Developing Antimicrobial Peptides Agents
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Antibiotics have gained great success in the battle between human and infectious diseases. However, the abuse of antibiotics has led to the emergence of the alarming number of resistance bacteria. Novel agents are urgently needed to treat these illnesses. Based on the the conserved sequences of antimicrobial peptides of insects (mainly Diptera) from different antimicrobial peptide databases, thirty-seven pairs of degenerate primers were designed, and the antimicrobial peptide genes from Hermetia illucens L were screened using these primers. Seven gene fragments for three types of antimicrobial peptides cecropin, sarcotoxin and stomoxyn were acquired. Bioinformatic analysis was done to predict the secondary structure and three-dimensional structure of these seven gene fragments that may exist. DNA sequence alignment analysis and the forecast-analysis of protein structure indicated that these seven antimicrobial peptide genes were not homologous to any other antimicrobial peptide genes in GenBank. A 189-bp antimicrobial peptide gene, which we named Stomoxyn – ZY, was selected to expression in the methylotrophic yeast Pichia pastoris; to acheive this aim the stomoxyn-zy gene was amplified by rapid amplification of cDNA ends (RACE) from Hermetia illucens L larvae stimulated with S. aureus, and cloned into the pPICZαA vector. The SacI-linearized plasmid pPICZαA-Stomoxyn -Zy was transformed into P. pastoris GS115 by electroporation. The expression of recombinant stomoxyn was induced with 0.5% methanol at pH 6.0 for 72 h at 28ËšC. Recombinant stomoxyn was purified using Ni-NTA HisTrap FF crude column chromatography. The antimicrobial activity of purified stomoxyn-zy assessment indicated that, it had an in vitro fungcidal and bacteriocidal effect against fungi (Magnaporthe grisea), Gram-positive bacteria (staphylococcus aureus) and the Gram-negative bacteria (E. coli). The exact mechanism through which stomoxyn-ZY kill targeted pathogens not well understood and need further studies.
Keywords: Hermetia illucens L.; insect antimicrobial peptides; genetic screening; yeast expression
Currently, the need for safe and effective antimicrobial peptides agents increases in parallel with the emergence of many antibiotic-resistant strains as a consequence of excessive use and widespread of the antibiotics . Many desirable features, such as low cost of production, rapidity, heat-tolerant, relatively broad antimicrobial spectrum and low toxicity to eukaryotic cells made of antimicrobial peptides to be a new alternative to the conventional antibiotic . The insects immune systems possess several features in common, does not have specific immune system as found in the higher animals , lacking of B and T lymphocytes and no immunoglobulins and complement production , this make insect developed effective and complex innate immune and antimicrobial peptides are a key factor of the insect’s immune system [4, 5]. Antimicrobial peptides (AMPs) have a variety of interesting biological functions including antibacterial, antifungal, antiparasitic, antitumoral, and antiviral activities [6-8].
