SaeR as a novel target for antivirulence therapy against Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen responsible for a wide range of clinical infections. SaeRS is one of the two-component systems in S. aureus that modulate multiple virulence factors. Although SaeR is required for S. aureus to develop an infection, inhibitors have not been reported. Using an in vivo knockdown method, we demonstrated that SaeR is targetable for the discovery of antivirulence agent. HR3744 was discovered through a high-throughput screening utilizing a GFP-Lux dual reporter system driven by saeP1 promoter. The antivirulence efficacy of HR3744 was tested using Western blot, Quantitative Polymerase Chain Reaction, leucotoxicity, and haemolysis tests. In electrophoresis mobility shift assay, HR3744 inhibited SaeR-DNA probe binding. WaterLOGSY-NMR test showed HR3744 directly interacted with SaeR's DNA-binding domain. When SaeR was deleted, HR3744 lost its antivirulence property, validating the target specificity. Virtual docking and mutagenesis were used to confirm the target's specificity. When Glu159 was changed to Asn, the bacteria developed resistance to HR3744. A structure-activity relationship study revealed that a molecule with a slight modification did not inhibit SaeR, indicating the selectivity of HR3744. Interestingly, we found that SAV13, an analogue of HR3744, was four times more potent than HR3744 and demonstrated identical antivirulence properties and target specificity. In a mouse bacteraemia model, both HR3744 and SAV13 exhibited in vivo effectiveness. Collectively, we identified the first SaeR inhibitor, which exhibited in vitro and in vivo antivirulence properties, and proved that SaeR could be a novel target for developing antivirulence drugs against S. aureus infections.

Construction of an attenuated glutamyl endopeptidase deletion strain of Nocardia seriolae.

The glutamyl endopeptidase homolog of Nocardia seriolae (GluNS) has been proved to be a potential virulence factor in our previous study. Present investigation was carried out to construct an attenuated N. seriolae strain by deletion with GluNS gene and evaluate its protective immunity in head snakehead. A deletion strain (NS-ΔGluNS) was established by knockout of gene GluNS from wild strain N. seriolae ZJ0503 via homologous recombination. The LD50 of NS-ΔGluNS in 3.41 × 106 cfu/mL was significantly increased than that of wild strain in 4.75 × 105 cfu/mL, indicating that the virulence of N. seriolae has been attenuated with the knockout of GluNS. Meanwhile, applying NS-ΔGluNS as an attenuated live vaccine to immune hybrid snakehead, the non-specific immunity parameters (serum LYZ, POD, ACP, and AKP activities), specific antibody (IgM) titers production and immune-related genes (MHCIα, CD4, and IL-8) expression were up-regulated in different tissues, which indicated that they were able to trigger humoral and cell-mediated immune responses. Furthermore, the protective efficacy in hybrid snakehead after vaccination with NS-ΔGluNS shown 73.53% relative percentage survival (RPS). Taken together, the attenuated NS-ΔGluNS was obtained successfully and it could elicit strong immune response and supply protective efficacy to hybrid snakehead against N. seriolae wild strain.

Dark secrets of phytomelatonin.

Phytomelatonin is a newly identified plant hormone, and its primary functions in plant growth and development remain relatively poorly appraised. Phytomelatonin is a master regulator of reactive oxygen species (ROS) signaling and acts as a darkness signal in circadian stomatal closure. Plants exhibit at least three interrelated patterns of interaction between phytomelatonin and ROS production. Exogenous melatonin can induce flavonoid biosynthesis, which might be required for maintenance of antioxidant capacity under stress, after harvest, and in leaf senescence conditions. However, several genetic studies have provided direct evidence that phytomelatonin plays a negative role in the biosynthesis of flavonoids under non-stress conditions. Phytomelatonin delays flowering time in both dicot and monocot plants, probably via its receptor PMTR1 and interactions with the gibberellin, strigolactone, and ROS signaling pathways. Furthermore, phytomelatonin signaling also functions in hypocotyl and shoot growth in skotomorphogenesis and ultraviolet B (UV-B) exposure; the G protein α-subunit (Arabidopsis GPA1 and rice RGA1) and constitutive photomorphogenic1 (COP1) are important signal components during this process. Taken together, these findings indicate that phytomelatonin acts as a darkness signal with important regulatory roles in circadian stomatal closure, flavonoid biosynthesis, flowering, and hypocotyl and shoot growth.

