Isolation and characterization of peptide antibiotics LI-F04 and polymyxin B6 produced by Paenibacillus polymyxa strain JSa-9

Paenibacillus polymyxa JSa-9 had been found to produce five cyclic LI-F type antibiotics which were released into culture medium in accordance with our previous report. In this study, another three kinds of antagonistic compounds were extracted from P. polymyxa JSa-9 cell pellets and (or) spores by methanol. Using high performance liquid chromatography (HPLC) method, two antagonistic fractions were separated and collected from the methanol extract. One showed inhibition against Escherichia coli and Staphylococcus aureus, while the other was active against Aspergillus niger and S. aureus. By means of electrospray ionization mass spectroscopy (ESI-MS), infrared spectroscopy (IR), and amino acid analysis, two kinds of compounds from fraction B with molecular masses of 901 and 915 Da were characterized as the linear lipopeptide analogs of antibiotics LI-F04a and LI-F04b, respectively. Another antimicrobial substance from fraction A could be attributed to polymyxin B₆.     

Anti-Legionella activity of staphylococcal hemolytic peptides

A collection of various Staphylococci was screened for their anti-Legionella activity. Nine of the tested strains were found to secrete anti-Legionella compounds. The culture supernatants of the strains, described in the literature to produce hemolytic peptides, were successfully submitted to a two step purification process. All the purified compounds, except one, corresponded to previously described hemolytic peptides and were not known for their anti-Legionella activity. By comparison of the minimal inhibitory concentrations, minimal permeabilization concentrations, decrease in the number of cultivable bacteria, hemolytic activity and selectivity, the purified peptides could be separated in two groups. First group, with warnericin RK as a leader, corresponds to the more hemolytic and bactericidal peptides. The peptides of the second group, represented by the PSMα from Staphylococcus epidermidis, appeared bacteriostatic and poorly hemolytic.     

Characterization and structure identification of an antimicrobial peptide, hominicin, produced by Staphylococcus hominis MBBL 2-9

Hominicin, antimicrobial peptide displaying potent activity against Staphylococcus aureus ATCC 25923, methicillin-resistant S. aureus (MRSA) ATCC 11435 and vancomycin-intermediate S. aureus (VISA) CCARM 3501, was purified by chloroform extraction, ion-exchange column chromatography and reverse-phase HPLC from culture supernatant of Staphylococcushominis MBBL 2-9. Hominicin exhibited heat stability up to 121 °C for 15 min and activity under both acidic and basic conditions (from pH 2.0 to 10.0). Hominicin was cleaved into two fragments after treatment with proteinase K, resulting in the loss of its antibacterial activity, while it was resistant to trypsin, α-chymotrypsin, pepsin and lipase. The molecular mass of hominicin determined by mass spectrometry was 2038.4 Da. LC-mass spectrometry and NMR spectroscopy analyses of the two fragments revealed the sequence of hominicin as DmIle-Dhb-Pro-Ala-Dhb-Pro-Phe-Dhb-Pro-Ala-Ile-Thr-Glu-Ile-Dhb-Ala-Ala-Val-Ile-Ala-Dmp, which had no similarity with other antimicrobial peptides previously reported. The present study is the first report of this novel antimicrobial peptide, which has uncommon amino acid residues like the ones in Class I group and shows potent activity against clinically relevant S. aureus, MRSA and VISA.     

Sesquiterpenes and diterpenes from Ambrosia arborescens

Six compounds, eudesm-11(13)-en-4β,9β-diol, 15R,16-dihydroxy-3-oxoisopimar-9(11)-ene, 15S,16-dihydroxy-3-oxoisopimar-9(11)-ene, 1α-hydroxy-7-oxo-iso-anhydrooplopanone, 10α-hydroxy-11,13-dihydro-5-epi-psilostachyin, and 4β-hydroxypseudoguaian-12,6-olide 4-O-β-d-glucopyranoside, together with 12 known sesquiterpenes, were isolated from the leaves of Ambrosia arborescens. Structures were elucidated by 1D and 2D NMR spectroscopy including 1D-TOCSY, DQF-COSY, 2D-ROESY, HSQC, and HMBC experiments, as well as by ESI mass spectrometry. The absolute configuration of the 15,16-diol moiety in 15R,16-dihydroxy-3-oxoisopimar-9(11)-ene and 15S,16-dihydroxy-3-oxoisopimar-9(11)-ene was determined using Snatzke’s method. All compounds were evaluated for antiproliferative activity.     

