Elongation Factor G Research Articles

Mycoplasma synoviae surface-located elongation factor G functions as a cytoadhesin to promote adhesion to synovial sheath cells through binding to vimentin

Mycoplasma synoviae infection has caused serious economic losses to the poultry industry worldwide. The molecular mechanism by which M. synoviae colonizes the synovium and induces synovitis is unclear. In this study, desthiobiotin pull-down and liquid chromatography‐tandem mass spectrometry analyses were used to screen M. synoviae membrane proteins that bind the membrane proteins of synovial sheath cells (SSCs). Among the 128 screened proteins, elongation factor G (EF-G) of M. synoviae was identified as a surface-located protein using colony blotting and dual fluorescence analyses. The immunogenicity of EF-G was confirmed by the preparation of a rabbit polyclonal antibody. EF-G was identified as a cytoadhesin that directly binds to SSCs using indirect immunofluorescence assay and ELISA plate binding assay. In addition, antibody adhesion inhibition and protein adhesion inhibition demonstrated that EF-G could significantly promote the adhesion of M. synoviae to SSCs. Co-IP, GST pull-down, bacterial two-hybridization, and ELISA plate binding assays were performed to demonstrate the binding of EF-G and vimentin in vivo and in vitro. Antibody adhesion inhibition, protein adhesion inhibition, and siRNA interference adhesion inhibition assays demonstrated that vimentin significantly affected M. synoviae adhesion to SSCs. These studies indicate that two interacting proteins, EF-G, a novel cytoadhesin, and vimentin, an important cell surface receptor, play important roles in the adhesion of M. synoviae to SSCs, laying a foundation for subsequent studies on the mechanism of M. synoviae-induced synovitis and providing meaningful targets for screening target drugs against M. synoviae infection.

Allosteric Effects of EF-G Domain I Mutations Inducing Ribosome Frameshifting Revealed by Multiplexed Force Spectroscopy.

Ribosome translocation catalyzed by elongation factor G (EF-G) is a critical step in protein synthesis where the ribosome typically moves along the mRNA by three nucleotides at each step. To investigate the mechanism of EF-G catalysis, it is essential to precisely resolve the ribosome motion at both ends of the mRNA, which, to our best knowledge, is only achieved with the magnetic-based force spectroscopy developed by our groups. Here, we introduce a novel multiplexed force spectroscopy technique that, for the first time, offers single-nucleotide resolution for multiple samples. This technique combines multiple acoustic force generators with the smallest atomic magnetometer designed for biological research. Utilizing this technique, we demonstrate that mutating EF-G at the GTP binding pocket results in the ribosome moving only two nucleotides on both ends of the mRNA, thereby compromising ribosome translocation. This finding suggests a direct link between GTP hydrolysis and ribosome translocation. Our results not only provide mechanistic insights into the role of GTP binding pocket but also illuminate how allosteric mutations can manipulate translocation. We anticipate broader applications of our technique in the ribosome field, leveraging its high efficiency and single-nucleotide resolution.

Structures of the Staphylococcus aureus ribosome inhibited by fusidic acid and fusidic acid cyclopentane

The antibiotic fusidic acid (FA) is used to treat Staphylococcus aureus infections. It inhibits protein synthesis by binding to elongation factor G (EF-G) and preventing its release from the ribosome after translocation. While FA, due to permeability issues, is only effective against gram-positive bacteria, the available structures of FA-inhibited complexes are from gram-negative model organisms. To fill this knowledge gap, we solved cryo-EM structures of the S. aureus ribosome in complex with mRNA, tRNA, EF-G and FA to 2.5 Å resolution and the corresponding complex structures with the recently developed FA derivative FA-cyclopentane (FA-CP) to 2.0 Å resolution. With both FA variants, the majority of the ribosomal particles are observed in chimeric state and only a minor population in post-translocational state. As expected, FA binds in a pocket between domains I, II and III of EF-G and the sarcin-ricin loop of 23S rRNA. FA-CP binds in an identical position, but its cyclopentane moiety provides additional contacts to EF-G and 23S rRNA, suggesting that its improved resistance profile towards mutations in EF-G is due to higher-affinity binding. These high-resolution structures reveal new details about the S. aureus ribosome, including confirmation of many rRNA modifications, and provide an optimal starting point for future structure-based drug discovery on an important clinical drug target.

