The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics.

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXYR) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXYR strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXYR as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and ß-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.

Integrated Succinylome and Metabolome Profiling Reveals Crucial Role of S-Ribosylhomocysteine Lyase in Quorum Sensing and Metabolism of Aeromonas hydrophila.

Protein modification by lysine succinylation is a newly identified post-translational modification (PTM) of lysine residues and plays an important role in diverse physiological functions, although their associated biological characteristics are still largely unknown. Here, we investigated the effects of lysine succinylation on the physiological regulation within a well-known fish pathogen, Aeromonas hydrophila A high affinity purification method was used to enrich peptides with lysine succinylation in A. hydrophila ATCC 7966, and a total of 2,174 lysine succinylation sites were identified on 666 proteins using LC-MS/MS. Gene ontology analysis indicated that these succinylated proteins are involved in diverse metabolic pathways and biological processes, including translation, protein export, and central metabolic pathways. The modifications of several selected candidates were further validated by Western blotting. Using site-directed mutagenesis, we observed that the succinylation of lysines on S-ribosylhomocysteine lyase (LuxS) at the K23 and K30 sites positively regulate the production of the quorum sensing autoinducer AI-2, and that these PTMs ultimately alter its competitiveness with another pathogen, Vibrio alginolyticus Moreover, subsequent metabolomic analyses indicated that K30 succinylation on LuxS may suppress the activated methyl cycle (AMC) and that both the K23 and K30 sites are involved in amino acid metabolism. Taken together, the results from this study provide significant insights into the functions of lysine succinylation and its critical roles on LuxS in regulating the cellular physiology of A. hydrophila.

Production of Biodiesel and High-Protein Feed from Fish Processing Wastes Using In Situ Transesterification.

Preparation of biodiesel using in situ transesterification has been extensively conducted for agricultural, microbial and algal biomass, while few works have been performed using aquatic animal tissue. In this work, fish processing wastes were collected to perform in situ transesterification using grass carp (Ctenopharyngodon idellus) biomass as a representative with which to optimize the reaction conditions. Under the optimum condition, the highest biodiesel purity reached up to 100% for sea bass wastes, which is higher than the 96.5% specified in the EN 14214-2008. The in situ method proposed here has the potential to save significant costs in biodiesel production compared to conventional methods, which usually require high-cost pretreatment of the raw materials. Additionally, the waste residue byproduct produced has a high protein content, and therefore the potential to be used for high-protein feed. This study is expected to inspire new strategies to prepare biodiesel and high-protein feed simultaneously from aquatic animal biomass using the novel in situ transesterification.

The characteristics of antibiotic resistance and phenotypes in 29 outer-membrane protein mutant strains in Aeromonas hydrophila.

Although many typical outer-membrane proteins (OMPs) have been well characterized, the biological functions of many OMPs remain largely elusive. In this study, we successfully constructed 29 OMP knockout strains in the pathogen Aeromonas hydrophila, which account for about 50% of all predicted OMPs in this bacterial species. We then further validated the antibiotics' susceptibility characteristics against 20 antimicrobial reagents in these mutants considering several phenotypes. Our results showed that a total of 22 OMP mutants affected the susceptibility to at least one antibiotic. The deletion of some OMPs, such as ΔlamB and ΔbamA, revealed very important roles in the resistance to certain antibiotics. However, not a single OMP mutant presented a constant behaviour to all of the tested antibiotics, suggesting the existence of a complex intercellular regulation mechanism and a protein-protein interaction network underlying the OMP homeostasis in the presence of antibiotics. Meanwhile, some OMP mutants also affected biofilm formation, ECPase and haemolytic activity, and carbon resources utilization. This report demonstrates the biological functions of OMPs on a large scale and most of results have not been reported in A. hydrophila.

The protective efficacy of four iron-related recombinant proteins and their single-walled carbon nanotube encapsulated counterparts against Aeromonas hydrophila infection in zebrafish.