A comprehensive metabolite profiling of Isatis tinctoria leaf extracts

A broad-based characterisation of a pharmacologically active dichloromethane extract from Isatis tinctoria leaves was carried out. For a comprehensive picture we also included the polar constituents of I. tinctoria (MeOH extract) and for comparative purposes, the taxonomically closely related plant I. indigotica. Diode array detector, evaporative light scattering detector, atmospheric pressure chemical ionisation and electrospray ionisation mass spectrometry, and electrospray ionisation time-of-flight mass spectrometry detectors were used in parallel to ensure a wide coverage of secondary metabolites with highly diverging analytical properties. Off-line microprobe nuclear magnetic resonance spectroscopy after peak purification by semi-preparative high-pressure liquid chromatography served for structure elucidation of some minor constituents.More than 65 compounds belonging to various structural classes such as alkaloids, flavonoids, fatty acids, porphyrins, lignans, carotenoids, glucosinolates and cyclohexenones were unambiguously identified, and tentative structures were proposed for additional compounds. Numerous compounds were identified for the first time in the genus Isatis, and an indolic alkaloid was discovered.     

Identification and characterization of a novel antimicrobial peptide from the venom of the ant Tetramorium bicarinatum

A novel antimicrobial peptide, named Bicarinalin, has been isolated from the venom of the ant Tetramorium bicarinatum. Its amino acid sequence has been determined by de novo sequencing using mass spectrometry and by Edman degradation. Bicarinalin contained 20 amino acid residues and was C-terminally amidated as the majority of antimicrobial peptides isolated to date from insect venoms. Interestingly, this peptide had a linear structure and exhibited no meaningful similarity with any known peptides. Antibacterial activities against Staphylococcus aureus and S. xylosus strains were evaluated using a synthetic replicate. Bicarinalin had a potent and broad antibacterial activity of the same magnitude as Melittin and other hymenopteran antimicrobial peptides such as Pilosulin or Defensin. Moreover, this antimicrobial peptide has a weak hemolytic activity compared to Melittin on erythrocytes, suggesting potential for development into an anti-infective agent for use against emerging antibiotic-resistant pathogens.     

Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator mississippiensis)

Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35 kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35 kDa protein was ~ 98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates.     

Identification of a potent Angiotensin-I converting enzyme inhibitory peptide from Black cumin seed hydrolysate using orthogonal bioassay-guided fractionations coupled with in silico screening

Black cumin (Nigella sativa) seed protein (BCSP) was individually hydrolyzed with pepsin, trypsin, and α-chymotrypsin. After ultrafiltration, the α-chymotrypsin hydrolysate (< 3 kDa) exhibited the highest ACE inhibitory (ACEI) activity with an IC₅₀ value of 34.4 ± 1.5 μg/mL. This hydrolysate was orthogonally fractionalized using reversed-phase high-performance liquid chromatography (RP-HPLC) and strong cation exchange (SCX) chromatography, and the most active RP-HPLC and SCX fractions (F7 and H4, respectively) were individually screened out by ACEI assay. These two fractions were analyzed with liquid chromatography-tandem mass spectrometry (LC–MS/MS) followed by automated de novo peptide sequencing, and totally 43 ACEI candidate peptides were identified. Three overlapping peptides (VTPVGVPKW, VVTPVGVPKW, and LVLTL) were simultaneously contained in both fractions, and VTPVGVPKW (VW-9) was speculated as to the most potent ACEI peptide based on the in silico analysis. Synthetic VW-9 was used to confirm the identity, and a remarkable IC₅₀ value of VW-9 (1.8 ± 0.09 μM) was determined. Preincubation and inhibition mechanism studies indicated that VW-9 was a true inhibitor as well as a non-competitive inhibitor on ACE, which was further illustrated with the molecular docking simulation. Our study revealed that the application of VW-9 to antihypertensive products is promising.     

Quantitative metabolome analysis using liquid chromatography-high-resolution mass spectrometry

In this report, we introduce a liquid chromatography single-mass spectrometry method for metabolome quantification, using the LTQ Orbitrap high-resolution mass spectrometer. Analytes were separated with hydrophilic interaction liquid chromatography. At a working resolution of 30,000 (at m/z 400), the limit of detection varied from 50 fmol to 5 pmol for 25 metabolites tested. In terms of metabolite concentration, the linearity was about 2 to 3 orders of magnitude for most compounds (R² > 0.99). To determine the accuracy of the system in complex sample matrices, the isotope dilution method was evaluated from mixtures of pure compounds and uniformly ¹³C-labeled cell extracts. With the application of this method, quantification was possible within single runs even when the pool sizes of individual metabolites varied from 0.13 to 55.6 μM. As a case study, intracellular concentrations of central metabolites were determined for Methylobacterium extorquens AM1 during growth on two different carbon sources, methanol and succinate. Reproducible results from technical and biological repetitions were obtained that revealed significant variations of intracellular metabolite pool sizes, depending on the carbon source. The LTQ Obitrap offers new perspectives and strategies for metabolome quantification.     

