Sylvie Rebuffat

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Structural changes of region 1-16 of the Alzheimer disease amyloid beta-peptide upon zinc binding and in vitro aging.

Amyloid deposits within the cerebral tissue constitute a characteristic lesion associated with Alzheimer disease. They mainly consist of the amyloid peptide Aβ and display an abnormal content in Zn2+ ions, together with many truncated, isomerized, and racemized forms of Aβ. The region 1-16 of Aβ can be considered the minimal zinc-binding domain and contains two aspartates subject to protein aging. The influence of zinc binding and protein aging related modifications on the conformation of this region of Aβ is of importance given the potentiality of this domain to constitute a therapeutic target, especially for immunization approaches. In this study, we determined from NMR data the solution structure of the Aβ-(1-16)-Zn2+ complex in aqueous solution at pH 6.5. The residues His6, His13, and His14 and the Glu11 carboxylate were identified as ligands that tetrahedrally coordinate the Zn(II) cation. In vitro aging experiments on Aβ-(1-16) led to the formation of truncated and isomerized species. The major isomer generated, Aβ-(1-16)-l-iso-Asp7, displayed a local conformational change in the His6-Ser8 region but kept a zinc binding propensity via a coordination mode involving l-iso-Asp7. These results are discussed here with regard to Aβ fibrillogenesis and the potentiality of the region 1-16 of Aβ to be used as a therapeutic target.

Microcins, gene-encoded antibacterial peptides from enterobacteria.

Microcins are gene-encoded antibacterial peptides, with molecular masses below 10 kDa, produced by enterobacteria. They are secreted under conditions of nutrient depletion and exert potent antibacterial activity against closely related species. Typical gene clusters encoding the microcin precursor, the self-immunity factor, the secretion proteins and frequently the post-translational modification enzymes are located either on plasmids or on the chromosome. In contrast to most of the antibiotics of microbial origin, which are non-ribosomally synthesized by multimodular enzymes termed peptide synthetases, microcins are ribosomally synthesized as precursors, which are further modified enzymatically. They form a restricted class of potent antibacterial peptides. Fourteen microcins have been reported so far, among which only seven have been isolated and characterized. Despite the low number of known representatives, microcins exhibit a diversity of structures and antibacterial mechanisms. This review provides an updated overview of microcin structures, antibacterial activities, genetic systems and biosyntheses, as well as of their mechanisms of action.

Isolation and Structural Characterization of Capistruin, a Lasso Peptide Predicted from the Genome Sequence of Burkholderia thailandensis E264

Lasso peptides are a structurally unique class of bioactive peptides characterized by a knotted arrangement, where the C-terminus threads through an N-terminal macrolactam ring. Although ribosomally synthesized, only the gene cluster for the best studied lasso peptide MccJ25 from Escherichia coli consisting of the precursor protein McjA and the processing and immunity proteins McjB, McjC, and McjD is known. Through genome mining studies, we have identified homologues of all four proteins in Burkholderia thailandensis E264 and predicted this strain to produce a lasso peptide. Here we report the successful isolation of the predicted peptide, named capistruin. Upon optimization of the fermentation conditions, mass spectrometric and NMR structural studies proved capistruin to adopt a novel lasso fold. Heterologous production of the lasso peptide in Escherichia coli showed that the identified genes are sufficient for the biosynthesis of capistruin, which exhibits antimicrobial activity against closely related Burkholderia and Pseudomonas strains. In general, our rational approach should be widely applicable for the isolation of new lasso peptides to explore their high structural stability and diverse biological activity.

Isolation and Characterization of Environmental Bacteria Capable of Extracellular Biosorption of Mercury

ABSTRACT Accumulation of toxic metals in the environment represents a public health and wildlife concern. Bacteria resistant to toxic metals constitute an attractive biomass for the development of systems to decontaminate soils, sediments, or waters. In particular, biosorption of metals within the bacterial cell wall or secreted extracellular polymeric substances (EPS) is an emerging process for the bioremediation of contaminated water. Here the isolation of bacteria from soil, effluents, and river sediments contaminated with toxic metals permitted the selection of seven bacterial isolates tolerant to mercury and associated with a mucoid phenotype indicative of the production of EPS. Inductively coupled plasma-optical emission spectroscopy and transmission electron microscopy in conjunction with X-ray energy dispersive spectrometry revealed that bacteria incubated in the presence of HgCl2 sequestered mercury extracellularly as spherical or amorphous deposits. Killed bacterial biomass incubated in the presence of HgCl2 also generated spherical extracellular mercury deposits, with a sequestration capacity (40 to 120 mg mercury per g [dry weight] of biomass) superior to that of live bacteria (1 to 2 mg mercury per g [dry weight] of biomass). The seven strains were shown to produce EPS, which were characterized by Fourier transform-infrared (FT-IR) spectroscopy and chemical analysis of neutral-carbohydrate, uronic acid, and protein contents. The results highlight the high potential of Hg-tolerant bacteria for applications in the bioremediation of mercury through biosorption onto the biomass surface or secreted EPS.

