Isolation And Purification Of Antimicrobial Peptides From Two Coleopteran Insects And Their Biological Activities

Antimicrobial peptides (AMPs) defined as small peptides of10to50amino acid residues,have played a particularly important role in innate immune system of insects. Generallyspeaking, most of AMPs are encoded by single genes that comprise highly homologous genefamilies. They are usually basic and cationic, with a net positive charge of+2to+7owing to anexcess of basic amino acids (artinine, lysine and histidine) over acidic amino acids. Besidestheir high antimicrobial activities against Gram-negative and/or Gram-positive bacteria, thesepeptides also have a diverse range of targets, including fungi, viruses, parasites such astrypanosomes and plasmodia, and even cancer cell. As the difference on the mechanisms ofaction against bacteria from conventional antibiotics, which targeted mainly cellular membrane,AMPs can be expected to develop as promising candidates against antibiotic-resistantpathogens that are often not sensitive to classical antibiotic treatment. Therefore the researchon AMPs is becoming one of the hottest fields in the world. Insects are not only the biggestgroup within the animal kingdom, but also a treasure of various antimicrobial peptides whichhas not been fully excavated. For decades, one major area of interest for the discovery andstudy of new antibiotics was the investigation of AMPs derived from abundant insectresources.In this disserdation, extraction, purification and identification of AMPs from the larvae oftwo Coleopteran insects, Tenebrio molitor Linnaeus (T. molitor) and Blaps rhynchopteraFairmaire (B. rhynchoptera), were systematically studied. Firstly we challenged the larvae withthe combined suspension of living Escherichia coli (E. coli) and Staphylococcus aureus (S.aureus) by pricking their abdomens with a stainless needle. Then extraction was carried outexactly24hours after the induction Four kinds of solutions were used to extract AMPs from T.molitor larvae. After repeated gel chromatography, reversed phase chromatography, higeperformance liquid chromatography (HPLC), a partly characterized small peptide was obtained from crude extracts of T. molitor, with strong anti-Gram-positive bacteria activities. Maincontents and results are listed as follows:(1) Screening of the optimum extraction solution of AMPs from T. molitorFour solutions,10%acetic acid,0.1%trifluoroacetic acid, Tris-HCl (pH8.0), andTris-HCl (pH6.5), were employed in extraction process of AMPs from T. molitor in order toselect the suitable extraction solution. The results indicated that10%acetic acid itself showedantimicrobial activity, leading to difficulty in judging inhibitory effect, and should not beselected as the extraction solution. Only0.1%trifluoroacetic acid extract among the rest threesolutions was found to display detectable inhibitory activity against bacteria afterpre-purification. Therefore,0.1%trifluoroacetic acid was the optimum extraction solution for T.molitor AMPs.Considering that both T. molitor and B. rhynchoptera belong to same order,0.1%trifluoroacetic acid was directly applied as extracted solution for B. rhynchoptera AMPs.(2) Pre-purification of two crude AMPs extracts by gel chromatography andTricine-SDS-PAGE analysisCrude extracts of T. molitor AMPs, coming from both induced and non-induced groupswere separated into two fractions after SephadexG50gel chromatography, and the second peak,marked as TFADZP2and TFAYDP2, respectively, showed obviously inhibitory activitiesagainst S. aureus and B. subtilis, but no anti-E. coli and anti-P. aeruginosa activities.Similarly, crude extracts of B. rhynchoptera AMPs from induced group and non-induced groupwere separated into three fractions after Superose12gel chromatography. The first peak,marked as HWDZP1and HWYDP1, respectively, had strong anti-S. aureus and anti-B. subtilisactivities, but no inhibitory effect against E. coli. In addition, HWYDP1from the inducedgroup displayed anti-P. aeruginosa activity. Tricine-SDS-PAGE analysis suggested thatTFADZP2and TFAYDP2mainly composed of small peptides with molecular mass below40kD, HWDZP1HWYDP1mainly consisted of small peptides with molecular mass less than20kD.(3) Comparison of antibacterial activity between induced group and non-induced group The antibacterial activities were detectable in induced group as well as the non-inducedfor these two kinds of insects. More interestingly, the fraction from the induced group hadbigger zone of inhibition against bacteria than the one from non-induced group at a lowerconcentration. The result implied that the AMPs encoded by gene in T. molitor and B.rhynchoptera were constitutively expressed and their activities could be enhanced greatly afterinduction with microbes. Additionally, the fraction from induced group of B. rhynchopteraexhibited anti-P. aeruginosa except anti-Gram-positive bacteria, suggesting a broader spectrumprobably occurred after challenge with miroorganisms.(4) Purification and Characterization of T. molitor AMPsBy two-step of Superdex Peptide, one-step of reversed-phase chromatography and highperformance liquid chromatography, a purified fraction with strong anti-Gram-positive bacteriaactivity was obtained from the induced group of T. molitor, named TFAYDP2-2-1-1-2. Thisfraction was partially characterized. The analysis of matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry (MALDI-TOF MS) indicated that its molecular mass was2552.5Da, a small molecule antimicrobial peptides. The fraction had no inhibitory effect onGram-negative bacteria E. coli and P. aeruginosa, but had apparent activity against S. aureusand B. subtilis and the minimal inhibitory concentrations were10μg/mL and20μg/mL,respectively. To the best of our knowledge, no antimicrobial peptide with ultra-low molecularmass has reported so far in the whole Coleopteran insects.(5) Purification of B. rhynchoptera AMPsBy one-step of reversed-phase chromatography, a component, named HWYDP1-1, wasgained from the induced group of B. rhynchoptera and displayed significantly inhibitoryactivity against S. aureus.In short, a homogeneous AMP from T. molitor larvae was achieved after the followingprocedures: extracted with acidic solution (0.1%trifluoroacetic acid), pre-purified by two stepsof gel chromatography, purified by one step of reversed-phase chromatography and one step ofhigh performance liquid chromatography. This AMP was proved to have strong inhibitory effects on S. aureus and B. subtilis, and the minimal inhibitory concentrations were10μg/mLand20μg/mL, respectively, but had no activity against E.coli and P aeruginosa. Its molecularmass was determined as2552.5Da by MALDI-TOF MS. It is probably a novel AMP fromColeoptera. Similar extraction and purification programs was carried out on AMPs of B.rhynchoptera. A fraction was gained with inhibitory activity against S. aureus.