In cyanobacteria, Elongation factor Tu (EF-Tu) plays a crucial role in the repair of photosystem II (PSII), which is highly susceptible to oxidative stress induced by light exposure and regulated by reactive oxygen species (ROS). However, the specific molecular mechanism governing the functional regulation of EF-Tu by ROS remains unclear. Previous research has shown that a mutated EF-Tu, where C82 is substituted with a Ser residue, can alleviate photoinhibition, highlighting the important role of C82 in EF-Tu photosensitivity. In this study, we elucidated how ROS deactivate EF-Tu by examining the crystal structures of EF-Tu in both wild-type and mutated form (C82S) individually at resolutions of 1.7 Å and 2.0 Å in Synechococcus elongatus PCC 7942 complexed with GDP. Specifically, the GDP-bound form of EF-Tu adopts an open conformation with C82 located internally, making it resistant to oxidation. Coordinated conformational changes in switches I and II create a tunnel that positions C82 for ROS interaction, revealing the vulnerability of the closed conformation of EF-Tu to oxidation. An analysis of these two structures reveals that the precise spatial arrangement of C82 plays a crucial role in modulating EF-Tu\'s response to ROS, serving as a regulatory element that governs photosynthetic biosynthesis.

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

Mycoplasma synoviae (MS) is a primary avian pathogen prevalent worldwide that causes airsacculitis and synovitis in birds. Vaccination is recommended as the most cost-effective strategy in the control of MS infection. Novel alternative vaccines are needed for eradicating and controlling MS infection in flocks. DnaK, enolase, elongation factor Tu (EF-Tu), MSPB, NADH oxidase and LP78 are the major immunogenic antigens of MS and are promising targets for subunit vaccine candidates. In the present study, genes encoding DnaK, enolase, EF-Tu, MSPB, LP78, and NADH oxidase were cloned and expressed in Escherichia coli. Enzyme-linked immunosorbent assay showed that the six recombinant proteins were recognized by convalescent sera, indicating that they were expressed during infection. Two injections of the six subunit vaccines induced a robust antibody response and increased the concentrations of IFN-γ and IL-4, especially rEnolase and rEF-Tu. The proliferation of peripheral blood lymphocytes was enhanced in all of the immunized groups. Chickens immunized with rEnolase, rEF-Tu, rLP78, and rMSPB conferred significant protection against MS infection, as indicated by significantly lower DNA copies in the trachea, lower scores of air sac lesions, and lesser tracheal mucosal thickness than that in the challenge control. Especially, rEnolase provided the best protective efficacy, followed by rEF-Tu, rMSPB, and rLP78. Our finds demonstrate that the subunit vaccines and bacterin can only reduce the lesions caused by MS infection, but not prevent colonization of the organism. Our findings may contribute to the development of novel vaccine agents against MS infection.

A soybean elongation factor Tu family (EF-Tu) protein, GmEF8, was determined to interact with GmCBL1, and GmEF8 expression was found to be induced by various abiotic stresses such as drought and heat. An ortholog of GmEF8 was identified in Arabidopsis, a T-DNA knockout line for which exhibited hypersensitivity to drought and heat stresses. Complementation with GmEF8 rescued the sensitivity of the Arabidopsis mutant to drought and heat stresses, and GmEF8 overexpression conferred drought and heat tolerance to transgenic Arabidopsis plants. In soybean, plants with GmEF8-overexpressing hairy roots (OE-GmEF8) exhibited enhanced drought and heat tolerance and had higher proline levels compared to plants with RNAi GmEF8-knockdown hairy roots (MR-GmEF8) and control hairy roots (EV). A number of drought-responsive genes, such as GmRD22 and GmP5CS, were induced in the OE-GmEF8 line compared to MR-GmEF8 and EV under normal growth conditions. These results suggest that GmEF8 has a positive role in regulating drought and heat stresses in Arabidopsis and soybean. This study reveals a potential role of the soybean GmEF8 gene in response to abiotic stresses, providing a foundation for further investigation into the complexities of stress signal transduction pathways.

