Elongation Factor Thermo Unstable Research Articles

Exploring a new phyto-derived nanoparticle for targeting bacterial protein EF-Tu: an integrated approach to develop antimicrobial drug

ABSTRACT The application of antibiotic medicine to save numerous lives is in jeopardy due to the alarming elevation in antibiotic-resistant organisms as a consequence of improper and imprudent usage of antibiotics. This study investigated the antibacterial efficacy of pelargonidin (PG) by employing a cost-effective nano-formulation-based targeted drug delivery system as an alternative to conventional strategies. The precise targeting of bacteria was made easier by synthesizing nano-particles of phyto-product pelargonidin (NPG) by employing poly-lactide-co-glycolide (PLGA), a biodegradable polymer. The physico-chemical characterization of NPGs was performed via AFM study and the antibacterial efficacy of NPGs tested against Escherichia coli and Staphylococcus aureus was assessed by agar disc diffusion method. Additionally, the docking platform explored in this study enabled the prediction of the ability of PG to occupy the binding pocket of EF-Tu (elongation factor thermo unstable), a vital bacterial protein that operates on a translational level. The overall results implicate that NPG was an effective drug candidate against the gram-negative bacteria E. coli and the gram-positive bacteria S. aureus according to the diameter of the inhibition zone (23 ± 0.82 mm: E. coli and 22.67 ± 1.25 mm: S. aureus). Furthermore, in silico analysis revealed that the NPG-EF-Tu complex had an excellent docking score of −9.13 Kcal/mol, indicating key amino acid residues required for interaction, and anticipating the signaling component/s involved that may be hindered by NPG, thereby leading to a decline of bacterial growth. The core phyto-compound of NPG (pelargonidin) could effectively bind to EF-Tu thereby exercising strong antimicrobial efficacy against both E. coli and S. aureus making it a potential alternative to the usage of commercial antibacterials with a history of drug-resistance.

Structures of the ribosome bound to EF-Tu-isoleucine tRNA elucidate the mechanism of AUG avoidance.

The frequency of errors upon decoding of messenger RNA by the bacterial ribosome is low, with one misreading event per 1 × 104 codons. In the universal genetic code, the AUN codon box specifies two amino acids, isoleucine and methionine. In bacteria and archaea, decoding specificity of the AUA and AUG codons relies on the wobble avoidance strategy that requires modification of C34 in the anticodon loop of isoleucine transfer RNAIleCAU (tRNAIleCAU). Bacterial tRNAIleCAU with 2-lysylcytidine (lysidine) at the wobble position deciphers AUA while avoiding AUG. Here we report cryo-electron microscopy structures of the Escherichia coli 70S ribosome complexed with elongation factor thermo unstable (EF-Tu) and isoleucine-tRNAIleLAU in the process of decoding AUA and AUG. Lysidine in tRNAIleLAU excludes AUG by promoting the formation of an unusual Hoogsteen purine-pyrimidine nucleobase geometry at the third position of the codon, weakening the interactions with the mRNA and destabilizing the EF-Tu ternary complex. Our findings elucidate the molecular mechanism by which tRNAIleLAU specifically decodes AUA over AUG.

Antibiotic that inhibits trans-translation blocks binding of EF-Tu to tmRNA but not to tRNA.

Elongation factor thermo-unstable (EF-Tu) is a universally conserved translation factor that mediates productive interactions between tRNAs and the ribosome. In bacteria, EF-Tu also delivers transfer-messenger RNA (tmRNA)-SmpB to the ribosome during trans-translation. We report the first small molecule, KKL-55, that specifically inhibits EF-Tu activity in trans-translation without affecting its activity in normal translation. KKL-55 has broad-spectrum antibiotic activity, suggesting that compounds targeted to the tmRNA-binding interface of EF-Tu could be developed into new antibiotics to treat drug-resistant infections.

Testosterone and estradiol modify the expression of adhesins and biofilm formation in Actinobacillus seminis.

