Da Teng

Expression of plectasin in Pichia pastoris and its characterization as a new antimicrobial peptide against Staphyloccocus and Streptococcus

Recombinant plectasin, the first fungus defensin, was expressed in Pichia pastoris and purified, and its physical, chemical and antimicrobial characteristics were studied. Following a 120 h induction of recombinant yeast, the amount of total secreted protein reached 748.63 μg/ml. The percentage of recombinant plectasin was estimated to be 71.79% of the total protein. After purification with a Sephadex G-25 column and RP-HPLC, the identity of plectasin was verified by MALDI-TOF MS. Plectasin exhibited strong antimicrobial activity against the Gram-positive bacteria Staphyloccocusaureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Streptococcus suis. At a concentration of 2560 μg/ml, this peptide showed approximately equal activity against S. aureus, S. epidermidis, S. suis, and S. pneumoniae, when compared to 320 μg/ml vancomycin, 640 μg/ml penicillin, 320 μg/ml vancomycin and 160 μg/ml vancomycin, respectively. In addition, plectasin showed anti-S. aureus activity over a wide pH range of 2.0 and 10.0, a high thermal stability at 100 °C for 1h and remarkable resistance to papain and pepsin. The expression and characterization of recombinant plectasin in P. pastoris has potential to treat Streptococcus and Staphyloccocus infections when most traditional antibiotics show no effect on them. Our results indicate that plectasin can be produced in large quantities, and that it has pharmaceutical importance for the prevention and clinical treatment of Staphyloccocus and Streptococcus infections.

Preparation of High-Purity Fructo-oligosaccharides by Aspergillus japonicus β-Fructofuranosidase and Successive Cultivation with Yeast

The short-chain FOSs with high purity were prepared using a two-step strategy: Aspergillus japonicus extracellular beta-fructofuranosidase-catalyzed synthesis of FOSs followed by cultivation with Pichia pastoris (P. pastoris). The higher FOSs content was obtained after 8 h under the catalysis of beta-fructofuranosidase at pH 5.5 and 55 degrees C. Successive P. pastoris cultivation exhausts almost all monosugars in 12 h at 30 degrees C, which increases the purity of FOSs, and also recovers beta-fructofuranosidase activity by ceasing the inhibition of glucose from catalysis of the enzyme, yielding more FOSs. Finally, the FOSs purity was increased from 56.55 to 84.45% (26.47% 1-kestose and 57.98% nystose).

Recombinant production of the antimicrobial peptide NZ17074 in Pichia pastoris using SUMO3 as a fusion partner

The antimicrobial peptide NZ17074, which is derived from arenicin‐3 isolated from Arenicola marina, displayed high activity against a broad range of pathogenic bacteria and fungi. However, NZ17074 has not been produced using fermentation technology. The aim of this work was to study the expression of difficult‐to‐express NZ17074 in Pichia pastoris by fusing with SUMO3. The DNA fragments of NZ17074 and SUMO3 were fused into SUMO3‐NZ17074 using overlap PCR and cloned into the pPICZαA vector to construct the pPICZ‐SUMO3‐NZ17074 expression vector. The rSUMO3‐NZ17074 fusion protein, purified by Ni2 + ‐chelating affinity chromatography, was cleaved by 50% formic acid at 50°C for 28 h to release recombinant NZ17074 (rNZ17074). After purification with second affinity column, 4·1 mg rNZ17074 peptide with the purity over 90% was obtained from per litre fermentation culture. The rNZ17074 peptide exhibited the significant inhibition activity against Gram‐negative bacteria: its minimal inhibitory concentrations (MICs) against Escherichia coli, Salmonella enteritidis and Pseudomonas aeruginosa were 2–4, 2 and 8–16 μg ml−1, respectively, which indicated that SUMO3 is a good fusion partner for the expression of the toxic peptide.

A dual mechanism involved in membrane and nucleic acid disruption of AvBD103b, a new avian defensin from the king penguin, against Salmonella enteritidis CVCC3377

