Identification and molecular characterization of an N-acetylmuramyl-L-alanine amidase Sle1 involved in cell separation of Staphylococcus aureus

We purified a peptidoglycan hydrolase involved in cell separation from a Staphylococcus aureus atl null mutant and identified its gene. Characterization of the gene product shows a 32 kDa N-acetylmuramyl-L-alanine amidase that we designated Sle1. Analysis of peptidoglycan digests showed Sle1 preferentially cleaved N-acetylmuramyl-L-Ala bonds in dimeric cross-bridges that interlink the two murein strands in the peptidoglycan. An insertion mutation of sle1 impaired cell separation and induced S. aureus to form clusters suggesting Sle1 is involved in cell separation of S. aureus. The Sle1 mutant revealed a significant decrease in pathogenesis using an acute infection mouse model. Atl is the major autolysin of S. aureus, which has been implicated in cell separation of S. aureus. Generation of an atl/sle1 double mutant revealed that the mutant cell separation was heavily impaired suggesting that S. aureus uses two peptidoglycan hydrolases, Atl and Sle1, for cell separation. Unlike Atl, Sle1 is not directly involved in autolysis of S. aureus.     

Characterization and functional analysis of atl, a novel gene encoding autolysin in Streptococcus suis

Streptococcus suis serotype 2 (S. suis 2) is an important swine and human pathogen responsible for septicemia and meningitis. A novel gene, designated atl and encoding a major autolysin of S. suis 2 virulent strain HA9801, was identified and characterized in this study. The Atl protein contains 1,025 amino acids with a predicted molecular mass of 113 kDa and has a conserved N-acetylmuramoyl-l-alanine amidase domain. Recombinant Atl was expressed in Escherichia coli, and its bacteriolytic and fibronectin-binding activities were confirmed by zymography and Western affinity blotting. Two bacteriolytic bands were shown in the sodium dodecyl sulfate extracts of HA9801, while both were absent from the atl inactivated mutant. Cell chains of the mutant strain became longer than that of the parental strain. In the autolysis assay, HA9801 decreased to 20% of the initial optical density (OD) value, while the mutant strain had almost no autolytic activity. The biofilm capacity of the atl mutant was reduced ～30% compared to the parental strain. In the zebrafish infection model, the 50% lethal dose of the mutant strain was increased up to 5-fold. Furthermore, the adherence to HEp-2 cells of the atl mutant was 50% less than that of the parental strain. Based on the functional analysis of the recombinant Atl and observed effects of atl inactivation on HA9801, we conclude that Atl is a major autolysin of HA9801. It takes part in cell autolysis, separation of daughter cells, biofilm formation, fibronectin-binding activity, cell adhesion, and pathogenesis of HA9801.     

Immune responses to the 18-kDa protein of Mycobacterium leprae. Similar B cell epitopes but different T cell epitopes seen by inbred strains of mice.

Antibody responses to the 18-kDa protein of Mycobacterium leprae have been analyzed in different strains of mice. High, intermediate, and low responder strains have been identified and these response patterns show clear linkage to genes encoded in the H-2 complex. Three peptides, residues 1-50, 51-100, and 101-148 have been synthesized, as well as a series of 20-mer peptides, which span the entire 18-kDa protein. Repeated immunization of different strains of mice with the 18-kDa protein resulted in IgG responses to epitopes found on all three synthetic peptides. Immunization of BALB/cJ and B10.BR mice, two high responder strains, with 18-kDa protein resulted in high levels of IgG antibody to epitopes found on peptides 1-20, 16-35, 31-50, 46-65, and 76-95. B10.BR mice also contained IgG that bound peptide 61-80 and BALB/cJ mice produced IgG that bound peptide 91-110. Although B10.BR mice produced IgG that bound the 50-mer peptide 101-148, this IgG was not detected by binding to peptides 91-110, 106-125, 121-140, and 131-148. Immunization of B10.BR mice with individual overlapping 20-mer peptides as Ag revealed that peptides 1-20, 16-35, 31-50, and 76-95 elicited high titers of IgG that bound both the immunizing peptide as well as 18-kDa protein. As these peptides induce antibody synthesis they must contain both B cell and T cell epitopes. By contrast, immunization of BALB/cJ mice with the same 20-mer peptides, all of which contain B cell epitopes for this strain, failed to elicit IgG responses with one exception. Peptide 91-110 induced IgG that bound peptide 91-110, but not the intact 18-kDa protein. We conclude that peptides 1-20, 16-35, 31-50, and 76-95 either lack T cell epitopes for BALB/cJ mice, or activate different T cell subpopulations in the two strains. We suggest that the induction of IgG responses to small peptide Ag is an in vivo assay of the activity of Th2 cell subpopulations.     

