Efficient production of a novel alkaline cold-active phospholipase C from Aspergillus oryzae by molecular chaperon co-expression for crude oil degumming.

A novel alkaline cold-active phospholipase C (PLC) gene (AoPC) from Aspergillus oryzae was cloned. AoPC exhibited the highest sequence similarity of 32.5% with that of a PLC from Arabidopsis thaliana. The gene was co-expressed in Pichia pastoris with molecular chaperone PDI (protein disulfide isomerases), and the highest PLC activity of 82, 782 U mL-1 was achieved in a 5-L fermentor. The recombinant enzyme (AoPC) was most active at pH 8.0 and 25 °C, respectively, and it was stable over a broad pH range of 4.5-9.0 and up to 40 °C. It is the first fungal alkaline PLC. The application of AoPC (with 25% citric acid, w/w) in oil degumming process significantly reduced the phosphorus of crude soybean oil by 93.3% to a commercially acceptable level (<10 mg kg-1). Therefore, the relatively high yield and excellent properties of AoPC may possess it great potential in crude oil refining industry.

Pharmacological evidence for transactivation within melatonin MT2 and serotonin 5-HT2C receptor heteromers in mouse brain.

Association of G protein-coupled receptors into heterodimeric complexes has been reported for over 50 receptor pairs in vitro but functional in vivo validation remains a challenge. Our recent in vitro studies defined the functional fingerprint of heteromers composed of Gi -coupled melatonin MT2 receptors and Gq -coupled serotonin 5-HT2C receptors, in which melatonin transactivates phospholipase C (PLC) through 5-HT2C . Here, we identified this functional fingerprint in the mouse brain. Gq protein activation was probed by [35 S]GTPγS incorporation followed by Gq immunoprecipitation, and PLC activation by determining the inositol phosphate levels in brain lysates of animals previously treated with melatonin. Melatonin concentration-dependently activated Gq proteins and PLC in the hypothalamus and cerebellum but not in cortex. These effects were inhibited by the 5-HT2C receptor-specific inverse agonist SB-243213, and were absent in MT2 and 5-HT2C knockout mice, fully recapitulating previous in vitro data and indicating the involvement of MT2 /5-HT2C heteromers. The antidepressant agomelatine had a similar effect than melatonin when applied alone but blocked the melatonin-promoted Gq activation due to its 5-HT2C antagonistic component. Collectively, we provide strong functional evidence for the existence of MT2 /5-HT2C heteromeric complexes in mouse brain. These heteromers might participate in the in vivo effects of agomelatine.

Immunization of rabbits with recombinant Clostridium perfringens alpha toxins CPA-C and CTB-CPA-C in a bicistronic design expression system confers strong protection against challenge.

The Clostridium perfringens alpha toxin (CPA), encoded by the plc gene, is the causative pathogen of gas gangrene, which is a lethal infection. In this study, we used an E. coli system for the efficient production of recombinant proteins and developed a bicistronic design (BCD) expression construct consisting of two copies of the C-terminal (247-370) domain of the alpha toxin (CPA-C) in the first cistron, followed by Cholera Toxin B (CTB) linked with another two copies of CPA-C in the second cistron that is controlled by a single promoter. Rabbits were immunized twice with purified proteins (rCPA-C rCTB-CPA-C) produced in the BCD expression system, with an inactivated recombinant E. coli vaccine (RE), C. perfringens formaldehyde-inactivated alpha toxoid (FA-CPA) and C. perfringensl-lysine/formaldehyde alpha toxoid (LF-CPA) vaccines. Following the second vaccination, 0.1 mL of pooled sera of the RE-vaccinated rabbits could neutralize 12× mouse LD100 (100% lethal dose) of CPA, while that of the rCPA-C rCTB-CPA-C-vaccinated rabbits could neutralize 6× mouse LD100 of CPA. Antibody titers against CPA were also assessed by ELISA, reaching titers as high as 1:2048000 in the RE group; this was significantly higher compared to the C. perfringens alpha toxoid vaccinated groups (FA-CPA and LF-CPA). Rabbits from all vaccinated groups were completely protected from a 2× rabbit LD100 of CPA challenge. These results demonstrate that the recombinant proteins are able to induce a strong immune responses, indicating that they may be potentially utilized as targets for novel vaccines specifically against the C. perfringens alpha toxin.