Actinobacillus seminis is the causal agent of epididymitis and has other effects on the reproductive tracts of small ruminants and bovines. This bacterium causes infection when luteinizing (LH) or follicle-stimulating hormones increase, and hosts reach sexual maturity. LH induces female ovulation and male testosterone production, suggesting that these hormones affect A. seminis pathogenicity. In the present study, we evaluated the effect of testosterone (1-5ng/ml) or estradiol (5-25 pg/ml) added to culture medium on the in vitro growth, biofilm production, and adhesin expression of A. seminis. Estradiol does not promote the growth of this bacterium, whereas testosterone increased A. seminis planktonic growth 2-fold. Both hormones induced the expression of the elongation factor thermo unstable (EF-Tu) and phosphoglycerate mutase (PGM), proteins that A. seminis uses as adhesins. Estradiol (5 or 10 pg/ml) decreased biofilm formation by 32%, whereas testosterone, even at 5ng/ml, showed no effect. Both hormones modified the concentrations of carbohydrates and eDNA in biofilms by 50%. Amyloid proteins are characterized by their capacity to bind Congo red (CR) dye. Actinobacillus seminis binds CR dye, and this binding increases in the presence of 5-20 pg/ml estradiol or 4ng/ml testosterone. The A. seminis EF-Tu protein was identified as amyloid-like protein (ALP). The effect of sexual hormones on the growth and expression of virulence factors of A. seminis seems to be relevant for its colonization and permanence in the host.

Ribosomal incorporation of negatively charged d-α- and N-methyl-l-α-amino acids enhanced by EF-Sep.

Ribosomal incorporation of d-α-amino acids (dAA) and N-methyl-l-α-amino acids (MeAA) with negatively charged sidechains, such as d-Asp, d-Glu, MeAsp and MeGlu, into nascent peptides is far more inefficient compared to those with neutral or positively charged ones. This is because of low binding affinity of their aminoacyl-transfer RNA (tRNA) to elongation factor-thermo unstable (EF-Tu), a translation factor responsible for accommodation of aminoacyl-tRNA onto ribosome. It is well known that EF-Tu binds to two parts of aminoacyl-tRNA, the amino acid moiety and the T-stem; however, the amino acid binding pocket of EF-Tu bearing Glu and Asp causes electric repulsion against the negatively charged amino acid charged on tRNA. To circumvent this issue, here we adopted two strategies: (i) use of an EF-Tu variant, called EF-Sep, in which the Glu216 and Asp217 residues in EF-Tu are substituted with Asn216 and Gly217, respectively; and (ii) reinforcement of the T-stem affinity using an artificially developed chimeric tRNA, tRNAPro1E2, whose T-stem is derived from Escherichia coli tRNAGlu that has high affinity to EF-Tu. Consequently, we could successfully enhance the incorporation efficiencies of d-Asp, d-Glu, MeAsp and MeGlu and demonstrated for the first time, to our knowledge, ribosomal synthesis of macrocyclic peptides containing multiple d-Asp or MeAsp. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Development of an indirect competitive enzyme linked immunosorbent assay for the quantitative detection of Mycoplasma hyopneumoniae during the vaccine production process

Inactivated Mycoplasma hyopneumoniae vaccine is used extensively to control M. hyopneumoniae infection worldwide. Quantification techniques are essential in the process of standardizing and validating vaccines. In this study, we developed and optimized an indirect competitive enzyme linked immunosorbent assay (ic-ELISA) for the rapid quantification of M. hyopneumoniae antigen during vaccine production. Briefly, whole M. hyopneumoniae antigen was coated onto microtiter plates, and a polyclonal antibody against M. hyopneumoniae recombinant elongation factor thermo unstable (EF-Tu) protein was prepared and added with the samples to be tested. The methods were optimized and showed significant reproducibility, with coefficients of variation of 4.01% and 6.14% for the intra-and inter-assays, respectively. Quantification of M. hyopneumoniae cultures at different growth stages using the ic-ELISA test showed a similar curve to that of the traditional color changing units (CCU) assay, with a delay in the time when the amount reached the peak and started to fall. In the inactivated vaccine production process, the cultures could be harvested later than that for the live vaccine, at about 12 h after the end of the logarithmic growth phase. Different batches of cultures were measured for their relative potency value compared with the in-house reference vaccine, which was used to determine whether the cultures met the antigen amount requirements for vaccine preparation. The curves of the CCU titer and ic-ELISA titer in the logarithmic phase correlated strongly and a linear regression equation was established to calculate the CCU values rapidly using the ic-ELISA results. In conclusion, an ic-ELISA method was established to rapidly assess the amount of antigen in an M. hyopneumoniae culture during the vaccine production process.

