Growth promotion in Arabidopsis thaliana by bacterial cyclodipeptides involves the TOR/S6K pathway activation.

Cyclodipeptides (CDPs) are the smallest peptidic molecules that can be produced by diverse organisms such as bacteria, fungi, and animals. They have multiple biological effects. In this paper, we examined the CDPs produced by the bacteria Pseudomonas aeruginosa PAO1, which are known as opportunistic pathogens of humans and plants on TARGET OF RAPAMYCIN (TOR) signaling pathways, and regulation of root system architecture. This bacterium produces the bioactive CDPs: cyclo(L-Pro-L-Leu), cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Tyr), and cyclo(L-Pro-L-Val). In a previous report, these molecules were found to modulate basic cellular programs not only via auxin mechanisms but also by promoting the phosphorylation of the S6 ribosomal protein kinase (S6K), a downstream substrate of the TOR kinase. In the present work, we found that the inoculation of Arabidopsis plants with P. aeruginosa PAO1, the non-pathogenic P. aeruginosa ΔlasI/Δrhll strain (JM2), or by direct exposure of plants to CDPs influenced growth and promoted root branching depending upon the treatment imposed, while genetic evidence using Arabidopsis lines with enhanced or decreased TOR levels indicated a critical role of this pathway in the bacterial phytostimulation.

Advancing d-amino acid-containing peptide discovery in the metazoan.

The discovery of enzyme-derived d-amino acid-containing peptides (DAACPs) that have physiological importance in the metazoan challenges previous assumptions about the homochirality of animal proteins while simultaneously revealing new analytical challenges in the structural and functional characterization of peptides. Most known DAACPs have been identified though laborious activity-guided purification studies or by homology to previously identified DAACPs. Peptide characterization experiments are increasingly dominated by high throughput mass spectrometry-based peptidomics, with stereochemistry rarely considered due to the technical challenges of identifying l/d isomerization. This review discusses the prevalence of enzyme-derived DAACPs among animals and the physiological consequences of peptide isomerization. Also highlighted are the analytical methods that have been applied for structural characterization/discovery of DAACPs, including results of several recent studies using non-targeted discovery methods for revealing novel DAACPs, strongly suggesting that more DAACPs remain to be uncovered.

Structural and Functional Insights into Iturin W, a Novel Lipopeptide Produced by the Deep-Sea Bacterium Bacillus sp. Strain wsm-1.

In the present study, a deep-sea bacterial strain designated Bacillus sp. strain wsm-1 was screened and found to exhibit strong antifungal activity against many plant-pathogenic fungi, and corresponding antifungal agents were thereby purified and determined by tandem mass spectrometry to be two cyclic lipopeptide homologs. These homologs, which were different from any previously reported lipopeptides, were identified to possess identical amino acid sequences of ß-amino fatty acid-Asn-Ser-Asn-Pro-Tyr-Asn-Gln and deduced as two novel lipopeptides designated C14 iturin W and C15 iturin W. Electron microscopy observation indicated that both iturin W homologs caused obvious morphological changes and serious disruption of plasma membrane toward fungal cells, while C15 iturin W exhibited more serious cell damages than C14 iturin W did, which was well consistent with the results of the antifungal activity assays. To improve the yield and antifungal activity of iturin W, the effects of different carbon and nitrogen sources and amino acids on production of C14 iturin W and C15 iturin W were investigated. The results indicated that supplements of most of the detected carbon and nitrogen sources could increase the yield of C14 iturin W, but inhibit the yield of C15 iturin W, while supplements of tryptone and most of the detected amino acids could increase the yield of both C14 iturin W and C15 iturin W.IMPORTANCE Plant disease caused by pathogenic fungi is one of the most devastating diseases, which affects the food safety of the whole world to a great extent. Biological control of plant diseases by microbial natural products is more desirable than traditional chemical control. In this study, we discovered a novel lipopeptide, iturin W, with promising prospects in biological control of plant diseases. Moreover, the effects of different carbon and nitrogen sources and amino acids on production of C14 iturin W and C15 iturin W would provide a reasonable basis for the optimization of the fermentation process of lipopeptides. Notably, the structure of iturin W was different from that of any previously reported lipopeptide, suggesting that deep-sea microorganisms might produce many novel natural products and have significant potential in the development of biological products in the future.

One Enzyme To Build Them All: Ring-Size Engineered Siderophores Inhibit the Swarming Motility of Vibrio.