Influence of the N- and C-terminal regions of leu-lys rich antimicrobial peptide on antimicrobial activity

P5 (KWKKLLKKPLLKKLLKKL-NH(2)) is an antibacterial 18-mer Leu-Lys rich peptide from CA (1-8)-MA (1-12) hybrid peptide (CA-MA). Here we show that decreasing the net hydrophobicity and charge of CA-MA by deleting Leu- or Lys- of the N- or C-terminal regions of P5 (P10 or P11). The antimicrobial activity of the peptides was measured by their growth inhibitory effect upon S. aureus, B. subtilis, P. aeruginosa, S. typhimurium, E. coli, T. beigelii and C. albicans. Antimicrobial activity required a full length C-terminus. Confocal microscopy showed that P11 was located in the plasma membrane. In this study, P11, K(3)K(4)L(5)L(6)-deleted peptide, acted independent on the ionic environment. Furthermore, P11 causes significant morphological alterations of the fungal surfaces as shown by scanning electron microscopy.     

Phylogeny of the staphylococcal major autolysin and its use in genus and species typing

The major staphylococcal autolysin Atl is an important player in cell separation and daughter cell formation. In this study, we investigated the amino acid sequences of Atl proteins derived from 15 staphylococcal and 1 macrococcal species representatives. The overall organization of the bifunctional precursor protein consisting of the signal peptide, a propeptide (PP), the amidase (AM), six repeat sequences (R(1) to R(6)), and the glucosaminidase (GL) was highly conserved in all of the species. The most-conserved domains were the enzyme domains AM and GL; the least-conserved regions were the PP and R regions. An Atl-based phylogenetic tree for the various species representatives correlated well with the corresponding 16S rRNA-based tree and also perfectly matched the phylogenetic trees based on core genome analysis. The phylogenetic distance analysis of 18 AtlA proteins of various Staphylococcus aureus strains and 15 AtlE proteins of S. epidermidis revealed that both species representatives formed a relatively homogeneous cluster. Two S. epidermidis strains, M23864:W1 and VCU116, were identified by Atl typing that clustered far more distantly and belonged to either S. caprae and S. capitis or a new subspecies. Here we show that Atl typing is a useful tool for staphylococcal genus and species typing by using either the highly conserved AM domain or the less-conserved PP domain.     

Reconstruction of peptidyltransferase activity on 50S and 70S ribosomal particles by peptide fragments of protein L16

Ribosomal protein L16 was digested with Staphylococcus aureus protease V8 and the resulting peptides were separated by reversed-phase high-performance liquid chromatography. One of the fragments, identified by sequence analysis as the N-terminal peptide of L16, was shown to exhibit partial peptide-bond-formation and transesterification activities of peptidyltransferase upon reconstitution with L16-depleted 50S core particles. However, several proteins enhanced these activities. L15 increased both reactions when added to the reconstitution mixture, suggesting a limited capacity of the L16 peptide to incorporate into 50S core particles. In contrast, the interaction of L11 with the N-terminal peptide stimulated the transesterification reaction but not the peptide-bond-forming activity of ribosomes, indicating a different topological domain for these reactions. Also, EF-P, a soluble protein which reconstructs the peptide-bond formation and transesterification reactions on 70S ribosomes, stimulated both peptidyltransferase activities exhibited by the L16 N-terminal peptide.     