Developing Multichannel smFRET Approach to Dissecting Ribosomal Mechanisms.

The ribosome, a 2.6 megadalton biomolecule measuring approximately 20 nm in diameter, coordinates numerous ligands, factors, and regulators to translate proteins with high fidelity and speed. Understanding its complex functions necessitates multiperspective observations. We developed a dual-FRET single-molecule Förste Resonance Energy Transfer method (dual-smFRET), allowing simultaneous observation and correlation of tRNA dynamics and Elongation Factor G (EF-G) conformations in the same complex, in a 10 s time window. By synchronizing laser shutters and motorized filter sets, two FRET signals are captured in consecutive 5 s intervals with a time gap of 50-100 ms. We observed distinct fluorescent emissions from single-, double-, and quadruple-labeled ribosome complexes. Through comprehensive spectrum analysis and correction, we distinguish and correlate conformational changes in two parts of the ribosome, offering additional perspectives on its coordination and timing during translocation. Our setup's versatility, accommodating up to six FRET pairs, suggests broader applications in studying large biomolecules and various biological systems.

Active role of the protein translation machinery in protecting against stress tolerance in Synechococcus elongatus PCC7942

In vivo protein synthesis is crucial for all domains of life. It is accomplished through translational machinery, and a key step is the translocation of tRNA-mRNA by elongation factor G (EF-G). Genome-based analysis revealed two EF-G encoding genes (S0885 and S2082) in the freshwater cyanobacterium model Synechococcus elongatus PCC7942. S0885 is the essential EF-G gene for photosynthesis. We generated a strain of S. elongatus PCC7942 that overexpressed S0885 (OX-S0885) to identify EF-G functionality. RT-PCR and Western blot analyses revealed increased transcriptional and translational levels in OX-S0885 at 10.5–13.5 and 2.0–3.0 fold, respectively. Overexpression of S0885 led to an increase in specific growth rate. Additionally, polysome-to-monosome ratio (P/M) and RNA-to-protein ratio (R/P) were elevated in OX-S0885 compared with the empty vector. Interestingly, R/P in OX-S0885 was retained at more than 70% under oxidative stress while R/P in the empty vector was severely depleted, suggesting the maintenance of translation. Thus, S0885 appeared to be the important target of oxidative stress because it was protected by the stress response system to maintain its function. These results suggest that cyanobacterial EF-G has a primary function in translation and an unrelated activity during stress conditions. These findings support the substantial role of EF-G in the formation and maintenance of cellular protein formation, and in the protection of the global translational mechanism under oxidative stress condition.

Comparing FRET Pairs that Report on Intersubunit Rotation in Bacterial Ribosomes

Mediated by elongation factor G (EF-G), ribosome translocation along mRNA is accompanied by rotational movement between ribosomal subunits. Here, we reassess whether the intersubunit rotation requires GTP hydrolysis by EF-G or can occur spontaneously. To that end, we employ two independent FRET assays, which are based on labeling either ribosomal proteins (bS6 and bL9) or rRNAs (h44 of 16S and H101 of 23S rRNA). Both FRET pairs reveal three FRET states, corresponding to the non-rotated, rotated and semi-rotated conformations of the ribosome. Both FRET assays show that in the absence of EF-G, pre-translocation ribosomes containing deacylated P-site tRNA undergo spontaneous intersubunit rotations between non-rotated and rotated conformations. While the two FRET pairs exhibit largely similar behavior, they substantially differ in the fraction of ribosomes showing spontaneous fluctuations. Nevertheless, instead of being an invariable intrinsic property of each FRET pair, the fraction of spontaneously fluctuating molecules changes in both FRET assays depending on experimental conditions. Our results underscore importance of using multiple FRET pairs in studies of ribosome dynamics and highlight the role of thermally-driven large-scale ribosome rearrangements in translation.

