Salmonella spvC gene suppresses macrophage/neutrophil antibacterial defense mediated by gasdermin D.

OBJECTIVE: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a representative model organism for investigating host-pathogen interactions. It was reported that S. Typhimurium spvC gene alleviated intestinal inflammation to aggravate systemic infection, while the precise mechanisms remain unclear. In this study, the influence of spvC on the antibacterial defense of macrophage/neutrophil mediated by gasdermin D (GSDMD) was investigated. METHODS: Mouse macrophage-like cell lines J774A.1 and RAW264.7, neutrophil-like cells derived from HL-60 cells (human promyletic leukemia cell lines) were infected with S. Typhimurium wild type, spvC deletion and complemented strains. Cell death was evaluated by LDH release and Annexin V-FITC/PI staining. Macrophage pyroptosis and neutrophil NETosis were detected by western blotting, live cell imaging and ELISA. Flow cytometry was used to assess the impact of spvC on macrophage-neutrophil cooperation in macrophage (dTHP-1)-neutrophil (dHL-60) co-culture model pretreated with GSDMD inhibitor disulfiram. Wild-type and Gsdmd-/- C57BL/6J mice were utilized for in vivo assay. The degree of phagocytes infiltration and inflammation were analyzed by immunofluorescence and transmission electron microscopy. RESULTS: Here we find that spvC inhibits pyroptosis in macrophages via Caspase-1/Caspase-11 dependent canonical and non-canonical pathways, and restrains neutrophil extracellular traps extrusion in GSDMD-dependent manner. Moreover, spvC could ameliorate macrophages/neutrophils infiltration and cooperation in the inflammatory response mediated by GSDMD to combat Salmonella infection. CONCLUSIONS: Our findings highlight the antibacterial activity of GSDMD in phagocytes and reveal a novel pathogenic mechanism employed by spvC to counteract this host defense, which may shed new light on designing effective therapeutics to control S. Typhimurium infection.

Evaluation of Thermal Fatigue Life and Crack Morphology in Brake Discs of Low-Alloy Steel for High-Speed Trains.

Effective braking in high-speed trains is one of the major bottlenecks in expediting the technology and possibilities to improve speed. Although substantial progress has been made to increase operating speed, perhaps, thermal fatigue cracking in brake discs is a primary constraint so far. Thermal fatigue cracking is the major cause of brake disc failure in high-speed trains, especially trains with a speed of 350 km/h or above. In this study, new material composition is proposed for brake discs of high-speed trains. A comprehensive investigation is presented based on fatigue crack initiation and propagation, along with wear and micro-hardness characterization. Thermal fatigue tests at various thermal cycles between 20 ℃ and 700 ℃ were performed and the experimental results are compared with fatigue properties of a commercial brake disc material. An experimental trial revealed that thermal cracks normally initiate and propagate along the oxidized grain boundaries; nevertheless, crack propagation is restricted by the fine precipitates and lath structure of martensitic. Moreover, crack length at the initiation and propagation stage is predicted through crack growth rate and favorable grain size in the crack vicinity. Thermal fatigue life can be improved by dictating the microstructure and precipitate morphology of cast steel by tailoring the alloying composition.

Salmonella spvC Gene Inhibits Autophagy of Host Cells and Suppresses NLRP3 as Well as NLRC4.

Salmonella spvC gene, encoding a phosphothreonine lyase on host mitogen-activated protein kinases, facilitates systemic infection of Salmonella while the precise mechanisms remain elusive. Autophagy and pyroptosis dependent on the activation of inflammasomes, as parts of innate immune response, contribute to host defense against Salmonella infection. Recently, we reported that spvC could inhibit pyroptosis. To explore the effect of spvC on autophagy and the relationship between its function in pyroptosis and autophagy, infection models of macrophages J774A.1 and epithelial HeLa cells co-cultured with Salmonella Typhimurium wild type, spvC deletion, site-directed mutant which lacks phosphothreonine lyase activity, or complemented strain were established. The levels of LC3 turnover and Beclin 1 of J774A.1 cells were determined by western blot. Confocal laser scanning microscopy was used to visualize the autophagic flux after being transfected with mRFP-GFP-LC3 plasmid in HeLa cells. Results showed that SpvC inhibited autophagosome formation through its phosphothreonine lyase activity. Additionally, analysis of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) and NLR with CARD domain-containing 4 (NLRC4) in J774A.1 cells indicated that spvC decreased the protein levels of NLRP3 and NLRC4, which were significantly changed by autophagy inhibitor Bafilomycin A1. Together, our observations reveal a novel mechanism of spvC in Salmonella pathogenesis and host inflammatory response via inhibiting autophagy and NLRP3 as well as NLRC4. These pathways and their subversion by diverse pathogen virulence determinants are expected to throw light on the design of anti-infective agents.