Melatonin increases leaf disease resistance and saponin biosynthesis in Panax notogiseng.

Panax notoginseng (Bruk.) FH Chen is a valuable traditional herb in China, with saponins being the main medicinal components in its roots. However, leaf diseases are a major factor limiting growth and production of P. notoginseng. Melatonin is a ubiquitous signaling molecule associated with abiotic stress resistance. In this study, we investigated the role of melatonin in leaf disease resistance of P. notoginseng in field conditions. Additionally, saponin concentrations were analyzed to evaluate the suitability of melatonin use in agricultural practice. Our results showed that exogenous application of melatonin promoted the endogenous phytomelatonin accumulation via upregulation of genes involved in its biosynthesis. The application of 10 µM melatonin decreased the incidence of leaf diseases (gray mold, round spot, and black spot) by about 40% compared with the solvent control, which might have been due to the increased expression of genes associated with immunity and disease resistance. Furthermore, concentrations of saponins and expression of their biosynthesis-related genes were significantly increased by melatonin. Taken together, the data presented here suggested that melatonin could be used in agricultural management of P. notoginseng because it increased leaf disease resistance and biosynthesis of saponins.

Identification of a secreted superoxide dismutase (SOD) from Nocardia seriolae which induces apoptosis in fathead minnow (FHM) cells.

Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is the main pathogen. The pathogenesis and virulence factors of N. seriolae are not fully understood. Secreted superoxide dismutase (SOD) may be a virulence factor found by a comparative bioinformatics analysis of the whole genome sequence of N. seriolae and the virulence factor database (VFDB). In order to determine the subcellular localization and study the preliminary function of SOD from N. seriolae (NsSOD), gene cloning, secreted protein identification, subcellular localization in fish cells, and apoptosis detection of NsSOD were carried out in this study. Subcellular localization research revealed that NsSOD-GFP fusion proteins were evenly distributed in the cytoplasm. Furthermore, apoptotic bodies were observed in the transfected FHM cells by the overexpression of protein NsSOD. Then, assays of mitochondrial membrane potential (ΔΨm) value, caspase-3 activity and apoptosis-related genes (Bax, Bid, Bad and Bcl-2) mRNA expression were conducted. The results showed that ΔΨm was decreased, and caspase-3 was significantly activated. The mRNA expression of the Bad gene showed significant up-regulated expression at 24 h.p.t., while Bid and Bax genes showed significant up-regulated expression at 72 and 96 h.p.t. and anti-apoptotic gene (Bcl-2) was down-regulated in NsSOD overexpressed cells. Taken together, the results indicated that the protein NsSOD might be involved in apoptosis regulation. This study may lay the foundations for further studies on the function of NsSOD and promote the understanding of the virulence factors and the pathogenic mechanisms of N. seriolae.

Characterization and function study of a glutamyl endopeptidase homolog from Nocardia seriolae.

Glutamic endopeptidases (Glu), belonging to the class of serine proteases, are a subfamily of chymotrypsin-like proteolytic enzymes, which are regarded as important virulence factors in bacteria. However, the roles of glutamic endopeptidases of Nocardia seriolae in pathogenic process still remain uncertain. Here, a glutamic endopeptidase homolog from N. seriolae (GluNS) was cloned and its function was elucidated. GluNS encoded a 414-aa protein which shared 93% identity to N. concava. In the phylogenetic tree, the glutamic endopeptidases of genus Nocardia clustered together firstly and then clustered with Streptomyces species. Moreover, GluNS was identified to be a secreted protein of N. seriolae and localized in the mitochondria of FHM cells. The transient overexpression of GluNS significantly induced increase in caspase-3 activity and decrease in ΔΨm values in FHM cells. The number of apoptotic bodies was remarkably higher than that in control group. Taken together, GluNS overexpression induced apoptotic characteristics in FHM cells. This study provided new insights into the function of glutamic endopeptidase from N. seriolae.

Identification of a cell-wall peptidase (NlpC/P60) from Nocardia seriolae which induces apoptosis in fathead minnow cells.

