Role of LC3-associated phagocytosis in regulating infection / 中华微生物学和免疫学杂志

LC3-associated phagocytosis (LAP) is a special phagocytosis occurring at the intersection of the two pathways of phagocytosis and autophagy. A hallmark event of the LAP process is the recruitment of microtubule-associated proteinⅠlight chain type 3-Ⅱ(LC3Ⅱ) to the phagosome surface of the monolayer membrane structure. The LAP pathway relies on the functions of the RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein (Rubicon) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The LC3-associated phagosome (LAPosome) binds to the lysosome to digest and degrade the contents. In recent years, increasing studies have found that LAP plays an important role in the infections caused by pathogenic microorganisms including fungi and bacteria. LAP is a crucial way in the host to resist and degrade the infection of pathogenic microorganisms. However, some pathogenic microorganisms can effectively escape from LAP in the host and even use LAPosome as a place for colonization and replication. This article summarized the recent progress in the role of LAP in host defense against pathogenic microorganism infection and the significance of it in the occurrence and development of diseases.

Progress in the mechanism of regulatory B cells and its role in human diseases / 中华微生物学和免疫学杂志

Regulatory B cells (Bregs) are a group of cells with immunoregulatory function. They can regulate the progression of diseases by inhibiting excessive inflammatory responses and serve as critical protective cells in immune disorders and other conditions. This article reviewed the source, function and mechanism of Bregs and its role in autoimmune diseases, infection and tumor.

Role of NOD-like receptor protein-3 in regulating phagocytosis in Vibrio vulnificus-infected murine macrophages / 中华微生物学和免疫学杂志

Objective:To investigate the role of NOD-like receptor protein 3 (NLRP3) in the regulation of phagocytosis in Vibrio vulnificus ( V. vulnificus)-infected macrophages. Methods:Expression profiles of phagocytosis-related genes in PBS- and V. vulnificus-infected J774A.1 cells were analyzed by RNA-Seq. NLRP3-knockout (NLRP3 KO) J774A.1 cells were constructed using CRISPR-Cas9 gene-editing system. The phagocytosis of V. vulnificus and pHrodo RED-labelled Escherichia coli ( E. coli) bioparticles in parental and NLRP3 KO J774A.1 cells was detected by flow cytometry. Real-time PCR was performed to measure the expression of Fgr2 b gene at mRNA level in PBS- and V. vulnificus-treated parental and NLRP3 KO J774A.1 cells. Results:The expression of 18 phagocytosis-related genes was upregulated in V. vulnificus-infected J774A.1 cells than in PBS-treated J774A.1 cells ( P<0.05). There was a 5 bp deletion in the exon 2 of NLRP3 gene in NLRP3 KO J774A.1 cells, resulting in frameshift mutation and complete loss of NLRP3 expression. NLRP3 KO J774A.1 cells exhibited enhanced phagocytosis of V. vulnificus and pHrodo RED-labelled E. coli bioparticles than parental J774A.1 cells ( P<0.05). Besides, the expression of Fgr2 b gene at mRNA level was significantly increased in V. vulnificus-infected NLRP3 KO J774A.1 cells than in parental J774A.1 cells ( P<0.05). Conclusions:The phagocytosis of V. vulnificus in macrophages could be negatively regulated by NLRP3, which was possibly mediated through the regulation of Fgr2 b gene expression.

Effects of Vibrio vulnificus cytolysin membrane pore-forming peptide on lysosome formation and ROS production in macrophages and its cytotoxicity / 中华微生物学和免疫学杂志

Objective:To investigate the cytotoxicity of a recombinant Vibrio vulnificus cytolysin membrane pore-forming peptide (rMpf) to J774A.1 cells as well as its influences on reactive oxygen species (ROS) and lysosomes, and to analyze the cellular localization of the rMpf. Methods:The rMpf was expressed using an Escherichia coli expression system and then used to treat murine J774A.1 cells in vitro. The viability of rMpf- and PBS-treated J774A.1 cells and the levels of ROS and lysosomes in these cells were analyzed by flow cytometry. Confocal microscopy was used to observe the cellular localization of rMpf in the J774A.1 cells. Results:No significant differences in cell viability, ROS production or lysosome formation in J774A.1 cells were found between low-dose (4 μg/ml) rMpf-treated and untreated groups. However, the cell viability, ROS production and lysosome formation were significantly decreased after treating J774A.1 cells with higher doses of rMpf (20 and 60 μg/ml). Moreover, the rMpf was found to localize in both the cytoplasm and nuclei of J774A.1 cells in a dose-dependent manner.Conclusions:The rMpf could localize in both the cytoplasm and nuclei of J774A.1 cells. It would inhibit the cellular ROS production and lysosome formation to damage macrophage function. This study provided a novel sight for understanding the cytotoxic domain and pathogenesis of Vibrio vulnificus cytolysin and other membrane pore-forming cytolysins.

