Anti-influenza activity of CPAVM1 protease secreted by Bacillus subtilis LjM2.

Bacillus spp. has been considered a promising source for identifying new antimicrobial substances, including anti-viral candidates. Here, we successfully isolated a number of bacteria strains from aged dry citrus peel (Chenpi). Of note, the culture supernatant of a new isolate named Bacillus subtilis LjM2 demonstrated strong inhibition of influenza A virus (IAV) infection in multiple experimental systems in vitro and in vivo. In addition, the anti-viral effect of LjM2 was attributed to its direct lysis of viral particles. Further analysis showed that a protease which we named CPAVM1 isolated from the culture supernatant of LjM2 was the key component responsible for its anti-viral function. Importantly, the therapeutic effect of CPAVM1 was still significant when applied 12 hours after IAV infection of experimental mice. Moreover, we found that the CPAVM1 protease cleaved multiple IAV proteins via targeting basic amino acid Arg or Lys. Furthermore, this study reveals the molecular structure and catalytic mechanism of CPAVM1 protease. During catalysis, Tyr75, Tyr77, and Tyr102 are important active sites. Therefore, the present work identified a special protease CPAVM1 secreted by a new strain of Bacillus subtilis LjM2 against influenza A virus infection via direct cleavage of critical viral proteins, thus facilitates future biotechnological applications of Bacillus subtilis LjM2 and the protease CPAVM1.

Roles and Mechanisms of NLRP3 in Influenza Viral Infection.

Pathogenic viral infection represents a major challenge to human health. Due to the vast mucosal surface of respiratory tract exposed to the environment, host defense against influenza viruses has perpetually been a considerable challenge. Inflammasomes serve as vital components of the host innate immune system and play a crucial role in responding to viral infections. To cope with influenza viral infection, the host employs inflammasomes and symbiotic microbiota to confer effective protection at the mucosal surface in the lungs. This review article aims to summarize the current findings on the function of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) in host response to influenza viral infection involving various mechanisms including the gut-lung crosstalk.

Pathobionts from chemically disrupted gut microbiota induce insulin-dependent diabetes in mice.

BACKGROUND: Dysbiotic gut microbiome, genetically predisposed or chemically disrupted, has been linked with insulin-dependent diabetes (IDD) including autoimmune type 1 diabetes (T1D) in both humans and animal models. However, specific IDD-inducing gut bacteria remain to be identified and their casual role in disease development demonstrated via experiments that can fulfill Koch's postulates. RESULTS: Here, we show that novel gut pathobionts in the Muribaculaceae family, enriched by a low-dose dextran sulfate sodium (DSS) treatment, translocated to the pancreas and caused local inflammation, beta cell destruction and IDD in C57BL/6 mice. Antibiotic removal and transplantation of gut microbiota showed that this low DSS disrupted gut microbiota was both necessary and sufficient to induce IDD. Reduced butyrate content in the gut and decreased gene expression levels of an antimicrobial peptide in the pancreas allowed for the enrichment of selective members in the Muribaculaceae family in the gut and their translocation to the pancreas. Pure isolate of one such members induced IDD in wildtype germ-free mice on normal diet either alone or in combination with normal gut microbiome after gavaged into stomach and translocated to pancreas. Potential human relevance of this finding was shown by the induction of pancreatic inflammation, beta cell destruction and IDD development in antibiotic-treated wildtype mice via transplantation of gut microbiome from patients with IDD including autoimmune T1D. CONCLUSION: The pathobionts that are chemically enriched in dysbiotic gut microbiota are sufficient to induce insulin-dependent diabetes after translocation to the pancreas. This indicates that IDD can be mainly a microbiome-dependent disease, inspiring the need to search for novel pathobionts for IDD development in humans. Video Abstract.

Microbiota-derived acetate enhances host antiviral response via NLRP3.

Pathogenic viral infections represent a major challenge to human health. Host immune responses to respiratory viruses are closely associated with microbiome and metabolism via the gut-lung axis. It has been known that host defense against influenza A virus (IAV) involves activation of the NLRP3 inflammasome, however, mechanisms behind the protective function of NLRP3 are not fully known. Here we show that an isolated bacterial strain, Bifidobacterium pseudolongum NjM1, enriched in the gut microbiota of Nlrp3-/- mice, protects wild-type but not Nlrp3 deficient mice against IAV infection. This effect depends on the enhanced production of type I interferon (IFN-I) mediated by NjM1-derived acetate. Application of exogenous acetate reproduces the protective effect of NjM1. Mechanistically, NLRP3 bridges GPR43 and MAVS, and promotes the oligomerization and signalling of MAVS; while acetate enhances MAVS aggregation upon GPR43 engagement, leading to elevated IFN-I production. Thus, our data support a model of NLRP3 mediating enhanced induction of IFN-I via acetate-producing bacterium and suggest that the acetate-GPR43-NLRP3-MAVS-IFN-I signalling axis is a potential therapeutic target against respiratory viral infections.