The food-borne bacterial gastrointestinal infection is a serious public health threat. Defensins are evolutionarily conserved innate immune components with broad-spectrum antibacterial activity that do not easily induce resistance. AvBD103b, an avian defensin with potent activity against Salmonella enteritidis, was isolated from the stomach contents of the king penguin (Aptenodytes patagonicus). To elucidate further the antibacterial mechanism of AvBD103b, its effect on the S. enteritidis CVCC3377 cell membrane and intracellular DNA was researched. The cell surface hydrophobicity and a N-phenyl-1-naphthylamine uptake assay demonstrated that AvBD103b treatment increased the cell surface hydrophobicity and outer membrane permeability. Atomic absorption spectrometry, ultraviolet spectrophotometry, flow cytometry, and transmission electron microscopy (TEM) indicated that AvBD103b treatment can lead to the release of the cellular contents and cell death through damage of the membrane. DNA gel retardation and circular dichroism analysis demonstrated that AvBD103b interacted with DNA and intercalated into the DNA base pairs. A cell cycle assay demonstrated that AvBD103b affected cellular functions, such as DNA synthesis. Our results confirmed that AvBD103b exerts its antibacterial activity by damaging the cell membrane and interfering with intracellular DNA, ultimately causing cell death, and suggested that AvBD103b may be a promising candidate as an alternative to antibiotics against S. enteritidis.

Detection of genetically modified crops using multiplex asymmetric polymerase chain reaction and asymmetric hyperbranched rolling circle amplification coupled with reverse dot blot

To meet the ever-increasing demand for detection of genetically modified crops (GMCs), low-cost, high-throughput and high-accuracy detection assays are needed. The new multiplex asymmetric polymerase chain reaction and asymmetric hyper-branched rolling circle amplification coupled with reverse dot blot (RDB) systems were developed to detect GMCs. Thirteen oligonucleotide probes were designed to identify endogenous targets (Lec1, Hmg and Sad1), event-specific targets (RRS-5C, RRS-3C, Bt176-3C and MON810-3C), screening targets (35S promoter and NOS terminator), and control targets (18S and PLX). Optimised conditions were as follows: tailed hybridization probes (1-2 pmol/l) were immobilized on a membrane by baking for 2h, and a 10:1 ratio of forward to reverse primers was used. The detection limits were 0.1 μg/l of 2% RRS and 0.5 ng/l of DNA from genetically modified (GM) soybean. These results indicate that the RDB assay could be used to detect multiplex target genes of GMCs rapidly and inexpensively.

Construction of a standard reference plasmid for detecting GM cottonseed meal.

A plasmid was constructed for quantification of genetically modified (GM) cottonseed meal in the gene-specific level. The Cry1Ab/c gene was connected with the Sad1 gene by fusion PCR. The fusion gene was cloned into the pMD®19-T Simple Vector. The plasmid DNA was then digested with a restriction endonuclease SmaI to reduce the characteristic differences between the plasmid DNA and genomic DNA. For a rough quantitative analysis of GM cotton meal contents, a rapid method for measurement of the copy numbers of the transgenic Cry and cotton endogenous Sad1 gene using a real-time PCR system with the plasmid DNA as a calibrator was established. The inter-run and intra-run coefficients of variation were less than 1.48% and 2.36%, respectively. The limits of detection and quantitation of the Cry and Sad1 genes were 9 and 91 copies of pMDCS, respectively. These results prove that the standard plasmid represents a valuable alternative to genomic DNA as a certified reference material for the quantification of GM cotton and is a useful tool to establish a feasible identification management for GM cottonseed meal content in the feed industry.

Detection of the soybean allergenic protein Gly m Bd 28K by an indirect enzyme-linked immunosorbent assay.

The full-length cDNA sequence of Gly m Bd 28K was chemically synthesized and expressed in Escherichia coli (E. coli) BL21 (DE3) as an inclusion body under the induction of 0.2 mmol/L of isopropyl β-D-1-thiogalactopyranoside (IPTG). The purity of the recombinant protein was over 90% following Ni-nitrilotriacetic acid (Ni-NTA) affinity chromatography, and its molecular weight was 29.71 kDa. The polyclonal antibody (pAB) against Gly m Bd 28K was prepared and referred to as pAB-28K, and it exhibited high specificity for the protein in soybean meal. We established an indirect enzyme-linked immunosorbent assay (iELISA) using the pAB-28K and the recombinant Gly m Bd 28K protein to determine the Gly m Bd 28K content in soybean products. The R(2) value of the standard curve was 0.9910, the average relative standard deviation (RSD) was 16.93%, and the average recovery was 95.50%, which indicated that the iELISA was highly reproducible and accurate. Therefore, the pAB-28K and the iELISA provide valuable tools for the rapid and sensitive detection of Gly m Bd 28K in food and feed products from soybeans. This protocol meets the technical requirements for quality control and food safety as related to soybean.

Biotechnical paving of recombinant enterocin A as the candidate of anti-Listeria agent.