Bacteria compete for ferric iron by producing siderophores, and some microbes engage in piracy by scavenging siderophores of their competitors. The macrocyclic hydroxamate siderophore avaroferrin of Shewanella algae inhibits swarming of Vibrio alginolyticus by evading this piracy. Avaroferrin, as well as related putrebactin and bisucaberin, are produced by the IucC-like synthetases AvbD, PubC, and BibCC. Here, we have established that they are capable of synthesizing not only their native product but also other siderophores. Exploiting this relaxed substrate specificity by synthetic precursors generated 15 different ring-size engineered macrocycles ranging from 18- to 28-membered rings, indicating unprecedented biosynthetic flexibility of the enzymes. Two of the novel siderophores could be obtained in larger quantities by precursor-directed biosynthesis in S. algae. Both inhibited swarming motility of Vibrio and, similar to avaroferrin, the most active one exhibited a heterodimeric architecture. Our results demonstrate the impact of minor structural changes on biological activity, which may trigger the evolution of siderophore diversity.

Key driving forces in the biosynthesis of autoinducing peptides required for staphylococcal virulence.

Staphylococci produce autoinducing peptides (AIPs) as quorum-sensing signals that regulate virulence. These AIPs feature a thiolactone macrocycle that connects the peptide C terminus to the side chain of an internal cysteine. AIPs are processed from ribosomally synthesized precursors [accessory gene regulator D (AgrD)] through two proteolytic events. Formation of the thiolactone is coupled to the first of these and involves the activity of the integral membrane protease AgrB. This step is expected to be thermodynamically unfavorable, and therefore, it is unclear how AIP-producing bacteria produce sufficient amounts of the thiolactone-containing intermediate to drive quorum sensing. Herein, we present the in vitro reconstitution of the AgrB-dependent proteolysis of an AgrD precursor from Staphylococcus aureus. Our data show that efficient thiolactone production is driven by two unanticipated features of the system: (i) membrane association of the thiolactone-containing intermediate, which stabilizes the macrocycle, and (ii) rapid degradation of the C-terminal proteolysis fragment AgrD(C), which affects the reaction equilibrium position. Cell-based studies confirm the intimate link between AIP production and intracellular AgrD(C) levels. Thus, our studies explain the chemical principles that drive AIP production, including uncovering a hitherto unknown link between quorum sensing and peptide turnover.

Cyclic lipopeptide iturin A structure-dependently induces defense response in Arabidopsis plants by activating SA and JA signaling pathways.

Iturin A is the most well studied antifungal cyclic lipopeptide produced by Bacillus species that are frequently utilized as biological control agents. Iturin A not only shows strong antifungal activity against phytopathogens but also induces defense response in plants, thereby reducing plant disease severity. Here we report the defense signaling pathways triggered by iturin A in Arabidopsis salicylic acid (SA) or jasmonic acid (JA)-insensitive mutants. Iturin A activated the transcription of defense genes PR1 and PDF1.2 through the SA and JA signaling pathways, respectively. The role of iturin A as an elicitor was dependent on the cyclization of the seven amino acids and/or the ß-hydroxy fatty acid chain. The iturin A derivative peptide, NH2-(L-Asn)-(D-Tyr)-(D-Asn)-(L-Gln)-(L-Pro)-(D-Asn)-(L-Ser)-COOH, completely suppressed PR1 and PDF1.2 gene expression in wild Arabidopsis plants. The identification of target molecules binding to iturin A and its derivative peptide is expected to shed new light on defense response in plants through the SA and JA signaling pathways.

Induced systemic resistance responses in perennial ryegrass against Magnaporthe oryzae elicited by semi-purified surfactin lipopeptides and live cells of Bacillus amyloliquefaciens.

The suppressive ability of several strains of cyclic lipopeptide-producing Bacillus rhizobacteria to grey leaf spot disease caused by Magnaporthe oryzae has been documented previously; however, the underlying mechanism(s) involved in the induced systemic resistance (ISR) activity in perennial ryegrass (Lolium perenne L.) remains unknown. Root-drench application of solid-phase extraction (SPE)-enriched surfactin and live cells of mutant Bacillus amyloliquefaciens strain FZB42-AK3 (produces surfactin, but not bacillomycin D and fengycin) significantly reduced disease incidence and severity on perennial ryegrass. The application of the treatments revealed a pronounced multilayered ISR defence response activation via timely and enhanced accumulation of hydrogen peroxide (H2O2), elevated cell wall/apoplastic peroxidase activity, and deposition of callose and phenolic/polyphenolic compounds underneath the fungal appressoria in naïve leaves, which was significantly more intense in treated plants than in mock-treated controls. Moreover, a hypersensitive response (HR)-type reaction and enhanced expression of LpPrx (Prx, peroxidase), LpOXO4 (OXO, oxalate oxidase), LpPAL (PAL, phenylalanine ammonia lyase), LpLOXa (LOX, lipoxygenase), LpTHb (putative defensin) and LpDEFa (DEFa, putative defensin) in perennial ryegrass were associated with SPE-enriched surfactin and live AK3 cell treatments, acting as a second layer of defence when pre-invasive defence responses failed. The results indicate that ISR activity following surfactin perception may sensitize H2O2 -mediated defence responses, thereby providing perennial ryegrass with enhanced protection against M. oryzae.