So far, hundreds of antimicrobial peptides have now been reported according to the antimicrobial Peptide Database [3, 9]. Nowadays, molecular biology researchs are featured a wide range of antimicrobial peptides (AMP)-related topics such as AMP genome library construction, development of antimicrobial peptides and their mimetics as therapeutic agents, structure/function analysis, cloning and expression, regulation, mechanisms of action, microbial escaping strategies and human disease associations. And these open the door widely to screening identifying and characterizing new antibacterial peptides. A growing body of research has focused on AMPs and antimicrobial proteins identification in insects. Initially, Steiner and his group successfully characterized the primary structures of two AMPs purified from hemolymph of immunized pupae of Hyalophora cecropia named cecropins A and B . Liucun Chen et al., have isolated an AMP that suppress pathogenic Staphylococcus aureu and Salmonella enteritidis-borne from the hemolymph of the housefly larvae  (Liucun Chen et al., 2001). Study conducted by Yamada and his team showed antimicrobial activity of the defensin isolated from the Japanese rhinoceros beetle against the pathogenic Staphylococcus aureus strains resistant to antibiotics such as methicillin . The black soldier fly is often associated with the outdoors and livestock, usually around decaying organic matter such as animal waste or plant material. Since black soldier fly larvae consume decaying matter, they have been used to reduce animal manure in commercial swine and poultry facilities. Although they are not known as a disease vector, adult soldier flies are a potential mechanical vector of various pathogen  and this indicats that the Hermetia illucens L. has its own unique immune defense mechanism. Recently, a novel antimicrobial peptide from the immunized hemolymph of Hermetia illucens larvae was discovered and its antimicrobial activity against Gram Positive bacteria is proven. Therefore, such kind of study suggests that screening the antimicrobial peptide from Hermetia illucens L is necessary and important. In this study, new AMP genes from Hermetia illucens L., were screened and the cDNA for Hermetia illucens L stomoxyn-zy was cloned and expressed in P. pastoris. The antibacterial property of the active expressed recombinant peptide is detected and characterizes.
Materials and methods
Straines, plasmid and reagents
E. coli DH5α was used for cloning the pPICZα-Stomoxyn plasmid. E. coli K88 and Staphylococcus aureus (Cowan I) were used for the antibacterial assay. P. pastoris GS115 and expression Plasmid pPICZαA were from Invitrogen (USA). TA cloning vector pMD18-T, T4 DNA ligase, and Taq DNA polymerase were purchased from TaKaRa Biotechnology (TaKaRa, Nanjing, China) TransScriptTM First-Strand cDNA Synthesis SuperMix (TransGenBiotech), AxyPrepTM DNA Gel Extraction Kit (Axygen Scientific Inc), the Ultrapure total RNA Rapid Extraction Kit (YuanPinghao Biotech Co.,Ltd).
Insects, immunization, tissue sample collection and total RNA extraction
Insect Hermetia illucens L. were reared on an artificial diet at 28°C and 62% humidity for 24, the fifth instar larvae were used for experiments. The larva was first soaked in 75% alcohol for 30 min, and then washed with ultrapure water for 3 times, excess water was wiped using towel paper, after which each larva was pricked deeply with a fine needle dipped into S. aureus (KCCM 40881; OD600 = 2.4), the tissue samples were cut with anatomical scissors and pestled in a RNase free mortar after treated with liquid nitrogen. The total RNA was extracted from Immunized H. illucens using Trizol (Invitrogen) according to the manufacturer’s protocol and dissolved in water treated with DEPC.
cDNA production, and cloning of the AMP genes
First strand cDNA was synthesized with a firrst-strand synthesis kit (Invitrogen) and used for reverse-transcription polymerase chain reactions (RT-PCR). Thirty-seven pairs degenerate primers (Table 1 & Table2) were designed based on the conserved amino acid sequences of the Hermetia illucens L and gradient PCR reactions were performed with 2 μL cDNA template (50 ngμl−1) prepared in 50 μl volumes which contains 34.5 μl ddH2O, 5.0 μl 10× PCR buffer, 4.0 μl dNTP mix (2.5 mM), 2 μl primer up (10 μM), 2 μl primer down (10 μM), 0.5 μl Taq DNA polymerase (1 Uμl−1). The cycling parameters used were: [94°C for 5min; 94 °C for 30 °C; (40-60°C) for 30 °C and 72 °C for 45 °C, for 30 cycles and 72 °C for 10 min.]. The amplified DNA products were analyzed electrophoretically by size fraction on agrose gels (2.5%) and stained with ethidium bromide; the amplified fragments were recovered from the gel by using the AxyPrep DNA Gel Extraction Kit, and cloned into a pMD18-T. Cloning and propagation of the recombinanat plasmids was carried out in the DH5α Escherichia coli strain. For colony PCR analysis transformants were initially grown on LB plates supplemented with 100µg/ml ampicilin and then 10 resistant colonies were selected to confirm the integration of amplified fragment into pMD18-T plasmid. The positive clones were sequenced by Sangon Biotecnology company (shanghai, China) using the ABI 337 sequencer.