BackgroundEnterocin A is a classic IIa bacteriocin isolated firstly from Enterococcus faecium CTC492 with selective antimicrobial activity against Listeria strains. However, the application of enterocin A as an anti-Listeria agent has been limited due to its very low native yield. The present work describes high production of enterocin A through codon optimization strategy and its character study.ResultsThe gene sequence of enterocin A was optimized based on preferential codon usage in Pichia pastoris to increase its expression efficiency. The highest anti-Listeria activity reached 51,200 AU/ml from 180 mg/l of total protein after 24 h of induction in a 5-L fermenter. Recombinant enterocin A (rEntA), purified by gel filtration chromatography, showed very strong activity against Listeria ivanovii ATCC 19119 with a low MIC of 20 ng/ml. In addition, the rEntA killed over 99% of tested L. ivanovii ATCC19119 within 4 h when exposed to 4 × MIC (80 ng/ml). Moreover, it showed high stability under a wide pH range (2-10) and maintained full activity after 1 h of treatment at 80°C within a pH range of 2-8. Its antimicrobial activity was enhanced at 25 and 50 mM NaCl, while 100-400 mM NaCl had little effect on the bactericidal ability of rEntA.ConclusionThe EntA was successfully expressed in P. pastoris, and this feasible system could pave the pre-industrial technological path of rEntA as a competent candidate as an anti-Listeria agent. Furthermore, it showed high stability under wide ranges of conditions, which could be potential as the new candidate of anti-Listeria agent.

Saccharomyces boulardii prevention of the hepatic injury induced by Salmonella Enteritidis infection

Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis) is the predominant cause of serovar-associated food-borne outbreaks in many countries and causes significant clinical symptoms of liver injury, enteritis, and diarrheal diseases. Saccharomyces boulardii is used in clinical application for prophylaxis and the treatment of a variety of diseases caused by bacterial infection. We used a mouse model of Salmonella Enteritidis infection, which included pretreatment with S. boulardii, to reveal the protection mechanisms of S. boulardii against Salmonella Enteritidis infection, including the translocation of Salmonella Enteritidis to the liver 10 days after Salmonella Enteritidis challenge, and the colonisation of Salmonella Enteritidis and the formation of hepatic tissue lesions in mice after Salmonella Enteritidis challenge on the 10th day. Compared with Salmonella Enteritidis infection in mice, S. boulardii decreased Salmonella Enteritidis translocation to the liver by 96%, and 99% of Salmonella Enteritidis colonised the cecum on the 10th day. Saccharomyces boulardii also abated hepatic tissue injury caused by the infiltration of neutrophilic granulocytes, lymphocytes, and plasmocytes by decreasing the translocation of Salmonella to the liver. These findings demonstrated that S. boulardii is an effective agent in the prevention of the hepatic injury induced by Salmonella Enteritidis infection in a mouse model.

Increased intracellular activity of MP1102 and NZ2114 against Staphylococcus aureus in vitro and in vivo

Treatment of Staphylococcus aureus infections remains very difficult due to its capacity to survive intracellularly and its multidrug resistance. In this study, the extracellular/intracellular activities of plectasin derivatives-MP1102/NZ2114 were investigated against three methicillin-susceptible/-resistant S. aureus (MSSA/MRSA) strains in RAW 264.7 macrophages and mice to overcome poor intracellular activity. Antibacterial activities decreased 4–16-fold under a mimic phagolysosomal environment. MP1102/NZ2114 were internalized into the cells via clathrin-mediated endocytosis and macropinocytosis and distributed in the cytoplasm; they regulated tumor necrosis factor-α, interleukin-1β and interleukin-10 levels. The extracellular maximal relative efficacy (Emax) values of MP1102/NZ2114 towards the three S. aureus strains were >5-log decrease in colony forming units (CFU). In the methicillin-resistant and virulent strains, MP1102/NZ2114 exhibited intracellular bacteriostatic efficacy with an Emax of 0.42–1.07-log CFU reduction. In the MSSA ATCC25923 mouse peritonitis model, 5 mg/kg MP1102/NZ2114 significantly reduced the bacterial load at 24 h, which was superior to vancomycin. In MRSA ATCC43300, their activity was similar to that of vancomycin. The high virulent CVCC546 strain displayed a relatively lower efficiency, with log CFU decreases of 2.88–2.91 (total), 3.41–3.50 (extracellular) and 2.11–2.51 (intracellular) compared with vancomycin (3.70). This suggests that MP1102/NZ2114 can be used as candidates for treating intracellular S. aureus.