Gaofu Qi

Lipopeptide induces apoptosis in fungal cells by a mitochondria-dependent pathway

Bacillus amyloliquefaciens WH1 inhibit the growth of fungi by producing a new surfactin called as WH1fungin. WH1fungin plays an anti-fungal role by two models: high concentration to elicit pores on cell membrane and low concentration to induce apoptosis. WH1fungin can also inhibits the glucan synthase resulting in a decreased synthesis of callose on fungal cell wall. Further detection revealed that classical apoptotic markers such as reactive oxygen species (ROS) accumulation, phosphatidylserine (PS) externalization, DNA strand breaks and caspase-like activities could be found in fungal cells after treated by WH1fungin. Oligomycin was used as an inhibitor to block the mitochondria-dependent apoptosis in fungal cells, and results showed it could not inhibit but enhance the apoptosis induced by WH1fungin. After isolation by affinity chromatography, WH1fungin was found to bind with ATPase on the mitochondrial membrane and result in a decreased ATPase activity in fungal cells. This was further verified by treating fungal cells with FITC-labeled WH1fungin, which could bind to the mitochondrial membrane showing green fluorescence in fungal cells. After that, cytochrome C was released from the mitochondria, which then acted with caspase 9 to induce apoptosis by an intracellular pathway. High caspase 8 activity was also detectable in apoptotic fungal cells, indicating that an extracellular pathway might also be responsible for apoptosis induced by WH1fungin. Taken together, we report that lipopeptide can induce apoptosis in fungal cells, and induction of apoptosis by lipopeptide might be a common anti-fungal mechanism of Bacillus in the natural habitat.

WH1fungin a surfactin cyclic lipopeptide is a novel oral immunoadjuvant.

WH1fungin, a surfactin lipopeptide from Bacillus amyloliquefaciens WH1, can be used as an adjuvant for eliciting strong immune response by parenteral immunization. In this study, WH1fungin was firstly reported as an oral immunoadjuvant. In mice, WH1fungin markedly enhanced the immune response to co-administered protein antigens (OVA or GST), similar to levels elicited by CTB, but no immune response was elicited to itself. Both IgG1 and IgG2a antibodies elicited from the immunizations indicating a mixed Th1/Th2 response. Splenocytes from mice immunized with OVA plus WH1fungin responded to OVA CTL peptide stimulation resulting in an increase in CD8(+)TNF-α(+) and CD8(+)IFN-γ(+) T cell populations. These results further suggested that WH1fungin helps to elicit both humoral and cellular responses to OVA. More studies revealed that the potential mechanism as oral immunoadjuvant was that WH1fungin could form co-precipitates with antigens in a pH value similar to gastric juice. The precipitation protected the antigens from degradation by pepsin providing an explanation for the antigens to withstand the acidic and proteolytic environments of the gastrointestinal tract when co-administered with WH1fungin. Moreover, WH1fungin promoted the uptake of OVA by the intestine and by cultured DC2.4 cells, and increased the expression of cell surface markers and cytokines in DC2.4 cells. Taken together, WH1fungin is a potent oral immunoadjuvant with the ability of protecting protein antigens from acidic and proteolytic degradation, suggesting its potential usage in oral vaccine development.

Controlling Myzus persicae with recombinant endophytic fungi Chaetomium globosum expressing Pinellia ternata agglutinin: using recombinant endophytic fungi to control aphids.

Aims:  Sap‐sucking insect pests have become the major threats to many crops in recent years; however, only a few biopesticides have been developed for controlling those pests. Here, we developed a novel pest management strategy, which uses endophytes to express anti‐pest plant lectins.

Immunomodulation therapy of diabetes by oral administration of a surfactin lipopeptide in NOD mice

Type 1 diabetes mellitus (T1DM) is considered an autoimmune disease, which can be attenuated by modulation of immune pathway from Th1- to Th2-type through vaccination. WH1fungin surfactin is a Bacillus-produced natural immunomodulator. NOD mice were orally treated with 5mg/kg or 25mg/kg WH1fungin once a week for total 4 weeks. After the final administration, the diabetes incidence and the anti-inflammatory roles of WH1fungin were investigated by immunohistochemistry, FACS and ELISA. The results showed oral WH1fungin obviously resulted in a WH1fungin-unspecific suppression of T1DM. Diabetes incidence was significantly reduced when compared to phosphate buffered saline (PBS) control. Mice in the control group began to be onset of diabetes at week 15, following with an increased mortality from week 16 to 28. At the end of observation, the diabetes incidence reached to 81% at week 30, while only 25% in WH1fungin groups. The splenocytes assay showed oral WH1fungin could suppress T cells proliferation, down-regulate amounts of activated CD8(+) T cells with the production of tumor necrosis factor (TNF)-α and interferon (IFN)-γ, and increase CD4(+)CD25(+)FOXP3(+) regulator T cells (Tregs). The serum assay revealed oral WH1fungin down-regulated TNF-α and IgG2a but increased interleukin (IL)-10 and IgG1 in mice. All of these data showed oral WH1fungin tended to switch the immune response from Th1- to Th2-type. The further surveys revealed that less IFN-γ but more transfer growth factor (TGF)-β were found in the islets of mice with oral WH1fungin when compared to that in the control group. As a result, the normal islet architecture and slight inflammatory cells infiltration was observed with a slight insulitis in the oral WH1fungin groups. These results demonstrate that oral WH1fungin might be a novel therapeutic approach for the prevention of T1DM.

