Edwardsiella tarda Attenuates Virulence upon Oxytetracycline Resistance.

The relationship between antibiotic resistance and bacterial virulence has not yet been fully explored. Here, we use Edwardsiella tarda as the research model to investigate the proteomic change upon oxytetracycline resistance (LTB4-ROTC). Compared to oxytetracycline-sensitive E. tarda (LTB4-S), LTB4-ROTC has 234 differentially expressed proteins, of which the abundance of 84 proteins is downregulated and 15 proteins are enriched to the Type III secretion system, Type VI secretion system, and flagellum pathways. Functional analysis confirms virulent phenotypes, including autoaggregation, biofilm formation, hemolysis, swimming, and swarming, are impaired in LTB4-ROTC. Furthermore, the in vivo bacterial challenge in both tilapia and zebrafish infection models suggests that the virulence of LTB4-ROTC is attenuated. Analysis of immune gene expression shows that LTB4-ROTC induces a stronger immune response in the spleen but a weaker response in the head kidney than that induced by LTB4-S, suggesting it's a potential vaccine candidate. Zebrafish and tilapia were challenged with a sublethal dose of LTB4-ROTC as a live vaccine followed by LTB4-S challenge. The relative percentage of survival of zebrafish is 60% and that of tilapia is 75% after vaccination. Thus, our study suggests that bacteria that acquire antibiotic resistance may attenuate virulence, which can be explored as a potential live vaccine to tackle bacterial infection in aquaculture.

PagJAZ5 regulates cambium activity through coordinately modulating cytokinin concentration and signaling in poplar.

Wood is resulted from the radial growth paced by the division and differentiation of vascular cambium cells in woody plants, and phytohormones play important roles in cambium activity. Here, we identified that PagJAZ5, a key negative regulator of jasmonate (JA) signaling, plays important roles in enhancing cambium cell division and differentiation by mediating cytokinin signaling in poplar 84K (Populus alba × Populus glandulosa). PagJAZ5 is preferentially expressed in developing phloem and cambium, weakly in developing xylem cells. Overexpression (OE) of PagJAZ5m (insensitive to JA) increased cambium activity and xylem differentiation, while jaz mutants showed opposite results. Transcriptome analyses revealed that cytokinin oxidase/dehydrogenase (CKXs) and type-A response regulators (RRs) were downregulated in PagJAZ5m OE plants. The bioactive cytokinins were significantly increased in PagJAZ5m overexpressing plants and decreased in jaz5 mutants, compared with that in 84K plants. The PagJAZ5 directly interact with PagMYC2a/b and PagWOX4b. Further, we found that the PagRR5 is regulated by PagMYC2a and PagWOX4b and involved in the regulation of xylem development. Our results showed that PagJAZ5 can increase cambium activity and promote xylem differentiation through modulating cytokinin level and type-A RR during wood formation in poplar.

Functional Proteomics Analysis of Norfloxacin-Resistant Edwardsiella tarda.

Multidrug-resistant Edwardsiella tarda threatens both sustainable aquaculture and human health, but the control measure is still lacking. In this study, we adopted functional proteomics to investigate the molecular mechanism underlying norfloxacin (NOR) resistance in E. tarda. We found that E. tarda had a global proteomic shift upon acquisition of NOR resistance, featured with increased expression of siderophore biosynthesis and Fe3+-hydroxamate transport. Thus, either inhibition of siderophore biosynthesis with salicyl-AMS or treatment with another antibiotic, kitasamycin (Kit), which was uptake through Fe3+-hydroxamate transport, enhanced NOR killing of NOR-resistant E. tarda both in vivo and in vitro. Moreover, the combination of NOR, salicyl-AMS, and Kit had the highest efficacy in promoting the killing effects of NOR than any drug alone. Such synergistic effect not only confirmed in vitro and in vivo bacterial killing assays but also applicable to other clinic E. tarda isolates. Thus, our data suggest a proteomic-based approach to identify potential targets to enhance antibiotic killing and propose an alternative way to control infection of multidrug-resistant E. tarda.

