Ubiquitin-based pathway acts inside chloroplasts to regulate photosynthesis.

Photosynthesis is the energetic basis for most life on Earth, and in plants it operates inside double membrane-bound organelles called chloroplasts. The photosynthetic apparatus comprises numerous proteins encoded by the nuclear and organellar genomes. Maintenance of this apparatus requires the action of internal chloroplast proteases, but a role for the nucleocytosolic ubiquitin-proteasome system (UPS) was not expected, owing to the barrier presented by the double-membrane envelope. Here, we show that photosynthesis proteins (including those encoded internally by chloroplast genes) are ubiquitinated and processed via the CHLORAD pathway: They are degraded by the 26S proteasome following CDC48-dependent retrotranslocation to the cytosol. This demonstrates that the reach of the UPS extends to the interior of endosymbiotically derived chloroplasts, where it acts to regulate photosynthesis, arguably the most fundamental process of life.

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.

Lipidomic profiling of the developing kernel clarifies the lipid metabolism of Paeonia ostii.

Lipid components in the developing kernel of Paeonia ostii were determined, and the fatty acid (FA) distributions in triacylglycerol and phospholipids were characterized. The lipids in the kernel were mainly phospholipids (43%), neutral glycerides (24%), fatty acyls (26%), and sphingolipids (4.5%). The dominant neutral glycerides were TAG and diacylglycerol. The PL components included phosphatidic acid, phosphatidyl glycerol, phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, and phosphatidyl ethanolamine. As the kernel developed, the profiles of the molecular species comprising TAG and PL changed, especially during the earlier phases of oil accumulation. During rapid oil accumulation, the abundances of sphingosine-1-phosphate, pyruvic acid, stearic acid, and alpha-linolenic acid changed significantly; the sphingolipid metabolism and unsaturated FAs biosynthesis pathways were significantly enriched in these differentially abundant metabolites. Our results improve our understanding of lipid accumulation in tree peony seeds, and provide a framework for the analysis of lipid metabolisms in other oil crops.

Transcriptomic analysis of α-linolenic acid content and biosynthesis in Paeonia ostii fruits and seeds.

BACKGROUND: Paeonia ostii is a potentially important oilseed crop because its seed yield is high, and the seeds are rich in α-linolenic acid (ALA). However, the molecular mechanisms underlying ALA biosynthesis during seed kernel, seed testa, and fruit pericarp development in this plant are unclear. We used transcriptome data to address this knowledge gap. RESULTS: Gas chromatograph-mass spectrometry indicated that ALA content was highest in the kernel, moderate in the testa, and lowest in the pericarp. Therefore, we used RNA-sequencing to compare ALA synthesis among these three tissues. We identified 227,837 unigenes, with an average length of 755 bp. Of these, 1371 unigenes were associated with lipid metabolism. The fatty acid (FA) biosynthesis and metabolism pathways were significantly enriched during the early stages of oil accumulation in the kernel. ALA biosynthesis was significantly enriched in parallel with increasing ALA content in the testa, but these metabolic pathways were not significantly enriched during pericarp development. By comparing unigene transcription profiles with patterns of ALA accumulation, specific unigenes encoding crucial enzymes and transcription factors (TFs) involved in de novo FA biosynthesis and oil accumulation were identified. Specifically, the bell-shaped expression patterns of genes encoding SAD, FAD2, FAD3, PDCT, PDAT, OLE, CLE, and SLE in the kernel were similar to the patterns of ALA accumulation in this tissue. Genes encoding BCCP, BC, KAS I- III, and FATA were also upregulated during the early stages of oil accumulation in the kernel. In the testa, the upregulation of the genes encoding SAD, FAD2, and FAD3 was followed by a sharp increase in the concentrations of ALA. In contrast, these genes were minimally expressed (and ALA content was low) throughout pericarp development. CONCLUSIONS: We used three tissues with high, moderate, and low ALA concentrations as an exemplar system in which to investigate tissue-specific ALA accumulation mechanisms in P. ostii. The genes and TFs identified herein might be useful targets for future studies of ALA accumulation in the tree peony. This study also provides a framework for future studies of FA biosynthesis in other oilseed plants.

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.

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.

Quantitative Proteomics Reveals Antibiotics Resistance Function of Outer Membrane Proteins in Aeromonas hydrophila.