A natural lipopeptide of surfactin for oral delivery of insulin

Abstract Surfactin, a natural lipopeptide produced by Bacillus, is gaining attention for potentially biomedical and pharmaceutical applications. Here, surfactin was assayed for oral delivery of insulin (INS) by its ability to bind to and promote protein to penetrate through the cell membrane. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, surfactin was found to form co-precipitates with INS to protect it from acidic and enzymatic attack in the gastrointestinal tract. Further analysis by non-reductive electrophoresis showed surfactin could bind to INS forming heteropolymers. Analysis with circular dichroism, we found this binding significantly influenced the INS structure with decreased rigid α-helix and β-turn, but with increased flexible β-sheet and random coil. The change with more flexible structure was favorable for INS to penetrate through the cell membrane. Fluorescence spectra analysis also showed surfactin could lead Phe and Tyr in the inner of INS exposed outside, further promoting INS permeabilization by improving the hydrophobic-lipophilic interactions between INS and cell membrane. As a result, the effective permeability (Peff) of INS plus surfactin was 4.3 times of that of INS alone. In vivo assay showed oral INS with surfactin displayed excellent hypoglycemic effects with a relative bioavailability of 12.48% and 5.97% in diabetic mice and non-diabetic dogs, respectively. Summary, surfactin is potential for oral delivery of INS by its role as an effective protease inhibitor and permeability enhancer.

ApoB-100 and HSP60 peptides exert a synergetic role in inhibiting early atherosclerosis in immunized ApoE-null mice.

Human heat shock protein 60 (hHSP60) and apolipoprotein B-100 (ApoB-100) in oxidized low density lipoproteins are considered pro-atherosclerotic factors by inducing autoimmunity response, and immunization with peptides from these two proteins can inhibit atherosclerosis in animal models. In this study, we constructed chimeric proteins containing ApoB-100 and/or hHSP60 peptides by human intestinal trefoil factor (ITF) as a scaffold and then fused with glutathionine-S transferase (GST) for expression in Escherichia coli. These purified chimeric proteins were used for immunizing apolipoprotein E (ApoE)-null mice fed on Western diet, and then the immune response and anti-atherosclerotic effect was assayed. Unexpectedly, neither anti-ApoB-100 nor anti-hHSP60 antibodies could be detected in serum. Histological analysis demonstrated the mice immunized with a chimeric protein containing both ApoB-100 and hHSP60 peptides showed the most significant reduction of atherosclerotic lesions (65.9%), and the mice immunized with the chimeric protein only containing ApoB-100 or hHSP60 peptide also showed a 26.7% (p<0.01) or 61.5% (p<0.001) reduction of atherosclerotic lesions when compared to GST control. The chimeric protein containing hHSP60 peptide was more efficient than that containing apoB-100 peptide for inhibiting atherosclerosis. This result was further supported by the in vitro assay that hHSP60 peptide could induce DCs and CD4(+) T cells to produce more TGF-beta (p<0.01) and less IFN-gamma (p<0.001) than ApoB-100 peptide. This result highlights a way for developing anti-atherosclerotic agents by construction of chimeric proteins containing hHSP60 and/or ApoB-100 peptides in the future.

A surfactin cyclopeptide of WH1fungin used as a novel adjuvant for intramuscular and subcutaneous immunization in mice

WH1fungin, a surfactin cyclopeptide from Bacillus amyloliquefaciens WH1, is firstly reported as a novel immunoadjuvant, which can markedly enhance the immune response when given in mixture with antigens. After intramuscular or subcutaneous immunization, WH1fungin can help to induce both of durable humoral and cellular immune response, even as strong as Freunds adjuvant. Both IgG1 and IgG2a antigen-specific antibodies were elicited from the immunizations indicating a mixed Th1/Th2 response. Splenocytes from mice intramuscularly immunized with OVA plus WH1fungin responded to OVA CTL peptide stimulation resulting in an increase in CD8(+)TNF-α(+) and CD8(+)IFN-γ(+) T cell populations, and also an increase in CD4(+)TNF-α(+) T cells and CD4(+)IFN-γ(+) T cell populations was found from mice subcutaneously immunized with OVA plus WH1fungin when responded to OVA Th peptide stimulation. These results further suggest that WH1fungin helps to elicit humoral and cellular responses to OVA. The potential mechanism of WH1fungin as an immunoadjuvant was investigated. In vitro assays showed that WH1fungin could enter into RAW 264.7 cells, induce ROS accumulation, and increase the expression of cell surface markers and cytokines in cells. Further investigation suggested that WH1fungin might exert its adjuvant activity by ligating with TLR-2 in antigen present cells such as RAW 264.7. Taken together, WH1fungin is very potent as a novel adjuvant for development of vaccines in the future.