The effects of probiotic Bacillus coagulans on the cytotoxicity and expression of alpha toxin gene of Clostridium perfringens type A.

Around the world, Clostridium perfringens type A is known to be a common foodborne pathogen. Therefore, the control and treatment of food poisoning caused by this pathogen are important. This study investigated, in vitro, the effects of Bacillus coagulans and its culture extracts on alpha toxin gene expression, growth inhibition, cytotoxicity, and apoptosis induced by C. perfringens spore, germinated spore and its enterotoxin. Flow cytometry was used to evaluate the apoptosis rate, and MTT test was used to evaluate cytotoxicity. Minimum inhibitory concentration was also used to measure the percentage of inhibition in the broth medium. Finally, RT-qPCR was used to evaluate alpha toxin gene expression. The results showed that the B. coagulans culture extract was able to inhibit the growth of the germinated spore of C. perfringens. Moreover, treating the extract with pepsin can reduce growth in the broth medium. MTT and flow cytometry showed that both B. coagulans and its extract can significantly reduce the cytotoxicity and apoptosis rate induced by C. perfringens type A. In addition, it was shown that the co-culture of B. coagulans and C. perfringens decreases alpha toxin gene expression. The findings of this study indicate that B. coagulans, with growth inhibition and reduced expression of alpha toxin in C. perfringens, can reduce the cytotoxicity and apoptosis rate induced on HT-29 cells.

RelA/DTD-mediated regulation of spore formation and toxin production by Clostridium perfringens type A strain SM101.

RelA is a global regulator for stationary phase development in the model bacterium Bacillus subtilis. The relA gene forms a bicistronic operon with the downstream dtd gene. In this study, we evaluated the significance of RelA and DTD proteins in spore formation and toxin production by an important gastrointestinal pathogen Clostridium perfringens. Our ß-glucuronidase assay showed that in C. perfringens strain SM101, relA forms a bicistronic operon with its downstream dtd gene, and the relA promoter is expressed during both vegetative and sporulation conditions. By constructing double relA dtd and single dtd mutants in C. perfringens SM101, we found that: (1) RelA is required for maintaining the efficient growth capacity of SM101 cells during vegetative conditions; (2) both RelA and DTD are required for spore formation and enterotoxin (CPE) production by SM101; (3) RelA/DTD activate CodY, which is known to activate spore formation and CPE production in SM101 by activating a key sporulation-specific σ factor F; (4) as expected, RelA/DTD activate sporulation-specific σ factors (σE, σF, σG and σK) by positively regulating Spo0A production; and finally (5) RelA, but not DTD, negatively regulates phospholipase C (PLC) production by repressing plc gene expression. Collectively, our results demonstrate that RelA modulates cellular physiology such as growth, spore formation and toxin production by C. perfringens type A strain SM101, although DTD also plays a role in these pleiotropic functions in coordination with RelA during sporulation. These findings have implications for the understanding of the mechanisms involved in the infectious cycle of C. perfringens.

Expression of the alpha toxin of Clostridium perfringens in Lactobacillus casei genome and evaluation of its immune effects in mice.

We previously developed a stable and marker-free Lactobacillus casei strain (PPαT Δupp) that contained a chromosomally integrated expression cassette (PPαT) that enabled the surface expression of the Clostridium perfringens alpha toxin. To measure immune responses against the alpha toxin, specific-pathogen-free BALB/c mice were inoculated with L. casei PPαT Δupp by oral gavage. Then, specific immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies and cytokines were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM). The results showed that alpha toxin-specific IgA and IgG antibodies and cytokines were markedly increased following immunization. Natural alpha toxin challenge and neutralization tests were performed. The results showed that immunized mice can fully resist 1.5 minimum lethal doses of toxin. These results indicated that the immunized mice can produce not only humoral immunity, but also cellular immunity. These results provide a new pathway for the development of a safe, effective, and food-grade vaccine.

