Development of a prototypic, field-usable diagnostic tool for the detection of gram-positive cocci-induced mastitis in cattle.

BACKGROUND: Bovine mastitis is one of the most widespread diseases affecting cattle, leading to significant losses for the dairy industry. Currently, the so-called gold standard in mastitis diagnosis involves determining the somatic cell count (SCC). Apart from a number of advantages, this method has one serious flaw: It does not identify the etiological factor causing a particular infection, making it impossible to introduce targeted antimicrobial therapy. This can contribute to multidrug-resistance in bacterial species. The diagnostic market lacks a test that has the advantages of SCC and also recognizes the species of pathogen causing the inflammation. Therefore, the aim of our study was to develop a lateral flow immunoassay (LFIA) based on elongation factor Tu for identifying most prevalent Gram-positive cocci responsible for causing mastitis including Streptococcus uberis, Streptococcus agalactiae and Staphylococcus aureus. RESULTS: As a result, we showed that the assay for S. uberis detection demonstrated a specificity of 89.02%, a sensitivity of 43.59%, and an accuracy of 80.3%. In turn, the second variant - assay for Gram-positive cocci reached a specificity of 95.59%, a sensitivity of 43.28%, and an accuracy of 78.33%. CONCLUSIONS: Our study shows that EF-Tu is a promising target for LFIA and we have delivered evidence that further evaluation could improve test parameters and fill the gap in the mastitis diagnostics market.

Engineered Agrobacterium improves transformation by mitigating plant immunity detection.

Agrobacterium tumefaciens microbe-associated molecular pattern elongation factor Tu (EF-Tu) is perceived by orthologs of the Arabidopsis immune receptor EFR activating pattern-triggered immunity (PTI) that causes reduced T-DNA-mediated transient expression. We altered EF-Tu in A. tumefaciens to reduce PTI and improved transformation efficiency. A robust computational pipeline was established to detect EF-Tu protein variation in a large set of plant bacterial species and identified EF-Tu variants from bacterial pathogen Pseudomonas syringae pv. tomato DC3000 that allow the pathogen to escape EFR perception. Agrobacterium tumefaciens strains were engineered to substitute EF-Tu with DC3000 variants and examined their transformation efficiency in plants. Elongation factor Tu variants with rarely occurred amino acid residues were identified within DC3000 EF-Tu that mitigates recognition by EFR. Agrobacterium tumefaciens strains were engineered by expressing DC3000 EF-Tu instead of native agrobacterial EF-Tu and resulted in decreased plant immunity detection. These engineered A. tumefaciens strains displayed an increased efficiency in transient expression in both Arabidopsis thaliana and Camelina sativa. The results support the potential application of these strains as improved vehicles to introduce transgenic alleles into members of the Brassicaceae family.

Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection.

The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu⋅GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.

Molecular evolutionary and 3D protein structural analyses of Lactobacillus fermentum elongation factor Tu, a novel brain health promoting factor.

The role of microbiota in gut-brain communication has led to the development of probiotics promoting brain health. Here we report a genomic study of a Lactobacillus fermentum PS150 and its patented bioactive protein, elongation factor Tu (EF-Tu), which is associated with cognitive improvement in rats. The L. fermentum PS150 circular chromosome is 2,238,401 bp and it consists of 2281 genes. Chromosome comparisons with other L. fermentum strains highlighted a cluster of glycosyltransferases as potential candidate probiotic factors besides EF-Tu. Molecular evolutionary analyses on EF-Tu genes (tuf) in 235 bacteria species revealed one to three copies of the gene per genome. Seven tuf pseudogenes were found and three species only possessed pseudogenes, which is an unprecedented finding. Protein variability analysis of EF-Tu showed five highly variable residues (40 K, 41G, 42 L, 44 K, and 46E) on the protein surface, which warrant further investigation regarding their potential roles as binding sites.

Analysis of heat-induced protein aggregation in human mitochondria.