Nocardia seriolae, a Gram-positive bacterium, is the main pathogen of fish nocardiosis. Protein NlpC/P60 is a cell-wall peptidase and a potential virulence factor of N. seriolae. Subcellular localization research revealed that both NlpC/P60-GFP and NlpC/P60Δsig-GFP fusion proteins were evenly distributed in the whole cell of fathead minnow (FHM) cells. Furthermore, typical apoptotic features, such as nuclear pyrosis and apoptotic bodies, were observed in the transfected FHM cells and grouper spleen cells by the overexpression of protein NlpC/P60. Then, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase-3 activity and apoptosis-related gene (Bax, BNIP3, TNF1 and TNF6) mRNA expression were conducted. The results showed that ΔΨm was decreased, caspase-3 was significantly activated, and the mRNA expression of pro-apoptotic genes (Bax and BNIP3) and tumour necrosis factors (TNF1 and TNF6) was up-regulated in NlpC/P60-overexpressed cells. Taken together, the results indicated that the protein NlpC/P60 of N. seriolae might involve in apoptosis regulation. This study may lay the foundation for further study on the function of N. seriolae NlpC/P60 and promote the understanding of the virulence factors and pathogenic mechanism of N. seriolae.

Development of DNA vaccines encoding ribosomal proteins (RplL and RpsA) against Nocardia seriolae infection in fish.

Nocardia seriolae, a Gram-positive pathogen, has been identified as the causative agent of fish nocardiosis. DNA vaccination has been proven to be effective in conferring protection against bacterial infection in fish. The 30S ribosomal protein S1 (RpsA) and 50S ribosomal protein L7/L12 (RplL) were identified to be the common immunodominant antigens of three fish pathogenic Nocardia (N. seriolae, N. salmonicida and N. asteroids) by immunoproteomics profiling in our previous study. In current study, the immunogenicity and protective efficacy of two DNA vaccines encoding RplL and RpsA were evaluated and compared in hybrid snakehead. The results showed vaccination of hybrid snakehead with the pcDNA-RplL and pcDNA-RpsA DNA vaccines provided protective efficacy with relative percentage survival (RPS) of 78.31% and 71.08%, respectively. Meanwhile, the immune response of hybrid snakehead induced by these two DNA vaccines were investigated, and it revealed that the non-specific immunity parameters (serum lysozyme (LYZ), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) activities), specific antibody (IgM) production and immune-related genes expression (MHCIα, MHCIIα, CD4, CD8α, IL-1ß and TNFα) were significantly increased compared with the corresponding control groups after immunization. Taken together, these results indicated that both pcDNA-RplL and pcDNA-RpsA DNA vaccines could boost the innate, humoral and cellular immune responses in hybrid snakehead and show highly protective efficacy against fish nocardiosis, suggesting that ribosomal proteins RplL and RpsA were promising candidates for DNA vaccines and it will promote the vaccine development against fish nocardiosis.

Identification of common antigens of three pathogenic Nocardia species and development of DNA vaccine against fish nocardiosis.

Fish nocardiosis is a chronic granulomatous bacterial disease and three pathogens have been reported so far, including Nocardia asteroids, N. seriolae and N. salmonicida. However, the absence of antigen markers is a bottleneck for developing effective vaccines against fish nocardiosis. In this study, the antigenicity of whole-cell protein of these three pathogenic Nocardia species were profiled by immunoproteomic analysis and 7 common immunogenic proteins were identified as follows: molecular chaperone DnaK (DnaK), molecular chaperone GroEL (GroEL), 30 S ribosomal protein S1 (RpsA), TerD family protein (TerD), FHA domain-containing protein (FHA), 50 S ribosomal protein L7/L12 (RplL) and PspA/IM30 family protein (PspA). Furthermore, the DNA vaccine encoding FHA gene against fish nocardiosis was developed and its efficacy was investigated in hybrid snakehead. The results suggested that it needed at least 7 d to transport pcDNA-FHA DNA vaccine from injected muscle to head kidney, spleen and liver and stimulate host's immune system for later protection. In addition, non-specific immunity paraments (serum lysozyme (LYZ), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) activities), specific antibody (IgM) titers production and immune-related genes (MHCIα, MHCIIα, CD4, CD8α, IL-1ß and TNFα) were used to evaluate the immune response induced in pcDNA-FHA vaccinated hybrid snakehead, it proved that all these mentioned immune activities were significantly enhanced after immunization. The results also showed hybrid snakehead vaccinated with pcDNA-FHA had higher survival rate (79.33%) compared with the controls after challenge with N. seriolae, indicating that the pcDNA-FHA DNA vaccine can supply immune protection against N. seriolae infection. Taken together, this study may warrant further development of these common immunogenic proteins as the antigens for vaccine or diagnosis and facilitate the prevention and treatment of fish nocardiosis.