Iron-related proteins play important roles in iron homeostasis, and they may be potential vaccine candidates against pathogenic Aeromonas hydrophila. In addition, the encapsulation of antigens in single-walled carbon nanotubes (SWCNTs) has recently been shown to effectively stimulate the host immune response. To investigate the immune response of zebrafish to iron-related proteins and SWCNT-encapsulated proteins, we overexpressed and purified four iron-related recombinant proteins (P55870, A0KGK5, A0KPP0, and A0KIY3) from A. hydrophila. We then vaccinated zebrafish with these proteins and their SWCNT-encapsulated counterparts via both intraperitoneal injection and bath immunization. The target proteins evoked an immune response in zebrafish after intraperitoneal injection, and SWCNT-encapsulation significantly increased the immune response after bath immunization. When challenged with virulent A. hydrophila, zebrafish administered 5 µg intraperitoneal injections of SWCNT-P55870, A0KGK5, A0KPP0, or A0KIY3 had remarkably high relative percent survivals (RPSs) (50%, 55.6%, 66.7%, and 94.44% respectively). The RPSs of zebrafish vaccinated via immunization bath with 40 mg/L SWCNT-encapsulated counterparts were also high (52.94%, 55.56%, 61.11%, and 86.11%, respectively). These results indicated that zebrafish vaccinated with P55870, A0KGK5, SWCNT-P55870, and SWCNT-A0KGK5 were partially protected, while A0KPP0 and A0KIY3 were promising vaccine candidates against pathogenic A. hydrophila infection.

Encapsulation of Phosphotungstic Acid into Metal-Organic Frameworks with Tunable Window Sizes: Screening of PTA@MOF Catalysts for Efficient Oxidative Desulfurization.

Clean fuels with extremely low sulfur content are highly desirable due to environmental concerns. Herein, three water-stable and eco-friendly metal-organic frameworks with tunable window diameters, denoted as MOF-808X, have been employed as PTA solid supports. An array of PTA@MOF-808X composites were facilely synthesized via the encapsulation strategy. With tunable window sizes and adjustable PTA loading amounts, the obtained PTA@MOF-808X composites were screened for catalytic oxidative desulfurization (ODS) with H2O2 serving as oxidant. The experiments found that 42%PTA@MOF-808A had the highest catalytic ODS activity and could completely remove dibenzothiophene (DBT) in a model fuel with an initial sulfur content of 1000 ppm within 30 min, which falls far below the acceptable limits for fuel standards (10 ppm). Further investigations revealed that this high catalytic activity could be attributed to the cooperative catalysis of metal clusters in the host framework and the guest PTA molecules. Moreover, 42%PTA@MOF-808A could be facilely recovered and reused for at least five runs without loss of catalytic activity. Having a combination of eco-sustainability, high stability, high catalytic activity, and good recyclability, 42%PTA@MOF-808A therefore represents a new benchmark material for catalytic ODS and provides a new perspective for ultradeep desulfurization.

Proteomics analysis reveals the effect of Aeromonas hydrophila sirtuin CobB on biological functions.

Our previous studies have profiled lysine acetylation and succinylation modifications in Aeromonas hydrophila protein and have found that CobB may be involved in lysine deacylation; however, its effects on bacterial biological function are still unknown. In this study, a data-independent acquisition (DIA)-based proteomics method was used to compare the protein abundance between cob-deleted mutants and wild-type strains. Of the total 2385 identified proteins, 385 were found to have increased abundance, while only 46 showed decreased abundance. Data analysis revealed that many proteins in six metabolic pathways, ribosome, the bacterial secretion system, protein export, RNA degradation, beta-Lactam resistance and oxidative phosphorylation, were affected by the deletion of cobB. Some proteins, such as outer membrane proteins, the two-component regulatory systems and transcriptional factor, were also regulated by cobB. The following phenotype assays confirmed that the ΔcobB mutant produced more biofilm, migrated farther in soft agar, and was more sensitive to oxidative stress than its WT parent. Taken together, the results presented herein provide insights into the behaviors of sirtuin protein CobB in bacteria and demonstrate its important biological functions in A. hydrophila. BIOLOGICAL SIGNIFICANCE: The sirtuin protein CobB play crucial roles on lysine deacylation, such as desuccinylation and deacetylation in many bacterial species, while the intrinsic behavior of CobB on bacteria remains elusive. The current DIA-based quantitative proteomics analysis showed that the deletion of A. hydrophila cobB significantly affect the intracellular biological processes. Further phenotype assays validated proteomics results. Overall, our data further confirmed the important roles of CobB on the complex protein-protein interaction network regulation in A. hydrophila.