Aim2 Couples With Ube2i for Sumoylation-Mediated Repression of Interferon Signatures in Systemic Lupus Erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) involves kidney damage, and the inflammasome-caspase-1 axis has been demonstrated to promote renal pathogenesis. The present study was designed to explore the function of the Absent in Melanoma 2 (Aim2) protein in SLE. METHODS: Female wild-type Aim2-/- , Aim2-/- Ifnar1-/- , Aim2-/- Rag1-/- , and Asc-/- mice ages 8-10 weeks received 1 intraperitoneal injection of 500 µl pristane or saline, and survival of mice was monitored twice a week for 6 months. RESULTS: The absence of Aim2, but not Asc, led to enhanced SLE in mice that received pristane treatment. Increased immune cell infiltration and type I interferon (IFN) signatures in the kidneys of Aim2-/- mice coincided with severity of lupus, which was alleviated by blockade of Ifnar1-mediated signal. Adaptive immune cells were also involved in the glomerular lesions of Aim2-/- mice after pristane challenge. Importantly, even in the absence of pristane, plasmacytoid dendritic cells in the kidneys of Aim2-/- mice were significantly increased compared to control animals. Accordingly, transcriptome analysis revealed that Aim2 deficiency led to enhanced expression of type I IFN-induced genes in the kidneys even at an early developmental stage. Mechanistically, Aim2 bound ubiquitin-conjugating enzyme 2i (Ube2i), which mediates sumoylation-based suppression of type I IFN expression deficiency of Aim2 decreased cellular sumoylation, resulting in an augmented type I IFN signature and kidney pathogenesis. CONCLUSION: The present study demonstrates a critical role for Aim2 in an optimal Ube2i-mediated sumoylation-based suppression of type I IFN generation and development of SLE. As such, the Aim2-Ube2i axis can thus be a novel target for intervention in SLE.

Antiviral effects of simeprevir on multiple viruses.

Simeprevir was developed as a small molecular drug targeting the NS3/4A protease of hepatitis C virus (HCV). Unexpectedly, our current work discovered that Simeprevir effectively promoted the transcription of IFN-ß and ISG15, inhibited the infection of host cells by multiple viruses including Zika virus (ZIKV), Enterovirus A71 (EV-A71), as well as herpes simplex virus type 1 (HSV-1). However, the inhibitory effects of Simeprevir on ZIKV, EV-A71 and HSV-1 were independent from IFN-ß and ISG15. This study thus demonstrates that the application of Simeprevir can be extended to other viruses besides HCV.

Hyperactivation of the NLRP3 inflammasome protects mice against influenza A virus infection via IL-1ß mediated neutrophil recruitment.

Host innate immune system is critical for combating invading microbes including Influenza A virus (IAV). As an important arm of the innate immunity, the NLRP3 inflammasome has been found essential for protecting host against IAV challenge, while the mechanism remained elusive. Here we found that mice carrying a gain-of-function mutation in the Nlrp3 gene (Nlrp3R258W) are strongly resistant to IAV infection. Upon H1N1 IAV infection, the Nlrp3R258W mice exhibited decreased weight loss, increased survival rate and attenuated lung damage compared with WT littermate controls. Mechanistically, the resistance of Nlrp3R258W mice to IAV infection was dependent on IL-1ß-mediated neutrophil recruitment. Upon IAV infection, mice carrying the Nlrp3R258W mutation produced more IL-1ß than WT mice in the lung, which enhanced neutrophil recruitment locally. The recruited neutrophils facilitated IAV clearance, so that the viral load in Nlrp3R258W mice was lower than that in control mice. Conversely, neutrophil depletion in Nlrp3R258W mice compromised IAV clearance. Taken together, our results demonstrate a previously undescribed mechanism by which hyperactivation of the NLRP3 Inflammasome protects mice from IAV infection through IL-1ß mediated neutrophil recruitment, thus suggest that positively fine tuning the physiological function of NLRP3 inflammasome can be beneficial for a mammalian host against IAV challenge.

Antiviral effects of ferric ammonium citrate.

Iron is an essential nutrient for cell survival and is crucial for DNA replication, mitochondrial function and erythropoiesis. However, the immunological role of iron in viral infections has not been well defined. Here we found the iron salt ferric ammonium citrate (FAC) inhibited Influenza A virus, HIV virus, Zika virus, and Enterovirus 71 (EV71) infections. Of note, both iron ion and citrate ion were required for the antiviral capability of FAC, as other iron salts and citrates did not exhibit viral inhibition. Mechanistically, FAC inhibited viral infection through inducing viral fusion and blocking endosomal viral release. These were further evidenced by the fact that FAC induced liposome aggregation and intracellular vesicle fusion, which was associated with a unique iron-dependent cell death. Our results demonstrate a novel antiviral function of FAC and suggest a therapeutic potential for iron in the control of viral infections.

Interleukin-18 protects mice from Enterovirus 71 infection.