Outer membrane proteins (OMPs) play essential roles in antibiotic resistance, particularly in Gram-negative bacteria; however, they still have many unidentified functions regarding their behavior in response to antibiotic stress. In the current work, quantitative tandem mass tag labeling-based mass spectrometry was used to compare the outer membrane related proteins between an oxytetracycline-resistant (OXY-R) and its original control stain (OXY-O) in Aeromonas hydrophila. Consequently, a total of 261 commonly altered proteins in two biological repeats were identified including 29 proteins that increased and 28 that decreased. Gene ontology analysis showed that the expression of transport proteins was significantly reduced, and translation-related proteins were downregulated in the OXY-R strain. After using western blotting to validate selected altered proteins, eight OMP-related genes were knocked out and their roles in antibiotic resistance were further evaluated. The survival assays showed that some mutants such as ΔAHA_4281, ΔAHA_2766, ΔAHA_2282, ΔAHA_1181, and ΔAHA_1280 affected the susceptibility of A. hydrophila to antimicrobials. Moreover, the minimum inhibitory concentration assay showed that these candidate mutants also respond differently to other types of antibiotics. Our results reveal several novel outer membrane related proteins of A. hydrophila that play important roles in antibiotic resistance, and as such, may be helpful for screening studies to identify novel drug targets.

In-Silico Prediction and Modeling of the Quorum Sensing LuxS Protein and Inhibition of AI-2 Biosynthesis in Aeromonas hydrophila.

luxS is conserved in several bacterial species, including A. hydrophila, which causes infections in prawn, fish, and shrimp, and is consequently a great risk to the aquaculture industry and public health. luxS plays a critical role in the biosynthesis of the autoinducer-2 (AI-2), which performs wide-ranging functions in bacterial communication, and especially in quorum sensing (QS). The prediction of a 3D structure of the QS-associated LuxS protein is thus essential to better understand and control A. hydrophila pathogenecity. Here, we predicted the structure of A. hydrophila LuxS and characterized it structurally and functionally with in silico methods. The predicted structure of LuxS provides a framework to develop more complete structural and functional insights and will aid the mitigation of A. hydrophila infection, and the development of novel drugs to control infections. In addition to modeling, the suitable inhibitor was identified by high through put screening (HTS) against drug like subset of ZINC database and inhibitor ((-)-Dimethyl 2,3-O-isopropylidene-l-tartrate) molecule was selected based on the best drug score. Molecular docking studies were performed to find out the best binding affinity between LuxS homologous or predicted model of LuxS protein for the ligand selection. Remarkably, this inhibitor molecule establishes agreeable interfaces with amino acid residues LYS 23, VAL 35, ILE76, and SER 90, which are found to play an essential role in inhibition mechanism. These predictions were suggesting that the proposed inhibitor molecule may be considered as drug candidates against AI-2 biosynthesis of A. hydrophila. Therefore, (-)-Dimethyl 2,3-O-isopropylidene-l-tartrate inhibitor molecule was studied to confirm its potency of AI-2 biosynthesis inhibition. The results shows that the inhibitor molecule had a better efficacy in AI-2 inhibition at 40 µM concentration, which was further validated using Western blotting at a protein expression level. The AI-2 bioluminescence assay showed that the decreased amount of AI-2 biosynthesis and downregulation of LuxS protein play an important role in the AI-2 inhibition. Lastly, these experiments were conducted with the supplementation of antibiotics via cocktail therapy of AI-2 inhibitor plus OXY antibiotics, in order to determine the possibility of novel cocktail drug treatments of A. hydrophila infection.

Reprint of: Quantitative proteomic analysis reveals that chemotaxis is involved in chlortetracycline resistance of Aeromonas hydrophila.

In recent years, Aeromonas hydrophila, which has been classified as a food borne pathogen, has presented with increased levels of antibiotic resistance, with the mechanisms of this resistance being poorly understood. In this study, iTRAQ coupled mass spectrometry was employed to compare differentially expressed proteins in chlortetracycline (CTC) resistant A. hydrophila relative to a control strain. Result showed that a total of 234 differential proteins including 151 down-regulated and 83 up-regulated were identified in chlortetracycline resistance strain. Bioinformatics analysis showed that chemotaxis related proteins, such as CheA-2, CheR-3, CheW-2, EnvZ, PolA, FliS and FliG were down-regulated in addition to previously reported tricarboxylic acid cycle (TCA) related proteins also being down-regulated. A subset of identified differentially expressed proteins was then further validated via Western blotting. Exogenous metabolite combined with CTC further enhanced the bacterial susceptibilities to CTC in A. hydrophila. Furthermore, a bacterial survival capability assay showed that several chemotaxis related mutants, such as ΔcheR-3 and ΔAHA_0305, may affect the antimicrobial susceptibility of A. hydrophila. Overall, these findings contribute to a further understanding of the mechanism of CTC resistance in A. hydrophila and may contribute to the development of more effective future treatments. BIOLOGICAL SIGNIFICANCE: A. hydrophila is a well-known fish pathogenic bacterium and has presented with increasing levels of antibiotic resistance, with the mechanisms of this resistance being poorly understood. Our current study compared the differentially expression proteins between chlortetracycline (CTC) resistant and control stains via an iTARQ-based quantitative proteomics method. Chemotaxis related proteins were down-regulated in CTC resistant strain but exogenous metabolite addition increased bacterial susceptibility in A.hydrophila. Significantly, chemotaxis related genes depletion affected antimicrobial susceptibilities of A.hydrophila indicating the role of chemotaxis process in antibiotics resistance.