Cyclic dipeptides: from bugs to brain.

Cyclic dipeptides (CDPs) are a group of hormone-like molecules that are evolutionarily conserved from bacteria to humans. In bacteria, CDPs are used in quorum sensing (QS) to communicate information about population size and to regulate a behavioural switch from symbiosis with their host to virulence. In mammals, CDPs have been shown to act on glial cells (macrophage-like cells) to control a conceptually homologous behavioural switch between homeostatic and inflammatory modes, with implications for the control of neurodegenerative disease. Here we argue that, because of their capacity to regulate inflammation via glial cells and induce a protective response in neuronal cells, CDPs have potential therapeutic utility in an array of inflammatory diseases.

A peptide from the extracellular region of the synaptic protein α Neurexin stimulates angiogenesis and the vascular specific tyrosine kinase Tie2.

Neurexin (NRXN) and Neuroligin (NLGN) are trans-synaptic proteins involved in vascular biology. NRXN is encoded in long (α) and short (ß) isoforms. We have shown that ßNRXN modulates blood vessel development in synergy with VEGFA and associates with NLGN. On the other hand αNRXN is also expressed in blood vessels but does not interact with NLGN or act in synergy with VEGFA, thus demonstrating a differential role. To find clues into the vascular functions of αNRXN, we chose a 7 aa motif that is part of its extracellular region and was formerly selected through a proteomic search for interactors of the vascular receptor Tie2. Next we (a) synthetized and modeled such peptide in order to determine its biological activity towards Tie2 in in vitro and in vivo angiogenesis assays and (b) evaluated if αNRXN and Tie2 physically associate in situ during vascular development. We used biochemical and cellular assays to prove that the synthetic αNRXN peptide (a) modulates the angiogenic phenotype of cultured endothelial cells and angiogenesis in vivo and (b) efficiently stimulates Tie2 phosphorylation and downstream mediators in endothelial cells. Moreover, we show that αNRXN and Tie2 can be reciprocally immunoprecipitated from chicken blood vessels at late stages of vascular development. These data have a double significance, i.e. provide a novel tool to modulate Tie2 and further suggest the involvement of the NRXN family of synaptic protein in the vascular system through their interaction with a fundamental vascular player.

θ-Defensins: cyclic peptides with endless potential.

θ-Defensins, the only cyclic peptides of animal origin, have been isolated from the leukocytes of rhesus macaques and baboons. Their biogenesis is unusual because each peptide is an 18-residue chimera formed by the head-to-tail splicing of nonapeptides derived from two separate precursors. θ-Defensins have multiple arginines and a ladder-like tridisulfide array spanning their two antiparallel ß-strands. Human θ-defensin genes contain a premature stop codon that prevents effective translation of the needed precursors; consequently, these peptides are not present in human leukocytes. Synthetic θ-defensins with sequences that correspond to those encoded within the human pseudogenes are called retrocyclins. Retrocyclin-1 inhibits the cellular entry of HIV-1, HSV, and influenza A virus. The rhesus θ-defensin RTD-1 protects mice from an experimental severe acute respiratory syndrome coronavirus infection, and retrocyclin-1 protects mice from infection by Bacillus anthracis spores. The small size, unique structure, and multiple host defense activities of θ-defensins make them intriguing potential therapeutic agents.

Thematic minireview series on circular proteins.

Circular proteins have now been discovered in all kingdoms of life and are characterized by their exceptional stability and the diversity of their biological activities, primarily in the realm of host defense functions. This thematic minireview series provides an overview of the distribution, evolution, activities, and biological synthesis of circular proteins. It also reviews approaches that biological chemists are taking to develop synthetic methods for making circular proteins in the laboratory. These approaches include solid-phase peptide synthesis based on an adaption of native chemical ligation technology and recombinant DNA approaches that are amenable to the in-cell production of cyclic peptide libraries. The thioester-mediated native chemical ligation approach mimics, to some extent, elements of the natural biosynthetic reaction, which, for disulfide-rich cyclic peptides, appears to involve asparaginyl endopeptidase-mediated processing from larger precursor proteins.

