Jon Nissen-Meyer

Hipposin, a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.)

A novel 51-residue antimicrobial peptide (AMP) from the skin mucus of Atlantic halibut (Hippoglossus hippoglossus L.) was isolated using acid extraction, and cationic exchange and reversed phase chromatography. The complete amino acid sequence of the AMP, termed hipposin, was determined by automated Edman degradation and mass spectrometry to be SGRGKTGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAHRVGAGAPVYL. The N-terminal amino group was acetylated. The theoretical mass of hipposin was calculated to be 5458.4 Da, which was in good agreement with the mass of 5459 Da determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Hipposin was shown to be derived from histone H2A by PCR amplifying the encoding sequences from Atlantic halibut genomic DNA. The peptide showed sequence similarity with the 39-mer AMP buforin I of Asian toad and the 19-mer AMP parasin I of catfish. Fifty of the fifty-one residues in hipposin were identical to the N-terminal region of histone H2A from rainbow trout. Hipposin showed strong antimicrobial activity against several Gram-positive and Gram-negative bacteria and activity could be detected down to hipposin concentrations of 0.3 microM (1.6 microg/ml). Hipposin without N-terminal acetylation was prepared by solid-phase peptide synthesis and shown to have the same antimicrobial activity as the natural acetylated peptide. Thus, hipposin is a new broad-spectrum histone-derived AMP found in the skin mucus of Atlantic halibut.

The Two-Peptide Class II Bacteriocins: Structure, Production, and Mode of Action

The two-peptide class II bacteriocins consist of two different unmodified peptides, both of which must be present in about equal amounts in order for these bacteriocins to exert optimal antimicrobial activity. These bacteriocins render the membrane of target cells permeable to various small molecules. The genes encoding the two peptides of two-peptide bacteriocins are adjacent to each other in the same operon and they are near the genes encoding (i) the immunity protein that protects the bacteriocin-producing bacteria from being killed by their own bacteriocin, (ii) a dedicated ABC transporter that transports the bacteriocin out of the bacteriocin-producing bacteria, and (iii) an accessory protein whose specific role is not known, but which also appears to be required for secretion of the bacteriocin. The production of some two-peptide bacteriocins is transcriptionally regulated through a three-component regulatory system that consists of a membrane-interacting peptide pheromone, a membrane-associated histidine protein kinase, and response regulators. Structure analysis of three two-peptide bacteriocins (plantaricin E/F, plantaricin J/K, and lactococcin G) by CD (and in part by NMR) spectroscopy reveal that these bacteriocins contain long amphiphilic α-helical stretches and that the two complementary peptides interact and structure each other when exposed to membrane-like entities. Lactococcin G shares about 55% sequence identity with enterocin 1071, but these two bacteriocins nevertheless kill different types of bacteria. The target-cell specificity of lactococcin G-enterocin 1071 hybrid bacteriocins that have been constructed by site-directed mutagenesis suggests that the β-peptide is important for determining the target-cell specificity.

Structure and Mode-of-Action of the Two-Peptide (Class-IIb) Bacteriocins

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15–30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure–function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix–helix structure involving helix–helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix–helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death.

Proline Conformation-Dependent Antimicrobial Activity of a Proline-Rich Histone H1 N-Terminal Peptide Fragment Isolated from the Skin Mucus of Atlantic Salmon

ABSTRACT A 30-residue N-terminally acetylated peptide derived from the N-terminal part of histone H1 was identified as the dominant antimicrobial peptide in skin mucus from Atlantic salmon (Salmo salar). The peptide (termed salmon antimicrobial peptide [SAMP H1]) was purified to homogeneity by a combination of reversed-phase and cation-exchange chromatographies. By Edman degradation of the deacetylated peptide and by sequencing of the PCR-amplified DNA that encodes the peptide, the complete amino acid sequence was determined to be AEVAPAPAAAAPAKAPKKKAAAKPKKAGPS. The theoretical molecular weight of N-terminally acetylated SAMP H1 was calculated to be 2,836, which is the same as that determined by matrix-assisted laser desorption ionization mass spectrometry. The peptide was active against both gram-negative and -positive bacteria. The N-terminal acetyl group was not necessary for activity since deacetylation did not reduce the activity. A synthetic peptide whose sequence was identical to that of the isolated fragment was initially inactive but could be activated by binding it to a cation-exchange column. Treatment of the synthetic peptide when it was bound to the exchange column with peptidylproline cis-trans-isomerase increased the amount of active peptide, indicating that isomerization of the proline peptide bond(s) was necessary for activation of the synthetic peptide. Comparison of the active and inactive forms by circular dichroism and chromatographic analyses suggests that the active form, both the natural and the synthetic forms, is more structured, condensed, and rigid than the inactive form, which has a more nonstructured conformation. This work shows for the first time the importance of proline isomers in the activity of an antimicrobial peptide.