Quantitative proteomic analysis reveals that chemotaxis is involved in chlortetracycline resistance of Aeromonas hydrophila.

In recent years, Aeromonas hydrophila, which has been classified as a food borne pathogen, has presented with increased levels of antibiotic resistance, with the mechanisms of this resistance being poorly understood. In this study, iTRAQ coupled mass spectrometry was employed to compare differentially expressed proteins in chlortetracycline (CTC) resistant A. hydrophila relative to a control strain. Result showed that a total of 234 differential proteins including 151 down-regulated and 83 up-regulated were identified in chlortetracycline resistance strain. Bioinformatics analysis showed that chemotaxis related proteins, such as CheA-2, CheR-3, CheW-2, EnvZ, PolA, FliS and FliG were down-regulated in addition to previously reported tricarboxylic acid cycle (TCA) related proteins also being down-regulated. A subset of identified differentially expressed proteins was then further validated via Western blotting. Exogenous metabolite combined with CTC further enhanced the bacterial susceptibilities to CTC in A. hydrophila. Furthermore, a bacterial survival capability assay showed that several chemotaxis related mutants, such as ΔcheR-3 and ΔAHA_0305, may affect the antimicrobial susceptibility of A. hydrophila. Overall, these findings contribute to a further understanding of the mechanism of CTC resistance in A. hydrophila and may contribute to the development of more effective future treatments. BIOLOGICAL SIGNIFICANCE: A. hydrophila is a well-known fish pathogenic bacterium and has presented with increasing levels of antibiotic resistance, with the mechanisms of this resistance being poorly understood. Our current study compared the differentially expression proteins between chlortetracycline (CTC) resistant and control stains via an iTARQ-based quantitative proteomics method. Chemotaxis related proteins were down-regulated in CTC resistant strain but exogenous metabolite addition increased bacterial susceptibility in A.hydrophila. Significantly, chemotaxis related genes depletion affected antimicrobial susceptibilities of A.hydrophila indicating the role of chemotaxis process in antibiotics resistance.

Global protein expression profile response of planktonic Aeromonas hydrophila exposed to chlortetracycline.

The antibiotics resistance phenomena of Aeromonas hydrophila has become serious economic and public health problems for the world aquaculture industry and human health care. In this study, to investigate the instinct antibiotics adaptive mechanism of this pathogen, iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) based quantitative proteomics technologies were performed to compare the differential expression of A. hydrophila in planktonic status in response to chlortetracycline (CTC) stress and then identified total 1552 proteins including 285 altered proteins with 90 increasing and 195 decreasing abundance proteins. The following bioinformatics analysis showed that many metabolic metabolism pathways such as carbon metabolism, pyruvate metabolism, and glycolysis/gluconeogenesis were trend to down-regulated whereas ß-Lactam resistance, RNA degradation, and amino acids biosynthesis processes were more likely to increase in CTC stress. The related pyruvate metabolism and ß-Lactam resistance processes in mRNA level were further measured using the q-PCR method. Thus, an understanding of the behaviors of A. hydrophila in response to CTC would be helpful to reveal the antibiotics adaptive mechanism and for the development of novel antibiotics therapy.

Proteomic Analysis Reveals That Metabolic Flows Affect the Susceptibility of Aeromonas hydrophila to Antibiotics.

The overuse of antibiotics results in the development of antibiotic resistance and limits the useful life of these drugs in fighting bacteria, including Aeromonas hydrophila, a well-known opportunistic pathogen that causes serious infections in fish and other animals. In this study, we investigated the adaptive resistance mechanism in A. hydrophila by multiple proteomic methods. Dimethyl labeling and label-free methods were performed to compare the differential expression of proteins in response to various doses of oxytetracycline (OXY). The results point to the conclusions that, in response to OXY stress, translational processes increase the abundance of these proteins whereas largely central metabolic pathways decrease their abundance. To confirm our hypothesis, various exogenous metabolites were compounded with OXY, and the resulting survival capabilities were measured. Results show that 7 metabolites (malic acid, serine, methionine, etc.) significantly decreased the survival capabilities of A. hydrophila in the presence of OXY, whereas 4 metabolites (arginine, lysine, tyrosine, etc.) did the opposite. Further investigation suggests that a compound comprising exogenous metabolites in combination with various antibiotics could have a significant bactericidal effect and might come into widespread use, especially together with tetracycline antibiotics. These findings may provide new clues to the antimicrobial treatment of A. hydrophila infection.

