Immunization with recombinant Salmonella expressing SspH2-EscI protects mice against wild type Salmonella infection.

BACKGROUND: Enhancing caspase-1 activation in macrophages is helpful for the clearance of intracellular bacteria in mice. Our previous studies have shown that EscI, an inner rod protein of type III system in E. coli can enhance caspase-1 activation. The purpose of this study was to further analyze the prospect of EscI in the vaccine design. RESULTS: A recombinant Salmonella expressing SspH2-EscI fusion protein using the promotor of Salmonella effector SspH2, X4550(pYA3334-P-SspH2-EscI), was constructed. A control recombinant Salmonella expressing SspH2 only X4550(pYA3334-P-SspH2) was also constructed. In the early stage of in vitro infection of mouse peritoneal macrophages, X4550(pYA3334-P-SspH2-EscI) could significantly (P < 0.05) enhance intracellular caspase-1 activation and pyroptotic cell death of macrophages, when compared with X4550(pYA3334-P-SspH2). Except for the intracellular pH value, the levels of reactive oxygen species, intracellular concentration of calcium ions, nitric oxide and mitochondrial membrane potential in macrophages were not significantly different between the cells infected with X4550(pYA3334-P-SspH2-EscI) and those infected with X4550(pYA3334-P-SspH2). Besides, only lower inflammatory cytokines secretion was induced by X4550(pYA3334-P-SspH2-EscI) than X4550(pYA3334-P-SspH2). After intravenous immunization of mice (1 × 106 cfu/mouse), the colonization of X4550(pYA3334-P-SspH2-EscI) in mice was significantly limited at one week post immunization (wpi), when compared with X4550(pYA3334-P-SspH2) (P < 0.05). The population of activated CD8+T lymphocytes in mouse spleens induced by X4550(pYA3334-P-SspH2-EscI) was lower than that induced by X4550(pYA3334-P-SspH2) at 2-3 wpi, and the ratio of CD4+T cells to CD8+T cells decreased. The blood coagulation assay indicated that no significant difference was found between X4550(pYA3334-P-SspH2-EscI) and uninfected control, while X4550(pYA3334-P-SspH2) could induce the quick coagulation. Notably, immunization of X4550(pYA3334-P-SspH2-EscI) could limit the colonization of challenged Salmonella strains in the early stage of infection and provide more effective protection. CONCLUSION: The activation of caspase-1 in macrophages by EscI can be used in the design of live attenuated Salmonella vaccine candidate.

Recombinant Salmonella expressing SspH2-EscI fusion protein limits its colonization in mice.

BACKGROUND: Activation of inflammasome contributes to the clearance of intracellular bacteria. C-terminus of E. coli EscI protein can activate NLRC4 (NLR family, CARD domain containing-4) inflammasome in macrophages. The purpose of this study was to determine if activation of NLRC4 inflammasome by EscI can reduce the colonization of Salmonella in mice. RESULTS: A recombinant S. typhimurium strain expressing fusion protein of the N-terminal SspH2 (a Salmonella type III secretion system 2 effector) and C-terminal EscI was constructed and designated as X4550(pYA3334-SspH2-EscI). In vitro assay showed that X4550(pYA3334-SspH2-EscI) significantly enhanced IL-1ß and IL-18 secretion (P < 0.05) and pyroptotic cell death of mouse peritoneal macrophages, compared with those infected with control strain, X4550(pYA3334-SspH2). In vivo studies showed that colonization of X4550(pYA3334-SspH2-EscI) in both spleen and liver were significantly lower than that of X4550(pYA3334-SspH2) (P < 0.05). The bacterial counts of X4550(pYA3334-SspH2-EscI) in mice decreased, while those of X4550(pYA3334-SspH2) increased over the time after infection. Additionally, X4550(pYA3334-SspH2-EscI) induced a less pathological alteration in spleen and liver than X4550(pYA3334-SspH2). CONCLUSION: Fusion protein SspH2-EscI may be translocated into macrophages and activate NLRC4 inflammasome, which limits Salmonella colonization in spleen and liver of mice.

[Inflammasome responses in macrophages induced by C-terminal peptides of Escherichia coli EscI protein].

OBJECTIVE: To analyze NLRC4 inflammasome responses in macrophages induced by C-terminal of Escherichia coli EscI protein. METHODS: NLRC4 inflammasome responses in mouse peritoneal macrophages were analyzed after delivery of the peptides containing C-terminal amino acid sequences of E. coli EscI protein in vitro. RESULTS: The peptides containing C-terminal 15 amino acids of EscI protein could significantly activate NLRC4 inflammasome responses in macrophages pre-stimulated with lipopolysaccharide. Intracellular caspase-1 was activated and pyroptotic dead cells were found after peptides delivery. The contents of cytokines, IL-1ß and IL-18, in supernatants were elevated significantly compared with that of the control (P < 0.05). Besides, through comparison of IL-1ß contents under different stimulation conditions, 4 h incubation after peptides delivery (peptides: lipofectamine 2000 = 70 µg/µL) could obviously promote the secretion of IL-1ß. CONCLUSION: Peptides containing C-terminal 15 amino acids of E. coli EscI protein can significantly induce NLRC4 inflammasome activation in macrophages.