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin lactococcin G

The three-dimensional structures of the two peptides, lactococcin G-alpha (LcnG-alpha; contains 39 residues) and lactococcin G-beta (LcnG-beta, contains 35 residues), that constitute the two-peptide bacteriocin lactococcin G (LcnG) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles and TFE. In DPC, LcnG-alpha has an N-terminal alpha-helix (residues 3-21) that contains a GxxxG helix-helix interaction motif (residues 7-11) and a less well defined C-terminal alpha-helix (residues 24-34), and in between (residues 18-22) there is a second somewhat flexible GxxxG-motif. Its structure in TFE was similar. In DPC, LcnG-beta has an N-terminal alpha-helix (residues 6-19). The region from residues 20 to 35, which also contains a flexible GxxxG-motif (residues 18-22), appeared to be fairly unstructured in DPC. In the presence of TFE, however, the region between and including residues 23 and 32 formed a well defined alpha-helix. The N-terminal helix between and including residues 6 and 19 seen in the presence of DPC, was broken at residues 8 and 9 in the presence of TFE. The N-terminal helices, both in LcnG-alpha and -beta, are amphiphilic. We postulate that LcnG-alpha and -beta have a parallel orientation and interact through helix-helix interactions involving the first GxxxG (residues 7-11) motif in LcnG-alpha and the one (residues 18-22) in LcnG-beta, and that they thus lie in a staggered fashion relative to each other.

Structure and Mode of Action of the Membrane-permeabilizing Antimicrobial Peptide Pheromone Plantaricin A

The three-dimensional structure in dodecyl phosphocholine micelles of the 26-mer membrane-permeabilizing bacteriocin-like pheromone plantaricin A (PlnA) has been determined by use of nuclear magnetic resonance spectroscopy. The peptide was unstructured in water but became partly structured upon exposure to micelles. An amphiphilic α-helix stretching from residue 12 to 21 (possibly also including residues 22 and 23) was then formed in the C-terminal part of the peptide, whereas the N-terminal part remained largely unstructured. PlnA exerted its membrane-permeabilizing antimicrobial activity through a nonchiral interaction with the target cell membrane because the d-enantiomeric form had the same activity as the natural l-form. This nonchiral interaction involved the amphiphilic α-helical region in the C-terminal half of PlnA because a 17-mer fragment that contains the amphiphilic α-helical part of the peptide had antimicrobial potency that was similar to that of the l- and d-enantiomeric forms of PlnA. Also the pheromone activity of PlnA depended on this nonchiral interaction because both the l- and d-enantiomeric forms of the 17-mer fragment inhibited the pheromone activity. The pheromone activity also involved, however, a chiral interaction between the N-terminal part of PlnA and its receptor because high concentrations of the l-form (but not the d-form) of a 5-mer fragment derived from the N-terminal part of PlnA had pheromone activity. The results thus reveal a novel mechanism whereby peptide pheromones such as PlnA may function. An initial nonchiral interaction with membrane lipids induces α-helical structuring in a segment of the peptide pheromone. The peptide becomes thereby sufficiently structured and properly positioned in the membrane interface, thus enabling it to engage in a chiral interaction with its receptor in or near the membrane water interface. This membrane-interacting mode of action explains why some peptide pheromones/hormones such as PlnA sometimes display antimicrobial activity in addition to their pheromone activity.

Sensitivity to the two-peptide bacteriocin lactococcin G is dependent on UppP, an enzyme involved in cell-wall synthesis

Most bacterially produced antimicrobial peptides (bacteriocins) are thought to kill target cells by a receptor‐mediated mechanism. However, for most bacteriocins the receptor is unknown. For instance, no target receptor has been identified for the two‐peptide bacteriocins (class IIb), whose activity requires the combined action of two individual peptides. To identify the receptor for the class IIb bacteriocin lactococcin G, which targets strains of Lactococcus lactis, we generated 12 lactococcin G‐resistant mutants and performed whole‐genome sequencing to identify mutations causing the resistant phenotype. Remarkably, all had a mutation in or near the gene uppP (bacA), encoding an undecaprenyl pyrophosphate phosphatase; a membrane protein involved in peptidoglycan synthesis. Nine mutants had stop codons or frameshifts in the uppP gene, two had point mutations in putative regulatory regions and one caused an amino acid substitution in UppP. To verify the receptor function of UppP, it was shown that growth of non‐sensitive Streptococcus pneumoniae could be inhibited by lactococcin G when L. lactisu2005uppP was expressed in this bacterium. Furthermore, we show that the related class IIb bacteriocin enterocin 1071 also uses UppP as receptor. The approach used here should be broadly applicable to identify receptors for other bacteriocins as well.

Mutational analysis of putative helix-helix interacting GxxxG-motifs and tryptophan residues in the two-peptide bacteriocin lactococcin G.