Effects of TNF-α knockout on liver and spleen neutrophil responses to Vibrio vulnificus bloodstream infection in a mouse model / 中华微生物学和免疫学杂志

Objective:To investigate the effects of TNF-α knockout on liver and spleen neutrophil responses to Vibrio vulnificus bloodstream infection in a mouse model. Methods:(1) TNF-α-knockout (TNF-α -/-) and wild-type (WT) C57BL/6J mice aged 6-8 weeks were randomly divided into four groups with six in each group: uninfected WT group, infected WT group, uninfected TNF-α -/- group and infected TNF-α -/- group. The mouse model of bloodstream infection was constructed by intraperitoneal injection of Vibrio vulnificus CGMCC1.1758 (2×10 8 CFU/200 μl), while the mice in the uninfected groups were injected intraperitoneally with equal amount of PBS. (2) Liver immune cells and splenocytes were isolated 4 h after infection and subjected to analyze the percentages and numbers of neutrophils, and the changes in cell viability, cellular reactive oxygen species (ROS) level and phagocytosis by flow cytometry. In addition, effects of Vibrio vulnificus bloodstream infection on mTOR signaling pathway in murine neutrophils were evaluated in vivo. Results:(1)Compared with the uninfected WT group, the percentages and numbers of neutrophils in liver and spleen tissues of the infected WT group increased significantly. The percentage and number of liver neutrophils were significantly higher in the infected TNF-α -/- group than in the infected WT group, but no significant difference in spleen neutrophils was detected between the two groups. (2) Compared with the infected WT group, the phagocytosis of liver neutrophils rather than that of spleen neutrophils was enhanced in the infected TNF-α -/- group. (3) The survival rates of neutrophils in both liver and spleen were decreased, while the cellular ROS level was significantly increased in the infected WT group compared with those of the uninfected WT group. Compared with the infected WT group, the infected TNF-α -/- group had increased survival rates of both liver and spleen neutrophils, but decreased level of ROS. (4) The levels of p-AKT (S473) in liver and spleen neutrophils of the infected WT group were lower than those of the uninfected WT group. Compared with the infected WT group, the infected TNF-α -/- group had lower level of p-AKT (S473) in liver neutrophils, but higher p-AKT (S473) level in spleen neutrophils. There were no significant differences in p-4E-BP1(T37/46) levels between the uninfected WT group and the infected WT group. The p-4E-BP1 (T37/46) level in liver neutrophils was lower in the infected TNF-α -/- group than in the infected WT group, but no significant difference in p-4E-BP1 (T37/46) levels in spleen neutrophils was observed between the two groups. Conclusions:TNF-α had different effects on the neutrophils in spleen and liver tissues of mice with Vibrio vulnificus bloodstream infection. It played a critical role in regulating the recruitment, phagocytic function and mTOR signaling of liver neutrophils after Vibrio vulnificus infection in vivo.

Effects of Vibrio vulnificus cytolysin on the numbers of mitochondria and the expression of CD62L in murine liver CD4+T cells / 中华微生物学和免疫学杂志

Objective To understand how Vibrio vulnificus hemolysin (VvhA) affects the viability of murine liver CD4+ T cells as well as its effects on the numbers of mitochondria and the expression of CD62L. Methods The primary murine liver monocytes (MNs) were isolated from C57BL/ 6 mice and then treated with recombinant VvhA (rVvhA) for 6 hours in vitro. The viability of murine liver CD4+T cells and the expression of CD62L were measured by staining with anti-mouse CD4, CD8, CD44, CD62L and cell via-bility fluorescent dye or fluorescent antibody. Moreover, the cells were simply incubated with MitoTracker or JC-1 probes to label mitochondria and mitochondrial membrane potential, which were further analyzed by using flow cytometry analysis. Results With the increase in the doses of rVvhA, the viability of murine liv-er CD4+T cells was decreased from 81. 5% to 15. 8% . The expression of CD62L on the surface of murine liver CD4+T cells was dramatically decreased. Both the murine liver na?ve and effector CD4+ T cells were sensitive to the cytotoxicity of rVvhA. Moreover, treating murine liver CD4+ T cells with rVvhA resulted in significantly decreased numbers of mitochondria and lower mitochondrial membrane potential. Conclusion The cytotoxicity of rVvhA to murine liver CD4+T cells might be achieved through inhibiting the expression of CD62L, decreasing the numbers of mitochondria and lowering mitochondrial membrane potential.