PagERF81 regulates lignin biosynthesis and xylem cell differentiation in poplar.

Lignin is a major component of plant cell walls and is essential for plant growth and development. Lignin biosynthesis is controlled by a hierarchical regulatory network involving multiple transcription factors. In this study, we showed that the gene encoding an APETALA 2/ethylene-responsive element binding factor (AP2/ERF) transcription factor, PagERF81, from poplar 84 K (Populus alba × P. glandulosa) is highly expressed in expanding secondary xylem cells. Two independent homozygous Pagerf81 mutant lines created by gene editing, produced significantly more but smaller vessel cells and longer fiber cells with more lignin in cell walls, while PagERF81 overexpression lines had less lignin, compared to non-transgenic controls. Transcriptome and reverse transcription quantitative PCR data revealed that multiple lignin biosynthesis genes including Cinnamoyl CoA reductase 1 (PagCCR1), Cinnamyl alcohol dehydrogenase 6 (PagCAD6), and 4-Coumarate-CoA ligase-like 9 (Pag4CLL9) were up-regulated in Pagerf81 mutants, but down-regulated in PagERF81 overexpression lines. In addition, a transient transactivation assay revealed that PagERF81 repressed the transcription of these three genes. Furthermore, yeast one hybrid and electrophoretic mobility shift assays showed that PagERF81 directly bound to a GCC sequence in the PagCCR1 promoter. No known vessel or fiber cell differentiation related genes were differentially expressed, so the smaller vessel cells and longer fiber cells observed in the Pagerf81 lines might be caused by abnormal lignin deposition in the secondary cell walls. This study provides insight into the regulation of lignin biosynthesis, and a molecular tool to engineer wood with high lignin content, which would contribute to the lignin-related chemical industry and carbon sequestration.

Development of a hyper-adhesive and attenuated Edwardsiella ictaluri strain as a novel immersion vaccine candidate in yellow catfish (Pelteobagrus fulvidraco).

Edwardsiella ictaluri, a Gram-negative intracellular pathogen, is the causative agent of enteric septicemia in channel catfish, and catfish aquaculture in China suffers heavy economic losses due to E. ictaluri infection. Vaccination is an effective control measure for this disease. In this study, an attenuated E. ictaluri strain was acquired through deletion mutation of the T3SS protein eseJei, and the ΔeseJei strain fails to replicate in the epithelioma papillosum of carp cells. The type 1 fimbria plays a pivotal role in the adhesion of E. ictaluri, and it was found in this study that deletion of -245 to -50 nt upstream of fimA increases its adhesion to around five times that of the WT strain. A hyper-adhesive and highly attenuated double mutant (ΔeseJeiΔfimA-245--50 strain) was constructed, and it was used as a vaccine candidate in yellow catfish via bath immersion at a dosage of 1 × 105 CFU/mL. It was found that this vaccine candidate can stimulate protection when challenged with E. ictaluri HSN-1 at 5 × 107 CFU/mL (∼20 × LD50). The survival rate was 83.61% for the vaccinated group and 33.33% for the sham-vaccinated group. The RPS (relative percent of survival) of the vaccination trial reached 75.41%. In conclusion, the ΔeseJeiΔfimA-245--50 strain developed in this study can be used as a vaccine candidate. It excels in terms of ease of delivery (via bath immersion) and is highly efficient in stimulating protection against E. ictaluri infection.

The role of senescence-associated gene101 (PagSAG101a) in the regulation of secondary xylem formation in poplar.

