A Comprehensive Analysis of the Lysine Acetylome in the Aquatic Animals Pathogenic Bacterium Vibrio mimicus

Protein lysine acetylation is an evolutionarily conserved post-translational modification (PTM), which is dynamic and reversible, playing a crucial regulatory role in almost every aspect of metabolism, of both eukaryotes and prokaryotes. Several global lysine acetylome studies have been carried out in various bacteria, but thus far, there have been no reports of lysine acetylation for the commercially important aquatic animal pathogen Vibrio mimicus. In the present study, we used anti-Ac-K antibody beads to highly sensitive immune-affinity purification and combined high-resolution LC-MS/MS to perform the first global lysine acetylome analysis in V. mimicus, leading to the identification of 1,097 lysine-acetylated sites on 582 proteins, and more than half (58.4%) of the acetylated proteins had only one site. The analysis of acetylated modified peptide motifs revealed six significantly enriched motifs, namely, KacL, KacR, L(-2) KacL, LKacK, L(-7) EKac, and IEKac. In addition, bioinformatic assessments state clearly that acetylated proteins have a hand in many important biological processes in V. mimicus, such as purine metabolism, ribosome, pyruvate metabolism, glycolysis/gluconeogenesis, the TCA cycle, and so on. Moreover, 13 acetylated proteins were related to the virulence of V. mimicus. To sum up, this is a comprehensive analysis whole situation protein lysine acetylome in V. mimicus and provides an important foundation for in-depth study of the biological function of lysine acetylation in V. mimicus

First Succinylome Profiling of Vibrio alginolyticus Reveals Key Role of Lysine Succinylation in Cellular Metabolism and Virulence

Recent studies have shown that a key strategy of many pathogens is to use post-translational modification (PTMs) to modulate host factors critical for infection. Lysine succinylation (Ksuc) is a major PTM widespread in prokaryotic and eukaryotic cells, and is associated with the regulation of numerous important cellular processes. Vibrio alginolyticus is a common pathogen that causes serious disease problems in aquaculture. Here we used the affinity enrichment method with LC-MS/MS to report the first identification of 2082 lysine succinylation sites on 671 proteins in V. alginolyticus, and compared this with the lysine acetylation of V. alginolyticus in our previous work. The Ksuc modification of SodB and PEPCK proteins were further validated by Co-immunoprecipitation combined with Western blotting. Bioinformatics analysis showed that the identified lysine succinylated proteins are involved in various biological processes and central metabolism pathways. Moreover, a total of 1,005 (25.4%) succinyl sites on 502 (37.3%) proteins were also found to be acetylated, which indicated that an extensive crosstalk between acetylation and succinylation in V. alginolyticus occurs, especially in three central metabolic pathways: glycolysis/gluconeogenesis, TCA cycle, and pyruvate metabolism. Furthermore, we found at least 50 (7.45%) succinylated virulence factors, including LuxS, Tdh, SodB, PEPCK, ClpP, and the Sec system to play an important role in bacterial virulence. Taken together, this systematic analysis provides a basis for further study on the pathophysiological role of lysine succinylation in V. alginolyticus and provides targets for the development of attenuated vaccines

Comparative transcriptomic analysis reveals the molecular mechanisms related to oxytetracycline- resistance in strains of Aeromonas hydrophila

Antibiotic resistance among aquatic bacterial pathogens has become a serious concern in aquaculture environments, which has increased research interest to develop solutions to overcome this problem. Moreover, the activities of several Aeromonas hydrophila gene pathways have remained elusive concerning antibiotic resistance evolution. Therefore, in this study, we have performed a transcriptomic analysis to compare differentially expressed genes between oxytetracycline (OXY) susceptible and resistant strains of A. hydrophila. Compared to the A. hydrophila susceptible strain, a total of 22 and 185 genes were differentially expressed in the 4-fold minimal inhibitory concentration (MIC) and 8-fold MIC resistant strains, respectively. Furthermore, the bioinformatics analysis revealed that the sulfur metabolism-related genes were down-regulated. The genes responsible for mannitol metabolism and the efflux pump system were up-regulated in resistant strains, compared to the susceptible strain. Therefore, it suggests that these three pathways may be involved in the OXY resistance evolution in A. hydrophila. The outcome of the transcriptomic data was further validated through quantitative reverse transcription-PCR (qRT-PCR) and Western blot analysis. Overall, the obtained data provides a deeper insight into the intrinsic molecular mechanism of OXY resistance evolution in A. hydrophila

