Abstract:The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome’s translocation on the messenger RNA, as well as by… Show more
“…9 . As validated through previous works as well 25 , the challenge presented by the introduction of aminoglycosides to E. coli is bactericidal and not bacteriostatic therefore growth effects are non-recoverable. Figure 9 ( A ) and ( B ) Calculated growth rate in absorbance increase per hour as determined by the first derivative of a growth curve for streptomycin and tobramycin respectively.…”
Section: Resultsmentioning
confidence: 61%
“…The prior consensus in the literature stated that aminoglycosides act through the disruption of protein synthesis, thereby preventing the translation of membrane proteins and leading to changes in the cell surface morphology and damage to the cytoplasmic membrane 20 – 24 . Recently, researchers have demonstrated, using single-molecule spectroscopic methods, that aminoglycosides only slow protein synthesis and don’t inhibit it fully, therefore suggesting that the bactericidal activity of aminoglycosides is not related to ribosomal inhibition 25 . Herein, we propose an alternative mechanism of action of aminoglycosides through the potent inhibition of HepI and disruption of lipopolysaccharide biosynthesis.…”
A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years because of its critical role in the biosynthesis of lipopolysaccharides on bacterial cell surfaces. While many labs have discovered or designed novel small molecule inhibitors, these compounds lacked the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. Structural inspection of Kdo2-lipid A suggested aminoglycoside antibiotics as potential inhibitors for HepI. Multiple aminoglycosides have been experimentally validated to be first-in-class nanomolar inhibitors of HepI, with the best inhibitor demonstrating a Ki of 600 ± 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and molecular dynamics simulations were used to corroborate kinetic experimental findings. While aminoglycosides have long been described as potent antibiotics targeting bacterial ribosomes’ protein synthesis leading to disruption of the stability of bacterial cell membranes, more recently researchers have shown that they only modestly impact protein production. Our research suggests an alternative and novel mechanism of action of aminoglycosides in the inhibition of HepI, which directly leads to modification of LPS production in vivo. This finding could change our understanding of how aminoglycoside antibiotics function, with interruption of LPS biosynthesis being an additional and important mechanism of aminoglycoside action. Further research to discern the microbiological impact of aminoglycosides on cells is warranted, as inhibition of the ribosome may not be the sole and primary mechanism of action. The inhibition of HepI by aminoglycosides may dramatically alter strategies to modify the structure of aminoglycosides to improve the efficacy in fighting bacterial infections.
“…9 . As validated through previous works as well 25 , the challenge presented by the introduction of aminoglycosides to E. coli is bactericidal and not bacteriostatic therefore growth effects are non-recoverable. Figure 9 ( A ) and ( B ) Calculated growth rate in absorbance increase per hour as determined by the first derivative of a growth curve for streptomycin and tobramycin respectively.…”
Section: Resultsmentioning
confidence: 61%
“…The prior consensus in the literature stated that aminoglycosides act through the disruption of protein synthesis, thereby preventing the translation of membrane proteins and leading to changes in the cell surface morphology and damage to the cytoplasmic membrane 20 – 24 . Recently, researchers have demonstrated, using single-molecule spectroscopic methods, that aminoglycosides only slow protein synthesis and don’t inhibit it fully, therefore suggesting that the bactericidal activity of aminoglycosides is not related to ribosomal inhibition 25 . Herein, we propose an alternative mechanism of action of aminoglycosides through the potent inhibition of HepI and disruption of lipopolysaccharide biosynthesis.…”
A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years because of its critical role in the biosynthesis of lipopolysaccharides on bacterial cell surfaces. While many labs have discovered or designed novel small molecule inhibitors, these compounds lacked the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. Structural inspection of Kdo2-lipid A suggested aminoglycoside antibiotics as potential inhibitors for HepI. Multiple aminoglycosides have been experimentally validated to be first-in-class nanomolar inhibitors of HepI, with the best inhibitor demonstrating a Ki of 600 ± 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and molecular dynamics simulations were used to corroborate kinetic experimental findings. While aminoglycosides have long been described as potent antibiotics targeting bacterial ribosomes’ protein synthesis leading to disruption of the stability of bacterial cell membranes, more recently researchers have shown that they only modestly impact protein production. Our research suggests an alternative and novel mechanism of action of aminoglycosides in the inhibition of HepI, which directly leads to modification of LPS production in vivo. This finding could change our understanding of how aminoglycoside antibiotics function, with interruption of LPS biosynthesis being an additional and important mechanism of aminoglycoside action. Further research to discern the microbiological impact of aminoglycosides on cells is warranted, as inhibition of the ribosome may not be the sole and primary mechanism of action. The inhibition of HepI by aminoglycosides may dramatically alter strategies to modify the structure of aminoglycosides to improve the efficacy in fighting bacterial infections.
