Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Inhibition of bacterial protein synthesis by elongation-factor-Tu-binding antibiotics MDL 62,879 and efrotomycin.

ABSTRACT: MDL 62,879 (formerly GE 2270 A) is a novel antibiotic active against Gram-positive bacteria by inhibiting protein synthesis. MDL 62,879 is not active against Gram-negative bacteria, but inhibits cell-free protein synthesis in extracts from Escherichia coli, and shows a high binding affinity for its elongation factor Tu (EF-Tu). We prepared ribosomes and protein-synthesis elongation factors from three sources: E. coli, Bacillus subtilis, and a strain of B. subtilis selected for resistance to MDL 62,879 (strain G1674). Homologous and heterologous reconstituted systems were used to compare the effects of MDL 62,879 and of efrotomycin, an EF-Tu inhibitor of the kirromycin class, which is inactive against both B. subtilis and E. coli. We showed that in cell-free protein synthesis: (a) E. coli was sensitive to both MDL 62,879 and efrotomycin; (b) B. subtilis was sensitive to MDL 62,879, but not to efrotomycin; (c) B. subtilis G1674 was resistant to both antibiotics. In the E. coli system and in the system from wild-type B. subtilis, inhibition by MDL 62,879 was reversed upon addition of purified EF-Tu from B. subtilis G1674. This demonstrates that the antibiotic acts by inhibition of EF-Tu. In contrast, extracts from B. subtilis failed to restore activity in an efrotomycin-inhibited E. coli system. Dominance or resistance to MDL 62,879 and of sensitivity to efrotomycin in heterologous cell-free protein synthesis confirms that inhibition of EF-Tu by the two antibiotics is mediated by different mechanisms of action.

SUBMITTER: Landini P

PROVIDER: S-EPMC1130934 | biostudies-other | 1992 May

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Engineering the elongation factor Tu for efficient selenoprotein synthesis.

Project description:Selenocysteine (Sec) is naturally co-translationally incorporated into proteins by recoding the UGA opal codon with a specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element). We have recently developed a SECIS-free selenoprotein synthesis system that site-specifically--using the UAG amber codon--inserts Sec depending on the elongation factor Tu (EF-Tu). Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis. A Sec-specific selection system was established by expression of human protein O(6)-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon. The formed hAGT selenoprotein repairs the DNA damage caused by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine, and thereby enables Escherichia coli to grow in the presence of this mutagen. An EF-Tu library was created in which codons specifying the amino acid binding pocket were randomized. Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library. The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.

| S-EPMC4150793 | biostudies-literature

Bacterial translation elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein.

Project description:We show that translation initiation factor EF-Tu plays a second important role in cell shape maintenance in the bacterium Bacillus subtilis. EF-Tu localizes in a helical pattern underneath the cell membrane and colocalizes with MreB, an actin-like cytoskeletal element setting up rod cell shape. The localization of MreB and of EF-Tu is interdependent, but in contrast to the dynamic MreB filaments, EF-Tu structures are more static and may serve as tracks for MreB filaments. In agreement with this idea, EF-Tu and MreB interact in vivo and in vitro. Lowering of the EF-Tu levels had a minor effect on translation but a strong effect on cell shape and on the localization of MreB, and blocking of the function of EF-Tu in translation did not interfere with the localization of MreB, showing that, directly or indirectly, EF-Tu affects the cytoskeletal MreB structure and thus serves two important functions in a bacterium.

| S-EPMC2840354 | biostudies-literature

Immunospecific responses to bacterial elongation factor Tu during Burkholderia infection and immunization.