Uniform affinity-tuning of N-methyl-aminoacyl-tRNAs to EF-Tu enhances their multiple incorporation.

In ribosomal translation, the accommodation of aminoacyl-tRNAs into the ribosome is mediated by elongation factor thermo unstable (EF-Tu). The structures of proteinogenic aminoacyl-tRNAs (pAA-tRNAs) are fine-tuned to have uniform binding affinities to EF-Tu in order that all proteinogenic amino acids can be incorporated into the nascent peptide chain with similar efficiencies. Although genetic code reprogramming has enabled the incorporation of non-proteinogenic amino acids (npAAs) into the nascent peptide chain, the incorporation of some npAAs, such as N-methyl-amino acids (MeAAs), is less efficient, especially when MeAAs frequently and/or consecutively appear in a peptide sequence. Such poor incorporation efficiencies can be attributed to inadequate affinities of MeAA-tRNAs to EF-Tu. Taking advantage of flexizymes, here we have experimentally verified that the affinities of MeAA-tRNAs to EF-Tu are indeed weaker than those of pAA-tRNAs. Since the T-stem of tRNA plays a major role in interacting with EF-Tu, we have engineered the T-stem sequence to tune the affinity of MeAA-tRNAs to EF-Tu. The uniform affinity-tuning of the individual pairs has successfully enhanced the incorporation of MeAAs, achieving the incorporation of nine distinct MeAAs into both linear and thioether-macrocyclic peptide scaffolds.

Recruitment of archaeal DTD is a key event toward the emergence of land plants.

Streptophyte algae emerged as a land plant with adaptations that eventually led to terrestrialization. Land plants encounter a range of biotic and abiotic stresses that elicit anaerobic stress responses. Here, we show that acetaldehyde, a toxic metabolite of anaerobic stress, targets and generates ethyl adducts on aminoacyl-tRNA, a central component of the translation machinery. However, elongation factor thermo unstable (EF-Tu) safeguards l-aminoacyl-tRNA, but not d-aminoacyl-tRNA, from being modified by acetaldehyde. We identified a unique activity of archaeal-derived chiral proofreading module, d-aminoacyl-tRNA deacylase 2 (DTD2), that removes N-ethyl adducts formed on d-aminoacyl-tRNAs (NEDATs). Thus, the study provides the molecular basis of ethanol and acetaldehyde hypersensitivity in DTD2 knockout plants. We uncovered an important gene transfer event from methanogenic archaea to the ancestor of land plants. While missing in other algal lineages, DTD2 is conserved from streptophyte algae to land plants, suggesting its role toward the emergence and evolution of land plants.

RAB GTPASE HOMOLOG8D is required for the maintenance of both the root stem cell niche and the meristem.

Previous studies have suggested that the plastid translation elongation factor, elongation factor thermo unstable (EF-Tu), encoded by RAB GTPASE HOMOLOG8D (RAB8D) is essential for plant growth. Here, through analyzing the root phenotypes of two knock-down alleles of RAB8D (rab8d-1 and rab8d-2), we further revealed a vital role for RAB8D in primary root development through the maintenance of both the stem cell niche (SCN) and the meristem. Our results showed that RAB8D deficiency affects the root auxin response and SCN maintenance signaling. RAB8D interacts with GENOMES UNCOUPLED1 (GUN1) invivo. Further analysis revealed that GUN1 is over-accumulated and is required for both stem cell death and maintenance of root architecture in rab8d Arabidopsis mutants. The ATAXIA-TELANGIECTASIA-MUTATED (ATM)-SUPPRESSOR OF GAMMA RESPONSE1 pathway is involved in the regulation of root meristem size through upregulating SIAMESE-RELATED5 expression in the rab8d-2 allele. Moreover, ETHYLENE RESPONSE FACTOR115 is highly expressed in rab8d-2, which plays a role in further quiescent center division. Our observations not only characterized the role of RAB8D in root development, but also uncovered functions of GUN1 and ATM in response to plastid EF-Tu deficiency.