Previous study has demonstrated that the NLRP3 inflammasome is essential for protecting murine host against Enterovirus 71 (EV71) infection. However, the underlying mechanism remained unknown. Here we discovered that the pleiotropic cytokine interleukin-18 (IL-18), an NLRP3 inflammasome-dependent effector protein, exhibits a protective capability against EV71 challenge. Deficiency of IL-18 in mice exacerbated EV71 infection, which was reflected by increased viral replication, elevated production of interferons (IFN-ß, IFN-γ), proinflammatory cytokines (TNF-α, IL-6) and chemokine CCL2,as well as decreased survival of experimental animals. Conversely, administration of recombinant IL-18 considerably restrained EV71 infection in IL-18 deficient mice. Thus, our results revealed a protective role for IL-18 against EV71 challenge, and indicated a novel therapeutic application for IL-18 in EV71 associated hand, foot, and mouth disease (HFMD).

Hyperactivation of the NLRP3 Inflammasome in Myeloid Cells Leads to Severe Organ Damage in Experimental Lupus.

Systemic lupus erythematosus (SLE) is an autoimmune syndrome associated with severe organ damage resulting from the activation of immune cells. Recently, a role for caspase-1 in murine lupus was described, indicating an involvement of inflammasomes in the development of SLE. Among multiple inflammasomes identified, the NLRP3 inflammasome was connected to diverse diseases, including autoimmune encephalomyelitis. However, the function of NLRP3 in SLE development remains elusive. In this study, we explored the role of NLRP3 in the development of SLE using the pristane-induced experimental lupus model. It was discovered that more severe lupus-like syndrome developed in Nlrp3-R258W mice carrying the gain-of-function mutation. Nlrp3-R258W mutant mice exhibited significantly higher mortality upon pristane challenge. Moreover, prominent hypercellularity and interstitial nephritis were evident in the glomeruli of Nlrp3-R258W mice. In addition, hyperactivation of the NLRP3 inflammasome in this mouse line resulted in proteinuria and mesangial destruction. Importantly, all of these phenotypes were largely attributed to the Nlrp3-R258W mutation expressed in myeloid cells, because Cre recombinase-mediated depletion of this mutant from such cells rescued mice from experimental lupus. Taken together, our study demonstrates a critical role for NLRP3 in the development of SLE and suggests that modulating the inflammasome signal may help to control the inflammatory damage in autoimmune diseases, including lupus.

[Composition diversity of the multifunctional bacterium community NSC-7].

The NSC-7 microbial community could decompose cellulose and lindan with high efficiency. In order to determine the bacterial composition of the community, 11 isolate strains were detected by plate isolation, while a community reset by the 11 isolate strains lost the capacity of degrading cellulose. The capacity of degrading of the filter paper in double deck plate and monolayer plate were determined, only the filter paper in double deck plate were degraded, that means the main or key microbe are anaerobic. The denaturing gradient gel electrophoresis (DGGE) and construction of 16S rDNA clone library were used to identify the composition diversity of NSC-7 community. 195 clones and 25 strains were detected in clone library, and about 60% closest relative among them was known the detailed information which were belonged to Clostridium, Petrobacter, Bacteria, Paenibacillus, Proteobacterium. Furthermore, there were 40% closest relative belonged to uncultured bacterium clone.

[Capability and stability of degrading rice straw of composite microbial system MC1].

The capability of degrading rice straw of lignocellulolytic composite microbial system MC1 was investigated under different methods of preservation and temperatures treatments of 80 degrees C to 95 degrees C, and stability of composite microbial system MC1 was studied through the method of Denaturing Gradient Gel Electrophoresis (DGGE). The results indicate that the rice straw of 2% dry weight of medium can be degraded completely at 50 degrees C within 10 days under static culture. After 9 days inoculating MC1, the dry weight of rice straw, cellulose, hemicellulose and lignin content was degraded by 81% , 99%, 74% and 51%, respectively. Capability of cellulose degrading and stability of composite microbial system MC1 is sustained under 4 years of continuing subculture, 4 years of dry preservation at room temperature, 4 years of preservation at - 20 degrees C, 1 year of liquid preservation at room temperature and at 4 degrees C, and treatment of 90 degrees C for 30 min, respectively. Plate culture results show that composite microbial system MC1 are consisted of bacteria. The main DNA bands are not changed by the method of 16S rDNA PCR-DGGE after culture of six months so that microbial composition of MC1 is very stable.

[Construction and function of a high-efficient complex microbial system to degrade cellulose and lindane in compost].

A complex microbial system capable of degrading cellulose and lindane with high efficiency was isolated from four compost heaps. It was selected and domesticated through two methods and by combination of different microbial communities. The results show that the complex microbial system can decompose filter paper, absorbent cotton, rice straw powder and sawdust effectively, especially has high degrading activity for the materials with higher native cellulose such as filter paper and absorbent cotton. As for both of them, the CMC saccharification activity is more than 40U and the degradation efficiency is more than 95% on the 5th day of inoculation. The complex microbial system can also keep a higher degrading capability in a wider range of pH. Filter paper and lindane can be degraded effectively by the complex microbial system during pH 7.0 - 9.0, and the degradation rates are more than 90 % and 45% respectively. Under pH 6.0 - 9.0, there is a good consistency between the degradation of Lindane and the decomposition of filter paper.