Salinicola zeshunii sp. nov., a moderately halophilic bacterium isolated from soil of a chicken farm.

The taxonomic status of a moderately halophilic bacterium, strain N4(T), isolated from soil of a chicken farm in China was determined. It was Gram-negative, non-spore-forming, motile, and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequence indicated that this strain belonged to the genus Salinicola, as it showed the highest sequence similarities to Salinicola salaries M27(T) (98.3 %), Salinicola socius SMB35(T) (98.1 %), and Salinicola halophilus CG4.1(T) (98.1 %). The major cellular fatty acids were C(16:0) (25.6 %), C(18:1)ω7c (35.0 %), and C(19:0) cyclo ω8c (11.9 %), which are properties shared by members of the genus Salinicola. The DNA G+C content of strain N4(T) was 69.1 mol %. The level of DNA-DNA relatedness between strain N4(T) and the other three type strains of the genus of Salinicola salaries M27(T), Salinicola socius SMB35(T), and Salinicola halophilus CG4.1(T) were 34.3, 28.7, and 26.9 %, respectively. Based on the results of phenotypic, chemotaxonomic, DNA-DNA relatedness, and phylogenetic analysis, strain N4(T) should be classified as a novel species of the genus Salinicola, for which the name Salinicola zeshunii sp. nov. is proposed, with strain N4(T) (=KACC 16602(T) = CCTCC AB 2012912(T)) as the type strain.

Sphingobium qiguonii sp. nov., a carbaryl-degrading bacterium isolated from a wastewater treatment system.

A Gram-staining-negative, catalase-positive, carbaryl-degrading, non-spore-forming, non-motile, rod-shaped bacterium, designated strain X23(T), was isolated from a wastewater treatment system. Phylogenetic analysis based on 16S rRNA gene sequence indicated that the strain belongs to the genus Sphingobium. The highest 16S rRNA gene sequence similarity observed for the isolate was 96.6 % with the type strain of Sphingobium amiense. Chemotaxonomic data [major ubiquinone: Q-10; major polar lipids: diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, sphingoglycolipid, phosphatidylethanolamine and unknown aminolipids and phospholipids; major fatty acids: summed feature 7 (C(18 : 1)ω7c, C(18 : 1)ω9t and/or C(18 : 1)ω12t), C(16 : 1)ω5c, C(14 : 0) 2-OH and C(16 : 0) 2-OH] as well as the inability to reduce nitrate and the presence of spermidine as the major polyamine supported the affiliation of the strain to the genus Sphingobium. Based on the phylogenetic analysis, whole-cell fatty acid composition and biochemical characteristics, the strain could be separated from all recognized species of the genus Sphingobium. Strain X23(T) should be classified as a novel species of the genus Sphingobium, for which the name Sphingobium qiguonii sp. nov. is proposed, with strain X23(T) (=CCTCC AB 208221(T) =DSM 21541(T)) as the type strain.

Cholesterol-Rich Lipid Rafts in the Cellular Membrane Play an Essential Role in Avian Reovirus Replication.

Cholesterol is an essential component of lipid rafts in cellular plasma membranes. Although lipid rafts have been reported to have several functions in multiple stages of the life cycles of many different enveloped viruses, the mechanisms by which non-enveloped viruses, which lack outer lipid membranes, infect host cells remain unclear. In this study, to investigate the dependence of non-enveloped avian reovirus (ARV) infection on the integrity of cholesterol-rich membrane rafts, methyl-ß-cyclodextrin (MßCD) was used to deplete cellular membrane cholesterol at the ARV attachment, entry, and post-entry stages. Treatment with MßCD significantly inhibited ARV replication at both the entry and post-entry stages in a dose-dependent manner, but MßCD had a statistically insignificant effect when it was added at the attachment stage. Moreover, MßCD treatment markedly reduced syncytium formation, which occurs at a relatively late stage of the ARV life cycle and is involved in cell-cell transmission and release. Furthermore, the addition of exogenous cholesterol reversed the effects mentioned above. Colocalization data also showed that the ARV proteins σC, µNS, and p10 prefer to localize to cholesterol-rich lipid raft regions during ARV infection. Altogether, these results suggest that cellular cholesterol in lipid rafts plays a critical role in ARV replication.

Isolation and characterization of a carbofuran-degrading strain Novosphingobium sp. FND-3.