Mycoplasma hyopneumoniae evades complement activation by binding to factor H via elongation factor thermo unstable (EF-Tu)

ABSTRACT Mycoplasmas persist in the host for a long time, suggesting that they possess mechanisms for immune evasion. Factor H is a negative regulator of the complement system, which binds to host cells to avoid unexpected complement activation. In this study, we revealed that many mycoplasmas, such as Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Mycoplasma gallisepticum, Mycoplasma pneumoniae, Mycoplasma genitalium, Mycoplasma flocculare, and Mycoplasma bovis could hijack factor H such that they present themselves as a host tissue and thus escape from complement attack. Furthermore, the mechanism of recruiting factor H was identified in M. hyopneumoniae. M. hyopneumoniae binds factor H via factor H binding proteins, such as elongation factor thermo unstable (EF-Tu), P146, pyruvate dehydrogenase (acetyl-transferring) E1 component subunit alpha (PdhA), P46, Pyruvate dehydrogenase E1 component subunit beta (PdhB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and three different hypothetical proteins. The binding of factor H by EF-Tu further contributes to decreased C3 deposition on the M. hyopneumoniae surface and ultimately blocks further complement activation. In fact, binding of factor H occurs in a multifactorial manner; factor H is not only exploited by M. hyopneumoniae via its regulator activity to help mycoplasmas escape from complement killing, but also increases M. hyopneumoniae adhesion to swine tracheal epithelial cells, partially through EF-Tu. Meanwhile, the high sequence identity among EF-Tu proteins in the above-mentioned mycoplasmas implied the universality of the mechanism. This is the first report that mycoplasmas can escape complement killing by binding to factor H.

Deciphering Additional Roles for the EF-Tu, l-Asparaginase II and OmpT Proteins of Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) causes outbreaks and sporadic cases of gastroenteritis. STEC O157:H7 is the most clinically relevant serotype in the world. The major virulence determinants of STEC O157:H7 are the Shiga toxins and the locus of enterocyte effacement. However, several accessory virulence factors, mainly outer membrane proteins (OMPs) that interact with the host cells may contribute to the virulence of this pathogen. Previously, the elongation factor thermo unstable (EF-Tu), l-asparaginase II and OmpT proteins were identified as antigens in OMP extracts of STEC. The known subcellular location of EF-Tu and l-asparaginase II are the cytoplasm and periplasm, respectively. Therefore, we investigate whether these two proteins may localize on the surface of STEC and, if so, what roles they have at this site. On the other hand, the OmpT protein, a well characterized protease, has been described as participating in the adhesion of extraintestinal pathogenic E. coli strains. Thus, we investigate whether OmpT has this role in STEC. Our results show that the EF-Tu and l-asparaginase II are secreted by O157:H7 and may also localize on the surface of this bacterium. EF-Tu was identified in outer membrane vesicles (OMVs), suggesting it as a possible export mechanism for this protein. Notably, we found that l-asparaginase II secreted by O157:H7 inhibits T-lymphocyte proliferation, but the role of EF-Tu at the surface of this bacterium remains to be elucidated. In the case of OmpT, we show its participation in the adhesion of O157:H7 to human epithelial cells. Thus, this study extends the knowledge of the pathogenic mechanisms of STEC.

Monoclonal Antibody Preparation and Epitope Identification for Brucella melitensis Elongation Factor Tu.