Quantitative proteomics reveals the molecular mechanism of Aeromonas hydrophila in enoxacin stress.

Aeromonas hydrophila is an aquatic pathogen that has been of increasing concern in cultivation fisheries because of antibiotics resistance; however, its intrinsic antibiotics resistance mechanism remains elusive. In this study, a label-free-based proteomics method was used to compare differential protein abundances in response to enoxacin (ENX) stress in A. hydrophila. The results showed a total of 96 proteins were altered, with 43 increased and 53 decreased. Subsequent bioinformatics analysis showed that 11 SOS response-related proteins increased and four chemotaxis proteins decreased in response to ENX stress. Further antibiotics susceptibility assay showed that SOS response inhibitors such as baicalein and curcumin significantly enhanced the bactericidal capacity when compounded with ENX but not with chlortetracycline. These findings indicate the importance of SOS response in quinolones resistance. Moreover, the survival rates of five related knock-out strains (ΔuvrA, ΔahyR, ΔompW, ΔcheV, and ΔhgpB) were compared in response to different doses of ENX. Finally, the auto-inducer 1 (AI-1) quorum sensing system may negatively regulate ENX resistance in A. hydrophila. Overall, our data provide insight into the quinolones resistance mechanism and indicate that an effective SOS response inhibitor compound with ENX may be a promising strategy for the treatment of quinolones-resistant A. hydrophila. BIOLOGICAL SIGNIFICANCE: Bacterial antibiotics resistance has become a very serious public health problem, whereas its intrinsic mechanism remains elusive. To investigate the antibiotics resistant characteristics of fish pathogenic Aeromonas hydrophila, we compared the differential protein abundances in response to enoxacin (ENX) stress using proteomics method in this study. Our bioinformatics analysis showed that SOS response-related proteins increased and chemotaxis proteins decreased in response to ENX stress. Further assays showed that SOS response inhibitors such as baicalein and curcumin significantly enhanced the bactericidal capacity when compounded with ENX but not with chlortetracycline. Moreover, the antibiotics susceptibility of five related knock-out strains were valued in different doses of ENX. Overall, our data provide insight into the quinolones resistance mechanism and indicate that SOS response process and quorum sensing may involve in ENX resistance in A. hydrophila.

The LysR-Type Transcriptional Regulator YeeY Plays Important Roles in the Regulatory of Furazolidone Resistance in Aeromonas hydrophila.

Aeromonas hydrophila is an aquatic pathogen of freshwater fish. The emergence of widespread antimicrobial-resistance strains of this pathogen has caused increasing rates of fish infections. Our previous research reported that A. hydrophila yeeY, a LysR-type transcriptional regulator (LTTR), negatively regulated furazolidone (FZ) resistance. Although, it's intrinsic regulatory mechanism is still unclear. In this study, a data-independent acquisition (DIA) quantitative proteomics method was used to compare the differentially expressed proteins (DEPs) between the ΔyeeY and wild-type strain under FZ treatment. When compared to the control, a total of 594 DEPs were identified in ΔyeeY. Among which, 293 and 301 proteins were substantially increased and decreased in abundance, respectively. Bioinformatics analysis showed that several biological pathways such as the secretion system and protein transport were mainly involved in FZ resistance. Subsequently, the antibiotics susceptibility assays of several gene deletion strains identified from the proteomics results showed that YeeY may regulate some important genes such as cysD, AHA_2766, AHA_3195, and AHA_4275, which affects the FZ resistance in A. hydrophila. Furthermore, 34 antimicrobial resistance genes (ARGs) from the bacterial drug resistance gene database (CARD) were found to be directly or indirectly regulated by YeeY. A subsequent assay of several ARGs mutants showed that ΔAHA_3222 increased the susceptibility of A. hydrophila to FZ, while ΔcysN and ΔAHA_3753 decreased the susceptibility rate. Finally, the chromatin immunoprecipitation (ChIP) PCR and an electrophoretic mobility shift assay (EMSA) have revealed that the genes such as AHA_3222 and AHA_4275 were directly and transcriptionally regulated by YeeY. Taken together, our findings demonstrated that YeeY may participate in antimicrobial resistance of A. hydrophila to FZ, which provides a new target for the development of novel antimicrobial agents in the future.