Proteins in mammalian cells exhibit optimal stability at physiological temperatures, and even small temperature variations may cause unfolding and nonspecific aggregation. Because this process leads to a loss of function of the affected polypeptides and to cytotoxic stress, formation of protein aggregates has been recognized as a major pathogenic factor in human diseases. In this study, we determined the impact of physiological heat stress on mitochondria isolated from HeLa cells. We found that the heat-stressed mitochondria had lower membrane potential and ATP level and exhibited a decreased production of reactive oxygen species. An analysis of the mitochondrial proteome by 2D PAGE showed that the overall solubility of endogenous proteins was only marginally affected by elevated temperatures. However, a small subset of polypeptides exhibited an high sensitivity to heat stress. The mitochondrial translation elongation factor Tu (Tufm), a protein essential for organellar protein biosynthesis, was highly aggregation-prone and lost its solubility already under mild heat-stress conditions. Moreover, mitochondrial translation and the import of cytosolic proteins were defective in the heat-stressed mitochondria. Both types of nascent polypeptides, produced by translation or imported into the mitochondria, exhibited a strong tendency to aggregate in the heat-exposed mitochondria. We propose that a fast and specific inactivation of elongation factors may prevent the accumulation of misfolded nascent polypeptides and may thereby attenuate proteotoxicity under heat stress.

Dynamic changes in lysine acetylation and succinylation of the elongation factor Tu in Bacillus subtilis.

Nε-lysine acetylation and succinylation are ubiquitous post-translational modifications in eukaryotes and bacteria. In the present study, we showed a dynamic change in acetylation and succinylation of TufA, the translation elongation factor Tu, from Bacillus subtilis. Increased acetylation of TufA was observed during the exponential growth phase in LB and minimal glucose conditions, and its acetylation level decreased upon entering the stationary phase, while its succinylation increased during the late stationary phase. TufA was also succinylated during vegetative growth under minimal citrate or succinate conditions. Mutational analysis showed that triple succinylation mimic mutations at Lys306, Lys308 and Lys316 in domain-3 of TufA had a negative effect on B. subtilis growth, whereas the non-acylation mimic mutations at these three lysine residues did not. Consistent with the growth phenotypes, the triple succinylation mimic mutant showed 67 % decreased translation activity in vitro, suggesting a possibility that succinylation at the lysine residues in domain-3 decreases the translation activity. TufA, including Lys308, was non-enzymatically succinylated by physiological concentrations of succinyl-CoA. Lys42 in the G-domain was identified as the most frequently modified acetylation site, though its acetylation was likely dispensable for TufA translation activity and growth. Determination of the intracellular levels of acetylating substrates and TufA acetylation revealed that acetyl phosphate was responsible for acetylation at several lysine sites of TufA, but not for Lys42 acetylation. It was speculated that acetyl-CoA was likely responsible for Lys42 acetylation, though AcuA acetyltransferase was not involved. Zn2+-dependent AcuC and NAD+-dependent SrtN deacetylases were responsible for deacetylation of TufA, including Lys42. These findings suggest the potential regulatory roles of acetylation and succinylation in controlling TufA function and translation in response to nutrient environments in B. subtilis.

[Protein Biosynthesis Proofreading Is Closely Associated with the Existence of Factor-Free Ribosomal Synthesis].

Despite protein biosynthesis being studied for decades, some major questions concerning this process are still to be addressed. We elucidate a close connection between proofreading of the emerging amino acid sequence during its normal, elongation factor-dependent ribosomal biosynthesis and the existence of the factor-free synthesis of a polypeptide chain on a ribosome. In this factor-free process, the biological role of proofreading is played by a process opposite to the factor-free attachment of Aa-tRNA to the ribosome, namely, the removal via the same pathway of that Aa-tRNA, which is not complementary to the mRNA codon exhibited by the ribosome.

Loop-mediated isothermal amplification-lateral-flow dipstick (LAMP-LFD) to detect Mycoplasma ovipneumoniae.