Systemic urocortin 2, but not urocortin 1 or stressin 1-A, suppresses feeding via CRF2 receptors without malaise and stress.

BACKGROUND AND PURPOSE: Infusion of corticotropin-releasing factor (CRF)/urocortin (Ucn) family peptides suppresses feeding in mice. We examined whether rats show peripheral CRF/Ucn-induced anorexia and determined its behavioural and pharmacological bases. EXPERIMENTAL APPROACH: Male Wistar rats (n= 5-12 per group) were administered (i.p.) CRF receptor agonists with different subtype affinities. Food intake, formation of conditioned taste aversion and corticosterone levels were assessed. In addition, Ucn 1- and Ucn 2-induced anorexia was studied in fasted CRF(2) knockout (n= 11) and wild-type (n= 13) mice. KEY RESULTS: Ucn 1, non-selective CRF receptor agonist, reduced food intake most potently (~0.32 nmol·kg(-1) ) and efficaciously (up to 70% reduction) in fasted and fed rats. The peptides' rank-order of anorexic potency was Ucn 1 ≥ Ucn 2 > >stressin(1) -A > Ucn 3, and efficacy, Ucn 1 > stressin(1) -A > Ucn 2 = Ucn 3. Ucn 1 reduced meal frequency and size, facilitated feeding bout termination and slowed eating rate. Stressin(1) -A (CRF(1) agonist) reduced meal size; Ucn 2 (CRF(2) agonist) reduced meal frequency. Stressin(1) -A and Ucn 1, but not Ucn 2, produced a conditioned taste aversion, reduced feeding efficiency and weight regain and elicited diarrhoea. Ucn 1, but not Ucn 2, also increased corticosterone levels. Ucn 1 and Ucn 2 reduced feeding in wild-type, but not CRF(2) knockout, mice. CONCLUSIONS AND IMPLICATIONS: CRF(1) agonists, Ucn 1 and stressin(1) -A, reduced feeding and induced interoceptive stress, whereas Ucn 2 potently suppressed feeding via a CRF(2) -dependent mechanism without eliciting malaise. Consistent with their pharmacological differences, peripheral urocortins have diverse effects on appetite.

Glycomimetic improves recovery after femoral injury in a non-human primate.

In adult mammals, restoration of function after peripheral nerve injury is often poor and effective therapies are not available. Previously we have shown in mice that a peptide which functionally mimics the human natural killer cell (HNK)-1 trisaccharide epitope significantly improves the outcome of femoral nerve injury. Here we evaluated the translational potential of this treatment using primates. We applied a linear HNK-1 mimetic or a functionally inactive control peptide in silicone cuffs used to reconstruct the cut femoral nerves of adult cynomolgus monkeys (Macaca fascicularis). Functional recovery was evaluated using video-based gait analysis over a 160-day observation period. The final outcome was further assessed using force measurements, H-reflex recordings, nerve histology, and ELISA to assess immunoreactivity to HNK-1 in the treated monkeys. Gait deficits were significantly reduced in HNK-1 mimetic-treated compared with control peptide-treated animals between 60 and 160 days after injury. Better outcome at 160 days after surgery in treated versus control animals was also confirmed by improved quadriceps muscle force, enhanced H-reflex amplitude, decreased H-reflex latency, and larger diameters of regenerated axons. No adverse reactions to the mimetic, in particular immune responses resulting in antibodies against the HNK-1 mimetic or immune cell infiltration into the damaged nerve, were observed. These results indicate the potential of the HNK-1 mimetic as an efficient, feasible, and safe adjunct treatment for nerve injuries requiring surgical repair in clinical settings.

A hormone-activated central pattern generator for courtship.

BACKGROUND: Medicinal leeches (Hirudo spp.) are simultaneous hermaphrodites. Mating occurs after a stereotyped twisting and oral exploration that result in the alignment of the male and/or female gonopores of one leech with the complementary gonopores of a partner. The neural basis of this behavior is presently unknown and currently impossible to study directly because electrophysiological recording techniques disrupt the behavior. RESULTS: Here we report that (Arg(8))-conopressin G and two other members of the oxytocin/vasopressin family of peptide hormones induce in Hirudo verbana a sequence of behaviors that closely mimic elements of spontaneous reproductive behavior. Through a series of progressively more reduced preparations, we show that one of these behaviors, a stereotyped twisting that is instrumental in aligning gonopores in preparation for copulation, is the product of a central pattern generator that consists of oscillators in ganglia M5 and M6 (the ganglia in the reproductive segments of the leech), and also in ganglion M4, which was not previously known to play a role in reproductive behavior. We find that the behavior is periodic, with a remarkably long cycle period of around five minutes, placing it among the slowest behavioral rhythms (other than diurnal and annual rhythms) yet described. CONCLUSION: These results establish the leech as a new model system for studying aspects of the neuronal basis of reproductive behavior.