“…In summary, our results show that the translation landscape in cells is reshaped by small molecules specifically binding to ribosomes, as well as to other targets. The presence of minor elongation states under antibiotic treatment is reminiscent of recent works showing ongoing slow translation in antibiotic-treated cells and context-dependent inhibition 50-52 . For example, Cm inhibition is affected by specific residues of the nascent peptide 50,51 .…”
Section: Antibiotics Alter Translation Landscapes In Cellsmentioning
Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here, we use cryo-electron tomography and sub-tomogram analysis to visualize the dynamics of translation inside the prokaryote Mycoplasma pneumoniae. We first obtain an in-cell atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves thirteen ribosome states that differ in conformation and composition and reflect intermediates during translation. Based on these states, we animate translation elongation and demonstrate how antibiotics reshape the translation landscape inside cells. During translation elongation, ribosomes often arrange in a defined manner to form polysomes. By mapping the intracellular three-dimensional organization of translating ribosomes, we show that their association into polysomes exerts a local coordination mechanism that is mediated by the ribosomal protein L9. Our work demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.
“…Aminoglycoside antibiotics such as kanamycin (Kan) bind to 16S rRNA in the A site of the 30S ribosomal subunit, interfering with the selection of cognate tRNAs [1]. Aberrant proteins are produced as a result, including proteins truncated by premature termination or with sequences garbled by frameshift events or misincorporation of amino acids [2]. By contrast, protein synthesis is completely halted by non-aminoglycoside antibiotics like tetracycline (Tc) [3,4] and chloramphenicol (Cm) [5].…”
Aminoglycoside antibiotics interfere with selection of cognate tRNAs during translation, resulting in the production of aberrant proteins that are the ultimate cause of the antibiotic bactericidal effect. To determine if these aberrant proteins are recognized as substrates by the cell’s protein quality control machinery, we studied whether the heat shock (HS) response was activated following exposure of Escherichia coli to the aminoglycoside kanamycin (Kan). Levels of the HS transcription factor σ32 increased about 10-fold after exposure to Kan, indicating that at least some aberrant proteins were recognized as substrates by the molecular chaperone DnaK. To investigate whether toxic aberrant proteins therefore might escape detection by the QC machinery, we studied model aberrant proteins that had a bactericidal effect when expressed in E. coli from cloned genes. As occurred following exposure to Kan, levels of σ32 were permanently elevated following expression of an acutely toxic 48-residue protein (ARF48), indicating that toxic activity and recognition by the QC machinery are not mutually exclusive properties of aberrant proteins, and that the HS response was blocked in these cells at some step downstream of σ32 stabilization. This block could result from halting of protein synthesis or from radial condensation of nucleoids, both of which occurred rapidly following ARF48 induction. Nucleoids were similarly condensed following expression of toxic aberrant secretory proteins, suggesting that transertion of inner membrane proteins, a process that expands nucleoids into an open conformation that promotes growth and gene expression, was disrupted in these cells. The 48-residue ARF48 protein would be well-suited for structural studies to further investigate the toxic mechanism of aberrant proteins.
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