In order to establish a rapid detection method for Mycoplasma ovipneumoniae, this study used the loop-mediated isothermal amplification (LAMP) technique to carry out nucleic acid amplification and chromatographic visualization via a lateral flow dipstick (LFD) assay. The M. ovipneumoniae elongation factor TU gene (EF-TU) was detected using a set of specific primers designed for the EF-TU gene, and the EF-TU FIP was detected by biotin labeling, which was used in the LAMP amplification reaction. The digoxin-labeled probe specifically hybridized with LAMP products, which were visually detected by LFD. Here, we established the M. ovipneumoniae LAMP-LFD rapid detection method and tested the specificity, sensitivity, and clinical application of this method. Results showed that the optimized LAMP performed at 60 °C for 60 min, and LFD can specifically and visually detect M. ovipneumoniae with a minimum detectable concentration at 1.0 × 102 CFU/mL. The sensitivity of LAMP-LFD was 1000 times that of the conventional PCR detection methods, and the clinical lung tissue detection rate was 86% of 50 suspected sheep infected with M. ovipneumoniae. In conclusion, LAMP-LFD was established in this study to detect M. ovipneumoniae, a method that was highly specific, sensitive, and easy to operate, and provides a new method for the prevention and diagnosis of M. ovipneumoniae infection.

[Unsolved Puzzles of Qß Replicase].

Qß phage replicase has been the first RNA-directed RNA polymerase purified to homogeneity and intensively studied in vitro. In the mid-sixties, papers on Qß and related replicases appeared in nearly every issue of the PNAS journal. By 1968, the mechanism of its action seemed to be almost completely understood. However, even now, a half of century later, a number of fundamental questions remains unanswered. How does the replicase manage to prevent the template and its complementary copy from annealing during the entire replication round? How does it recognize its templates? What is the function of the translation factors present in the replicase molecule? What is the mechanism the replicase uses to join (recombine) separate RNA molecules? Even the determination of the crystal structure of Qß replicase did not help much. Certainly, there remains a lot to discover in the replication of Qß phage, one of the smallest viruses known.

Secretomic Analysis of Host-Pathogen Interactions Reveals That Elongation Factor-Tu Is a Potential Adherence Factor of Helicobacter pylori during Pathogenesis.

The secreted proteins of bacteria are usually accompanied by virulence factors, which can cause inflammation and damage host cells. Identifying the secretomes arising from the interactions of bacteria and host cells could therefore increase understanding of the mechanisms during initial pathogenesis. The present study used a host-pathogen coculture system of Helicobacter pylori and monocytes (THP-1 cells) to investigate the secreted proteins associated with initial H. pylori pathogenesis. The secreted proteins from the conditioned media from H. pylori, THP-1 cells, and the coculture were collected and analyzed using SDS-PAGE and LC-MS/MS. Results indicated the presence of 15 overexpressed bands in the coculture. Thirty-one proteins were identified-11 were derived from THP-1 cells and 20 were derived from H. pylori. A potential adherence factor from H. pylori, elongation factor-Tu (EF-Tu), was selected for investigation of its biological function. Results from confocal microscopic and flow cytometric analyses indicated the contribution of EF-Tu to the binding ability of H. pylori in THP-1. The data demonstrated that fluorescence of EF-Tu on THP-1 cells increased after the addition of the H. pylori-conditioned medium. This study reports a novel secretory adherence factor in H. pylori, EF-Tu, and further elucidates mechanisms of H. pylori adaptation for host-pathogen interaction during pathogenesis.

The C-terminal Helix of Pseudomonas aeruginosa Elongation Factor Ts Tunes EF-Tu Dynamics to Modulate Nucleotide Exchange.