High-level in vitro resistance to gentamicin acquired in a stepwise manner in Neisseria gonorrhoeae.

Gentamicin is used in several alternative treatments for gonorrhoea. Verified clinical Neisseria gonorrhoeae isolates with gentamicin resistance are mainly lacking and understanding the mechanisms for gonococcal gentamicin resistance is imperative. We selected gentamicin resistance in gonococci in vitro, identified the novel gentamicin-resistance mutations, and examined the biofitness of a high-level gentamicin-resistant mutant. Low- and high-level gentamicin resistance was selected in WHO X (gentamicin MIC = 4 mg/L) on gentamicin-gradient agar plates. Selected mutants were whole-genome sequenced. Potential gentamicin-resistance fusA mutations were transformed into WT strains to verify their impact on gentamicin MICs. The biofitness of high-level gentamicin-resistant mutants was examined using a competitive assay in a hollow-fibre infection model. WHO X mutants with gentamicin MICs of up to 128 mg/L were selected. Primarily selected fusA mutations were further investigated, and fusAR635L and fusAM520I + R635L were particularly interesting. Different mutations in fusA and ubiM were found in low-level gentamicin-resistant mutants, while fusAM520I was associated with high-level gentamicin resistance. Protein structure predictions showed that fusAM520I is located in domain IV of the elongation factor-G (EF-G). The high-level gentamicin-resistant WHO X mutant was outcompeted by the gentamicin-susceptible WHO X parental strain, suggesting lower biofitness. We describe the first high-level gentamicin-resistant gonococcal isolate (MIC = 128 mg/L), which was selected in vitro through experimental evolution. The most substantial increases of the gentamicin MICs were caused by mutations in fusA (G1560A and G1904T encoding EF-G M520I and R635L, respectively) and ubiM (D186N). The high-level gentamicin-resistant N. gonorrhoeae mutant showed impaired biofitness.

The many faces of ribosome translocation along the mRNA: reading frame maintenance, ribosome frameshifting and translational bypassing.

In each round of translation elongation, the ribosome translocates along the mRNA by precisely one codon. Translocation is promoted by elongation factor G (EF-G) in bacteria (eEF2 in eukaryotes) and entails a number of precisely-timed large-scale structural rearrangements. As a rule, the movements of the ribosome, tRNAs, mRNA and EF-G are orchestrated to maintain the exact codon-wise step size. However, signals in the mRNA, as well as environmental cues, can change the timing and dynamics of the key rearrangements leading to recoding of the mRNA into production of trans-frame peptides from the same mRNA. In this review, we discuss recent advances on the mechanics of translocation and reading frame maintenance. Furthermore, we describe the mechanisms and biological relevance of non-canonical translocation pathways, such as hungry and programmed frameshifting and translational bypassing, and their link to disease and infection.

Interplay between Inter-Subunit Rotation of the Ribosome and Binding of Translational GTPases.

Translational G proteins, whose release from the ribosome is triggered by GTP hydrolysis, regulate protein synthesis. Concomitantly with binding and dissociation of protein factors, translation is accompanied by forward and reverse rotation between ribosomal subunits. Using single-molecule measurements, we explore the ways in which the binding of translational GTPases affects inter-subunit rotation of the ribosome. We demonstrate that the highly conserved translation factor LepA, whose function remains debated, shifts the equilibrium toward the non-rotated conformation of the ribosome. By contrast, the catalyst of ribosome translocation, elongation factor G (EF-G), favors the rotated conformation of the ribosome. Nevertheless, the presence of P-site peptidyl-tRNA and antibiotics, which stabilize the non-rotated conformation of the ribosome, only moderately reduces EF-G binding. These results support the model suggesting that EF-G interacts with both the non-rotated and rotated conformations of the ribosome during mRNA translocation. Our results provide new insights into the molecular mechanisms of LepA and EF-G action and underscore the role of ribosome structural dynamics in translation.