Elongation factor thermo-unstable (EF-Tu), an abundant multifunctional protein, is pivotal during protein synthesis and is an important antigen. Few studies have addressed the role of this protein in Brucella species, and the epitopes of this protein have not been reported. Here, we describe a monoclonal antibody (McAb), BD6, for EF-Tu in Brucella melitensis. Using western blotting involving a series of partially overlapping recombinant EF-Tu truncation peptides, a novel linear B-cell epitope, 110QTREHIL116 (EF), was identified. Alanine-scanning mutagenesis revealed that residues Q110, T111, R112, I115, and L116 were core residues involved in recognition. Sequence alignment suggested that the epitope peptide was conserved among bacterial species but differed by one amino acid residue (I115) from the host sequence. The epitope peptide was recognized by sera from B. melitensis-infected mice, and while recombinant epitope peptide induced a strong humoral immune response, the corresponding mouse peptide, QTREHLL, did not. These results suggested that I115 may be the key residue for the host immune system to distinguish between bacterial and self epitope EF sequences. Indirect immunofluorescence and western blotting assays showed that epitope peptide could be used in Saccharomyces cerevisiae, human embryonic kidney cell (HEK-293), and chicken fibroblast cell (DF1) expression systems and immunoprecipitation assay. Together, our results suggested that the McAb BD6 is a useful tool for further investigation of the potential functions of the EF-Tu protein in pathogen-host interactions, and that the epitope tag may be useful for application as a novel affinity tag to identify other bacterial pathogens, especially convenient for the identification of intracellular bacteria.

The unique dependence of cancer cells on mitochondrial metabolism has been exploited therapeutically in various cancers but not osteosarcoma. In this work, we demonstrate that inhibition of mitochondrial translation is effective and selective in targeting osteosarcoma. We firstly showed that tigecycline at pharmacological achievable concentrations inhibited growth and induced apoptosis of multiple osteosarcoma cell lines while sparing normal osteoblast cells. Similarly, tigecycline at effective doses that delayed osteosarcoma growth did not cause significant toxicity to mice. We next showed that tigecycline specifically inhibits mitochondrial translation, resulting in defective mitochondrial respiration in both osteosarcoma and normal osteoblast cells. We further confirm mitochondrial respiration as the target of tigecycline using three independent approaches. In addition, we demonstrate that compared to normal osteoblasts, osteosarcoma cells have higher mitochondrial biogenesis. We finally show that specific inhibition of mitochondrial translation via EF-Tu depletion produces the similar anti-osteosarcoma effects of tigecycline. Our work highlights the therapeutic value of targeting mitochondrial metabolism in osteosarcoma and tigecycline as a useful addition to the treatment of osteosarcoma.

Chloroplasts are semiautonomous organelles, retaining their own genomes and gene expression apparatuses but controlled by nucleus genome encoded protein factors during evolution. To analyze the genetic regulatory network of FtsH-mediated chloroplast development in Arabidopsis, a set of suppressor mutants of yellow variegated (var2) have been identified. In this research, we reported the identification of another new var2 suppressor locus, SUPPRESSOR OF VARIEGATION11 (SVR11), which encodes a putative chloroplast-localized prokaryotic type translation elongation factor EF-Tu. SVR11 is likely essential to chloroplast development and plant survival. GUS activity reveals that SVR11 is abundant in the juvenile leaf tissue, lateral roots, and root tips. Interestingly, we found that SVR11 and SVR9 together regulate leaf development, including leaf margin development and cotyledon venation patterns. These findings reinforce the notion that chloroplast translation state triggers retrograde signals regulate not only chloroplast development but also leaf development.