Wood is produced by the accumulation of secondary xylem via proliferation and differentiation of the cambium cells in woody plants. Identifying the regulators involved in this process remains a challenging task. In this study, we isolated PagSAG101a, the homolog of Arabidopsis thaliana SAG101, from a hybrid poplar (Populus alba × Populus glandulosa) clone 84K and investigated its role in secondary xylem development. PagSAG101a was expressed predominantly in lignified stems and localized in the nucleus. Compared with non-transgenic 84K plants, transgenic plants overexpressing PagSAG101a displayed increased plant height, internode number, stem diameter, xylem width, and secondary cell wall thickness, while opposite phenotypes were observed for PagSAG101a knock-out plants. Transcriptome analyses revealed that differentially expressed genes were enriched for those controlling cambium cell division activity and subsequent secondary cell wall deposition during xylem formation. In addition, the tandem CCCH zinc finger protein PagC3H17, which positively regulates secondary xylem width and secondary wall thickening in poplar, could bind to the promoter of PagSAG101a and mediate the regulation of xylem differentiation. Our results support that PagSAG101a, downstream of PagC3H17, functions in wood development.

Edwardsiella piscicida type III protein EseJ suppresses apoptosis through down regulating type 1 fimbriae, which stimulate the cleavage of caspase-8.

The type III secretion system effector EseJ plays a regulatory role inside bacteria. It suppresses the adherence of Edwardsiella piscicida (E. piscicida) to host epithelial cells by down regulating type 1 fimbriae. In this study, we observed that more macrophages infected with ΔeseJ strain of E. piscicida detached as compared with those infected with the wild-type (WT) strain. Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining and cleaved caspase-3 examination revealed that the detachment is due to increased apoptosis, suggesting that EseJ suppresses macrophage apoptosis. However, apoptosis inhibition by EseJ is not relative to a type III secretion system (T3SS) and is not related to EseJ's translocation. Since EseJ negatively regulates type 1 fimbriae, murine J774A.1 cells were infected with ΔeseJΔfimA or ΔeseJΔfimH strains. It was demonstrated that ΔeseJ stimulates macrophage apoptosis through type 1 fimbriae. Moreover, we found that infecting J774A.1 cells with the ΔeseJ strain increased levels of cleaved caspase-8, caspase-9, and caspase-3, demonstrating that EseJ inhibits apoptosis through either an extrinsic or a combination of extrinsic and intrinsic pathways. Pre-treatment of macrophages with caspase-8 inhibitor prior to infection with the ΔeseJ strain decreased the levels of cleaved caspase-8, caspase-9, and caspase-3, indicating that the ΔeseJ strain stimulates apoptosis, mainly through an extrinsic pathway by up regulating type 1 fimbriae. Zebrafish larvae or blue gourami fish infected with the ΔeseJ strain consistently exhibited higher apoptosis than those infected with the E. piscicida WT strain or ΔeseJΔfimA strain. Taken together, we revealed that the T3SS protein EseJ of E. piscicida inhibits host apoptosis, mainly through an extrinsic pathway by down regulating type 1 fimbriae.

The Impact of Hypertension Definition Based on Two-visit Strategy on Estimate of Hypertension Burden: Results From the China Health and Nutrition Survey 1989-2011.

BACKGROUND: The diagnosis of hypertension should be based on the mean of two or more properly measured BP readings on each of two visits for clinical practice, but a one-visit strategy was applied in most epidemiological surveys. The impact of hypertension definition based on two visits on estimates of hypertension burden is unknown. This study aims to assess the impact of hypertension diagnosis based on a two-visit strategy for estimating hypertension burden in China. METHODS: The one-visit and two-visit strategies were applied to investigate the incidence of hypertension in a cohort study based on the China Health and Nutrition Survey (CHNS) 1989-2011. Additionally the prevalence of hypertension was investigated in a cross-sectional study based on the CHNS 2006-2009/2011 and the hypertension burden in China was estimated with data from the 2012-2015 China hypertension survey. RESULTS: Overall, the age-adjusted incidence of hypertension based on the two-visit strategy (1.82%; 95% confidence interval [CI], 1.74-1.90%) was 62.1% lower than estimation based on the one-visit strategy (4.80%; 95% CI, 4.68-4.93%). Similar results were found in the prevalence of hypertension (one-visit: 18.13% [95% CI, 17.34-18.92%]; two-visit: 9.47% [95% CI, 8.87-10.07%]). When the two-visit strategy was applied to the 2012-2015 China hypertension survey, the hypertension burden was predicted to be overestimated by 25.5-47.8% (based on JNC 7) and 23.5-48.2% (based on the 2017 ACC/AHA). CONCLUSION: The hypertension burden would decrease from 244.5 million persons to 127.5-182.3 million persons in China if the two-visit strategy was applied.

