Synthesis,preferred conformation,protease stability,and membrane activity of heptaibin,a medium‐length peptaibiotic

The medium‐length peptaibiotics are characterized by a primary structure of 14–16 amino acid residues. Despite the interesting antibiotic and antifungal properties exhibited by these membrane‐active peptides, their exact mechanism of action is still unknown. Here, we present our results on heptaibin, a 14‐amino acid peptaibiotic found to exhibit antimicrobial activity against Staphylococcus aureus. We carried out the very challenging synthesis of heptaibin on solid phase and a detailed conformational analysis in solution. The peptaibiotic is folded in a mixed 3₁₀‐/α‐helix conformation which exhibits a remarkable amphiphilic character. We also find that it is highly stable toward degradation by proteolytic enzymes and nonhemolytic. Finally, fluorescence leakage experiments using small unilamellar vesicles of three different compositions revealed that heptaibin, although uncharged, is a selective compound for permeabilization of model membranes mimicking the overall negatively charged surface of Gram‐positive bacteria. This latter finding is in agreement with the originally published antimicrobial activity data. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Sequences of stilboflavin C: towards the peptaibiome of the filamentous fungus Stilbella (= Trichoderma) flavipes

Filamentous fungi of the genus Stilbella are recognized as an abundant source of naturally occurring α‐aminoisobutyric acid‐containing peptides. The culture broth of Stilbella (Trichoderma) flavipes CBS 146.81 yielded a mixture of peptides named stilboflavins (SF), and these were isolated and separated by preparative TLC into groups named SF‐A, SF‐B, and SF‐C. Although all three of these groups resolved as single spots on thin‐layer chromatograms, HPLC analysis revealed that each of the groups represents very microheterogeneous mixtures of closely related peptides. Here, we report on the sequence analysis of SF‐C peptides, formerly isolated by preparative TLC. HPLC coupled to QqTOF‐ESI‐HRMS provided the sequences of 10 16‐residue peptides and five 19‐residue peptides, all of which were N‐terminally acetylated. In contrast to the previously described SF‐A and SF‐B peptaibols, SF‐C peptaibols contain Ser‐Alaol or Ser‐Leuol, which are rarely found as C‐termini, and repetitive Leu‐Aib‐Gly sequences, which have not been detected in peptaibols before. Taking the previously determined sequences of SF‐A and SF‐B into account, the entirety of peptides produced by S. flavipes (the ‘peptaibiome’) approaches or exceeds 100 non‐ribosomally biosynthesized peptaibiotics. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

CropSNPdb: a database of SNP array data for Brassica crops and hexaploid bread wheat

Advances in sequencing technology have led to a rapid rise in the genomic data available for plants, driving new insights into the evolution, domestication and improvement of crops. Single nucleotide polymorphisms (SNPs) are a major component of crop genomic diversity, and are invaluable as genetic markers in research and breeding programs. High‐throughput SNP arrays, or ‘SNP chips’, can generate reproducible sets of informative SNP markers and have been broadly adopted. Although there are many public repositories for sequencing data, which are routinely uploaded, there are no formal repositories for crop SNP array data. To make SNP array data more easily accessible, we have developed CropSNPdb ( http://snpdb.appliedbioinformatics.com.au ), a database for SNP array data produced by the Illumina Infinium? hexaploid bread wheat (Triticum aestivum) 90K and Brassica 60K arrays. We currently host SNPs from datasets covering 526 Brassica lines and 309 bread wheat lines, and provide search, download and upload utilities for users. CropSNPdb provides a useful repository for these data, which can be applied for a range of genomics and molecular crop‐breeding activities.     

Solution Synthesis,Conformational Analysis,and Antimicrobial Activity of Three Alamethicin F50/5 Analogs Bearing a Trifluoroacetyl Label

We prepared, by solution‐phase methods, and fully characterized three analogs of the membrane‐active peptaibiotic alamethicin F50/5, bearing a single trifluoroacetyl (Tfa) label at the N‐terminus, at position 9 (central region) or at position 19 (C‐terminus), and with the three Gln at positions 7, 18, and 19 replaced by Glu(OMe) residues. To add the Tfa label at position 9 or 19, a γ‐trifluoroacetylated α,γ‐diaminobutyric acid (Dab) residue was incorporated as a replacement for the original Val⁹ or Glu(OMe)¹⁹ amino acid. We performed a detailed conformational analysis of the three analogs (using FT‐IR absorption, CD, 2D‐NMR, and X‐ray diffraction), which clearly showed that Tfa labeling does not introduce any dramatic backbone modification in the predominantly α‐helical structure of the parent peptaibiotic. The results of an initial solid‐state ¹⁹F‐NMR study on one of the analogs favor the conclusion that the Tfa group is a very promising reporter for the analysis of peptaibiotic? membrane interactions. Finally, we found that the antimicrobial activities of the three newly synthesized analogs depend on the position of the Tfa label in the peptide sequence.     

