A natural prodrug activation mechanism in nonribosomal peptide synthesis

We have identified a new mechanism for the cleavage and activation of nonribosomally made peptides and peptide-polyketide hybrids that are apparently operational in several different bacteria. This process includes the cleavage of a precursor molecule by a membrane-bound and D-asparagine-specific peptidase, as shown here in the biosynthesis of the antibiotic xenocoumacin from Xenorhabdus nematophila.     

Advances in antimicrobial peptide immunobiology

Antimicrobial peptides are ancient components of the innate immune system and have been isolated from organisms spanning the phylogenetic spectrum. Over an evolutionary time span, these peptides have retained potency, in the face of highly mutable target microorganisms. This fact suggests important coevolutionary influences in the host-pathogen relationship. Despite their diverse origins, the majority of antimicrobial peptides have common biophysical parameters that are likely essential for activity, including small size, cationicity, and amphipathicity. Although more than 900 different antimicrobial peptides have been characterized, most can be grouped as belonging to one of three structural classes: (1) linear, often of alpha-helical propensity; (2) cysteine stabilized, most commonly conforming to beta-sheet structure; and (3) those with one or more predominant amino acid residues, but variable in structure. Interestingly, these biophysical and structural features are retained in ribosomally as well as nonribosomally synthesized peptides. Therefore, it appears that a relatively limited set of physicochemical features is required for antimicrobial peptide efficacy against a broad spectrum of microbial pathogens.During the past several years, a number of themes have emerged within the field of antimicrobial peptide immunobiology. One developing area expands upon known microbicidal mechanisms of antimicrobial peptides to include targets beyond the plasma membrane. Examples include antimicrobial peptide activity involving structures such as extracellular polysaccharide and cell wall components, as well as the identification of an increasing number of intracellular targets. Additional areas of interest include an expanding recognition of antimicrobial peptide multifunctionality, and the identification of large antimicrobial proteins, and antimicrobial peptide or protein fragments derived thereof. The following discussion highlights such recent developments in antimicrobial peptide immunobiology, with an emphasis on the biophysical aspects of host-defense polypeptide action and mechanisms of microbial resistance.     

Lantibiotics, class I bacteriocins from the genus Bacillus

Antimicrobial peptides exhibit high levels of antimicrobial activity against a broad range of spoilage and pathogenic microorganisms. Compared with bacteriocins produced by lactic acid bacteria, antimicrobial peptides from the genus Bacillus have been relatively less recognized despite their broad antimicrobial spectra. These peptides can be classified into two different groups based on whether they are ribosomally (bacteriocins) or nonribosomally (polymyxins and iturins) synthesized. Because of their broad spectra and high activity, antimicrobial peptides from Bacillus spp. may have great potential for applications in the food, agricultural, and pharmaceutical industries to prevent or control spoilage and pathogenic microorganisms. In this review, we introduce ribosomally synthesized antimicrobial peptides, the lantibiotic bacteriocins produced by members of Bacillus. In addition, the biosynthesis, genetic organization, mode of action, and regulation of subtilin, a well-investigated lantibiotic from Bacillus subtilis, are discussed.     

Antimicrobial lipopolypeptides composed of palmitoyl Di- and tricationic peptides: in vitro and in vivo activities, self-assembly to nanostructures, and a plausible mode of action

Antimicrobial lipopeptides are produced nonribosomally in bacteria and fungi during cultivation. They are composed of a cationic or an anionic peptide covalently bound to a specifically modified aliphatic chain. Most of the peptidic moieties have complex cyclic structures. Here we report that conjugation of a palmitic acid to the N-terminus of very short cationic di- and tripeptides composed of all l- and d, l-amino acids endowed them with potent antimicrobial activities. Interestingly, cell specificity was determined by the sequence of the short peptidic chain. Palmitoyllysine served as a control and was inactive toward all microorganisms tested. Replacing an l-amino acid with its d-enantiomer did not affect the activity of the corresponding lipopeptides. Importantly, selected lipopeptides were also potent in vivo in a mouse model of Candida albicans infection. Bacterial leakage experiments and negative staining electron microscopy suggest that their mode of action involves permeation and disintegration of the microorganism's membrane, similar to many long antimicrobial peptides and lipopeptides. Interestingly, each lipopeptide assembled in solution into a nanostructure with a unique morphology which could partially explain differences in their biological activity. Besides adding important information on the parameters necessary for antimicrobial lipopeptides to kill microorganisms, the simple composition of these minilipopeptides and their diverse cell specificities make them attractive candidates for various applications.     

