
enterobactin scaffold via a stable poly(ethylene glycol) linker are
reported. Under conditions of iron limitation, these siderophore-modified
antibiotics provide enhanced antibacterial activity against Escherichia coli strains, including uropathogenic E. coli CFT073 and UTI89, enterohemorrhagic E. coli O157:H7, and enterotoxigenic E. coli O78:H11, compared to the parent β-lactams. Studies with E. coli K-12 derivatives defective in ferric enterobactin
transport reveal that the enhanced antibacterial activity observed
for this strain requires the outer membrane ferric enterobactin transporter
FepA. A remarkable 1000-fold decrease in minimum inhibitory concentration
(MIC) value is observed for uropathogenic E. coli CFT073 relative to Amp/Amx, and time-kill kinetic studies demonstrate
that Ent-Amp/Amx kill this strain more rapidly at 10-fold lower concentrations
than the parent antibiotics. Moreover, Ent-Amp and Ent-Amx selectively
kill E. coli CFT073 co-cultured with other bacterial
species such as Staphylococcus aureus, and Ent-Amp
exhibits low cytotoxicity against human T84 intestinal cells in both
the apo and iron-bound forms. These studies demonstrate that the native
enterobactin platform provides a means to effectively deliver antibacterial
cargo across the outer membrane permeability barrier of Gram-negative
pathogens utilizing enterobactin for iron acquisition.
The design and syntheses of monofunctionalized enterobactin (Ent, L- and D-isomers) scaffolds where one catecholate moiety of enterobactin houses an alkene, aldehyde, or carboxylic acid at the C5 position are described. These molecules are key precursors to a family of 10 enterobactin-cargo conjugates presented in this work, which were designed to probe the extent to which the Gram-negative ferric enterobactin uptake and processing machinery recognizes, transports, and utilizes derivatized enterobactin scaffolds. A series of growth recovery assays employing enterobactin-deficient E. coli ATCC 33475 (ent-) revealed that six conjugates based on L-Ent having relatively small cargos promoted E. coli growth under iron-limiting conditions whereas negligible-to-no growth recovery was observed for four conjugates with relatively large cargos. No growth recovery was observed for the enterobactin receptor-deficient strain of E. coli H1187 (fepA-) or the enterobactin esterase-deficient derivative of E. coli K-12 JW0576 (fes-), or when the D-isomer of enterobactin was employed. These results demonstrate that the E. coli ferric enterobactin transport machinery identifies and delivers select cargo-modified scaffolds to the E. coli cytoplasm. Pseudomonas aeruginosa PAO1 K648 (pvd-, pch-) exhibited greater promiscuity than that of E. coli for the uptake and utilization of the enterobactin-cargo conjugates, and growth promotion was observed for eight conjugates under iron-limiting conditions. Enterobactin may be utilized for delivering molecular cargos via its transport machinery to the cytoplasm of E. coli and P. aeruginosa thereby providing a means to overcome the Gram-negative outer membrane permeability barrier.
Summary
Phosphopantetheine-modified carrier domains play a central role in the template-directed, biosynthesis of several classes of primary and secondary metabolites. Fatty acids, polyketides and nonribosomal peptides are constructed on multidomain enzyme assemblies using phosphopantetheinyl thioester-linked carrier domains to traffic and activate building blocks. The carrier domain is a dynamic component of the process, shuttling pathway intermediates to sequential enzyme active sites. Here we report an approach to structurally fix carrier domain/enzyme constructs suitable for X-ray crystallographic analysis. The structure of a two-domain construct of E. coli EntF was determined with a conjugated phosphopantetheinyl-based inhibitor. The didomain structure is locked in an active orientation relevant to the chemistry of nonribosomal peptide biosynthesis. This structure provides details into the interaction of phosphopantetheine arm with the carrier domain and the active site of the thioesterase domain.
A novel
Co-based metal–organic framework (MOF) with the formula of
{[Co3(BIBT)3(BTC)2(H2O)2]·solvents}
n
(JXUST-2, where JXUST denotes Jiangxi University of Science
and Technology, BIBT = 4,7-bi(1H-imidazol-1-yl)benzo-[2,1,3]thiadiazole,
and H3BTC = 1,3,5-benzenetricarboxylic acid) has been solvothermally
prepared, which takes 3D structure with a rare 3,4,6-c topology and
contains intramolecular hydrogen bonds. Interestingly, the sensing
investigations suggest that JXUST-2 could be considered
as a multifunctional fluorescence sensor toward Fe3+, Cr3+, and Al3+ via a turn-on effect with good reusability
and detection limits of 0.13, 0.10, and 0.10 μM, respectively.
The turn-on effect of JXUST-2 could be ascribed to an
absorbance caused enhancement (ACE) mechanism. Notably, JXUST-2 is the first turn-on MOF fluorescent sensor for Fe3+,
Cr3+, and Al3+ simultaneously.
A Zn II -based metal−organic framework (MOF) with a rare tcj topology has been solvothermally synthesized and displays relatively good thermal and chemical stabilities. Interestingly, the MOF can sensitively and selectively sense acetylacetone (acac) via a fluorescence enhancement effect with a detection limit of 0.10 ppm and good reusability, which demonstrates the first example of a MOF-based turn-on fluorescent sensor for acac.
Infections with Gram-negative pathogens pose a serious threat to public health. This scenario is exacerbated by increases in antibiotic resistance and the limited availability of vaccines and therapeutic tools to combat these infections. Here, we report an immunization approach that targets siderophores, which are small molecules exported by enteric Gram-negative pathogens to acquire iron, an essential nutrient, in the host. Because siderophores are nonimmunogenic, we designed and synthesized conjugates of a native siderophore and the immunogenic carrier protein cholera toxin subunit B (CTB). Mice immunized with the CTB-siderophore conjugate developed anti-siderophore antibodies in the gut mucosa, and when mice were infected with the enteric pathogen Salmonella, they exhibited reduced intestinal colonization and reduced systemic dissemination of the pathogen. Moreover, analysis of the gut microbiota revealed that reduction of Salmonella colonization in the inflamed gut was accompanied by expansion of Lactobacillus spp., which are beneficial commensal organisms that thrive in similar locales as Enterobacteriaceae. Collectively, our results demonstrate that anti-siderophore antibodies inhibit Salmonella colonization. Because siderophore-mediated iron acquisition is a virulence trait shared by many bacterial and fungal pathogens, blocking microbial iron acquisition by siderophore-based immunization or other siderophore-targeted approaches may represent a novel strategy to prevent and ameliorate a broad range of infections.
Siderophores are low-molecular-weight iron chelators that are produced and exported by bacteria and fungi during periods of nutrient deprivation. The structures, biosynthetic logic, and coordination chemistry of these molecules have fascinated chemists for decades. Studies of such fundamental phenomena guide the use of siderophores and siderophore conjugates in a variety of medicinal applications that include iron-chelation therapies and drug delivery. Sensing applications constitute another important facet of siderophore-based technologies. The high affinities of siderophores for both ferric ions and siderophore receptors, proteins expressed on the cell surface that are required for ferric siderophore import, indicate that these small molecules may be employed for the selective capture of metal ions, proteins, and live bacteria.This minireview summaries progress in methods that utilize native bacterial siderophore scaffolds for the detection of Fe(III) or microbial pathogens.2
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