The neuropeptidome of Rhodnius prolixus brain

We show a sensitive and straightforward off‐line nano‐LC‐MALDI‐MS/MS workflow that allowed the first comprehensive neuropeptidomic analysis of an insect disease vector. This approach was applied to identify neuropeptides in the brain of Rhodnius prolixus, a vector of Chagas disease. This work will contribute to the annotation of genes in the ongoing R. prolixus genome sequence project. Peptides were identified by de novo sequencing and comparisons to known neuropeptides from different organisms by database search. By these means, we were able to identify 42 novel neuropeptides from R. prolixus. The peptides were classified as extended FMRF‐amide‐related peptides, sulfakinins, myosuppressins, short neuropeptide F, long neuropeptide F, SIF‐amide‐related peptides, tachykinins, orcokinins, allatostatins, allatotropins, calcitonin‐like diuretic hormones, corazonin, and pyrokinin. Some of them were detected in multiple isoforms and/or truncated fragments. Interestingly, some of the R. prolixus peptides, as myosuppressin and sulfakinins, are unique in their characteristic C‐terminal domain among insect neuropeptides identified so far.     

Improving the genetic representation of rare taxa within complex microbial communities using DNA normalization methods

Complex microbial communities typically contain a large number of low abundance species, which collectively, comprise a considerable proportion of the community. This ‘rare biosphere’ has been speculated to contain keystone species and act as a repository of genomic diversity to facilitate community adaptation. Many environmental microbes are currently resistant to cultivation, and can only be accessed via culture‐independent approaches. To enhance our understanding of the role of the rare biosphere, we aimed to improve their metagenomic representation using DNA normalization methods, and assess normalization success via shotgun DNA sequencing. A synthetic metagenome was constructed from the genomic DNA of five bacterial species, pooled in a defined ratio spanning three orders of magnitude. The synthetic metagenome was fractionated and thermally renatured, allowing the most abundant sequences to hybridize. Double‐stranded DNA was removed either by hydroxyapatite chromatography, or by a duplex‐specific nuclease (DSN). The chromatographic method failed to enrich for the genomes present in low starting abundance, whereas the DSN method resulted in all genomes reaching near equimolar abundance. The representation of the rarest member was increased by approximately 450‐fold. De novo assembly of the normalized metagenome enabled up to 18.0% of genes from the rarest organism to be assembled, in contrast to the un‐normalized sample, where genes were not able to be assembled at the same sequencing depth. This study has demonstrated that the application of normalization methods to metagenomic samples is a powerful tool to enrich for sequences from rare taxa, which will shed further light on their ecological niches.     

A peptide resource for the analysis of Staphylococcus aureus in host–pathogen interaction studies

Staphylococcus aureus is an opportunistic human pathogen, which can cause life‐threatening disease. Proteome analyses of the bacterium can provide new insights into its pathophysiology and important facets of metabolic adaptation and, thus, aid the recognition of targets for intervention. However, the value of such proteome studies increases with their comprehensiveness. We present an MS–driven, proteome‐wide characterization of the strain S. aureus HG001. Combining 144 high precision proteomic data sets, we identified 19 109 peptides from 2088 distinct S. aureus HG001 proteins, which account for 72% of the predicted ORFs. Peptides were further characterized concerning pI, GRAVY, and detectability scores in order to understand the low peptide coverage of 8.7% (19 109 out of 220 245 theoretical peptides). The high quality peptide‐centric spectra have been organized into a comprehensive peptide fragmentation library (SpectraST) and used for identification of S. aureus‐typic peptides in highly complex host–pathogen interaction experiments, which significantly improved the number of identified S. aureus proteins compared to a MASCOT search. This effort now allows the elucidation of crucial pathophysiological questions in S. aureus‐specific host–pathogen interaction studies through comprehensive proteome analysis. The S. aureus‐specific spectra resource developed here also represents an important spectral repository for SRM or for data‐independent acquisition MS approaches. All MS data have been deposited in the ProteomeXchange with identifier PXD000702 ( http://proteomecentral.proteomexchange.org/dataset/PXD000702 ).     