Quantitative proteomic analysis of cell envelope preparations under iron starvation stress in Aeromonas hydrophila.

BACKGROUND: Iron homeostasis is an essential process over the entire lives of both hosts and bacterial pathogens, and also plays roles in many other metabolic functions. Currently, knowledge is limited on the iron scavenging mechanism of the cell envelope in the aquatic pathogen, Aeromonas hydrophila. To understand the iron homeostasis mechanism in A. hydrophila, a dimethyl labelling based quantitative proteomics method was used to compare the differential expression of cell envelope proteins under iron starvation. RESULTS: A total of 542 cell envelope proteins were identified by LC-MS/MS, with 66 down-regulated and 104 up-regulated proteins. Bioinformatics analysis showed that outer membrane siderophores, heme and iron receptors, periplasmic iron binding proteins, inner membrane ABC transporters and H(+)-ATP synthase subunits increased in abundance while iron-cluster proteins, electron transport chain and redox proteins were down-regulated. Further q-PCR validation, in vivo addition of exogenous metabolites, and an enzyme inhibition assay revealed that redox, the energy generation process, and ATP synthase elevated the susceptibility of A. hydrophila to iron starvation. CONCLUSIONS: Our study demonstrates that the redox and energy generation process, and ATP synthase in A. hydrophila may play critical roles in iron acquisition under conditions of iron-stress. An understanding of the iron scavenging mechanism may be helpful for the development of strategies for preventing and treating A. hydrophila infection.

Proteomics Analysis Reveals a Potential Antibiotic Cocktail Therapy Strategy for Aeromonas hydrophila Infection in Biofilm.

Antibiotic fitness and acquired resistance are the two critical factors when bacteria respond to antibiotics, and the correlations and mechanisms between these two factors remain largely unknown. In this study, a TMT-labeling-based quantitative proteomics method was used to compare the differential expression of proteins between the fitness and acquired resistance to chlortetracycline in Aeromonas hydrophila biofilm. Bioinformatics analysis showed that translation-related ribosomal proteins, such as 30s ribosome subunits, increased in both factors; fatty acid biosynthesis related proteins, such as FabB, FabD, FabG, AccA, and AccD, increased in biofilm fitness, and some pathways (including propanoate-metabolism-related protein, such as PrpB, AtoB, PflB, AcsA, PrpD, and GabT) displayed decreased abundance in acquired resistance biofilm. The varieties of selected proteins involved in fatty acid biosynthesis and propanoate metabolism were further validated by q-PCR assay or Western blotting. Furthermore, the antibiotic-resistance-function assays showed that fatty-acid biosynthesis should be a protective antibiotics-resistance mechanism and a cocktail of chlortetracycline and triclosan, a fatty-acid-biosynthesis inhibitor, exhibited more efficient antimicrobial capability than did each antibiotic individually on biofilm, specifically on chlortetracycline-sensitive biofilm. We therefore demonstrate that the up-regulation of fatty acid biosynthesis may play an important role in antibiotic resistance and suggest that a cocktail of chlortetracycline and triclosan may be a potential cocktail therapy for pathogenic infections in biofilm.

An integrated quantitative and targeted proteomics reveals fitness mechanisms of Aeromonas hydrophila under oxytetracycline stress.

To date, above ten thousand tons of antibiotics are used in aquaculture each year that lead to the deterioration of natural resources. However, knowledge is limited on the molecular biological behavior of common aquatic pathogens against antibiotics stress. In this study, proteomics profiles of Aeromonas hydrophila, which were exposed to different levels of oxytetracycline (OXY) stress, were displayed and compared using iTRAQ labeling and SWATH-MS based LC-MS/MS methods. A total 1383 proteins were identified by SWATH-MS method, and 2779 proteins were identified from iTRAQ labeling samples. There are 152 up-regulated and 52 down-regulated proteins overlapped in 5 µg/mL OXY stress and both 83 up- and down-regulated proteins overlapped in 10 µg/mL OXY stress in both methods, respectively. Results show that many protein synthesis and translation related proteins increased, while energy generation related proteins decreased in OXY stress. The varieties of selected proteins involved in both pathways were further validated by sMRM(HR), q-PCR, and enzyme activity assay. Furthermore, the concentrations of NAD+ and NADH were measured to verify the characteristic of energy generation process in OXY stress and OXY resistance strain. We demonstrate that the down-regulation of energy generation related metabolic pathways and up-regulation of translation may play an important role in antibiotics fitness or resistance of aquatic pathogens.