The letters Y, R, W, and S in degenerate primers mean nucleotide mixtures of CT, AG, AT, and GC, respectively.
The predicted protein sequences, secondary structure, protein 3D structure and active sites were analyzed using bioinformation professional analysis website includes http://www.imtech.res.in/raghava/antibp/;http://amp.biosino.org/;http://zhanglab.ccmb.med.umich.edu/I-TASSER/output/S90438/.
Construction of recombinant expression plasmid
First the DNA fragment containing the stomoxyn amino acids, about 189 coding sequence, was amplified by PCR using the plasmid pMD18-T/stomoxyn as a template under the following conditions: 94°C for 5 min, 94°C for 30 s, 57°C for 30 s, 72°C for 45 s (30 cycles) and 72°C for 10 min.. The forward primers 5-CCGGAATTCAGAGGATTTCGTAAGCA-3, contain an EcoRI site, and reverse primer, 5-AATGCGGCCGCAGTAGCAGCAACAACAGCA- 3, which contains a Notâ… restriction site. The amplified fragment, which encodes the mature peptide of stomoxyn was digested with EcoRI and NotI enzymes and the digested fragment was ligated into the EcoRI/NotI-digested pPICzαA in-frame to the α-factor secretion signal and down stream of the alcohol oxidase1 (AOX1) promoter. The recombinant plasmid (pPICzαAα/stomoxyn) was transformed into Ecoli DH5α.
Identification of recombinant plasmid
pPICzαA/stomoxyn recombinant plasmid was extracted from E.coli DH5α with Mini Plasmid Purification Kit according to the instructions, restriction enzyme analysis was peformed as following ; 1 µg of recombinant plasmid ws digested by restriction enzyme EcoR I and Not I at 37°C, for 8h; 5 µl of digestion product was analysed with 1% agarose gel electrophoresis; colony PCR was conducted for Zeocin resistance transformants and the insert was sequenced to ensure that the coding sequence of H.illucens L stomoxyn was correct and inframe with the α-factor secretion signal the positive plasmid was sequenced by TaKaRa Biotechnology Co, Ltd (Dalian, China).
Transformation of P. pastoris and selection of transformants Approximately 10 μg of recombinant plasmid were linearized by SaI at 37°C for 1 h, the digested plasmid was gel-purified and transformed into Pichia pastoris GS115 by electroporation following the manufacturer’s instructions (Bio-Rad, USA). As a negative control pPICzαA vector alone was also linearized and transformed into P. pastoris GS115 cells. The transformants were grown in YPD medium (1% yeast extract, 2% peptone, 2% dextrose, 2% agar, and 100 g/ml Zeocin) The resistance colonies were obtained from YPD plates and the inserts were verified by PCR using genomic DNA as a template and 5AOX1 (5-CGA CTG GTT CCA ATT GAC AAG C-3) and 3AOX1 (5- GGC AAA TGG CAT TCT GAC ATC C-3) as primers. The positive clones were used for suspension culture.