Cordyceps militaris Immunomodulatory Protein Promotes the Phagocytic Ability of Macrophages through the TLR4-NF-κB Pathway.

Enhancing the phagocytosis of immune cells with medicines provides benefits to the physiological balance by removing foreign pathogens and apoptotic cells. The fungal immunomodulatory protein (FIP) possessing various immunopotentiation functions may be a good candidate for such drugs. However, the effect and mechanism of FIP on the phagocytic activity is limitedly investigated. Therefore, the present study determined effects of Cordyceps militaris immunomodulatory protein (CMIMP), a novel FIP reported to induce cytokines secretion, on the phagocytosis using three different types of models, including microsphere, Escherichia Coli and Candida albicans. CMIMP not only significantly improved the phagocytic ability (p < 0.05), but also enhanced the bactericidal activity (p < 0.05). Meanwhile, the cell size, especially the cytoplasm size, was markedly increased by CMIMP (p < 0.01), accompanied by an increase in the F-actin expression (p < 0.001). Further experiments displayed that CMIMP-induced phagocytosis, cell size and F-actin expression were alleviated by the specific inhibitor of TLR4 (p < 0.05). Similar results were observed in the treatment with the inhibitor of the NF-κB pathway (p < 0.05). In conclusion, it could be speculated that CMIMP promoted the phagocytic ability of macrophages through increasing F-actin expression and cell size in a TLR4-NF-κB pathway dependent way.

TMT-MS/MS proteomic analysis of the carbohydrate-active enzymes in the fruiting body of Pleurotus tuoliensis during storage.

BACKGROUND: The fruiting body of Pleurotus tuoliensis deteriorates rapidly after harvest, causing a decline in its commercial value and a great reduction in its shelf life. According to the present research, carbohydrate-active enzymes (CAZymes) may cause the softening, liquefaction and autolysis of mature mushrooms after harvest. To further understand the in vivo molecular mechanism of CAZymes affecting the postharvest quality of P. tuoliensis fruiting bodies, a tandem mass tags labelling combined liquid chromatography-tandem mass spectrometry (TMT-MS/MS) proteomic analysis was performed on P. tuoliensis fruiting bodies during storage at 25 °C. RESULTS: A total of 4737 proteins were identified, which had at least one unique peptide and had a confidence level above 95%. Consequently, 1307 differentially expressed proteins (DEPs) were recruited using the criteria of abundance fold change (FC) >1.5 or < 0.67 and P < 0.05. The identified proteins were annotated by dbCAN2, a meta server for automated CAZymes annotation. Subsequently, 222 CAZymes were obtained. Several CAZymes participating in the cell wall degradation process, including ß-glucosidase, glucan 1,3-ß-glucosidase, endo-1,3(4)-ß-glucanase and chitinases, were significantly upregulated during storage. The protein expression level of CAZymes, such as xylanase, amylase and glucoamylase, were upregulated significantly, which may participate in the P. tuoliensis polysaccharide degradation. CONCLUSIONS: The identified CAZymes degraded the polysaccharides and lignin, destroying the cell wall structure, preventing cell wall remodeling, causing a loss of nutrients and the browning phenomenon, accelerating the deterioration of P. tuoliensis fruiting body. © 2020 Society of Chemical Industry.