Mutagenesis of Escherichia coli for sm-FRET and FIRMS study of domain IV EF-G studies.

The translation of mRNA into protein is performed by a ribonucleoprotein complex which consists of two subunits which come together to form three tRNA binding sites; the A-, P-, and E-site. The complex is associated with several proteins, one of which is Elongation Factor G (EF-G) which advances the mRNA sequence through the ribosome in a process known as translocation. During translocation tRNAs move from the A- and P-sites to the P- and E-sites, respectively, and the ribosome slides the mRNA exactly three nucleotides. Two key residues (Q508 and H584) in the domain IV region of EF-G form interactions with nucleotides of the A-site tRNA during translocation. The primary goal of this project will be to reveal the function of EF-G mediated translocation via mutagenesis of these two residues. Single molecule fluorescent resonance energy transfer (sm-FRET) will be used to determine the confirmational change of mutant EF-G. Force production of these mutants will also be measured using force-induced remnant magnetization spectroscopy (FIRMS) to determine the overall effect the domain IV mutants have on power-stroke force. We expect that the EF-G mutants will only be able to complete partial translocation and will have a reduction in force exertion, while GTP hydrolysis will not compromised, which will imply an active role in the translocation mechanism by these residues. These findings together will visualize the ribosome translocation mechanism with sub-nucleotide resolution, which only the FIRMS method can do to our best knowledge.

Ligand and structure-based approaches for the exploration of structure-activity relationships of fusidic acid derivatives as antibacterial agents.

Introduction: Fusidic acid (FA) has been widely applied in the clinical prevention and treatment of bacterial infections. Nonetheless, its clinical application has been limited due to its narrow antimicrobial spectrum and some side effects. Purpose: Therefore, it is necessary to explore the structure-activity relationships of FA derivatives as antibacterial agents to develop novel ones possessing a broad antimicrobial spectrum. Methods and result: First, a pharmacophore model was established on the nineteen FA derivatives with remarkable antibacterial activities reported in previous studies. The common structural characteristics of the pharmacophore emerging from the FA derivatives were determined as those of six hydrophobic centers, two atom centers of the hydrogen bond acceptor, and a negative electron center around the C-21 field. Then, seven FA derivatives have been designed according to the reported structure-activity relationships and the pharmacophore characteristics. The designed FA derivatives were mapped on the pharmacophore model, and the Qfit values of all FA derivatives were over 50 and FA-8 possessed the highest value of 82.66. The molecular docking studies of the partial target compounds were conducted with the elongation factor G (EF-G) of S. aureus. Furthermore, the designed FA derivatives have been prepared and their antibacterial activities were evaluated by the inhibition zone test and the minimum inhibitory concentration (MIC) test. The derivative FA-7 with a chlorine group as the substituent group at C-25 of FA displayed the best antibacterial property with an MIC of 3.125µM. Subsequently, 3D-QSAR was carried on all the derivatives by using the CoMSIA mode of SYBYL-X 2.0. Conclusion: Hence, a computer-aided drug design model was developed for FA, which can be further used to optimize FA derivatives as highly potent antibacterial agents.

Staphylococcus aureus-Cure-Associated Antigens Elicit Type 3 Immune Memory T Cells.