Siderophore peptide, a new type of post-translationally modified antibacterial peptide with potent activity

Microcin E492 (MccE492, 7886 Da), the 84-amino acid antimicrobial peptide from Klebsiella pneumoniae, was purified in a post-translationally modified form, MccE492m (8717 Da), from culture supernatants of either the recombinant Escherichia coli VCS257 strain harboring the pJAM229 plasmid or the K. pneumoniae RYC492 strain. Chymotrypsin digestion of MccE492m led to the MccE492m-(74–84) C-terminal fragment that carries the modification and that was analyzed by mass spectrometry and nuclear magnetic resonance at natural abundance. The 831-Da post-translational modification consists of a trimer of N-(2,3-dihydroxybenzoyl)-l-serine linked via a C-glycosidic linkage to a β-d-glucose moiety, itself linked to the MccE492m Ser-84-carboxyl through an O-glycosidic bond. This modification, which mimics a catechol-type siderophore, was shown to bind ferric ions by analysis of the collision-induced dissociation pattern obtained for MccE492m-(74–84) by electrospray ion trap mass spectrometry experiments in the presence of FeCl3. By using a series of wild-type and mutant isogenic strains, the three catechol-type siderophore receptors Fiu, Cir, and FepA were shown to be responsible for the recognition of MccE492m at the outer membrane of sensitive bacteria. Because MccE492m shows a broader spectrum of antibacterial activity and is more potent than MccE492, we propose that by increasing the microcin/receptor affinity, the modification leads to a better recognition and subsequently to a higher antimicrobial activity of the microcin. Therefore, MccE492m is the first member of a new class of antimicrobial peptides carrying a siderophore-like post-translational modification and showing potent activity, which we term siderophore-peptides.

Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state

Significance ATP-binding cassette (ABC) exporters transport substrates by an alternating access mechanism that is driven by ATP binding and hydrolysis. The general mechanism is a motion from an inward to an outward state, with a different intertwining of the half-transporters in both states. In this study we determined the function and crystal structure of the ABC exporter McjD that exports the antibacterial peptide microcin J25. Our structure represents a novel nucleotide-bound, outward-occluded state. It does not possess subunit intertwining and shows a well-defined binding cavity that is closed to all sides, consistent with it being an intermediate between the inward- and outward-facing state. Our structure provides valuable insights in a transition state of an ABC exporter. Enterobacteriaceae produce antimicrobial peptides for survival under nutrient starvation. Microcin J25 (MccJ25) is an antimicrobial peptide with a unique lasso topology. It is secreted by the ATP-binding cassette (ABC) exporter McjD, which ensures self-immunity of the producing strain through efficient export of the toxic mature peptide from the cell. Here we have determined the crystal structure of McjD from Escherichia coli at 2.7-Å resolution, which is to the authors’ knowledge the first structure of an antibacterial peptide ABC transporter. Our functional and biochemical analyses demonstrate McjD-dependent immunity to MccJ25 through efflux of the peptide. McjD can directly bind MccJ25 and displays a basal ATPase activity that is stimulated by MccJ25 in both detergent solution and proteoliposomes. McjD adopts a new conformation, termed nucleotide-bound outward occluded. The new conformation defines a clear cavity; mutagenesis and ligand binding studies of the cavity have identified Phe86, Asn134, and Asn302 as important for recognition of MccJ25. Comparisons with the inward-open MsbA and outward-open Sav1866 structures show that McjD has structural similarities with both states without the intertwining of transmembrane (TM) helices. The occluded state is formed by rotation of TMs 1 and 2 toward the equivalent TMs of the opposite monomer, unlike Sav1866 where they intertwine with TMs 3–6 of the opposite monomer. Cysteine cross-linking studies on the McjD dimer in inside-out membrane vesicles of E. coli confirmed the presence of the occluded state. We therefore propose that the outward-occluded state represents a transition intermediate between the outward-open and inward-open conformation of ABC exporters.

The iron–siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11–Pro16 β-hairpin region in the recognition mechanism

The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA(-) strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a K(d) of 1.2 microM. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand-receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val11-Pro16 beta-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.

Structural and functional diversity of microcins, gene-encoded antibacterial peptides from enterobacteria.

Microcins are a peculiar class of gene-encoded low-molecular-mass antibacterial peptides secreted by enterobacteria. They contribute to the regulation of microbial competitions within the intestinal microbiota. The genetic systems involved in microcin biosynthesis share a conserved organization. Similar to bacteriocins of Gram-positive bacteria, microcins exert potent antibacterial activity directed against phylogenetically-related bacterial strains, with minimal inhibitory concentrations in the nanomolar range. In contrast to bacteriocins, they display a great structural diversity among the few representatives well characterized until now, that makes difficult the description of microcin subclasses. This review focuses on three microcins, MccE492m that carries a C-terminal posttranslational modification containing a catechol-type siderophore, MccJ25, a cyclic peptide with a unique ‘lasso-type’ structure and MccC7 or C51, with a common N-formylated heptapeptide-nucleotide structure. We show these microcins exhibit ‘Trojan horse’ mechanisms of antibacterial activity: either (i) the microcin structure is a mime of an essential element, permitting its recognition by outer membrane receptors used for vital functions in bacteria and further translocation into the periplasmic space, or (ii) it is secreted as a harmless molecule and further processed in susceptible bacteria to form the toxic entity. When inside target bacteria, microcins bind essential enzymes or interact with the inner membrane to form a bacterial killing structure.

Prokaryotic Antimicrobial Peptides

Prokaryotic antimicrobial peptides , Prokaryotic antimicrobial peptides , کتابخانه مرکزی دانشگاه علوم پزشکی تهران