Inhibition of hemoglobin S polymerization in vitro by a novel 15-mer EF-helix beta73 histidine-containing peptide

Our mutational studies on Hb S showed that the Hb S beta73His variant (beta6Val and beta73His) promoted polymerization, while Hb S beta73Leu (beta6Val and beta73Leu) inhibited polymerization. On the basis of these results, we speculated that EF-helix peptides containing beta73His interact with beta4Thr in Hb S and compete with Hb S, resulting in inhibition of Hb S polymerization. We, therefore, studied inhibitory effects of 15-, 11-, 7-, and 3-mer EF-helix peptides containing beta73His on Hb S polymerization. The delay time prior to Hb S polymerization increased only in the presence of the 15-mer His peptide; the higher the amount, the longer the delay time. DIC image analysis also showed that the fiber elongation rate for Hb S polymers decreased with increasing concentration of the 15-mer His peptide. In contrast, the same 15-mer peptide containing beta73Leu instead of His and peptides shorter than 11 amino acids containing beta73His including His alone showed little effect on the kinetics of polymerization and elongation of polymers. Analysis by protein-chip arrays showed that only the 15-mer beta73His peptide interacted with Hb S. CD spectra of the 15-mer beta73His peptide did not show a specific helical structure; however, computer docking analysis suggested a lower energy for interaction of Hb S with the 15-mer beta73His peptide compared to peptides containing other amino acids at this position. These results suggest that the 15-mer beta73His peptide interacts with Hb S via the beta4Thr in the betaS-globin chain in Hb S. This interaction may influence hydrogen bond interaction between beta73Asp and beta4Thr in Hb S polymers and interfere in hydrophobic interactions of beta6Val, leading to inhibition of Hb S polymerization.     

Synthesis of peptide-oligonucleotide phosphorothioate conjugates by convergent or stepwise solid-phase strategies

A convergent strategy was employed to link eight 10-27-mer peptides to oligonucleotide phosphorothioates, resulting in twenty-six various conjugates. A stepwise synthesis strategy for the preparation of peptide-oligonucleotide phosphorothioate conjugates, employing Fmoc peptide chemistry, was developed and applied to the synthesis of four conjugates. Three of these conjugates contained either a 10 or 16-mer peptide, incorporating either 2 or 3 arginine residues, respectively.     

Interplay between PEO tether length and ligand spacing governs cell spreading on RGD-modified PMMA-g-PEO comb copolymers

The effects of tether length on cell adhesion to poly(methyl methacrylate)-graft-poly(ethylene oxide), PMMA-g-PEO, comb copolymer films functionalized with the adhesion peptide RGD were investigated. Copolymers having PEO tether lengths of 10 and 22 EO segments were synthesized and coupled with a synthetic peptide that contained both RGD and the synergy sequence PHSRN. Cell spreading assays revealed that the longer polymer tethers increased the rate of spreading and reduced the time required for fibroblasts to form focal adhesions. Fluorescence resonance energy transfer (FRET) measurements indicated a mean separation between integrin-bound peptides of 15.6 +/- 1.4 nm for combs with long (22-mer) tethers, compared with 17.5 +/- 1.3 nm for short (10-mer) tethers, on films of comparable peptide density (approximately 2500 peptides/microm2). The results suggest that the added mobility afforded by the more extensible tethers encouraged the formation of focal adhesions by allowing cells to reorganize tethered peptides on the nanometer length scale. In addition, adhesion peptides were selectively coupled to 10-mer or 22-mer PEO tethers within a bimodal brush to investigate stratification effects on cell adhesion. Peptides bound by short tethers in a bed of long unsubstituted chains resulted in surfaces that resisted, rather than promoted, cell adhesion. By contrast, when long peptide tethers were employed with short unsubstituted chains, cell attachment and spreading were comparable to that found on a monomodal brush of long chains at equivalent peptide density.     