Expression of recombinant Stomoxyn in P. pastoris
A single purified Pichia colony was inoculated in 25 ml of buffered methanol-complex medium (BMGY, 1% yeast extract, 2% peptone, 100mM potassium phosphate buffer, pH 6.0, 1.34% YNB, 4×10-5 % biotin, and 1% glycerol) and grown at 28Cο in a shaking incubator (250-300 rpm) until culture reaches an OD600= 2-6, the cells were harvested by centrifugation at 3000 xg for 5 minutes at room temperature, and resuspended to OD600= 1 in 100-200 ml of buffered methanol-complex medium [BMMY, 1% yeast extract, 2% peptone, 100mM potassium phosphate buffer, pH 6.0, 1.34% YNB, 4×10-5 % biotin, and 1% methanol] in a 1L baffled flask and grown at 28Cο with shaking, to maintain induction of the recombinant protein expression, 0.5% sterilized pure methanol was added every 24 hours, 1 ml of the culture was collected at certain time points and Centrifuged at 1300 x g for 2.5 minutes at room temperature the supernatants and the cell pellets were analyzed by Tricine- SDS-PAGE to to established the optimal time period for protein expression. After optimized the expression conditions of 28ËšC with 0.5% methanol for 72 h, Scale-up expression was performed in a 1 L baffled flask. P.pastoris harboring the recombinant expression plasmid was cultivated in 200ml BMGY at 28°C with constant vigorous shaking till reached the optimum OD600; cells were pelleted and resuspended in 100 ml BMMY, then cultured for 72hours. Methanol was added in 24h interval period.The culture was collected and centrifugated at 3000xg for 5 mints at room temprature, the cell was harvested and proceed for protein purification. P. pastoris transformed with naked pPICZαA was used as a negative control.
After 72h incubation period, the 100ml culture medium was collected by centrifugation at 3000 g for 10 min. The cell pellets were resuspend in ice-cold phosphate buffer and the cell suspension was passed through the Emulsiflex-C3 cell disrupter fitted with a chilled heat exchanger for three times. The cell lysate was cenrifugated at 10000g for 15 min; the supernatant was preequilibrated with phosphate buffer and then applied to a nickel chelating Sepharose column. Target protein was eluted with a gradient of 0.02–0.5 M imidazole in phosphate buffer. Tricine SDS-PAGE was applied to analyze the eluted proteins. The purified stomoxyn was dialyzed overnight against PBS, pH 7.5 and finally lyophilized.
Antimicrobial activity assay
The antibacterial activity of expressed stomoxyn was determined with the agar dilution method (inhibition zone assay), experiments was conducted in Petri plates, where 100 µl of suspension containing 108CFU/ml of bacteria and 105spore/ml of fungi, spread evenly on the surface of the nutrient agar and PDA plates, respectively. Six holes with 5 mm diameter was applied to each plate surface, to which either 80 µl of expressed stomoxyn, positive control or empty vector as negative control were added, incubated at 37C° for 24 hours for bacteria and fungi at 28°C for about 2 days. Plates exhibiting zones of growth inhibition was scored as showing antimicrobial activity. Plates with inhibition zones were photographed against a black background to accurately measure the diameter of the inhibition zone. All experiments were performed in triplicate.
Results and Discussion
Screening and cDNA cloning of AMP gene
Using corresponding degenerate primers and the cDNA of Hermetia illucens L. as template, a 160,160 and 180 base pairs (bp) DNA fragments were obtained by the gradient PCR amplification which named Cecrobin, sacrtoxin and stomoxyn respectively as shown in figure 1.
Figure1: Gel electophorosis analysis of H.illucens L antimicrobial genes screening: (a) Cecropin amplification. M: 50 DNA Markers; Lane 1 cecropin PCR Product about 160 bp (b) Sarcotoxin amplification. M: 50 DNA Markers; Lane 1 cecropin PCR Product about 160 bp (c) Stomoxyn amplification. M: 50 DNA Markers; Lane 1 cecropin PCR Product about 180 bp.
These DNA fragments were ligated into pMD18-T vector and transformed into DH5α; positive clones from each ligation were selected for sequencing and according to amin acids sequences seven genes were screened. Four isoform were detected for Sarcotoxin named Sarcotoxin1, Sarcotoxin (a), Sarcotoxin (b) and Sarcotoxin3, while for Cecropin only one form was found. On the otherhand, two isoform were screened for stomoxyn named, Stomoxyn and stomoxyn (a). Table 1 summarized the screened gene sequencing and their optimum anneling temperature.
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