In silico analysis of nonribosomal peptide synthetases of Xanthomonas axonopodis pv. citri: identification of putative siderophore and lipopeptide biosynthetic genes

The genomes of the plant pathogens Xanthomonas axonopodis (Xac) and Xanthomonas campestris (Xcc) were analysed with the aim of deducing their ability to produce nonribosomal peptides. Nonribosomal peptide synthetase (NRPS) genes were identified in two separate loci of Xac. While the genes of locus 1 are common to both strains, locus 2 was only found in Xac. Dissection and phylogenetic analysis of the condensation and thioesterase domains of the NRPSs of loci 1 and 2 of Xac revealed homology, respectively, with siderophore and lipopeptide synthetases. Further analysis of locus 1 revealed genes related to polyketide and polyamine biosynthesis that could be involved in the assembly of substrates for siderophore biosynthesis in both strains. In vitro production of siderophores by both Xac and Xcc was confirmed. Since bacterial siderophores and lipopeptides can be pathogenic and are typically produced nonribosomally, these results suggest that the identified genes could be involved in phytotoxin production.     

Global regulatory systems operating in Bacilysin biosynthesis in Bacillus subtilis

In Bacillus subtilis, bacilysin is a nonribosomally synthesized dipeptide antibiotic composed of L-alanine and L-anticapsin. The biosynthesis of bacilysin depends on the bacABCDEywfG operon (bac operon)and the adjacent ywfH gene. To elucidate the effects of global regulatory genes on the expression of bac operon, we used the combination of lacZ fusion analysis and the gel mobility shift assays. The cell density-dependent transition state induction of the bac operon was clearly shown. The basal expression level of the bac operon as well as transition state induction of bac is directly ComA dependent. Three Phr peptides, PhrC, PhrF and PhrK, are required for full-level expression of ComA-dependent bac operon expression, but the most important role seemed to be played by PhrC in stimulating bac expression through a RapC-independent manner. Spo0A is another positive regulator which participates in the transition state induction of bac both directly by interacting with the bac promoter and indirectly by repressing abrB expression. AbrB and CodY proteins do not only directly repress the bac promoter, but they also mutually stimulate the transition state induction of bac indirectly, most likely by antagonizing their repressive effects without preventing each other's binding since both proteins can bind to the bac promoter simultaneously.     

Biomedicinals from the phytosymbionts of marine invertebrates: a molecular approach

Marine invertebrate animals such as sponges, gorgonians, tunicates and bryozoans are sources of biomedicinally relevant natural products, a small but growing number of which are advancing through clinical trials. Most metazoan and anthozoan species harbour commensal microorganisms that include prokaryotic bacteria, cyanobacteria (blue-green algae), eukaryotic microalgae, and fungi within host tissues where they reside as extra- and intra-cellular symbionts. In some sponges these associated microbes may constitute as much as 40% of the holobiont volume. There is now abundant evidence to suggest that a significant portion of the bioactive metabolites thought originally to be products of the source animal are often synthesized by their symbiotic microbiota. Several anti-cancer metabolites from marine sponges that have progressed to pre-clinical or clinical-trial phases, such as discodermolide, halichondrin B and bryostatin 1, are thought to be products derived from their microbiotic consortia. Freshwater and marine cyanobacteria are well recognised for producing numerous and structurally diverse bioactive and cytotoxic secondary metabolites suited to drug discovery. Sea sponges often contain dominant taxa-specific populations of cyanobacteria, and it is these phytosymbionts (= photosymbionts) that are considered to be the true biogenic source of a number of pharmacologically active polyketides and nonribosomally synthesized peptides produced within the sponge. Accordingly, new collections can be pre-screened in the field for the presence of phytobionts and, together with metagenomic screening using degenerate PCR primers to identify key polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes, afford a biodiscovery rationale based on the therapeutic prospects of phytochemical selection. Additionally, new cloning and biosynthetic expression strategies may provide a sustainable method for the supply of new pharmaceuticals derived from the uncultured phytosymbionts of marine organisms.     