SYNTHESIS OF PEPTIDE-OLIGONUCLEOTIDE PHOSPHOROTHIOATE CONJUGATES BY CONVERGENT OR STEPWISE SOLID-PHASE STRATEGIES

A convergent strategy was employed to link eight 10–27-mer peptides to oligonucleotide phosphorothioates, resulting in twenty-six various conjugates. A stepwise synthesis strategy for the preparation of peptide-oligonucleotide phosphorothioate conjugates, employing Fmoc peptide chemistry, was developed and applied to the synthesis of four conjugates. Three of these conjugates contained either a 10 or 16-mer peptide, incorporating either 2 or 3 arginine residues, respectively.     

Role of staphylococcal wall teichoic acid in targeting the major autolysin Atl

Staphylococcal cell separation depends largely on the bifunctional autolysin Atl that is processed to amidase‐R_1,2 and R₃‐glucosaminidase. These murein hydrolases are targeted via repeat domains (R) to the septal region of the cell surface, thereby allowing localized peptidoglycan hydrolysis and separation of the dividing cells. Here we show that targeting of the amidase repeats is based on an exclusion strategy mediated by wall teichoic acid (WTA). In Staphylococcus aureus wild‐type, externally applied repeats (R_1,2) or endogenously expressed amidase were localized exclusively at the cross‐wall region, while in ΔtagO mutant that lacks WTA binding was evenly distributed on the cell surface, which explains the increased fragility and autolysis susceptibility of the mutant. WTA prevented binding of Atl to the old cell wall but not to the cross‐wall region suggesting a lower WTA content. In binding studies with ConcanavalinA‐fluorescein (ConA‐FITC) conjugate that binds preferentially to teichoic acids, ConA‐FITC was bound throughout the cell surface with the exception of the cross wall. ConA binding suggest that either content or polymerization of WTA gradually increases with distance from the cross‐wall. By preventing binding of Atl, WTA directs Atl to the cross‐wall to perform the last step of cell division, namely separation of the daughter cells.     

Inhibition of Staphylococcus aureus pathogenesis in vitro and in vivo by RAP-binding peptides

Staphylococcus aureus cause many diseases by producing toxins, whose synthesis is regulated by quorum-sensing mechanisms. S. aureus secretes a protein termed RNAIII activating protein (RAP) which autoinduces toxin production via the phosphorylation of is target protein TRAP. Mice vaccinated with RAP were protected from S. aureus infection, suggesting that RAP is an useful target for selecting potential therapeutic molecules to inhibit S. aureus pathogenesis. We show here that RAP (native and recombinant) was used to select RAP-binding peptides (RBPs) from a random 12-mer phage-displayed peptide library. Two RBPs were shown to inhibit RNAIII production in vitro (used a marker for pathogenesis). The peptide WPFAHWPWQYPR, which had the strongest inhibitory activity, was chemically synthesized and also expressed in Escherichia coli as a GST-fusion. Both synthetic peptide and GST-fusion peptide decreased RNAIII levels in a dose-dependent manner. The GST-fusion peptide was also shown to protect mice from a S. aureus infection in vivo (tested in a murine cutaneous S. aureus infection model). Our results suggest the potential use of RAP-binding proteins in treating clinical S. aureus infections.     

Three-dimensional structure in lipid micelles of the pediocin-like antimicrobial peptide curvacin A

The 3D structure of the membrane-permeabilizing 41-mer pediocin-like antimicrobial peptide curvacin A produced by lactic acid bacteria has been studied by NMR spectroscopy. In DPC micelles, the cationic and hydrophilic N-terminal half of the peptide forms an S-shaped beta-sheet-like domain stabilized by a disulfide bridge and a few hydrogen bonds. This domain is followed by two alpha-helices: a hydrophilic 6-mer helix between residues 19 and 24 and an amphiphilic/hydrophobic 11-mer helix between residues 29 and 39. There are two hinges in the peptide, one at residues 16-18 between the N-terminal S-shaped beta-sheet-like structure and the central 6-mer helix and one at residues 26-28 between the central helix and the 11-mer C-terminal helix. The latter helix is the only amphiphilic/hydrophobic part of the peptide and is thus presumably the part that penetrates into the hydrophobic phase of target-cell membranes. The hinge between the two helices may introduce the flexibility that allows the helix to dip into membranes. The helix-hinge-helix structure in the C-terminal half of curvacin A clearly distinguishes this peptide from the other pediocin-like peptides whose structures have been analyzed and suggests that curvacin A along with the structural homologues enterocin P and carnobacteriocin BM1 belong to a subgroup of the pediocin-like family of antimicrobial peptides.     