Comparison of protective efficacy between two DNA vaccines encoding DnaK and GroEL against fish nocardiosis.

Fish nocardiosis is a chronic granulomatous bacterial disease mainly caused by three pathogenic bacteria, including Nocardia seriolae, N. asteroids and N. salmonicida. Molecular chaperone DnaK and GroEL were identified to be the common antigens of the three pathogenic Nocardia species in our previous studies. To evaluate the immune protective effect of two DNA vaccines encoding DnaK or GroEL against fish nocardiosis, hybrid snakehead were vaccinated and the immune responses induced by these two vaccines were comparatively analyzed. The results suggested it needed at least 7 d to transport DnaK or GroEL gene from injected muscle to head kidney, spleen and liver and stimulate host's immune system for later protection after immunization by DNA vaccines. Additionally, non-specific immunity parameters (serum lysozyme (LYZ), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) activities), specific antibody (IgM) production and immune-related genes (MHCIα, MHCIIα, CD4, CD8α, IL-1ß and TNFα) were used to evaluate the immune responses induced in vaccinated hybrid snakehead. It proved that all the above-mentioned immune activities were significantly enhanced after immunization with these two DNA vaccines. The protective efficacy of pcDNA-DnaK and pcDNA-GroEL DNA vaccines, in terms of relative percentage survival (RPS), were 53.01% and 80.71% respectively. It demonstrated that these two DNA vaccines could increase the survival rate of hybrid snakehead against fish nocardiosis, albeit with variations in immunoprotective effects. Taken together, these results indicated that both pcDNA-DnaK and pcDNA-GroEL DNA vaccines could boost the innate, humoral and cellular immune response in hybrid snakehead and show highly protective efficacy against fish nocardiosis, suggesting that DnaK and GroEL were promising vaccine candidates. These findings will promote the development of DNA vaccines against fish nocardiosis in aquaculture.

Identification of a mitochondrial-targeting secretory protein from Nocardia seriolae which induces apoptosis in fathead minnow cells.

Nocardia seriolae is the main pathogen responsible for fish nocardiosis. A mitochondrial-targeting secretory protein (MTSP) 3141 with an N-terminal transit peptide (TP) from N. seriolae was predicted by bioinformatic analysis based on the genomic sequence of the N. seriolae strain ZJ0503. However, the function of the MTSP3141 and its homologs remains totally unknown. In this study, mass spectrometry analysis of the extracellular products from N. seriolae proved that MTSP3141 was a secretory protein, subcellular localization research showed the MTSP3141-GFP fusion protein co-localized with mitochondria in fathead minnow (FHM) cells, the TP played an important role in mitochondria targeting, and only the TP located at N-terminus but not C-terminus can lead to mitochondria directing. Moreover, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase-3 activity and apoptosis-related gene (Bcl-2, Bax, Bad, Bid and p53) mRNA expression suggested that cell apoptosis was induced in FHM cells by the overexpression of both MTSP3141 and MTSP3141ΔTP (with the N-terminal TP deleted) proteins. Taken together, the results of this study indicated that the MTSP3141 of N. seriolae was a secretory protein, might target mitochondria, induce apoptosis in host cells and function as a virulence factor.

Identification and functional characterization of Histone-like DNA-binding protein in Nocardia seriolae (NsHLP) involved in cell apoptosis.