Role of rVvhA in inducing THP-1 cells damage / 中华微生物学和免疫学杂志

Objective To investigate the role of recombinant Vibrio vulnificus cytolysin (rVvhA) in inducing THP-1 cells damage and study the pathway of associated calcium influx .Methods Inverted mi-croscope, CCK-8 cell proliferation kit, Fluo3/AM staining and caspase activity detection were performed to analyze the damage of THP-1 cells induced by rVvhA and the pathway of calcium influx .Results rVvhA had cytotoxic effects on THP-1 cells in a dose-dependent manner .The concentrations of extracellular K +and LDH were respectively up-regulated after 1 h and 6 h of 12 μg/ml rVvhA intervention .Verapamil , Mibe-fradil and SKF-96365 could not prevent the influx of free Ca 2+induced by rVvhA .The activities of caspase-3 and caspase-9 were singanificantly enhanced by rVvhA in a time-dependant manner .Conclusion rVvhA can induce THP-1 cells damage through triggering extracellular calcium influx via porous channel on cell membrane.Moreover, rVvhA might induce THP-1 cell apoptosis through activating caspase-9/3-dependent pathway .

Prediction, cloning, expression and identification of the functional motif of the recombinant Ricin B / 中华微生物学和免疫学杂志

Objective To determine the functional motif of the recombinant Ricin B(rRicin B) in Vibrio vulnificus cytolysin (VVC) and understand its molecule pathogenic mechanism.Methods The motif of VVC was predicted through bioinformatics analysis and cloned into a procaryotic expression vector pET28a-rRicin B.The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by IPTG to express rRicin B.The expressed protein was further analyzed by SDS-PAGE and purified by Ni2+-NTA agarose.Renaturation of the rRicin B were also carried out for further analysis.ELISA assay and confocal microscope was applied to identify the activity of the rRicin B on human Hela cells.Results Ricin B motif located in the 336-465 amino acids of Vibrio vulnificus cytolysin with a relative molecular weight of 20×103.The result of ELISA showed that the antigenicity of rRicin B was 28.71 U/L after renaturation.FITC labeled rRicin B could bind to the cell membrane and enter the cytoplasm of human Hela cells.Conclusion The Ricin B motif in Vibrio vulnificus cytolysin bearing the similar ability with the natural Ricin B can bind to the cell membrane and enter the cytoplasm.This feature may play an important role in the activity of pore-forming and the cytotoxicity of Vibrio vulnificus cytolysin.

The role of calcium in the rVvhA induced THP-1 cells apoptosis / 中华微生物学和免疫学杂志

Objective To study the role of Vibrio vulnificus cytolysin(rVvhA) induced THP-1 apoptosis and calcium influx.Methods CCK-8 cell proliferation kit,Fluo3/AM staining and AnnexinV/PI staining were performed to identify the apoptosis and calcium influx induced by rVvhA in THP-1 cells.Results rVvhA could induce THP-1 apoptosis and up-regulate the cellular calcium concentration.BAPTAAM could enhance the calcium influx induced by rVvhA in THP-1.Conclusion rVvhA had cytotoxic to THP-1 cells by inducing apoptosis and triggering extracellular calcium influx.

Study on transcription and expression of IL-8 in human Caco-2 cells induced by VvhA recombinant protein / 中华微生物学和免疫学杂志

Objective To investigate the effect of VvhA recombinant protein to the expression of IL-8 in human intestinal epithelial Caco-2 cells. Methods The VvhA recombinant protein(rVvhA) was ex-pressed by prokaryotic expression vector pET-28a (+)-whA in E. coli BL21 (DE3) , purified by Ni~(2+)-NTA affinity chromatography refoldod by stepwise deliquation together with dialysis methods and identified by Western blot. The cytotoxic of rVvhA to human Caco-2 cells was measured by CCK-8. The transcription of IL-8 mRNA in human Caco-2 cells induced by rVvhA was determined by RT-PCR, and the expression of IL-8 in human Caco-2 cells induced by rVvhA was determined by ELISA assay. Results rVvhA was purified with high purity up to 95%. The viability of human Caco-2 cells treated with 1.5 HU/ml rVvhA was inhibi-ted significantly (P < 0.05). The rVvhA can induce human Caco-2 cells to increase the transcription and expression of IL-8 in dose- and time-dependent manner. The transcription of IL-8 gene in human Caco-2 cells treated with 0.6 HU/ml rVvhA in 30 min can be up-regulated significantly, and the expression of IL-8 in human Caco-2 cells treated with rVvhA in 4 h can be increased significantly. Conclusion rVvhA has cy-totoxic to human Caco-2 and can increase the expression of IL-8, it might play a major role in the inflamma-tory reaction of rVvhA-exposed cells.