Corrosion Crack Morphology and Creep Analysis of Members Based on Meso-Scale Corrosion Penetration

In this paper, to study the development of load-carrying capacity and long-term creep performance of reinforced concrete beams under different corrosion patterns, the rate-dependent model of concrete is used as the basis to consider the creep development process from the meso-scale level. The porosity mechanics method is used to simulate the generation and penetration process of corrosion products. Three corrosion conditions are set: bottom longitudinal reinforcement corrosion, top longitudinal reinforcement corrosion and all reinforcement corrosion. The corrosion rate is used as the variable in each corrosion condition. The results show that: (1) the greater the corrosion rate in all conditions, the lower the bearing capacity. In addition, the corrosion of top longitudinal reinforcement causes the damage form of the beam to change to brittle damage; (2) the creep coefficient decreases with the increase in corrosion rate in all working conditions, but the main factor for this phenomenon is the obvious increase in initial deformation. Consequently, it is not suitable to follow the conventional creep concept (deformation development/initial deformation) for the development of plastic deformation of damaged members. It is more reasonable to use the global deflection to describe the long-term deformation of corrosion-damaged members

Corrosion-Induced Cracking Pattern Analysis of RC Beam under Sustained Load Considering the Poromechanical Characteristics of Corrosion Products

Concrete cracking is the significant stage of RC structural deterioration induced by steel corrosion. To predict the corrosion-induced cracking of the loaded RC structure, a multi-scale model is proposed. The formation and transport of corrosion products, which affect the volumetric expansion at the steel–concrete interface, are considered in this model. Then, based on poro-mechanics, the calculation of corrosion-induced stress in the pore structure of concrete is enabled. The corrosion-induced cracking of the practical component obtained from the proposed model shows a satisfactory agreement with the experimental observations. Then, the corrosion-induced cracking under different loading conditions are investigated. The results show that the effect of external loads on the time-to-cracking is moderate; the steel corrosion varies by no more 9% under different loading conditions at the surface cracking moment, whereas significant effects of the loading condition on the cracking pattern of reinforced concrete beams are found. Furthermore, the higher the load level, the more rapidly the corrosion-induced cracks develop; the maximum corrosion-induced crack widths on the surface of the beam subjected to 60% ultimate load is 1.14 and 1.22 times that of the 30% and 0% loaded beams, respectively, when reaching the serviceability limit state

Cloning and Bioinformatics Analysis of pepck Gene in Vibrio alginolyticus

[Objectives] To clone the pepck gene of Vibrio alginolyticus strain HY9901 and analyze its sequence by bioinformatics. [Methods] According to the complete gene sequence of V. alginolyticus on GenBank, specific primers were designed to amplify the target gene pepck by PCR. The sequence of the pepck gene was analyzed using bioinformatics. The phylogenic tree of pepck gene and the corresponding single-subunit three-dimensional structure were constructed. [Results] The pepck gene of V. alginolyticus strain HY9901 has a full length of 1 629 bp, with theoretical molecular weight of 60.12 kD. The prediction results show that there is no signal peptide or transmembrane region at the N-terminus of the sequence, the amino acid sequence contains 11 phosphorylation sites of casein kinase II. The prediction results of protein subcellular localization indicate that PEPEK protein is localized in the cytoplasm. The protein is stable and hydrophobic. The tertiary structure of the PEPCK protein of V. alginolyticus is similar to that of Vibrio parahaemolyticus. It is predicted that PEPCK has a major functional domain PEPCK_ATP. In the secondary structure, alpha helix, random coil, and extended strand accounted for 21.96%, 52.03% and 26.01%, respectively. The PEPCK homology between V. alginolyticus and Vibrio diabolicus is as high as 99%. [Conclusions] This study lays the foundation for further understanding the function of pepck gene in V. alginolyticus

Bioinformatics Analysis of DNA-binding Response Regulator PhoP in Vibrio alginolyticus