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 Laminin Interactome: A Multifactorial Laminin-Binding Strategy by Nontypeable Haemophilus influenzae for Effective Adherence and Colonization.

Laminin is a well-defined component of the airway basement membrane (BM). Efficient binding of laminin via multiple interactions is important for nontypeable Haemophilus influenzae (NTHi) colonization in the airway mucosa. In this study, we identified elongation factor thermo-unstable (EF-Tu), l-lactate dehydrogenase (LDH), protein D (PD), and peptidoglycan-associated lipoprotein P6 as novel laminin-binding proteins (Lbps) of NTHi. In parallel with other well-studied Lbps (protein 4 [P4], protein E [PE], protein F [PF], and Haemophilus adhesion and penetration protein [Hap]), EF-Tu, LDH, PD, and P6 exhibited interactions with laminin, and mediated NTHi laminin-dependent adherence to pulmonary epithelial cell lines. More importantly, the NTHi laminin interactome consisting of the well-studied and novel Lbps recognized laminin LG domains from the subunit α chains of laminin-111 and -332, the latter isoform of which is the main laminin in the airway BM. The NTHi interactome mainly targeted multiple heparin-binding domains of laminin. In conclusion, the NTHi interactome exhibited a high plasticity of interactions with different laminin isoforms via multiple heparin-binding sites.

Engineering Translation Components Improve Incorporation of Exotic Amino Acids.

Methods of genetic code manipulation, such as nonsense codon suppression and genetic code reprogramming, have enabled the incorporation of various nonproteinogenic amino acids into the peptide nascent chain. However, the incorporation efficiency of such amino acids largely varies depending on their structural characteristics. For instance, l-α-amino acids with artificial, bulky side chains are poorer substrates for ribosomal incorporation into the nascent peptide chain, mainly owing to the lower affinity of their aminoacyl-tRNA toward elongation factor-thermo unstable (EF-Tu). Phosphorylated Ser and Tyr are also poorer substrates for the same reason; engineering EF-Tu has turned out to be effective in improving their incorporation efficiencies. On the other hand, exotic amino acids such as d-amino acids and β-amino acids are even poorer substrates owing to their low affinity to EF-Tu and poor compatibility to the ribosome active site. Moreover, their consecutive incorporation is extremely difficult. To solve these problems, the engineering of ribosomes and tRNAs has been executed, leading to successful but limited improvement of their incorporation efficiency. In this review, we comprehensively summarize recent attempts to engineer the translation systems, resulting in a significant improvement of the incorporation of exotic amino acids.

Anti-EF-Tu IgG titers increase with age and may contribute to protection against the respiratory pathogen Haemophilus influenzae.

Non‐typeable Haemophilus influenzae (NTHi) is a pathogen that commonly colonizes the nasopharynx of preschool children, causing opportunistic infections including acute otitis media (AOM). Patients suffering from chronic obstructive pulmonary disease (COPD) are persistently colonized with NTHi and occasionally suffer from exacerbations by the bacterium leading to increased morbidity. Elongation‐factor thermo unstable (EF‐Tu), a protein critical for bacterial protein synthesis, has been found to moonlight on the surface of several bacteria. Here, we show that antibodies against NTHi EF‐Tu were present in children already at 18 months of age, and that the IgG antibody titers increased with age. Children harboring NTHi in the nasopharynx also displayed significantly higher IgG concentrations. Interestingly, children suffering from AOM had significantly higher anti‐EF‐Tu IgG levels when NTHi was the causative agent. Human sera recognized mainly the central and C‐terminal part of the EF‐Tu molecule and peptide‐based epitope mapping confirmed similar binding patterns for sera from humans and immunized mice. Immunization of BALB/c and otitis‐prone Junbo (C3H/HeH) mice promoted lower infection rates in the nasopharynx and middle ear, respectively. In conclusion, our results suggest that IgG directed against NTHi EF‐Tu may play an important role in the host immune response against NTHi.

EF-Tu From Non-typeable Haemophilus influenzae Is an Immunogenic Surface-Exposed Protein Targeted by Bactericidal Antibodies.