抗菌肽Fengycins抑制串珠镰刀菌的初步机制

Fengycins是枯草芽孢杆菌非核糖体合成的环状脂肽类抗生素,本文从其特性入手,研究了其抑制串珠镰刀菌的初步作用机制。普通显微观察结果显示,Fengycins处理能使部分串珠镰刀菌菌丝顶端破裂,进一步通过PI染色与荧光显微观察发现,Fengycins处理会导致串珠镰刀菌菌丝膜的损伤。在添加几丁质、壳聚糖、β-1,3葡聚糖、甾醇、胆固醇的平板内,Fengycins的抑菌活性没有受到太大影响;而在添加卵磷脂的平板,Fengycins的抑菌活性受到明显的拮抗。这些结果说明卵磷脂很可能是Fengycins在膜上的作用靶标。此外,研究还发现Fengycins能够抑制串珠镰刀菌分泌的磷脂酶A2的活性,该性质很可能也在Fengycins的抑菌活性中起到了一定作用。     

Method for purification of large cyanogen bromide peptides by carboxymethyl cellulose chromatography in 8 m urea: Application to the purification of cyanogen bromide peptides from staphylcoccal enterotoxin A,phosphoglycerate kinase,and glucose-6-phosphate dehydrogenase

A method for purification of large cyanogen bromide peptides from proteins by means of carboxymethyl cellulose chromatography in the presence of 8 m urea is described. Chromatography of a number of large cyanogen bromide peptides which could not be separated by gel filtration showed that the resolution of the system was sufficient to enable large cyanogen bromide peptides to be separated from one another. The use of this method to purify cyanogen bromide peptides of a protein as a first step is also discussed.     

Degenerate T-cell recognition of peptides on MHC molecules creates large holes in the T-cell repertoire

The cellular immune system screens peptides presented by host cells on MHC molecules to assess if the cells are infected. In this study we examined whether the presented peptides contain enough information for a proper self/nonself assessment by comparing the presented human (self) and bacterial or viral (nonself) peptides on a large number of MHC molecules. For all MHC molecules tested, only a small fraction of the presented nonself peptides from 174 species of bacteria and 1000 viral proteomes ([Formula: see text]0.2%) is shown to be identical to a presented self peptide. Next, we use available data on T-cell receptor-peptide-MHC interactions to estimate how well T-cells distinguish between similar peptides. The recognition of a peptide-MHC by the T-cell receptor is flexible, and as a result, about one-third of the presented nonself peptides is expected to be indistinguishable (by T-cells) from presented self peptides. This suggests that T-cells are expected to remain tolerant for a large fraction of the presented nonself peptides, which provides an explanation for the "holes in the T-cell repertoire" that are found for a large fraction of foreign epitopes. Additionally, this overlap with self increases the need for efficient self tolerance, as many self-similar nonself peptides could initiate an autoimmune response. Degenerate recognition of peptide-MHC-I complexes by T-cells thus creates large and potentially dangerous overlaps between self and nonself.     