Little is known about the conservation of critical kinetic parameters and the mechanistic strategies of elongation factor (EF) Ts-catalyzed nucleotide exchange in EF-Tu in bacteria and particularly in clinically relevant pathogens. EF-Tu from the clinically relevant pathogen Pseudomonas aeruginosa shares over 84% sequence identity with the corresponding elongation factor from Escherichia coli Interestingly, the functionally closely linked EF-Ts only shares 55% sequence identity. To identify any differences in the nucleotide binding properties, as well as in the EF-Ts-mediated nucleotide exchange reaction, we performed a comparative rapid kinetics and mutagenesis analysis of the nucleotide exchange mechanism for both the E. coli and P. aeruginosa systems, identifying helix 13 of EF-Ts as a previously unnoticed regulatory element in the nucleotide exchange mechanism with species-specific elements. Our findings support the base side-first entry of the nucleotide into the binding pocket of the EF-Tu·EF-Ts binary complex, followed by displacement of helix 13 and rapid binding of the phosphate side of the nucleotide, ultimately leading to the release of EF-Ts.

Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins.

The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc.

Identification of proteins derived from Listeria monocytogenes inducing human dendritic cell maturation.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that can promote antitumor immunity when pulsed with tumor antigens and then matured by stimulatory agents. Despite apparent progress in DC-based cancer immunotherapy, some discrepancies were reported in generating potent DCs. Listeria monocytogenes as an intracellular microorganism is able to effectively activate DCs through engaging pattern-recognition receptors (PRRs). This study aimed to find the most potent components derived from L. monocytogenes inducing DC maturation. The preliminary results demonstrated that the ability of protein components is higher than DNA components to promote DC maturation and activation. Protein lysate fractionation demonstrated that fraction 2 HIC (obtained by hydrophobic interaction chromatography) was able to efficiently mature DCs. F2HIC-matured DCs are able to induce allogeneic CD8(+) T cells proliferation better than LPS-matured DCs and induce IFN-γ producing CD8(+) T cells. Mass spectrometry results showed that F2HIC contains 109 proteins. Based on the bioinformatics analysis for these 109 proteins, elongation factor Tu (EF-Tu) could be considered as a PRR ligand for stimulating DC maturation.

Structural outline of the detailed mechanism for elongation factor Ts-mediated guanine nucleotide exchange on elongation factor Tu.

Translation elongation factor EF-Tu belongs to the superfamily of guanine-nucleotide binding proteins, which play key cellular roles as regulatory switches. All G-proteins require activation via exchange of GDP for GTP to carry out their respective tasks. Often, guanine-nucleotide exchange factors are essential to this process. During translation, EF-Tu:GTP transports aminoacylated tRNA to the ribosome. GTP is hydrolyzed during this process, and subsequent reactivation of EF-Tu is catalyzed by EF-Ts. The reaction path of guanine-nucleotide exchange is structurally poorly defined for EF-Tu and EF-Ts. We have determined the crystal structures of the following reaction intermediates: two structures of EF-Tu:GDP:EF-Ts (2.2 and 1.8Å resolution), EF-Tu:PO4:EF-Ts (1.9Å resolution), EF-Tu:GDPNP:EF-Ts (2.2Å resolution) and EF-Tu:GDPNP:pulvomycin:Mg(2+):EF-Ts (3.5Å resolution). These structures provide snapshots throughout the entire exchange reaction and suggest a mechanism for the release of EF-Tu in its GTP conformation. An inferred sequence of events during the exchange reaction is presented.

Direct evidence of an elongation factor-Tu/Ts·GTP·Aminoacyl-tRNA quaternary complex.

During protein synthesis, elongation factor-Tu (EF-Tu) bound to GTP chaperones the entry of aminoacyl-tRNA (aa-tRNA) into actively translating ribosomes. In so doing, EF-Tu increases the rate and fidelity of the translation mechanism. Recent evidence suggests that EF-Ts, the guanosine nucleotide exchange factor for EF-Tu, directly accelerates both the formation and dissociation of the EF-Tu-GTP-Phe-tRNA(Phe) ternary complex (Burnett, B. J., Altman, R. B., Ferrao, R., Alejo, J. L., Kaur, N., Kanji, J., and Blanchard, S. C. (2013) J. Biol. Chem. 288, 13917-13928). A central feature of this model is the existence of a quaternary complex of EF-Tu/Ts·GTP·aa-tRNA(aa). Here, through comparative investigations of phenylalanyl, methionyl, and arginyl ternary complexes, and the development of a strategy to monitor their formation and decay using fluorescence resonance energy transfer, we reveal the generality of this newly described EF-Ts function and the first direct evidence of the transient quaternary complex species. These findings suggest that EF-Ts may regulate ternary complex abundance in the cell through mechanisms that are distinct from its guanosine nucleotide exchange factor functions.