Non-typeable Haemophilus influenzae (NTHi), a commensal organism in pre-school children, is an opportunistic pathogen causing respiratory tract infections including acute otitis media. Adults suffering from chronic obstructive pulmonary disease (COPD) are persistently colonized by NTHi. Previous research has suggested that, in some bacterial species, the intracellular elongation factor thermo-unstable (EF-Tu) can moonlight as a surface protein upon host encounter. The aim of this study was to determine whether EF-Tu localizes to the surface of H. influenzae, and if such surface-associated EF-Tu is a target for bactericidal antibodies. Using flow cytometry, transmission immunoelectron microscopy, and epitope mapping, we demonstrated that EF-Tu is exposed at the surface of NTHi, and identified immunodominant epitopes of this protein. Rabbits immunized with whole-cell NTHi produced significantly more immunoglobulin G (IgG) directed against EF-Tu than against the NTHi outer membrane proteins D and F as revealed by enzyme-linked immunosorbent assays. Chemical cleavage of NTHi EF-Tu by cyanogen bromide (CNBr) followed by immunoblotting showed that the immunodominant epitopes were located within the central and C-terminal regions of the protein. Peptide epitope mapping by dot blot analysis further revealed four different immunodominant peptide sequences; EF-Tu41−65, EF-Tu161−185, EF-Tu221−245, and EF-Tu281−305. These epitopes were confirmed to be surface-exposed and accessible by peptide-specific antibodies in flow cytometry. We also analyzed whether antibodies raised against NTHi EF-Tu cross-react with other respiratory tract pathogens. Anti-EF-Tu IgG significantly detected EF-Tu on unencapsulated bacteria, including the Gram-negative H. parainfluenzae, H. haemolyticus, Moraxella catarrhalis and various Gram-positive Streptococci of the oral microbiome. In contrast, considerably less EF-Tu was observed at the surface of encapsulated bacteria including H. influenzae serotype b (Hib) and Streptococcus pneumoniae (e.g., serotype 3 and 4). Removal of the capsule, as exemplified by Hib RM804, resulted in increased EF-Tu surface density. Finally, anti-NTHi EF-Tu IgG promoted complement-dependent bacterial killing of NTHi and other unencapsulated Gram-negative bacteria as well as opsonophagocytosis of Gram-positive bacteria. In conclusion, our data demonstrate that NTHi EF-Tu is surface-exposed and recognized by antibodies mediating host innate immunity against NTHi in addition to other unencapsulated respiratory tract bacteria.

Elongation Factor Thermo Unstable (EF-Tu) Moonlights as an Adhesin on the Surface of Mycoplasma hyopneumoniae by Binding to Fibronectin.

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.

Structural and Dynamics Comparison of Thermostability in Ancient, Modern, and Consensus Elongation Factor Tus

Rationally engineering thermostability in proteins would create enzymes and receptors that function under harsh industrial applications. Several sequence-based approaches can generate thermostable variants of mesophilic proteins. To gain insight into the mechanisms by which proteins become more stable, we use structural and dynamic analyses to compare two popular approaches, ancestral sequence reconstruction (ASR) and the consensus method, used to generate thermostable variants of Elongation Factor Thermo-unstable (EF-Tu). We present crystal structures of ancestral and consensus EF-Tus, accompanied by molecular dynamics simulations aimed at probing the strategies employed to enhance thermostability. All proteins adopt crystal structures similar to extant EF-Tus, revealing no difference in average structure between the methods. Molecular dynamics reveals that ASR-generated sequences retain dynamic properties similar to extant, thermostable EF-Tu from Thermus aquaticus, while consensus EF-Tu dynamics differ from evolution-based sequences. Thiswork highlights the advantage of ASR for engineering thermostability while preserving natural motions in multidomain proteins.

Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase.

D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD’s invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD’s chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2′-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD’s activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting.

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1-IF3). Here, we describe a novel type of initiation termed "70S-scanning initiation," where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine-Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.