Staphylococcus aureus is one of the most frequently major mastitis pathogens that cause clinical and subclinical mastitis worldwide. Current antimicrobial treatments are usually ineffective, and the commercially available vaccines lack proven effectiveness. The immunological response elicited by the recombinant S. aureus-cure-associated proteins phosphoglycerate kinase (PGK), enolase (ENO), and elongation factor-G (EF-G) in combination with the granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA vaccination was studied in this work. Here, twenty-three C57BL/6 mice were divided into four groups and vaccinated with: G1: none (control); G2: GM-CSF DNA plasmid DNA vaccine; G3: the combination of EF-G+ENO+PGK; and G4: the combinations of EF-G+ENO+PGK proteins plus GM-CSF plasmid DNA vaccine. After 44 days, spleen cells were collected for immunophenotyping and lymphocyte proliferation evaluation by flow cytometry upon S. aureus stimulus. Immunization with the three S. aureus recombinant proteins alone resulted in a higher percentage of IL-17A+ cells among CD8+ T central memory cells, as well as the highest intensity of IL-17A production by overall lymphocytes indicating that the contribution of the combined lymphocyte populations is crucial to sustaining a type 3 cell immunity environment. The immunization with three S. aureus-cure-associated recombinant proteins triggered type 3 immunity, which is a highly interesting path to pursue an effective bovine S. aureus mastitis vaccine.

Gut colonization by Bacteroides requires translation by an EF-G paralog lacking GTPase activity.

Protein synthesis is crucial for cell growth and survival yet one of the most energy-consuming cellular processes. How, then, do cells sustain protein synthesis under starvation conditions when energy is limited? To accelerate the translocation of mRNA-tRNAs through the ribosome, bacterial elongation factor G (EF-G) hydrolyzes energy-rich guanosine triphosphate (GTP) for every amino acid incorporated into a protein. Here, we identify an EF-G paralog-EF-G2-that supports translocation without hydrolyzing GTP in the gut commensal bacterium Bacteroides thetaiotaomicron. EF-G2's singular ability to sustain protein synthesis, albeit at slow rates, is crucial for bacterial gut colonization. EF-G2 is ~10-fold more abundant than canonical EF-G1 in bacteria harvested from murine ceca and, unlike EF-G1, specifically accumulates during carbon starvation. Moreover, we uncover a 26-residue region unique to EF-G2 that is essential for protein synthesis, EF-G2 dissociation from the ribosome, and responsible for the absence of GTPase activity. Our findings reveal how cells curb energy consumption while maintaining protein synthesis to advance fitness in nutrient-fluctuating environments.

Screening and Molecular Docking of Bioactive Metabolites of the Red Sea Sponge Callyspongia siphonella as Potential Antimicrobial Agents.

Marine sponges create a wide range of bioactive secondary metabolites, as documented throughout the year. Several bioactive secondary metabolites were isolated from different members of Callyspongia siphonella species. This study aimed for isolation and structural elucidation of major metabolites in order to investigate their diverse bioactivities such as antimicrobial and anti-biofilm activities. Afterwards, a molecular docking study was conducted, searching for the possible mechanistic pathway of the most bioactive metabolites. Extraction, fractionation, and metabolomics analysis of different fractions was performed in order to obtain complete chemical profile. Moreover, in vitro assessment of different bioactivities was performed, using recent techniques. Additionally, purification, structural elucidation of high features using recent chromatographic and spectroscopic techniques was established. Finally, AutoDock Vina software was used for the Pharmacophore-based docking-based analysis. As a result, DCM (dichloromethane) fraction exerted the best antibacterial activity using disc diffusion method; particularly against S. aureus with an inhibition zone of 6.6 mm. Compound 11 displayed a considerable activity against both MRSA (Methicillin-resistant Staphyllococcus aureus) and Staphyllococcus aureus with inhibition ratios of 50.37 and 60.90%, respectively. Concerning anti-biofilm activity, compounds 1 and 2 displayed powerful activity with inhibition ratios ranging from 39.37% to 70.98%. Pharmacophore-based docking-based analysis suggested elongation factor G (EF-G) to be a probable target for compound 11 (siphonellinol C) that showed the best in vitro antibacterial activity, offering unexplored potential for new drugs and treatment candidates.