Autolysis in Staphylococcus aureus: Preferential Release of Old Cell Walls

The kinetics of release of old versus new cell wall in two strains of Staphylococcus aureus were studied during autolysis. In both strains the autolytic enzyme is an amidase. Cells were double labeled with (3)H and (14)C, and the distribution of radioactivity in the cell walls was monitored during autolysis. In all cases the rate of release of steady-state lable from peptidoglycan was significantly higher than that of pulse label. Identical results were obtained with whole cells or isolated cell walls. The results suggest that in S. aureus the old cell wall is preferentially released during autolysis.     

Enhancing the affinity of SEB-binding peptides by repeating their sequence

The utilization of peptide ligands in biosensors and bioassays is dependent on achieving high affinity of these peptides toward their targets. In a previous report, we identified 12-mer peptides that could selectively bind to Staphylococcal enterotoxin B (SEB) using a phage-display library. In this study, we explore for new modification approaches to enhance the affinity of two different SEB-binding peptides. In order to identify the binding regions of selected peptides, the charged residues and the ones, critical for the structure of peptide, were replaced with alanine. However, a specific binding region could not be suggested as all mutant peptides have lost their affinities toward SEB completely. The modifications for the affinity enhancement were done by repeating the 12-mer peptide sequences. A 10-fold increase was observed in the binding affinity of one of the two-repeated peptides, while this modification did not affect the affinity of the other tested peptide. The peptide, with enhanced affinity, was further modified as three repeats; however the affinity of the peptide decreased. The structural basis of the affinity difference between modified peptides was examined by molecular dynamics simulation. The results showed that the conformational differences hold the key for affinity of peptides modified by repeating the sequence. This high affinity peptide with increased affinity is a promising molecular recognition agent to be used in the detection of SEB to be utilized in biosensing systems.     

Discovery of potent antimicrobial peptide analogs of Ixosin-B

Antimicrobial peptides (AMPs) represent the first defense line against infection when organisms are infected by pathogens. These peptides are generally good targets for the development of antimicrobial agents. Peptide amide analogs of Ixosin-B, an antimicrobial peptide with amino acid sequence of QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY, were designed, synthesized and examined for antimicrobial activities against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Within the peptides synthesized, we discovered an 11-mer peptide, KRLRRVWRRWR-amide, which exhibited potent antimicrobial activity while very little hemolytic activity in human erythrocytes was observed even at high dose level (100 μM). With further modifications, this peptide could be developed into a potent antimicrobial agent in the future.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris.

Six peptides with antimicrobial activity were isolated from an extract of freeze-dried skin of the Japanese mountain brown frog Rana ornativentris. Two structurally related peptides (brevinin-20a GLFNVFKGALKTAGKHVAGSLLNQLKCKVSGGC, 11 nmol/g dried tissue, and brevinin-20b GIFNVFKGALKTAGKHVAGSLLNQLKCKVSGEC, 170 nmol/g) belong to the brevinin-2 family, previously identified in Asian and European, but not North American, Ranid frogs. Four peptides (temporin-10a FLPLLASLFSRLL.NH2, 13 nmol/g; temporin-10b FLPLIGKILGTI L.NH2, 350 nmol/g; temporin-10c FLPLLASLFSRLF.NH2, 14 nmol/g; and temporin-10d FLPLLASLFSGLF.NH2, 8 nmol/g) are members of the temporin family first identified in the European common frog Rana temporaria but also found in the skins of North American Ranids. The brevinin-2 peptides showed broad-spectrum activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli and the yeast Candida albicans, whereas the temporins showed potent activity only against S. aureus. The brevinins and temporins belong to the class of cationic antimicrobial peptides that adopt an amphipathic alpha-helical conformation but it is significant that temporin-10d, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 microM compared with 2 microM for temporin-10a). This suggests that strong electrostatic interaction between the peptide and the negatively charged phospholipids of the cell membrane is not an absolute prerequisite for antimicrobial activity.     