[Objectives] To amplify the DNA-binding response regulator PhoP in Vibrio alginolyticus and analyze its sequence characteristics and subunit structure. [Methods] According to the sequence of the DNA-binding response regulator PhoP in V. alginolyticus, a pair of specific primers was designed for PCR amplification, and the bioinformatics of the sequence amplified was analyzed. Using MEGA 5.0 software, the phoP phylogenetic tree was constructed by the neighbor-joining method. Using SWISS-MODEL software, the three-dimensional structural model of the PhoP subunit was simulated. [Results] The full-length phoP gene was 732 bp, encoding a total of 243 amino acids. The predicted theoretical molecular weight of the protein is about 27.67 kD, and the isoelectric point is 5.09. The prediction results of protein subcellular localization, SignalP 4.0, TMHMM Server 2.0 and SoftBerry-Psite show that PhoP is located in the cytoplasm, and is stable and hydrophobic; there is a signal peptide cleavage site between amino acids 29 and 30, and there is no transmembrane region. The amino acid sequence contains one Asn-glycosylation site, one protein kinase C phosphorylation site, seven casein kinase II phosphorylation sites, one tyrosine kinase phosphorylation site, three myristoylation sites, and seven C-terminal microbody targeting signal sites. The PhoP of V. alginolyticus has high homology with that of Vibrio campbellii. The PhoP subunit of V. alginolyticus has similar configuration to the single-subunit RegX3 protein of Mycobacterium tuberculosis. [Conclusions] This study has a positive effect on the prevention and control of vibriosis and the improvement of the current aquatic economic animal breeding environment

Functional characterization of Vibrio alginolyticus T3SS regulator ExsA and evaluation of its mutant as a live attenuated vaccine candidate in zebrafish (Danio rerio) model

Vibrio alginolyticus, a Gram-negative bacterium, is an opportunistic pathogen of both marine animals and humans, resulting in significant losses in the aquaculture industry. Type III secretion system (T3SS) is a crucial virulence mechanism of V. alginolyticus. In this study, the T3SS regulatory gene exsA, which was cloned from V. alginolyticus wild-type strain HY9901, is 861 bp encoding a protein of 286 amino acids. The ΔexsA was constructed by homologous recombination and Overlap-PCR. Although there was no difference in growth between HY9901 and ΔexsA, the ΔexsA exhibited significantly decreased extracellular protease activity and biofilm formation. Besides, the ΔexsA showed a weakened swarming phenotype and an ~100-fold decrease in virulence to zebrafish. Antibiotic susceptibility testing showed the HY9901ΔexsA was more sensitive to kanamycin, minocycline, tetracycline, gentamicin, doxycycline and neomycin. Compared to HY9901 there were 541 up-regulated genes and 663 down-regulated genes in ΔexsA, screened by transcriptome sequencing. qRT-PCR and β-galactosidase reporter assays were used to analyze the transcription levels of hop gene revealing that exsA gene could facilitate the expression of hop gene. Finally, Danio rerio, vaccinated with ΔexsA through intramuscular injection, induced a relative percent survival (RPS) value of 66.7% after challenging with HY9901 wild type strain. qRT-PCR assays showed that vaccination with ΔexsA increased the expression of immune-related genes, including GATA-1, IL6, IgM, and TNF-α in zebrafish. In summary, these results demonstrate the importance of exsA in V. alginolyticus and provide a basis for further investigations into the virulence and infection mechanism

Cloning and Bioinformatics Analysis of TpiA Gene of Vibrio alginolyticus HY9901

[Objectives] To clone and analyze the TpiA gene of Vibrio alginolyticus HY9901. [Methods] According to the TpiA gene sequence of V. alginolyticus, a pair of specific primers was designed, and its full length was amplified by PCR. [Results] The full length of TpiA gene is 771 bp, encoding 256 amino acid residues in total, and the NCBI accession number is OM906798. According to the deduced amino acid sequence, its molecular weight was predicted to be about 26.975 48 kDa, and its isoelectric point was 4.78. The amino acid sequence of the N-terminal signal peptide structure was predicted, and it was found that there was no obvious signal peptide cleavage site, no signal peptide, and no transmembrane region; the amino acid sequence contained 3 N-glycosylation sites, 4 protein kinase C phosphorylation sites, 2 casein kinase II phosphorylation sites, 6 N-myristoylation sites, 7 microbody C - terminal target signal site, and 1 triose phosphate isomerase active site. The prediction results of protein subcellular localization showed that TpiA may be located in mitochondria or cytoplasm, with probability of 39.1% and 34.8%, respectively. The amino acid sequence of the TpiA gene of V. alginolyticus shared 98.83%-99.61% homology with other Vibrio species, and it was clustered into the same subfamily with Vibrio parahaemolyticus and had a close relationship. In the secondary structure prediction, the proportions of α-helix, random coil and extended chain were 44.53%, 41.41% and 14.06%, respectively, and the similarity of its tertiary structure model to template 1aw1.1.A was 85.16%. [Conclusions] This study is intended to provide a basis for further research on the role of TpiA gene in the type III secretion system and related research on antibiotic resistance