Nocardia seriolae, a facultative intracellular bacterium, is the main pathogen of fish nocardiosis. Bioinformatic analysis showed that the histone-like DNA-binding protein (HLP) gene of N. seriolae (nshlp) encoded a secreted protein and might target the mitochondria in the host cell. To further study the preliminary function of HLP in N. seriolae (NsHLP), the gene cloning, extracellular products identification, subcellular localization, overexpression and apoptosis detection assay were carried out in this study. Mass spectrometry analysis of the extracellular products from N. seriolae showed that NsHLP was a secreted protein. Subcellular localization of HLP-GFP fusion proteins mainly assembled in the nucleus, which indicated that the NsHLP was co-located with the nucleus rather than mitochondria in fathead minnow (FHM) cells. Notably, the expression of NsHLP had changed the distribution of mitochondria into lumps in the FHM cell. In addition, apoptotic features were found in the transfected FHM cells by overexpression of NsHLP. Quantitative assays of mitochondrial membrane potential value, caspase-3 activity and pro-apoptotic genes mRNA (Bad, Bid and Bax) expression level demonstrated that the cell apoptosis was induced in the transfected FHM cells. All the results presented in this study provided insight on the function of NsHLP, which suggested that it may participate in the cell apoptosis regulation and play an important role in the pathogenesis of N. seriolae.

Transcriptome analysis of immune-related gene expression in hybrid snakehead (Channa maculata ♀ × Channa argus ♂) after challenge with Nocardia seriolae.

Hybrid snakehead fish (Channa maculata ♀ × Channa argus ♂), a new species used in freshwater aquaculture in China, is the common host of an epizootic bacterial infection by Nocardia seriolae. However, the information on the functions and mechanisms of hybrid snakehead immune pathways with the N. seriolae infection is limited. Thus, the peripheral blood lymphocytes from hybrid snakehead were used for transcriptome analysis to understand the host immune response after challenge with N. seriolae. A total of 49,839,332 and 50,059,283 raw reads were obtained from the N. seriolae-challenged group (Ns group) and phosphate-buffered saline control group (Ctr group), respectively. The 75.50% and 74.25% reads from the Ns and Ctr groups were matched to reference genomic sequence after cleaning the raw reads, respectively. Additionally, there were 2892 significant differentially expressed genes (DEGs) among the 17,196 expressed genes between the Ns and Ctr groups, including 1387 upregulated and 1505 downregulated genes. All the DEGs were classified into three gene ontology categories, and 2502 DEGs had significant matches, which were allocated to 246 Kyoto Encyclopedia of Genes and Genomes pathways. Immune-related genes were detected from immune system pathways among the top 20 enriched pathways. Moreover, the regulation of several observed effective genes was confirmed by real-time quantitative polymerase chain reaction. Altogether, this study offers deep-sequence data of hybrid snakehead peripheral blood lymphocyte via transcriptome analysis and lays the foundation for further study on the immunogenetics of hybrid snakehead. Moreover, it provides insights into the pathogenic mechanism of N. seriolae, facilitating the prevention and treatment of fish nocardiosis.

Complex-surfactant-assisted hydrothermal synthesis and properties of hierarchical worm-like cobalt sulfide microtubes assembled by hexagonal nanoplates.

The preparation of exquisite hierarchical worm-like Co(1-x)S (x = 0.75) microtubes by a one-pot complex-surfactant-assisted hydrothermal method is successfully achieved for the first time. The hierarchical structures of the microtube wall are assembled from numerous interleaving hexagonal nanoplates. X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy, transmission electron microscopy, and high-resolution transmission electron microscopy were used to characterize the samples. The experimental results indicate that the "soft template" surfactant cetyltrimethylammonium bromide and the chelating ethylenediamine both play important roles for the formation of hierarchical Co(1-x)S microtubes. A possible formation mechanism for the growth processes is proposed. Additionally, the electrochemical and magnetic properties of Co(1-x)S microtubes were systematically studied.

Solvothermal synthesis and magnetic properties of 3D flower-like NiHPO(3).H(2)O superstructures.

A self-assembled three-dimensional (3D) flower-like NiHPO(3).H(2)O micro/nanocomposite structure has been successfully synthesized for the first time by a simple solvothermal method, employing Na(6)P(6)O(18) as the phosphor source without the assistance of any template or surfactant. The as-obtained product was characterized by X-ray powder diffraction, scanning electron microscopy and transmission electron microscopy. The formation mechanism was proposed based on the evolution of this morphology as a function of solvothermal time. Some factors influencing the morphology of the NiHPO(3).H(2)O flower-like micro/nanocomposite structures were systematically investigated. The magnetic measurements demonstrated that the flower-like 3D superstructures exhibit ferromagnetic property.