Mechanism of activation of elongation factor Tu by ribosome: catalytic histidine activates GTP by protonation.

Elongation factor Tu (EF-Tu) is central to prokaryotic protein synthesis as it has the role of delivering amino-acylated tRNAs to the ribosome. Release of EF-Tu, after correct binding of the EF-Tu:aa-tRNA complex to the ribosome, is initiated by GTP hydrolysis. This reaction, whose mechanism is uncertain, is catalyzed by EF-Tu, but requires activation by the ribosome. There have been a number of mechanistic proposals, including those spurred by a recent X-ray crystallographic analysis of a ribosome:EF-Tu:aa-tRNA:GTP-analog complex. In this work, we have investigated these and alternative hypotheses, using high-level quantum chemical/molecular mechanical simulations for the wild-type protein and its His85Gln mutant. For both proteins, we find previously unsuggested mechanisms as being preferred, in which residue 85, either His or Gln, directly assists in the reaction. Analysis shows that the RNA has a minor catalytic effect in the wild-type reaction, but plays a significant role in the mutant by greatly stabilizing the reaction's transition state. Given the similarity between EF-Tu and other members of the translational G-protein family, it is likely that these mechanisms of ribosome-activated GTP hydrolysis are pertinent to all of these proteins.

Enhancement of innate immune system in monocot rice by transferring the dicotyledonous elongation factor Tu receptor EFR.

The elongation factor Tu (EF-Tu) receptor (EFR) in cruciferous plants specifically recognizes the N-terminal acetylated elf18 region of bacterial EF-Tu and thereby activates plant immunity. It has been demonstrated that Arabidopsis EFR confers broad-spectrum bacterial resistance in the EFR transgenic solanaceous plants. Here, the transgenic rice plants (Oryza sativa L. ssp. japonica cv. Zhonghua 17) and cell cultures with constitutive expression of AtEFR were developed to investigate whether AtEFR senses EF-Tu and thus enhances bacterial resistance in the monocot plants. We demonstrated that the Xanthomonas oryzae-derived elf18 peptide induced oxidative burst and mitogen-activated protein kinase activation in the AtEFR transgenic rice cells and plants, respectively. Pathogenesis-related genes, such as OsPBZ1, were upregulated dramatically in transgenic rice plant and cell lines in response to elf18 stimulation. Importantly, pretreatment with elf18 triggered strong resistance to X. oryzae pv. oryzae in the transgenic plants, which was largely dependent on the AtEFR expression level. These plants also exhibited enhanced resistance to rice bacterial brown stripe, but not to rice fungal blast. Collectively, the results indicate that the rice plants with heterologous expression of AtEFR recognize bacterial EF-Tu and exhibit enhanced broad-spectrum bacterial disease resistance and that pattern recognition receptor-mediated immunity may be manipulated across the two plant classes, dicots and monocots.

Elongation factor Ts directly facilitates the formation and disassembly of the Escherichia coli elongation factor Tu·GTP·aminoacyl-tRNA ternary complex.

BACKGROUND: Aminoacyl-tRNA (aa-tRNA) enters the ribosome in a ternary complex with the G-protein elongation factor Tu (EF-Tu) and GTP. RESULTS: EF-Tu·GTP·aa-tRNA ternary complex formation and decay rates are accelerated in the presence of the nucleotide exchange factor elongation factor Ts (EF-Ts). CONCLUSION: EF-Ts directly facilitates the formation and disassociation of ternary complex. SIGNIFICANCE: This system demonstrates a novel function of EF-Ts. Aminoacyl-tRNA enters the translating ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here, we describe bulk steady state and pre-steady state fluorescence methods that enabled us to quantitatively explore the kinetic features of Escherichia coli ternary complex formation and decay. The data obtained suggest that both processes are controlled by a nucleotide-dependent, rate-determining conformational change in EF-Tu. Unexpectedly, we found that this conformational change is accelerated by elongation factor Ts (EF-Ts), the guanosine nucleotide exchange factor for EF-Tu. Notably, EF-Ts attenuates the affinity of EF-Tu for GTP and destabilizes ternary complex in the presence of non-hydrolyzable GTP analogs. These results suggest that EF-Ts serves an unanticipated role in the cell of actively regulating the abundance and stability of ternary complex in a manner that contributes to rapid and faithful protein synthesis.

