Uptake Dynamics of Cubosome Nanocarriers at Bacterial Surfaces and the Routes for Cargo Internalization
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Abstract:
Antibiotic-resistant bacteria pose a significant threat to humanity. Gram-negative strains have demonstrated resistance to last resort antibiotics, partially due to their outer membrane, which hinders transport of antimicrobials into the bacterium. Nanocarrier (NC)-mediated drug delivery is one proposed strategy for combating this emerging issue. Here, the uptake of self-assembled lipid nanocarriers of cubic symmetry (cubosomes) into bacteria revealed fundamental differences in the uptake mechanism between Gram-positive and Gram-negative bacteria. For Gram-positive bacteria, the NCs adhere to the outer peptidoglycan layers and slowly internalize to the bacterium. For Gram-negative bacteria, the NCs interact in two stages, fusion with the outer lipid membrane and then diffusion through the inner wall. The self-assembled nature of the cubosomes imparts a unique ability to transfer payloads via membrane fusion. Remarkably, the fusion uptake mechanism allowed rapid NC internalization by the Gram-negative bacteria, overcoming the outer membrane responsible for their heightened resilience. Here this is demonstrated by the marked reduction in the minimal inhibition concentration required for antibiotics against a pathogenic strain of Gram-negative bacteria, Escherichia coli. These results provide mechanistic insight for the development of lipid NCs as a new tool to combat bacteria.Keywords:
Nanocarriers
Gram-Negative Bacteria
Internalization
Lipid II
An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.
Lipid II
Tunicamycin
Bacitracin
Cell envelope
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Lipid II
Lantibiotics
Cell envelope
Twin-arginine translocation pathway
Cell membrane
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An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.
Lipid II
Tunicamycin
Bacitracin
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Lipid II
Bacitracin
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Lipid II
Tunicamycin
Bacitracin
Cell envelope
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Lipid II
POPC
Cell envelope
Lipid microdomain
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Lipid II
Translocase
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For a long time, colicin M was known for killing susceptible Escherichia coli cells by interfering with cell wall peptidoglycan biosynthesis, but its precise mode of action was only recently elucidated: this bacterial toxin was demonstrated to be an enzyme that catalyzes the specific degradation of peptidoglycan lipid intermediate II, thereby provoking the arrest of peptidoglycan synthesis and cell lysis. The discovery of this activity renewed the interest in this colicin and opened the way for biochemical and structural analyses of this new class of enzyme (phosphoesterase). The identification of a few orthologs produced by pathogenic strains of Pseudomonas further enlarged the field of investigation. The present article aims at reviewing recently acquired knowledge on the biology of this small family of bacteriocins.
Colicin
Lipid II
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Lipid II
POPC
Cell envelope
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