Project description:Burkholderia pseudomallei is the etiological agent of melioidosis, a disease endemic in parts of Southeast Asia and Northern Australia. Currently there is no licensed vaccine against infection with this biological threat agent. In this study, we employed an immunoproteomic approach and identified bacterial Elongation factor-Tu (EF-Tu) as a potential vaccine antigen. EF-Tu is membrane-associated, secreted in outer membrane vesicles (OMVs), and immunogenic during Burkholderia infection in the murine model of melioidosis. Active immunization with EF-Tu induced antigen-specific antibody and cell-mediated immune responses in mice. Mucosal immunization with EF-Tu also reduced lung bacterial loads in mice challenged with aerosolized B. thailandensis. Our data support the utility of EF-Tu as a novel vaccine immunogen against bacterial infection.

| S-EPMC3003680 | biostudies-literature

Tetracycline does not directly inhibit the function of bacterial elongation factor Tu.

Project description:Understanding the molecular mechanism of antibiotics that are currently in use is important for the development of new antimicrobials. The tetracyclines, discovered in the 1940s, are a well-established class of antibiotics that still have a role in treating microbial infections in humans. It is generally accepted that the main target of their action is the ribosome. The estimated affinity for tetracycline binding to the ribosome is relatively low compared to the actual potency of the drug in vivo. Therefore, additional inhibitory effects of tetracycline on the translation machinery have been discussed. Structural evidence suggests that tetracycline inhibits the function of the essential bacterial GTPase Elongation Factor (EF)-Tu through interaction with the bound nucleotide. Based on this, tetracycline has been predicted to impede the nucleotide-binding properties of EF-Tu. However, detailed kinetic studies addressing the effect of tetracycline on nucleotide binding have been prevented by the fluorescence properties of the antibiotic. Here, we report a fluorescence-based kinetic assay that minimizes the effect of tetracycline autofluorescence, enabling the detailed kinetic analysis of the nucleotide-binding properties of Escherichia coli EF-Tu. Furthermore, using physiologically relevant conditions, we demonstrate that tetracycline does not affect EF-Tu's intrinsic or ribosome-stimulated GTPase activity, nor the stability of the EF-Tu•GTP•Phe-tRNAPhe complex. We therefore provide clear evidence that tetracycline does not directly impede the function of EF-Tu.

| S-EPMC5446176 | biostudies-literature

Elongation factor Tu is a multifunctional and processed moonlighting protein.

Project description:Many bacterial moonlighting proteins were originally described in medically, agriculturally, and commercially important members of the low G?+?C Firmicutes. We show Elongation factor Tu (Ef-Tu) moonlights on the surface of the human pathogens Staphylococcus aureus (SaEf-Tu) and Mycoplasma pneumoniae (MpnEf-Tu), and the porcine pathogen Mycoplasma hyopneumoniae (MhpEf-Tu). Ef-Tu is also a target of multiple processing events on the cell surface and these were characterised using an N-terminomics pipeline. Recombinant MpnEf-Tu bound strongly to a diverse range of host molecules, and when bound to plasminogen, was able to convert plasminogen to plasmin in the presence of plasminogen activators. Fragments of Ef-Tu retain binding capabilities to host proteins. Bioinformatics and structural modelling studies indicate that the accumulation of positively charged amino acids in short linear motifs (SLiMs), and protein processing promote multifunctional behaviour. Codon bias engendered by an A?+?T rich genome may influence how positively-charged residues accumulate in SLiMs.

| S-EPMC5593925 | biostudies-literature

Elfamycins: inhibitors of elongation factor-Tu.

Project description:Elfamycins are a relatively understudied group of antibiotics that target the essential process of translation through impairment of EF-Tu function. For the most part, the utility of these compounds has been as laboratory tools for the study of EF-Tu and the ribosome, as their poor pharmacokinetic profile and solubility has prevented implementation as therapeutic agents. However, due to the slowing of the antibiotic pipeline and the rapid emergence of resistance to approved antibiotics, this group is being reconsidered. Some researchers are using screens for novel naturally produced variants, while others are making directed, systematic chemical improvements on publically disclosed compounds. As an example of the latter approach, a GE2270 A derivative, LFF571, has completed phase 2 clinical trials, thus demonstrating the potential for elfamycins to become more prominent antibiotics in the future.

| S-EPMC5701666 | biostudies-literature

The Diverse Functional Roles of Elongation Factor Tu (EF-Tu) in Microbial Pathogenesis.