Polymicrobial infections involving clinically relevant Gram-negative bacteria and fungi.

Interactions between fungi and bacteria and their relevance to human health and disease have recently attracted increased attention in biomedical fields. Emerging evidence shows that bacteria and fungi can have synergistic or antagonistic interactions, each with important implications for human colonization and disease. It is now appreciated that some of these interactions may be strategic and helps promote the survival of one or both microorganisms within the host. This review will shed light on clinically relevant interactions between fungi and Gram-negative bacteria. Mechanism of interaction, host immune responses, and preventive measures will also be reviewed.

Chronic Psychological Stress Disrupted the Composition of the Murine Colonic Microbiota and Accelerated a Murine Model of Inflammatory Bowel Disease.

The effect of psychological stress on the gastrointestinal microbiota is widely recognized. Chronic psychological stress may be associated with increased disease activity in inflammatory bowel disease, but the relationships among psychological stress, the gastrointestinal microbiota, and the severity of colitis is not yet fully understood. Here, we examined the impact of 12-week repeated water-avoidance stress on the microbiota of two inbred strains of T cell receptor alpha chain gene knockout mouse (background, BALB/c and C57BL/6) by means of next-generation sequencing of bacterial 16S rRNA genes. In both mouse strains, knockout of the T cell receptor alpha chain gene caused a loss of gastrointestinal microbial diversity and stability. Chronic exposure to repeated water-avoidance stress markedly altered the composition of the colonic microbiota of C57BL/6 mice, but not of BALB/c mice. In C57BL/6 mice, the relative abundance of genus Clostridium, some members of which produce the toxin phospholipase C, was increased, which was weakly positively associated with colitis severity, suggesting that expansion of specific populations of indigenous pathogens may be involved in the exacerbation of colitis. However, we also found that colitis was not exacerbated in mice with a relatively diverse microbiota even if their colonic microbiota contained an expanded phospholipase C-producing Clostridium population. Exposure to chronic stress also altered the concentration of free immunoglobulin A in colonic contents, which may be related to both the loss of bacterial diversity in the colonic microbiota and the severity of the colitis exacerbation. Together, these results suggest that long-term exposure to psychological stress induces dysbiosis in the immunodeficient mouse in a strain-specific manner and also that alteration of microbial diversity, which may be related to an altered pattern of immunoglobulin secretion in the gastrointestinal tract, might play a crucial role in the development of chronic stress-induced colitis.

Recent research progress with phospholipase C from Bacillus cereus.

Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce phosphate monoesters and diacylglycerol. It has many applications in the enzymatic degumming of plant oils. PLC Bc , a bacterial PLC from Bacillus cereus, is an optimal choice for this activity in terms of its wide substrate spectrum, high activity, and approved safety. Unfortunately, its large-scale production and reliable high-throughput screening of PLC Bc remain challenging. Herein, we summarize the research progress regarding PLC Bc with emphasis on the screening methods, expression systems, catalytic mechanisms and inhibitor of PLC Bc . This review hopefully will inspire new achievements in related areas, to promote the sustainable development of PLC Bc and its application.

High-level production of Bacillus cereus phospholipase C in Corynebacterium glutamicum.

Enzymatic oil degumming (removal of phospholipids) using phospholipase C (PLC) is a well-established and environmentally friendly process for vegetable oil refining. In this work, we report the production of recombinant Bacillus cereus PLC in Corynebacterium glutamicum ATCC 13869 in a high cell density fermentation process and its performance in soybean oil degumming. A final concentration of 5.5g/L of the recombinant enzyme was achieved when the respective gene was expressed from the tac promoter in a semi-defined medium. After treatment with trypsin to cleave the propeptide, the mature enzyme completely hydrolyzed phosphatidylcholine and phosphatidylethanolamine, which represent 70% of the phospholipids present in soybean oil. The results presented here show the feasibility of using B. cereus PLC for oil degumming and provide a manufacturing process for the cost effective production of this enzyme.