Cell surface-associated protein elongation factor Tu interacts with fibronectin mediating the adhesion of Lactobacillus plantarum HC-2 to Penaeus vannamei intestinal epithelium and inhibiting the apoptosis induced by LPS and pathogen in Caco-2 cells.

The adherent colonization of lactic acid bacteria in the animal intestine is the basis for their probiotic effect, and their bacteria surface proteins play an important role in this process. Previous work has demonstrated that Lactobacillus plantarum HC-2 can adhere and colonize the intestine of Penaeus vannamei, modulate the intestinal immune response and microbial diversity, protect the intestinal tissues from pathogenic damage, and improve the protection rate of shrimp. The aim of this work was to identify adhesion molecules on the surface of HC-2 and its adhesion receptors in the intestinal epithelium of shrimp. The elongation factor Tu (EF-Tu) on the surface of HC-2 was found to interact with Fibronectin (Fib) in the shrimp intestine by immunoblotting and yeast two-hybrid assays, and this interaction relationship was verified by immunoprecipitation (Co-IP). The adhesion of HC-2 to Caco-2 cells could be blocked via EF-Tu antibody confinement, and the adhesion of Fib to HC-2 could be blocked by Fib antibody confinement. Expression of Fib on the surface of HEK293T cells revealed a significant increase in the adhesion rate of HC-2 to HEK293T cells. Using immunofluorescence, a significant reduction in HC-2 adhesion to the intestine of shrimp was observed after blocking the Fib site in the shrimp intestine, particularly in Vibrio parahaemolyticus E1-infected intestines. In addition, the recombinant protein rEF-Tu was found to promote the growth of Caco-2 cells in a certain concentration range and significantly inhibit the apoptosis induced by LPS, Staphylococcus aureus and V. parahaemolyticus E1. Our results indicate that EF-Tu might participate in gut immunity and homeostasis, through its binding to the shrimp intestinal cells and inhibiting apoptosis.

Proteomic Analysis Revealed Metabolic Inhibition and Elongation Factor Tu Deamidation by p-Coumaric Acid in Cronobacter sakazakii.

Screening drugs and compounds to fight against Cronobacter sakazakii (C. sakazakii), one of the most common pathogens that can cause fatal necrotizing enterocolitis, septicema and meningitis, is still needed. We found that p-coumaric acid (pCA) has an inhibitory effect on C. sakazakii in vitro and in vivo. Proteomic changes of C. sakazakii BAA-894 exposed to pCA were studied to reveal the antibacterial mechanisms involved. A total of 1,553 proteins were identified in C. sakazakii BAA-894 by label-free proteomics analysis. Fuzzy cluster analysis showed that 33 were up-regulated, and 110 were down-regulated with pCA treatment. Gene Ontology (GO) analysis concluded that pCA caused the change of metabolic state of bacteria and generally in the state of metabolic inhibition. KEGG Enrichment Analysis (KEGG) analysis showed that pCA inhibited energy metabolism and distorted the balance of amino acid metabolism. Posttranslational modification analysis showed that pCA affected the deamidation of three proteins, including Elongation factor Tu, one of the vital proteins in bacteria. Molecular docking suggested the hydrogen bond between the pCA carboxyl group and Elongation factor Tu Asn-64 might contribute to deamidation. Overall, we found that pCA interfered with cellular energy and amino acid metabolism and promoted elongation factor Tu deamidation, suggesting that pCA can be an effective natural substitute to control C. sakazakii.