Project description:Elongation factor thermal unstable Tu (EF-Tu) is a G protein that catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome inside living cells. Structural and biochemical studies have described the complex interactions needed to effect canonical function. However, EF-Tu has evolved the capacity to execute diverse functions on the extracellular surface of both eukaryote and prokaryote cells. EF-Tu can traffic to, and is retained on, cell surfaces where can interact with membrane receptors and with extracellular matrix on the surface of plant and animal cells. Our structural studies indicate that short linear motifs (SLiMs) in surface exposed, non-conserved regions of the molecule may play a key role in the moonlighting functions ascribed to this ancient, highly abundant protein. Here we explore the diverse moonlighting functions relating to pathogenesis of EF-Tu in bacteria and examine putative SLiMs on surface-exposed regions of the molecule.

| S-EPMC6822514 | biostudies-literature

Epitope Mapping of Streptococcus agalactiae Elongation Factor Tu Protein Recognized by Human Sera.

Project description:The elongation factor Tu has been identified as one of the most immunoreactive proteins that was recognized by human sera of GBS (group B streptococcus) positive patients. In this paper, we present the polypeptide-specific epitopes of the bacterial protein that are recognized by human antibodies: 28LTAAITTVLARRLP41 (peptide no. 3) and 294GQVLAKPGSINPHTKF309 (peptide no. 21). To determine the shortest amino acid sequence recognized by antibodies, truncation peptide libraries were prepared using the PEPSCAN method. The analysis of immunoreactivity of peptides with sera of GBS positive and negative women revealed that the most immunoreactive sequence was 306HTKF309. Moreover, we observed that this sequence also showed the highest specificity which was based on ratio of reactivity with sera of GBS positive relative to sera of GBS negative patients. Epitope was synthetized on Wang resin with the Fmoc strategy. Our results open the possibility to use 306HTKF309 peptide in diagnostic assays to determine Streptococcus agalactiae infection in humans.

| S-EPMC5808131 | biostudies-literature

Dynamics of Recognition between tRNA and elongation factor Tu.

Project description:Elongation factor Tu (EF-Tu) binds to all standard aminoacyl transfer RNAs (aa-tRNAs) and transports them to the ribosome while protecting the ester linkage between the tRNA and its cognate amino acid. We use molecular dynamics simulations to investigate the dynamics of the EF-Tu.guanosine 5'-triphosphate.aa-tRNA(Cys) complex and the roles played by Mg2+ ions and modified nucleosides on the free energy of protein.RNA binding. Individual modified nucleosides have pronounced effects on the structural dynamics of tRNA and the EF-Tu.Cys-tRNA(Cys) interface. Combined energetic and evolutionary analyses identify the coevolution of residues in EF-Tu and aa-tRNAs at the binding interface. Highly conserved EF-Tu residues are responsible for both attracting aa-tRNAs as well as providing nearby nonbonded repulsive energies that help fine-tune molecular attraction at the binding interface. In addition to the 3' CCA end, highly conserved tRNA nucleotides G1, G52, G53, and U54 contribute significantly to EF-Tu binding energies. Modification of U54 to thymine affects the structure of the tRNA common loop resulting in a change in binding interface contacts. In addition, other nucleotides, conserved within certain tRNA specificities, may be responsible for tuning aa-tRNA binding to EF-Tu. The trend in EF-Tu.Cys-tRNA(Cys) binding energies observed as the result of mutating the tRNA agrees with experimental observation. We also predict variations in binding free energies upon misacylation of tRNA(Cys) with d-cysteine or O-phosphoserine and upon changing the protonation state of l-cysteine. Principal components analysis in each case reveals changes in the communication network across the protein.tRNA interface and is the basis for the entropy calculations.

| S-EPMC3232051 | biostudies-literature

Rewiring translation for elongation factor Tu-dependent selenocysteine incorporation.

Project description: Not available

| S-EPMC3776052 | biostudies-literature