Surfactin inducing mitochondria-dependent ROS to activate MAPKs, NF-κB and inflammasomes in macrophages for adjuvant activity

Surfactin, a natural lipopeptide, can be used both as parenteral and non-parenteral adjuvant for eliciting immune response. However, the mechanisms that confer its adjuvant properties have not been fully explored. By staining with NHS-Rhodamine B labeled surfactin and Mito-Tracker Green, we found surfactin could penetrate into macrophages to bind with mitochondria, following induce ROS that could be inhibited by mitochondria-dependent ROS inhibitor. ROS enhanced p38 MAPK and JNK expression, as well their phorsphorylation, following activated NF-κB nuclear translocation in macrophages that was obviously inhibited by mitochondria-dependent ROS inhibitor. However, inhibition of ROS production only weakened p38 MAPK and JNK expression, but not their phosphorylation in macrophages. As a result, surfaction could activate NF-κB to release TNF-α by the mitochondria-dependent ROS signalling pathway. ROS also induced macrophages apoptosis to release endogenous danger signals, following activated inflammasomes of NLRP1, NLRP3, IPAF and AIM2 in vitro and only NLRP1 in vivo, as well caspase-1 and IL-1 in macrophages, which were significantly inhibited by pre-treatment with ROS inhibitors. Collectively, surfactin as a kind of non-pathogen-associated molecular patterns, modulates host innate immunity by multiple signalling pathways, including induction of mitochondria-dependent ROS, activating MAPKs and NF-κB, and inducing cell apoptosis to realease endogenous danger signals for activation of inflammasomes.

Constructing Tumor Vaccines Targeting for Vascular Endothelial Growth Factor (VEGF) by DNA Shuffling.

Most of tumor antigens are self-proteins with poor antigenicity because of immune tolerance. Here, we describe DNA shuffling for overcoming the tolerance of tumor antigens such as vascular endothelial growth factor (VEGF), a growth factor associated with tumor angiogenesis. VEGF genes from mouse, rat, human, and chicken were randomly assembled to chimeric genes by DNA shuffling for constructing an expression library, then screened by PCR, SDS-PAGE, and immunization. A chimeric protein named as No. 46 was selected from the library with the strongest immunotherapy effects on mouse H22 hepatocellular carcinoma, which could induce long-lasted and high level of antibodies recognizing VEGF in mice. Immunization with this chimeric protein could significantly inhibit tumor angiogenesis, slow down tumor growth, increase the survival rate of tumor-bearing mice, and inhibit the lung metastases of tumor in mouse. Treatment with the anti-VEGF IgG induced by this chimeric protein also significantly inhibited tumor growth and improved the survival rate of tumor-bearing mice, by blocking the tyrosine phosphorylation of ERK1/2 pathway of VEGF-VEGFR interaction. Our study provides an efficient approach to overcome the immune tolerance of self-antigens for developing novel tumor vaccines.

A Surfactin Lipopeptide Adjuvanted Hepatitis B Vaccines Elicit Enhanced Humoral and Cellular Immune Responses in Mice

WH1fungin (WF), a lipopeptide surfactin, has been verified as an immunoadjuvant previously. In this study, mice were intranasally or parenterally immunized with WF plus Hepatitis B surface antigen (HBsAg), then the immune responses were detected. The results showed 50 µg WF plus 20 μg HBsAg for intranasal and 10 μg WF plus 1 µg HBsAg for parenteral immunization was efficient inducing strong immune response against HBsAg in mice. A high titer and longterm anti-HBsAg IgG was observed for more than 19 weeks in intranasal or parenteral immunizations, much higher than that induced by CpG or Alum adjuvant. The anti-HBsAg IgA was also induced in intestine and lung, indicating that mucosal as well as systemic immune response was elicited for intranasal immunization. The IgG isotype in serum revealed WF induced a Th2-bias immune response with a higher titer of anti-HBsAg IgG1 than IgG2a in mice. Moreover, WF also induced more Th1 cells producing interferon (IFN)-γ and stronger cytotoxic T lymphocytes response than controls for intramuscular administration. These data further confirmed that WF induced Th1- as well as Th2- type immune response toward HBsAg. Taken together, WF-adjuvanted HBsAg elicits more effective immune response than that adjuvanted by Alum or CpG, suggesting its potential for development of more efficient HBV vaccines in the future.