Integrating phylogeny,geographic niche partitioning and secondary metabolite synthesis in bloom-forming Planktothrix

Toxic freshwater cyanobacteria form harmful algal blooms that can cause acute toxicity to humans and livestock. Globally distributed, bloom-forming cyanobacteria Planktothrix either retain or lose the mcy gene cluster (encoding the synthesis of the secondary metabolite hepatotoxin microcystin or MC), resulting in a variable spatial/temporal distribution of (non)toxic genotypes. Despite their importance to human well-being, such genotype diversity is not being mapped at scales relevant to nature. We aimed to reveal the factors influencing the dispersal of those genotypes by analyzing 138 strains (from Europe, Russia, North America and East Africa) for their (i) mcy gene cluster composition, (ii) phylogeny and adaptation to their habitat and (iii) ribosomally and nonribosomally synthesized oligopeptide products. Although all the strains from different species contained at least remnants of the mcy gene cluster, various phylogenetic lineages evolved and adapted to rather specific ecological niches (for example, through pigmentation and gas vesicle protein size). No evidence for an increased abundance of specific peptides in the absence of MC was found. MC and peptide distribution rather depended on phylogeny, ecophysiological adaptation and geographic distance. Together, these findings provide evidence that MC and peptide production are primarily related to speciation processes, while within a phylogenetic lineage the probability that strains differ in peptide composition increases with geographic distance.     

Bacteriocins from plant pathogenic bacteria

Many bacteria produce antimicrobial substances such as nonribosomally synthesized antibiotics and ribosomally synthesized proteinaceous compounds referred to as bacteriocins. Secretion of antimicrobials is generally thought to contribute to the competitiveness of the producing organism, but there are indications that these compounds in some cases may have regulatory roles too. Bacteriocins most often act on closely related species only and are thus of interest for application as targeted narrow-spectrum antimicrobials with few side effects. Although the application of bacteriocins in plant disease control is an attractive option, very little is known about the occurrence and roles of these compounds in plant pathogenic bacteria and their natural competitors occurring in the same biotopes. This study presents an overview of current knowledge of bacteriocins from plant pathogenic bacteria.     

Functional analysis of fengycin synthetase FenD

Fengycin is a cyclic lipopeptidic antibiotic produced nonribosomally by Bacillus subtilis. A fengycin synthetase mutant of B. subtilis F29-3 was generated with Tn917lux, which contains a transposon inserted in a 7716-bp gene, fenD. The mutation can be genetically complemented by transforming a plasmid carrying a wild-type fenD, confirming the participation of the gene in fengycin synthesis. Sequencing and biochemical analysis reveal that this gene encodes an enzyme that includes two amino acid-activating modules, FenD1 and FenD2, which activate l-Tyr and l-Thr, the third and the fourth amino acids in fengycin, respectively.     

Required structure of cationic peptide for oligonucleotide-binding and -delivering into cells.

Improvement of the methods for oligonucleotide delivery into cells is necessary for the development of antisense therapy. In the present work, a new strategy for oligonucleotide delivery into cells was tested using cationic peptides as a vector. At first, to understand what structure of the peptide is required for binding with an oligonucleotide, several kinds of alpha-helical and non-alpha-helical peptides containing cationic amino acids were employed. As a result, the amphiphilic alpha-helix peptides were best for binding with the oligonucleotide, and the long chain length and large hydrophobic region in the amphiphilic structure of the peptide were necessary for the binding and forming of aggregates with the oligonucleotide. In the case of non-alpha-helical peptides, no significant binding ability was observed even if their chain lengths and number of cationic amino acid residues were equal to those of the alpha-helical peptides. The remarkable ability of oligonucleotide delivery into COS-7 cells was observed in the alpha-helical peptides with a long chain length and large hydrophobic region in the amphiphilic structure, but was not observed in the non-alpha-helical peptides. It is considered that such alpha-helical peptides could form optimum aggregates with the ODN for uptake into cells. Based on these results, the alpha-helical peptide with a long chain length and large hydrophobic region is applicable as a vector for the delivery of oligonucleotides into cells.     