The membrane-permeabilizing two-peptide bacteriocin lactococcin G consists of two different peptides, LcnG-alpha and LcnG-beta. The bacteriocin contains several tryptophan and tyrosine residues and three putative helix-helix interacting GxxxG-motifs, G 7xxxG 11 and G 18xxxG 22 in LcnG-alpha and G 18xxxG 22 in LcnG-beta. The tryptophan and tyrosine residues and residues in the GxxxG-motifs were altered by site-directed mutagenesis to analyze the structure and membrane-orientation of lactococcin G. Substituting the glycine residues at position 7 or 11 in the G 7xxxG 11-motif in LcnG-alpha with large hydrophobic or hydrophilic residues was highly detrimental, whereas small residues were tolerated. Qualitatively similar results were obtained for the G 18xxxG 22-motif in LcnG-beta. In contrast, replacement of the glycine residues in the middle of these two motifs with large hydrophilic residues was tolerated. All mutations in the G 18xxxG 22-motif in LcnG-alpha were relatively well-tolerated, indicating that this motif is not involved in helix-helix interactions. The four aromatic residues in the N-terminal part of LcnG-beta could individually be replaced by other aromatic residues, a hydrophilic positive residue, and a hydrophobic residue without a marked reduced activity, indicating that this region is structurally flexible and not embedded in a strictly hydrophobic or hydrophilic environment. The results are in accordance with a structural model where the G 7xxxG 11-motif in LcnG-alpha and the G 18xxxG 22-motif in LcnG-beta interact and allow the two peptides to form a parallel transmembrane helix-helix structure, with the tryptophan-rich N-terminal part of LcnG-beta positioned in the outer membrane interface and the cationic C-terminal end of LcnG-alpha inside the cell.

1.6-A Crystal Structure of Enta-Im: A Bacterial Immunity Protein Conferring Immunity to the Antimicrobial Activity of the Pediocin-Like Bacteriocin Enterocin A

Many Gram-positive bacteria produce ribosomally synthesized antimicrobial peptides, often termed bacteriocins. Genes encoding pediocin-like bacteriocins are generally cotranscribed with or in close vicinity to a gene encoding a cognate immunity protein that protects the bacteriocin-producer from their own bacteriocin. We present the first crystal structure of a pediocin-like immunity protein, EntA-im, conferring immunity to the bacteriocin enterocin A. Determination of the structure of this 103-amino acid protein revealed that it folds into an antiparallel four-helix bundle with a flexible C-terminal part. The fact that the immunity protein conferring immunity to carnobacteriocin B2 also consists of a four-helix bundle (Sprules, T., Kawulka, K. E., and Vederas, J. C. (2004) Biochemistry 43, 11740–11749) strongly indicates that this is a conserved structural motif in all pediocin-like immunity proteins. The C-terminal half of the immunity protein contains a region that recognizes the C-terminal half of the cognate bacteriocin, and the flexibility in the C-terminal end of the immunity protein might thus be an important characteristic that enables the immunity protein to interact with its cognate bacteriocin. By homology modeling of three other pediocin-like immunity proteins and calculation of the surface charge distribution for EntA-im and the three structure models, different charge distributions were observed. The differences in the latter part of helix 3, the beginning of helix 4, and the loop connecting these helices might also be of importance in determining the specificity.

Structure-Function Analysis of Immunity Proteins of Pediocin-Like Bacteriocins: C-Terminal Parts of Immunity Proteins Are Involved in Specific Recognition of Cognate Bacteriocins

ABSTRACT The immunity proteins of pediocin-like bacteriocins show a high degree of specificity with respect to the pediocin-like bacteriocin they recognize and confer immunity to. The aim of this study was to identify regions of the immunity proteins that are involved in this specific recognition. Six different hybrid immunity proteins were constructed from three different pediocin-like bacteriocin immunity proteins that have similar sequences but confer resistance to different bacteriocins. These hybrid immunity proteins were then tested for their ability to confer immunity to various pediocin-like bacteriocins. The specificities of the hybrid immunity proteins proved to be similar to those of the immunity proteins from which the C-terminal halves were derived, thus revealing that the C-terminal half of immunity proteins for pediocin-like bacteriocins contains a domain that is involved in specific recognition of the bacteriocins they confer immunity to. Moreover, the results also revealed that the effectiveness of an immunity protein is strain dependent and that its functionality thus depends in part on interplay with strain-dependent factors. To further investigate the structure-function relationship of these immunity proteins, the enterocin A and leucocin A immunity proteins (EntA-im and LeuA-im) were purified to homogeneity and structurally analyzed under various conditions by Circular dichroism (CD) spectroscopy. The results revealed that both immunity proteins are α-helical and well structured in an aqueous environment, the denaturing temperature being 78.5°C for EntA-im and 58.0°C for LeuA-im. The CD spectra also revealed that there was no further increase in the structuring or α-helical content when the immunity proteins were exposed to dodecylphosphocholine micelles or dioleoyl-l-α-phosphatidyl-dl-glycerol (DOPG) liposomes, indicating that the immunity proteins, in contrast to the bacteriocins, do not interact extensively with membranes. They may nevertheless be loosely associated with the membrane, possibly as peripheral membrane proteins, thus enabling them to interact with their cognate bacteriocin.