The role of GTP hydrolysis by EF-G in ribosomal translocation

Translocation of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome is catalyzed by the GTPase elongation factor G (EF-G) in bacteria. Although guanosine-5'-triphosphate (GTP) hydrolysis accelerates translocation and is required for dissociation of EF-G, its fundamental role remains unclear. Here, we used ensemble Förster resonance energy transfer (FRET) to monitor how inhibition of GTP hydrolysis impacts the structural dynamics of the ribosome. We used FRET pairs S12-S19 and S11-S13, which unambiguously report on rotation of the 30S head domain, and the S6-L9 pair, which measures intersubunit rotation. Our results show that, in addition to slowing reverse intersubunit rotation, as shown previously, blocking GTP hydrolysis slows forward head rotation. Surprisingly, blocking GTP hydrolysis completely abolishes reverse head rotation. We find that the S13-L33 FRET pair, which has been used in previous studies to monitor head rotation, appears to report almost exclusively on intersubunit rotation. Furthermore, we find that the signal from quenching of 3'-terminal pyrene-labeled mRNA, which is used extensively to follow mRNA translocation, correlates most closely with reverse intersubunit rotation. To account for our finding that blocking GTP hydrolysis abolishes a rotational event that occurs after the movements of mRNA and tRNAs are essentially complete, we propose that the primary role of GTP hydrolysis is to create an irreversible step in a mechanism that prevents release of EF-G until both the tRNAs and mRNA have moved by one full codon, ensuring productive translocation and maintenance of the translational reading frame.

The cyclic octapeptide antibiotic argyrin B inhibits translation by trapping EF-G on the ribosome during translocation

Argyrins are a family of naturally produced octapeptides that display promising antimicrobial activity against Pseudomonas aeruginosa. Argyrin B (ArgB) has been shown to interact with an elongated form of the translation elongation factor G (EF-G), leading to the suggestion that argyrins inhibit protein synthesis by interfering with EF-G binding to the ribosome. Here, using a combination of cryo-electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET), we demonstrate that rather than interfering with ribosome binding, ArgB rapidly and specifically binds EF-G on the ribosome to inhibit intermediate steps of the translocation mechanism. Our data support that ArgB inhibits conformational changes within EF-G after GTP hydrolysis required for translocation and factor dissociation, analogous to the mechanism of fusidic acid, a chemically distinct antibiotic that binds a different region of EF-G. These findings shed light on the mechanism of action of the argyrin-class antibiotics on protein synthesis as well as the nature and importance of rate-limiting, intramolecular conformational events within the EF-G-bound ribosome during late-steps of translocation.

AP Profiling: Resolving Co‐Translational Protein Folding Pathways and Chaperone Interactions In Vivo

Co‐translational folding and molecular chaperone action are crucial for productive protein folding in the cell, but how these processes shape folding pathways remains largely unknown. We have utilized translation arrest peptides (APs) to monitor co‐translational folding in live bacterial cells, combined with single‐molecule optical tweezers experiments, to define the co‐translational folding pathway of the GTPase domain (G‐domain) from E. coli elongation factor G (EF‐G). Surprisingly, the 293 amino acid long domain remains unfolded, without forming stable intermediate structures, until it is fully extruded from the ribosome. The full‐length G‐domain transitions to its stable native structure via obligate folding intermediates both in isolation and while bound to the ribosome. Folding therefore follows a strictly sequential pathway that initiates at the very C‐terminus, which is likely imposed by the structure and topology of the G‐domain from EF‐G. Consequently, folding and synthesis proceed in opposite directions. G‐domains represent a common element in a number of multi‐domain proteins. To determine whether their folding pathways are conserved, we have combined our AP approach with cell sorting and deep sequencing into a method that we term “AP profiling”. Homologous G‐domains exhibited distinct folding patterns that are conserved across distant bacterial species. Individual deletion of the primary nascent chain‐binding chaperones, trigger factor and DnaK (Hsp70), resulted in numerous localized changes to co‐translational folding while preserving overall G‐domain folding, highlighting the functional redundancy of cellular chaperone systems. In summary, we have developed AP profiling as a technique for monitoring co‐translational folding in the native cellular environment with high throughput. Combined with single‐molecule force spectroscopy, AP profiling yields a unique view of co‐translational folding and chaperone function.