A gram-negative Novosphingobium sp. strain FND-3 capable of degrading carbofuran was isolated and characterized. The carbofuran-degrading ability of strain FND-3 was investigated under various culture conditions. Strain FND-3 showed a high average carbofuran-degrading rate of 28.6 mg L(-1) h(-1) in mineral salts medium with 100 mg L(-1) carbofuran. GC/MS analysis pointed out the presence of several unknown metabolites. One hydrolyzate was identified as 2-hydroxy-3-(3-methypropan-2-ol) phenol via hydrolysis of carbofuran phenol. The appearance of another metabolite with M(+) of 180 m/z indicated that the hydroxylation of carbofuran occurred at the aromatic ring. One novel degrading product with M(+) of 239 m/z was identified as 2-hydroxy-3-(3-methylpropan-2-ol) benzene-N-methylcarbamate via hydrolyzing at the ether bond of furanyl ring of carbofuran. Strain FND-3 was also able to degrade other N-methylcarbamate pesticides.

A facile and green method for synthesis of reduced graphene oxide/Ag hybrids as efficient surface enhanced Raman scattering platforms.

Reduced graphene oxide/Ag nanoparticles hybrids (rGO/AgNPs) were fabricated via a green and facile hydrothermal method. The as-synthesized materials were characterized in detail using various spectroscopic and microscopic techniques. Under a suitable dosage of silver ions, well-dispersed AgNPs on the reduced graphene oxide sheets were obtained. The surface plasmon resonance properties of AgNPs on graphene show that there is an interaction between AgNPs and graphene. Trace detection of organic dyes is studied based on rGO/AgNPs hybrids as efficient surface enhanced Raman scattering platforms. It has been found that the suitable experiment parameter is crucial to trace detection of organic dyes molecules. This work is of importance in the practical application in device-design based on the SERS effect of noble metal/reduced oxide graphene (or oxide graphene) hybrids.

[Relationship between interferon-α conformation and its anti-viral activity determined by circular dichroism and flow cytometry].

The relationship between the conformation of interferon-α (IFN-α) and its anti-viral activity were analyzed by circular dichroism (CD) and flow cytometry (FCM) techniques. The recombinant human IFN-α (rIFN-α2b and rIFN-α2a) were used. CD spectra from 190 nm to 240 nm indicated that two the IFN-α showed stable secondary structure at 65 degrees C, but unstable when the temperature was above 65 degrees C, and the change was irreversible. FCM data of the anti-viral activity of IFN-α indicated that the change of its secondary structures partly weakened its anti-viral activity. The rIFN-α2b and rIFN-α2a showed the same phenomenon. These data indicated that the conformation of IFN-α is one of the factors to influence its anti-viral activity and the combination of CD and FCM is a good method to analyze the relationship between the conformation of protein drugs and their biological activities in single cell level.

Rhizobium phenanthrenilyticum sp. nov., a novel phenanthrene-degrading bacterium isolated from a petroleum residue treatment system.

Strain F11(T), a phenanthrene-degrading bacterium, was isolated from a petroleum residue treatment system, and classified under the genus Rhizobium based on the similarity analysis of its 16S rRNA and recA gene sequences. Strain F11(T) falls into the same phylogenetic clade with Rhizobium oryzae Alt 505(T) (96.8% 16S rRNA gene sequence similarity) and Rhizobium pseudoryzae J34A-127(T) (96.2%). Major cellular fatty acids of strain F11(T) are C(16:0) (6.24%) and summed feature 8 (C(18:1ω7c) and/or C(18:1ω6c), 76.59%), which are also the major fatty acids of R. oryzae Alt 505(T) and R. pseudoryzae J34A-127(T). The DNA G+C content of strain F11(T) was 59.3±0.4 mol%. Based on the phylogenetic analysis as well as biochemical and physiological characteristics, strain F11(T) could be separated from all recognized Rhizobium species. Strain F11(T) (=DSM 21882(T) =CCTCC AB 209029(T)) was considered to be representative of a novel species of Rhizobium, for which the name Rhizobium phenanthrenilyticum sp. nov. is proposed.

Analysis of the role of LinA and LinB in biodegradation of delta-hexachlorocyclohexane.

Commercial formulations of hexachlorocyclohexane (HCH) consist of a mixture of four isomers, alpha, beta, gamma and delta. All these four isomers are toxic and recalcitrant pollutants. Sphingobium (formerly Sphingomonas) sp. strain BHC-A is able to degrade all four HCH isomers. Eight lin genes responsible for the degradation of gamma-HCH in BHC-A were cloned and analysed for their role in the degradation of delta-HCH, and the initial conversion steps in delta-HCH catabolism by LinA and LinB in BHC-A were found. LinA dehydrochlorinated delta-HCH to produce 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN) via delta-pentachlorocyclohexene (delta-PCCH). Subsequently, both 1,4-TCDN and delta-PCCH are catalysed by LinB via two successive rounds of hydrolytic dechlorinations to form 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) and 2,3,5-trichloro-5-cyclohexene-1,4-diol (2,3,5-TCDL) respectively. LinB could also catalyse the hydrolytic dechlorination of delta-HCH to 2,3,5,6-tetrachloro-1,4-cyclohexanediol (TDOL) via 2,3,4,5,6-pentachlorocyclohexanol (PCHL).