Comparative metabolomics shows the metabolic profiles fluctuate in multi-drug resistant Escherichia coli strains.

In this study, two susceptible strains and two multi-drug resistant clinical Escherichia coli strains were obtained by Kirby-Bauer method, and then a GC-MS-based metabolomics method was used to compare the differential expression of metabolites between two drug sensitive (CK1 and CK2) and two multidrug-resistant (MDR1 and MDR2) clinical strains of E. coli. We characterized a total of 273 metabolites, including 77 commonly altered metabolites, between MDR vs. antibiotic sensitive strains. Interestingly, the PCA score plot clearly discriminated drug sensitive and MDR strains. The following bioinformatics analysis showed that biosynthesis of amino acids, biosynthesis of phenylpropanoids and purine metabolism were commonly enriched in MDR strains. Moreover, microbial metabolism in diverse environments, carbon metabolism，and pyrimidine metabolism pathways were more likely to be enriched MDR1 strain, while ABC transporters, and cysteine and methionine metabolism pathways were enriched in MDR2 strains. The enzyme activities in several involved metabolic pathways were further measured and metabolite candidates were validated by GC-MS-SIM method. These results indicated that antibiotic resistance affects the metabolite profiles of bacteria. In general, our study provides evidence on the study and prediction of MDR characteristics and mechanisms in bacteria at the metabolite level. BIOLOGICAL SIGNIFICANCE: Overuse and abuse of antibiotics has led to the emergence of antibiotic-resistant strains of bacteria; however, relatively little is known about their resistance mechanisms. In this study, metabolomics method was used to compare the differential expression of metabolites between sensitive and multidrug-resistant clinical strains of E. coli. Results show that the PCA score plot clearly discriminated sensitive and MDR strains, indicating that they had different metabolic profiles. Further, bioinformatics analysis showed that biosynthesis of amino acids, biosynthesis of phenylpropanoids and purine metabolism may be related to resistance. Finally, the enzyme activities in several involved metabolic pathways were further measured and metabolite candidates were validated by GC-MS-SIM method. In general, our study provides evidence on the study and prediction of MDR characteristics and mechanisms in bacteria at the metabolite level.

Comprehensive analysis of the lysine acetylome in Aeromonas hydrophila reveals cross-talk between lysine acetylation and succinylation in LuxS.

Lysine acetylation and succinylation are both prevalent protein post-translational modifications (PTMs) in bacteria species, whereas the effect of the cross-talk between both PTMs on bacterial biological function remains largely unknown. Our previously study found lysine succinylated sites on proteins play important role on metabolic pathways in fish pathogenic Aeromonas hydrophila. A total of 3189 lysine-acetylation sites were further identified on 1013 proteins of this pathogen using LC-MS/MS in this study. Functional examination of these PTMs peptides showed associations with basal biological processes, especially metabolic pathways. Additionally, when comparing the obtained lysine acetylome to a previously obtained lysine succinylome, 1198 sites in a total of 547 proteins were found to be in common and associated with various metabolic pathways. As the autoinducer-2 (AI-2) synthase involved in quorum sensing of bacteria, the site-directed mutagenesis of LuxS at the K165 site was performed and revealed that the cross-talk between lysine acetylation and succinylation exerts an inverse influence on bacterial quorum sensing and on LuxS enzymatic activity. In summary, this study provides an in-depth A. hydrophila lysine acetylome profile and for the first time reveals the role of cross-talk between lysine acetylation and succinylation, and its potential impact on bacterial physiological functions.