Anti-obesity and anti-tumor pro-apoptotic peptides are sufficient to cause release of cytochrome c from vesicles

Peptides that target tissue for an apoptotic death have potential as therapeutics in a variety of disease conditions. The class of peptides described herein enters the cell through a specific receptor-mediated interaction. Once inside the cell, the peptide migrates toward the mitochondria, where the membrane barrier is disrupted. These experiments demonstrate that upon treatment with these short peptides large unilamellar vesicles are not lysed, a graded mode of leakage is observed and the transient pores formed by these peptides are large enough to release entrapped cytochrome c from the vesicles.     

δ-(<Emphasis Type="SmallCaps">l</Emphasis>-α-aminoadipyl)-<Emphasis Type="SmallCaps">l</Emphasis>-cysteinyl-<Emphasis Type="SmallCaps">d</Emphasis>-valine synthetase (ACVS): discovery and perspectives

The δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV) tripeptide is the first dedicated intermediate in the biosynthetic pathway leading to the penicillin and cephalosporin classes of β-lactam natural products in bacteria and fungi. It is synthesized nonribosomally by the ACV synthetase (ACVS) enzyme, which has been purified and partially characterized from many sources. Due to its large size and instability, many details regarding the reaction mechanism of ACVS are still not fully understood. In this review we discuss the chronology and associated methodology that led to the discovery of ACVS, some of the main findings regarding its activities, and some recent/current studies being conducted on the enzyme. In addition, we conclude with perspectives on what can be done to increase our understating of this very important protein in the future.     

Phosphorylation of polyomavirus large T antigen: effects of viral mutations and cell growth state.

Phosphorylation is responsible for the shift in electrophoretic mobility of polyomavirus large T antigen observed in pulse-chase or continuous-labeling experiments. Phosphorylated forms migrated more slowly than newly synthesized [35S]methionine large T antigen, and alkaline phosphatase treatment reversed the mobility shift. Analysis of phosphopeptides with Staphylococcus aureus V8 protease showed that large T antigen forms of intermediate mobility were enriched in peptides 1 to 4, 8, and 9, while the slower migrating species had all nine phosphopeptides, including peptides 5 and 7. The phosphorylations represented by phosphopeptides 5 and 7 were of particular interest. These phosphopeptides were entirely lacking in large T antigen from tsa mutants such as ts616 labeled at the nonpermissive temperature. Also, the phosphorylation of peptides 5 and 7 depends on the growth state of the cell. Early in infection of quiescent cells intermediate mobility forms of large T antigen with little or no phosphorylation, particularly of peptides 5 and 7, were seen, whereas peptides 5 and 7 were well represented at the same time in patterns from growing cells. Later in infection of growth-arrested cells, these phosphorylations were observed, suggesting that infection stimulates the relevant kinase. Because large T antigen of hrt mutants, which lack middle and small T antigens, showed phosphorylation of peptides 5 and 7, large T antigen was apparently responsible for the stimulation. Because some differences in the distribution of phosphopeptides were noted between hrt mutants and the wild type, middle T antigen, small T antigen, or both may play a modulating role in large T antigen phosphorylation.     

Transposon mutagenesis and cloning of the genes encoding the enzymes of fengycin biosynthesis inBacillus subtilis

A total of 20Bacillus subtilis F29-3 mutants defective in fengycin biosynthesis was obtained by Tn917 mutagenesis. Cloning and mapping results showed that the transposon in these mutants was inserted in eleven different locations on the chromosome. We were able to use the chromosomal sequence adjacent to the transposon as a probe to screen for cosmid clones containing the fengycin biosynthesis genes. One of the clones obtained, pFC660, was 46 kb long. Eight transposon insertion sites were mapped within this plasmid. Among the eleven different mutants analyzed, four mutants had Tn917 inserted in regions which encoded peptide sequences similar to part of gramicidin S synthetase, surfactin synthetase, and tyrocidine synthetase. Our results suggest that fengycin is synthesized nonribosomally by the multienzyme thiotemplate mechanism.