Identification of an antifungal peptide from Trapa natans fruits with inhibitory effects on Candida tropicalis biofilm formation

Due to recent emergence of fungal pathogens resistant to current antifungal therapies, several studies have been focused on screening of plant peptides to find novel compounds having antifungal activities. Here, a novel antifungal plant peptide, with molecular mass of 1230 Da was purified from fruits of Trapa natans by reverse phase high performance liquid chromatography using 300SB-C18 column and named as Tn-AFP1. Determination of complete amino acid sequences of this peptide by tandem mass spectrometry showed to contain following eleven amino acid residues: LMCTHPLDCSN. Purified Tn-AFP1 showed the inhibition of Candida tropicalis growth in vitro and disrupted the biofilm formation in a concentration dependent manner. It also showed downregulation of MDR1 and ERG11 gene expression in real time-PCR analysis. In silico molecular modeling predicted the structure of Tn-AFP1 as a single coil attached by a unique disulfide bond. Characterization of Tn-AFP1 could contribute in designing novel derivative(s) of this peptide for the development of more effective antimycotic compounds.     

Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa

Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280 nm by circular dichroism spectroscopy. Four of the Pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria.     

A novel myotropic peptide from the skin secretions of the tree frog, Polypedates pingbianensis

A novel myotropic peptide, polypedatein, was purified and characterized from the skin secretions of the tree frog, Polypedates pingbianensis. Its primary structure, TLLCKYFAIC, was determined by Edman degradation and mass spectrometry. Polypedatein was subjected to bioassays including myotropic, antimicrobial, and serine protease inhibitory activities, which are related with many amphibian skin bioactive peptides. It was found to elicit concentration-dependent contractile effects on isolated rat ileum. cDNA clones encoding the precursor of polypedatein were isolated by screening a skin cDNA library of P. pingbianensis and then sequenced. The amino acid sequence deduced from the cDNA sequences matches well with the result from Edman degradation. BLAST search revealed that the sequence of polypedatein did not show similarity to known protein or peptide sequences. Especially, polypedatein does not contain conserved structural motifs of other amphibian myotropic peptides, such as bradykinins, bombesins, cholecystokinin (CCK), and tachykinins, indicating that polypedatein belongs to a novel amphibian myotropic peptide family. The signal peptide of the precursor encoding polypedatein shows significant sequence identity to that of other amphibian skin defensive peptides, such as antimicrobial peptides, bradykinins, lectins, and serine protease inhibitors, suggesting that polypedatein belongs to a novel amphibian myotropic peptide family. Polypedatein is also the first bioactive peptide from the genus of the frog, Polypedates.     

A family of small cyclic amphipathic peptides (SCAmpPs) genes in citrus

Background

Citrus represents a crop of global importance both in economic impact and significance to nutrition. Citrus production worldwide is threatened by the disease Huanglongbing (HLB), caused by the phloem-limited pathogen Candidatus Liberibacter spp.. As a source of stable HLB-resistance has yet to be identified, there is considerable interest in characterization of novel disease-associated citrus genes.

Results

A gene family of Small Cyclic Amphipathic Peptides (SCAmpPs) in citrus is described. The citrus genomes contain 100–150 SCAmpPs genes, approximately 50 of which are represented in the citrus EST database. These genes encode small ~50 residue precursor proteins that are post-translationally processed, releasing 5–10 residue cyclic peptides. The structures of the SCAmpPs genes are highly conserved, with the small coding domains interrupted by a single intron and relatively extended untranslated regions. Some family members are very highly transcribed in specific citrus tissues, as determined by representation in tissue-specific cDNA libraries. Comparison of the ESTs of related SCAmpPs revealed an unexpected evolutionary profile, consistent with targeted mutagenesis of the predicted cyclic peptide domain. The SCAmpPs genes are displayed in clusters on the citrus chromosomes, with apparent association with receptor leucine-rich repeat protein arrays. This study focused on three SCAmpPs family members with high constitutive expression in citrus phloem. Unexpectedly high sequence conservation was observed in the promoter region of two phloem-expressed SCAmpPs that encode very distinct predicted cyclic products. The processed cyclic product of one of these phloem SCAmpPs was characterized by LC-MS-MS analysis of phloem tissue, revealing properties consistent with a K⁺ ionophore.

Conclusions

The SCAmpPs amino acid composition, protein structure, expression patterns, evolutionary profile and chromosomal distribution are consistent with designation as ribosomally synthesized defense-related peptides.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1486-4) contains supplementary material, which is available to authorized users.     

Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae

The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus.     

Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry

A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC. Uniformly ¹³C-labeled isotopes of nucleotides were used as internal standards for both extraction and quantification of intracellular nucleotides. The method was validated by determining the linearity, sensitivity, and repeatability.     

Quantitative Trait Loci Mapping of the Mouse Plasma Proteome (pQTL)

A current challenge in the era of genome-wide studies is to determine the responsible genes and mechanisms underlying newly identified loci. Screening of the plasma proteome by high-throughput mass spectrometry (MALDI-TOF MS) is considered a promising approach for identification of metabolic and disease processes. Therefore, plasma proteome screening might be particularly useful for identifying responsible genes when combined with analysis of variation in the genome. Here, we describe a proteomic quantitative trait locus (pQTL) study of plasma proteome screens in an F₂ intercross of 455 mice mapped with 177 genetic markers across the genome. A total of 69 of 176 peptides revealed significant LOD scores (≥5.35) demonstrating strong genetic regulation of distinct components of the plasma proteome. Analyses were confirmed by mechanistic studies and MALDI-TOF/TOF, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the two strongest pQTLs: A pQTL for mass-to-charge ratio (m/z) 3494 (LOD 24.9, D11Mit151) was identified as the N-terminal 35 amino acids of hemoglobin subunit A (Hba) and caused by genetic variation in Hba. Another pQTL for m/z 8713 (LOD 36.4; D1Mit111) was caused by variation in apolipoprotein A2 (Apoa2) and cosegregated with HDL cholesterol. Taken together, we show that genome-wide plasma proteome profiling in combination with genome-wide genetic screening aids in the identification of causal genetic variants affecting abundance of plasma proteins.     

Qualitative and quantitative determination of major saponins in Paris and Trillium by HPLC-ELSD and HPLC–MS/MS

High-performance liquid chromatographic (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization multistage tandem mass spectrometry (HPLC–ESI-MSⁿ) were used to identify and quantify steroid saponins in Paris and Trillium plants. The content of the known saponins such as Paris I, II, III, V, VI, VII, H, gracillin and protodioscin in Paris and Trillium plants was determined simultaneously using the developed HPLC-ELSD method. Furthermore, other 12 steroid saponins were identified by HPLC–ESI(+/−)-MSⁿ detection. In the end, a developed analytical procedure was proved to be a reliable and rapid method for the quality control of Paris and Trillium plants. In addition, the alternative resources for Paris yunnanensis used as a traditional Chinese medicine were discovered according to the hierarchical clustering analysis of the saponin fraction of these plants.     

Metabolic profiling of Dactylopius (Hemiptera: Dactylopiidae) species pigments by geographical origin and hosts using multivariate data analysis

The genus Dactylopius includes the group of insects historically used in Mexico as a source of natural red colorant, the cochineal color. Five species of Dactylopius collected in thirteen states of Mexico and two provinces of Argentina were analyzed using high-performance liquid chromatography with a photodiode array detector. This analysis allowed each species to be identified on the basis of differences in their metabolic profiles. Cluster analysis and principal component analysis differentiated the species by localities and host plant. These two multivariate data analysis techniques were complementary and confirmed the grouping of all analyzed Dactylopius samples. For all species, carminic acid, identified by reference to a commercial sample, was the major compound present in significant amounts, making all five species potential sources of colorant. In addition, each species could be differentiated by the presence of other minor compounds.     

Repellent activity of alligator pepper, Aframomum melegueta, and ginger, Zingiber officinale, against the maize weevil, Sitophilus zeamais

The repellent activity of alligator pepper, Aframomum melegueta, and ginger, Zingiber officinale (Zingiberaceae), against the maize weevil, Sitophilus zeamais (Coleoptera: Curculionidae), was investigated in four-way olfactometer bioassays. Results showed that vacuum distilled A. melegueta and Z. officinale extracts were repellent towards adult S. zeamais both in the absence and the presence of maize, Zea mays, grains. Bioassay-guided liquid chromatographic fractionation of the distillates showed that fractions containing oxygenated compounds accounted for the repellent activity. Coupled gas chromatography-mass spectrometry (GC-MS), followed by GC peak enhancement and enantioselective GC using authentic compounds, identified 3 major compounds in the behaviourally active fractions of A. melegueta and Z. officinale to be (S)-2-heptanol, (S)-2-heptyl acetate and (R)-linalool in a ratio of 1:6:3, and 1,8-cineole, neral and geranial in a ratio of 5.48:1:2.13, respectively. The identification of these behaviourally active compounds provides the scientific basis for the observed repellent properties of A. melegueta and Z. officinale, and demonstrates the potential for their use in stored-product protection at the small-scale farmer level in Africa.