Mycoplasma hyopneumoniae is a colonizing respiratory pathogen that can cause great economic losses to the pig industry worldwide. Although putative virulence factors have been reported, the pathogenesis of this species remains unclear. Here, we used the virulent M. hyopneumoniae strain 168 to infect swine tracheal epithelial cells (STEC) to identify the infection-associated factors by two-dimensional electrophoresis (2-DE). Whole proteins of M. hyopneumoniae were obtained and compared with samples cultured in broth. Six differentially expressed proteins with an increase in abundance of ≥1.5 in the cell infection group were successfully identified. A String network of virulence-associated proteins showed that all the six differential abundance proteins were involved in virulence of M. hyopneumoniae. One of the most important upregulated hubs in this network, elongation factor thermo unstable (EF-Tu), which showed a relatively higher expression in M. hyopneumoniae-infected STEC and obtained a higher score on mass spectrometry was successfully recombined. In addition to its canonical enzymatic activities in protein synthesis, EF-Tu was also reported to be located on the cell surface as an important adhesin in many other pathogens. The cell surface location of EF-Tu was then observed in M. hyopneumoniae with flow cytometry. Recombinant EF-Tu (rEF-Tu) was found to be able to adhere to STEC and anti-rEF-Tu antibody enclosed M. hyopneumoniae decreased adherence to STEC. In addition, surface plasmon resonance (SPR) analysis showed that rEF-Tu could bind to fibronectin with a specific and moderately strong interaction, a dissociation constant (KD) of 605 nM. Furthermore, the block of fibronectin in STEC also decreased the binding of M. hyopneumoniae to the cell surface. Collectively, these data imply EF-Tu as an important adhesin of M. hyopneumoniae and fibronectin as an indispensable receptor on STEC. The binding between EF-Tu with fibronectin contributes to the adhesion of M. hyopneumoniae to STEC. HIGHLIGHTS Elongation factor thermo unstable (EF-Tu) exists on the cell surface of M. hyopneumoniae.EF-Tu moonlights as an adhesin of M. hyopneumoniae.The adhesive effect of EF-Tu is partly meditated by fibronectin.

Vaccine development efforts against Streptococcus suis serotype 2 (S. suis 2) are often constrained by strain/serotype antigen variability. Bioinformatics analyses revealed two highly conserved S. suis 2 factors, EF-Tu and FtsZ. Murine immunization with recombinant proteins emulsified in white oil adjuvant or eukaryotic DNA vaccine vectors provided significant protection against lethal S. suis 2 challenge. Immune responses elicited by recombinant protein immunization revealed the robust generation of humoral immune responses, with a mixed induction of Th1-type and Th2-type responses. Furthermore, the antiserum from mice immunized with recombinant proteins significantly inhibited the growth of S. suis 2 in healthy pig whole blood, suggesting the triggering of a strong opsonizing response. Histological examination found that immunizing mice with purified recombinant proteins reduced neutrophil and macrophage accumulation in brain and lung tissues after challenge with virulent S. suis. Taken together, these findings reveal that EF-Tu and FtsZ may be promising targets for subunit and DNA vaccine candidates against S. suis 2 infection.

The functional importance of mitochondrial protein translation has been recently documented in the context of various cancers but not renal cell carcinoma (RCC). In lines with these efforts, our work demonstrates that mitochondrial translation inhibition by tigecycline or depletion of EF-Tu mitochondrial translation factor effectively targets RCC and significantly sensitizes RCC response to chemotherapy. We show that antibiotic tigecycline inhibits multiple biological functions of RCC, including growth, colony formation and survival. It also significantly enhances in vitro and in vivo efficacy of paclitaxel in RCC. Tigecycline preferentially inhibits translation of mitochondrial DNA-encoded proteins, activities of mitochondrial respiratory complexes that contain mitochondrially encoded subunits. As a consequence of mitochondrial respiratory chain inhibition, decreased mitochondrial respiration is observed in RCC cells exposed to tigecycline. In contrast, tigecycline is ineffective in RCC ρ0 cells that lack mitochondrial DNA and subsequent mitochondrial respiration, further confirm mitochondrial translation inhibition as the mechanism of tigecycline\'s action in RCC. Importantly, genetic inhibition of mitochondrial translation by EF-Tu knockdown reproduced the inhibitory effects of tigecycline. Finally, we show the association between mitochondrial translation inhibition and suppression of PI3K/Akt/mTOR signaling pathway. Our work used pharmacological and genetic strategies to demonstrate the important roles of mitochondrial translation in RCC and emphasize the therapeutic value of sensitizing RCC to chemotherapy.