Database search engines and target database features impinge upon the identification of post-translationally cis-spliced peptides in HLA class I immunopeptidomes

Unconventional epitopes presented by HLA class I complexes are emerging targets for T cell targeted immunotherapies. Their identification by mass spectrometry (MS) required development of novel methods to cope with the large number of theoretical candidates. Methods to identify post-translationally spliced peptides led to a broad range of outcomes. We here investigated the impact of three common database search engines – that is, Mascot, Mascot+Percolator, and PEAKS DB – as final identification step, as well as the features of target database on the ability to correctly identify non-spliced and cis-spliced peptides. We used ground truth datasets measured by MS to benchmark methods’ performance and extended the analysis to HLA class I immunopeptidomes. PEAKS DB showed better precision and recall of cis-spliced peptides and larger number of identified peptides in HLA class I immunopeptidomes than the other search engine strategies. The better performance of PEAKS DB appears to result from better discrimination between target and decoy hits and hence a more robust FDR estimation, and seems independent to peptide and spectrum features here investigated.     

Translating olfactomes into attractants: shared volatiles provide attractive bridges for polyphagy in fruit flies

Tephritid flies are serious fruit pests. Despite clear niche differences, many species show considerable overlap in fruit preferences, of which we here analysed the olfactory correlate. Using the volatiles of four unrelated fruit species, antennal responses were quantified to construct a fruit‐odour response database for four tephritid species. Although responses were distinct with a significant niche‐correlated bias, the analyses show that the probability of detection of a volatile strongly increased with its sharedness across fruits. This also held for the unrelated fruit fly Drosophila melanogaster (DoOR repository‐based analyses). We conjectured that shared volatiles signify ‘host’ to the fly ‘nose’ and induce attraction. Indeed, blends of volatiles shared by fruit and detected by all four species were very attractive for tephritid species, more than fruits. Quantitative whole antennal recordings en lieu of, or complementing bottom‐up molecular neurogenetic approaches, enables comparative olfactomics in non‐model species, and facilitate interpretation of olfaction in evolutionary, ecological, and applied contexts.     

Research note: Analysis of expressed sequence tags from the chrysophycean alga Ochromonas danica (Heterokontophyta)

A cDNA library was constructed from the chrysophycean alga, Ochromonas danica E. G. Pringsheim. 5′‐end sequencing of about 600 cDNA clones yielded 476 authentic expressed sequence tags (EST) of which 275 showed significant matches (E‐value <10^?4) to sequences in a public database. The annotation of these ESTs was carried out to assess subcellular localization of the putative proteins using several internet‐accessible prediction programs for subcellular localization. These analyses revealed that putative plastid proteins in Ochromonas possess N‐terminal bipartite presequences with a conserved phenylalanine at the N‐terminus of the predicted transit peptide‐like domains, similar to other ‘red‐lineage’ secondary symbiotic organisms. The examination of sequences of 3′‐UTR revealed that, similarly to chlorophyte algae, UGUAA may represent a putative polyadenylation signal in O. danica.     

Clonostachys rosea BAFC3874 as a Sclerotinia sclerotiorum antagonist: mechanisms involved and potential as a biocontrol agent

Aims: To establish the modes of action of the antagonistic fungal strain Clonostachys rosea BAFC3874 isolated from suppressive soils against Sclerotinia sclerotiorum and to determine its potential as a biocontrol agent. Methods and Results: The antagonistic activity of C. rosea BAFC3874 was determined in vitro by dual cultures. The strain effectively antagonized S. sclerotiorum in pot‐grown lettuce and soybean plants. Antifungal activity assays of C. rosea BAFC3874 grown in culture established that the strain produced antifungal compounds against S. sclerotiorum associated with secondary metabolism. High mycelial growth inhibition coincided with sclerotia production inhibition. The C. rosea strain produced a microheterogeneous mixture of peptides belonging to the peptaibiotic family. Moreover, mycoparasitism activity was observed in the dual culture. Conclusions: Clonostachys rosea strain BAFC3874 was proved to be an effective antagonist against the aggressive soil‐borne pathogen S. sclerotiorum in greenhouse experiments. The main mechanisms involve peptaibiotic metabolite production and mycoparasitism activity. Significance and Impact of the Study: Clonostachys rosea BAFC3874 may be a good fungal biological control agent against S. sclerotiorum. In addition, we were also able to isolate and identify peptaibols, an unusual family of compounds in this genus of fungi.     