Microbial interactions: bacteria talk to (some of) their neighbors.

A recent study reports that Bacillus subtilis biofilm formation depends upon paracrine signaling where the signal-producing and target-responsive cells are different.

Dentigerumycin: a bacterial mediator of an ant-fungus symbiosis.

Fungus-growing ants engage in mutualistic associations with both the fungus they cultivate for food and actinobacteria (Pseudonocardia spp.) that produce selective antibiotics to defend that fungus from specialized fungal parasites. We have analyzed one such system at the molecular level and found that the bacterium associated with the ant Apterostigma dentigerum produces dentigerumycin, a cyclic depsipeptide with highly modified amino acids, to selectively inhibit the associated parasitic fungus (Escovopsis sp.).

Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens.

The genome of plant-associated Bacillus amyloliquefaciens FZB42 harbors an array of giant gene clusters involved in synthesis of lipopeptides and polyketides with antifungal, antibacterial and nematocidal activity. Five gene clusters, srf, bmy, fen, nrs, dhb, covering altogether 137 kb, were shown to direct synthesis of the cyclic lipopeptides surfactin, bacillomycin, fengycin, an unknown peptide, and the iron-siderophore bacillibactin. In addition, one gene cluster encoding enzymes involved in synthesis and export of the antibacterial dipeptide bacilysin is also functional in FZB42. Three gene clusters, mln, bae, and dfn, with a total size of 199 kb were shown to direct synthesis of the antibacterial acting polyketides macrolactin, bacillaene, and difficidin. In total, FZB42 dedicates about 340 kb, corresponding to 8.5% of its total genetic capacity, to synthesis of secondary metabolites. On the contrary, genes involved in ribosome-dependent synthesis of lantibiotics and other peptides are scarce. Apart from two incomplete gene clusters directing immunity against mersacidin and subtilin, only one peptide-like compound has been detected in the culture fluid that inhibits the growth of B. subtilis lacking the alternative sigma factor W.

Bacterial quorum sensing: a new target for anti-infective immunotherapy.

BACKGROUND: Cell-to-cell communication via exchange of small molecules, 'autoinducers', is a widespread phenomenon among Gram-negative and -positive bacteria. This intercellular signaling that synchronizes population-wide gene expression in a cell-density-dependent manner is termed 'quorum sensing' (QS). The discovery that Gram-negative bacteria employ non-peptide structures, N-acyl homoserine lactones, to globally regulate production of secondary metabolites and proteins, initiated a new area of research. Subsequently, other quorum-sensing systems and small signaling molecules were identified. With the emergence of antibiotic-resistant bacteria, most prominently methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, new approaches for combating infections are needed. Inhibition of QS results in attenuation of virulence rather than direct killing of microbes. OBJECTIVE: We highlight current trends in preventing bacterial infections using quorum-quenching strategies. METHODS: We mainly focus on P. aeruginosa and S. aureus and their QS systems as targets for intervention. RESULTS/CONCLUSION: New research strongly suggests that QS systems represent attractive targets for discovery of novel anti-infective agents, including immunotherapeutic strategies.

Cortistatin--functions in the central nervous system.

Cortistatin (CST) is a neuropeptide from the somatostatin (SRIF)/urotensin (UII) family named after its predominantly cortical expression and ability to depress cortical activity, which was discovered a decade ago. In vitro assays show CST is able to bind all five cloned somatostatin receptors and shares many pharmacological and functional properties with SRIF. However, distinct from SRIF, CST has been shown to induce slow-wave sleep, reduce locomotor activity, and activate cation selective currents not responsive to somatostatin. Different lines of evidence also indicate that CST, like SRIF, is involved in learning and memory processes. CST-14 may also function as an endogenous anti-convulsant. In addition to its role in cortical synchronization, CST-14 has emerged as an important mediator of immunity and inflammation. This review will cover some of the basic properties of CST in the brain, and will discuss new data on the role of CST in cortical activity.