Effects of Group 3 LEA protein model peptides on desiccation-induced protein aggregation

Group 3 late embryogenesis abundant (G3LEA) proteins have amino acid sequences with characteristic 11-mer motifs and are known to reduce aggregation of proteins during dehydration. Previously, we clarified the structural and thermodynamic properties of the 11-mer repeating units in G3LEA proteins using synthetic peptides composed of two or four tandem repeats originating from an insect (Polypedilum vanderplanki), nematodes and plants. The purpose of the present study is to test the utility of such 22-mer peptides as protective reagents for aggregation-prone proteins. For lysozyme, desiccation-induced aggregation was abrogated by low molar ratios of a 22-mer peptide, PvLEA-22, derived from a P. vanderplanki G3LEA protein sequence. However, an unexpected behavior was noted for the milk protein, α-casein. On drying, the resultant aggregation was significantly suppressed in the presence of PvLEA-22 with its molar ratios>25 relative to α-casein. However, when the molar ratio was <10, aggregation occurred on addition of PvLEA-22 to aqueous solutions of α-casein. Other peptides derived from nematode, plant and randomized G3LEA protein sequences gave similar results. Such an anomalous solubility change in α-casein was shown to be due to a pH shift to ca. 4, a value nearly equal to the isoelectric point (pI) of α-casein, when any of the 22-mer peptides was mixed. These results demonstrate that synthetic peptides derived from G3LEA protein sequences can reduce protein aggregation caused both by desiccation and, at high molar ratios, also by pH effects, and therefore have potential as stabilization reagents.     

The murine cytomegalovirus pp89 immunodominant H-2Ld epitope is generated and translocated into the endoplasmic reticulum as an 11-mer precursor peptide

The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitopes is known to be generated as precursor peptides requiring trimming either before or after translocation into the endoplasmic reticulum (ER). In this study, we have followed the proteasomal processing and TAP-dependent ER translocation of the immunodominant epitope of the murine CMV immediate early protein pp89. For the first time, we experimentally linked peptide generation by the proteasome system and TAP-dependent ER translocation. Our experiments show that the proteasome generates both an N-terminally extended 11-mer precursor peptide as well as the correct H2-L(d) 9-mer epitope, a process that is accelerated in the presence of PA28. Our direct peptide translocation assays, however, demonstrate that only the 11-mer precursor peptide is transported into the ER by TAPs, whereas the epitope itself is not translocated. In consequence, our combined proteasome/TAP assays show that the 11-mer precursor is the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.     

High-throughput generation of small antibacterial peptides with improved activity

Cationic antimicrobial peptides are able to kill a broad variety of Gram-negative and Gram positive bacteria and thus are good candidates for a new generation of antibiotics to treat multidrug-resistant bacteria. Here we describe a high-throughput method to screen large numbers of peptides for improved antimicrobial activity. The method relies on peptide synthesis on a cellulose support and a Pseudomonas aeruginosa strain that constitutively expresses bacterial luciferase. A complete substitution library of 12-amino-acid peptides based on a linearized variant (RLARIVVIRVAR-NH(2)) of the bovine peptide bactenecin was screened and used to determine which substitutions at each position of the peptide chain improved activity. By combining the most favorable substitutions, we designed optimized 12-mer peptides showing broad spectrum activities with minimal inhibitory concentrations (MIC) as low as 0.5 microg/ml against Escherichia coli. Similarly, we generated an 8-mer substituted peptide that showed broad spectrum activity, with an MIC of 2 microg/ml, against E. coli and Staphylococcus aureus.