Analyzing protease specificity and detecting in vivo proteolytic events using tandem mass spectrometry

Although trypsin remains the most commonly used protease in MS, other proteases may be employed for increasing peptide coverage or generating overlapping peptides. Knowledge of the accurate specificity rules of these proteases is helpful for database search tools to detect peptides, and becomes crucial when label‐free MS is used to discover in vivo proteolytic cleavages. Since in vivo cleavages are inferred by subtracting digestion‐induced cleavages from all observed cleavages, it is important to ensure that the specificity rule used to identify digestion‐induced cleavages are broad enough to capture even minor cleavages produced in digestion, to avoid erroneously identifying them as in vivo cleavages. In this study, we describe MS‐Proteolysis, a software tool for identifying putative sites of in vivo proteolytic cleavage using label‐free MS. The tool is used in conjunction with digestion by trypsin and three other proteases, whose specificity rules are revised and extended before inferring proteolytic cleavages. Finally, we show that comparative analysis of multiple proteases can be used to detect putative in vivo proteolytic sites on a proteome‐wide scale.     

Seasonal selection and resource dynamics in a seasonally polyphenic butterfly

Seasonal polyphenisms are widespread in nature, yet the selective pressures responsible for their evolution remain poorly understood. Previous work has largely focussed either on the developmental regulation of seasonal polyphenisms or putative ‘top‐down’ selective pressures such as predation that may have acted to drive phenotypic divergence. Much less is known about the influence of seasonal variation in resource availability or seasonal selection on optimal resource allocation. We studied seasonal variation in resource availability, uptake and allocation in Araschnia levana L., a butterfly species that exhibits a striking seasonal colour polyphenism consisting of predominantly orange ‘spring form’ adults and black‐and‐white ‘summer form’ adults. ‘Spring form’ individuals develop as larvae in the late summer, enter a pupal diapause in the fall and emerge in the spring, whereas ‘summer form’ individuals develop directly during the summer months. We find evidence for seasonal declines in host plant quality, and we identify similar reductions in resource uptake in late summer, ‘spring form’ larvae. Further, we report shifts in the body composition of diapausing ‘spring form’ pupae consistent with a physiological cost to overwintering. However, these differences do not translate into detectable differences in adult body composition. Instead, we find minor seasonal differences in adult body composition consistent with augmented flight capacity in ‘summer form’ adults. In comparison, we find much stronger signatures of sex‐specific selection on patterns of resource uptake and allocation. Our results indicate that resource dynamics in A. levana are shaped by seasonal fluctuations in host plant nutrition, climatic conditions and intraspecific interactions.     

Novel M‐Superfamily and T‐Superfamily conotoxins and contryphans from the vermivorous snail Conus figulinus

The venom of Conus figulinus, a vermivorous cone snail, found in the south east coast of India, has been studied in an effort to identify novel peptide toxins. The amino acid sequences of seven peptides have been established using de novo mass spectrometric based sequencing methods. Among these, three peptides belong to the M‐Superfamily conotoxins, namely, Fi3a, Fi3b, and Fi3c, and one that belongs to the T‐Superfamily, namely, Fi5a. The other three peptides are contryphans, namely, contryphans fib, fic, and fid. Of these Fi3b, Fi3c, Fi5a, and contryphan fib are novel and are reported for the first time from venom of C. figulinus. The details of the sequencing methods and the relationship of these peptides with other ‘M’‐Superfamily conotoxins from the fish hunting and mollusk hunting clades are discussed. These novel peptides could serve as a lead compounds for the development of neuropharmacologically important drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Total Synthesis of the Peptaibols Hypomurocin A3 and Hypomurocin A5, and Their Conformation Analysis

The total syntheses of hypomurocin A3 and hypomuricin A5 (HM A3 and HM A5, resp.) in solution phase are described. These syntheses have been successfully achieved by applying the ‘azirine/oxazolone method’ to introduce the two Aib‐Pro units into the backbone of these undecapeptaibols in one step with methyl 2,2‐dimethyl‐2H‐azirine‐3‐prolinate as the ‘Aib‐Pro synthon’. The coupling of Z‐protected (Z=(benzyloxy)carbonyl) amino acids or peptide acids with amino acid tert‐butyl esters and of peptide segments was carried out according to the TBTU (=O‐(benzotriazol‐1‐yl)‐N,N,N′,N′‐tetramethyluronium tetrafluoroborate) and HOBt (=1‐hydroxybenzotriazole) protocol. Purification by reversed‐phase HPLC gave the peptides in pure form. The products were characterized by optical rotation, NMR and IR spectroscopy, mass spectrometry, and elemental analysis. The crystal structures of HM A3 and of an octapeptide fragment of HM A5 could be obtained. An NMR analysis was also carried out with HM A3 and HM A5 to determine their conformations in solution. A global structural comparison between the three sequences of HM A1, HM A3, and HM A5 was performed, as well as the HPLC correlation of the natural HM A family and the synthetic samples.