Construction of EF-G knockdown strain of Mycobacterium smegmatis and drug resistance analysis

As the only translational factor that plays a critical role in two translational processes (elongation and ribosome regeneration), GTPase elongation factor G (EF-G) is a potential target for antimicrobial agents. Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes, MsmEFG1 (MSMEG_1400) and MsmEFG2 (MSMEG_6535), fusA1 (Rv0684) and fusA2 (Rv0120c), respectively. MsmEFG1 (MSMEG_1400) and fusA1 (Rv0684) were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis. To investigate the biological function and characteristics of EF-G in mycobacterium, two induced EF-G knockdown strains (Msm-ΔEFG1(KD) and Msm-ΔEFG2(KD)) from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference (CRISPRi) technique. EF-G2 knockdown had no effect on bacterial growth, while EF-G1 knockdown significantly retarded the growth of mycobacterium, weakened the film-forming ability, changed the colony morphology, and increased the length of mycobacterium. It was speculated that EF-G might be involved in the division of bacteria. Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin, isoniazid, erythromycin, fucidic acid, capreomycin and other antibacterial agents, suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.

Extraintestinal Pathogenic Escherichia coli Utilizes Surface-Located Elongation Factor G to Acquire Iron from Holo-Transferrin.

ABSTRACTExtraintestinal pathogenic Escherichia coli (ExPEC) can cause systemic infections in both humans and animals. As an essential nutrient, iron is strictly sequestered by the host. Circumventing iron sequestration is a determinant factor for ExPEC infection. However, the ExPEC iron acquisition mechanism, particularly the mechanism of transferrin (TF) acquisition, remains unclear. This study reports that iron-saturated holo-TF can be utilized by ExPEC to promote its growth in culture medium and survival in macrophages. ExPEC specifically bound to holo-TF instead of iron-free apo-TF via the surface located elongation factor G (EFG) in both culture medium and macrophages. As a moonlighting protein, EFG specifically bound holo-TF and also released iron in TF. These two functions were performed by different domains of EFG, in which the N-terminal domains were responsible for holo-TF binding and the C-terminal domains were responsible for iron release. The functions of EFG and its domains have also been further confirmed by surface-display vectors. The surface overexpression of EFG bound significantly more holo-TF in macrophages and significantly improved bacterial intracellular survival ability. Our findings reveal a novel iron acquisition mechanism involving EFG, which suggests novel research avenues into the molecular mechanism of ExPEC resistance to nutritional immunity.IMPORTANCE Extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen causing systemic infections in humans and animals. The competition for iron between ExPEC and the host is a determinant for ExPEC to establish a successful infection. Here, we sought to elucidate the role of transferrin (TF) in the interaction between ExPEC and the host. Our results revealed that holo-TF could be utilized by ExPEC to enhance its growth in culture medium and survival in macrophages. Furthermore, the role of elongation factor G (EFG), a novel holo-TF-binding and TF-iron release protein, was confirmed in this study. Our work provides insights into the iron acquisition mechanism of ExPEC, deepens understanding of the interaction between holo-TF and pathogens, and broadens further researches into the molecular mechanism of ExPEC pathogenicity.

Measuring the mechanical forces of elongation factor G mediated translocation

The ribosome is the complex molecular machine found in all living cells that is responsible for the synthesis of all proteins. The ribosome is associated with various protein factors, including the GTPase Elongation Factor G (EF-G). EF-G is responsible for catalyzing tRNA and mRNA translocation on the ribosome, however, the mechanism of this translocation remains elusive. Recent studies implied large conformational changes of EF-G during translocation, through the observation of a compact, pre-translocation state.