Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M405884200 on June 1, 2004

J. Biol. Chem., Vol. 279, Issue 35, 36250-36258, August 27, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/35/36250    most recent
M405884200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kulanthaivel, P.
Right arrow Articles by Peng, S.-B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kulanthaivel, P.
Right arrow Articles by Peng, S.-B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Novel Lipoglycopeptides as Inhibitors of Bacterial Signal Peptidase I*

Palaniappan Kulanthaivel, Adam J. Kreuzman, Mark A. Strege, Matthew D. Belvo, Tim A. Smitka, Matthew Clemens, James R. Swartling, Kristina L. Minton, Feng Zheng, Eddie L. Angleton, Deborah Mullen, Louis N. Jungheim, Valentine J. Klimkowski, Thalia I. Nicas, Richard C. Thompson, and Sheng-Bin Peng{ddagger}

From the Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285

Received for publication, May 26, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Signal peptidase (SPase) I is responsible for the cleavage of signal peptides of many secreted proteins in bacteria. Because of its unique physiological and biochemical properties, it serves as a potential target for development of novel antibacterial agents. In this study, we report the production, isolation, and structure determination of a family of structurally related novel lipoglycopeptides from a Streptomyces sp. as inhibitors of SPase I. Detailed spectroscopic analyses, including MS and NMR, revealed that these lipoglycopeptides share a common 14-membered cyclic peptide core, an acyclic tripeptide chain, and a deoxy-{alpha}-mannose sugar, but differ in the degree of oxidation of the N-methylphenylglycine residue and the length and branching of the fatty acyl chain. Biochemical analysis demonstrated that these peptides are potent and competitive inhibitors of SPase I with Ki 50 to 158 nM. In addition, they showed modest antibacterial activity against a panel of pathogenic Gram-positive and Gram-negative bacteria with minimal inhibitory concentration of 8–64 µM against Streptococcus pneumonniae and 4–8 µM against Escherichia coli. Notably, they mechanistically blocked the protein secretion in whole cells as demonstrated by inhibiting {beta}-lactamase release from Staphylococcus aureus. Taken together, the present discovery of a family of novel lipoglycopeptides as potent inhibitors of bacterial SPase I may lead to the development of a novel class of broad-spectrum antibiotics.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteins destined for secretion in both prokaryotic and eukaryotic organisms are initially synthesized as precursors with an amino-terminal extension known as signal (or leader) peptide. The signal sequence is removed by a signal peptidase (SPase)1 that is localized in the cytoplasmic membrane in bacteria. Cleavage of precursors by SPase leads to the release of secreted proteins from the outer surface of cytoplasmic membrane. In bacteria, two major SPases, SPase I and SPase II with different cleavage specificities, have been identified. SPase I is responsible for processing the majority of secreted proteins (1–3), and SPase II is exclusively involved in processing glyceride-modified lipoproteins (4).

SPase I is an attractive target for development of antibacterial agents because of its unique biochemical and physiological properties. It is essential for bacterial viability and growth as demonstrated by gene knockout and other genetic experiments (5–8). It is widely distributed in both Gram-positive and Gram-negative bacteria, as well as in Chlamydia. Genes encoding SPase I have been cloned and sequenced from different bacterial species, including many of clinically relevant bacteria (8, 9). The active domain of bacterial SPase I is exposed to the surface of cytoplasmic membrane as revealed by sequence and topological analysis (10–12), and thus is accessible to potential inhibitors. In addition, SPases from bacteria and eukaryotic cells are different in composition, location, and possibly catalytic mechanism (13–17). These differences make it possible to identify selective bacterial SPase I inhibitors without toxicity to mammalian cells.

SPase I belongs to a novel class of serine protease that utilize a serine and a lysine to form a unique catalytic dyad for peptide hydrolysis (14–16). Because of this unique catalytic mechanism, they are not sensitive to the classic protease inhibitors (2, 3, 18). The first effective inhibitor of bacterial SPase I was described in 1994 by Kuo and colleagues (19), who reported that {beta}-lactams could inhibit Escherichia coli SPase I in a pH- and time-dependent manner (19). Certain {beta}-lactams have also been shown to be effective irreversible inhibitors of a number of serine proteases and hydrolases, such as elastase (20, 21), phospholipase A2 (22), and {beta}-lactamase (23). Researchers at Smith-Kline Beecham have extensively studied {beta}-lactam (or penem)-type inhibitors against E. coli SPase I (24, 25). To our knowledge, allyl (5S,6S)-6-((R)-acetoxyethyl)-penem-3-carboxylate (5S,6S-penem) is one of the most potent inhibitors of E. coli SPase I reported thus far.

In previous studies, we cloned the gene encoding SPase I of Streptococcus pneumoniae and identified the precursor of streptokinase as a native substrate of the enzyme (9). Consequently, we developed a fluorescent peptide substrate, KLTF-GTVK(Abz)PVQAIAGY(NO2)EWL and a continuous fluorimetric assay for this enzyme (26). In this report we identify a family of novel lipoglycopeptides from a microbial source, which are potent inhibitors of bacterial SPase I and are able to inhibit the growth of clinically relevant bacteria.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains—S. pneumoniae R6 was an unencapsulated D39 derivative (27, 28). Staphylococcus aureus ATCC 33592 was a gentamicin and methicillin-resistant, {beta}-lactamase-producing strain (29). Haemophilus influenzae ATCC 49247 and S. aureus ATCC 13709 were commonly used bacterial strains for MIC studies. E. coli K12, EL683 (K12MG1655, envA1 zac3051::Tn10), and EL744 (K12MG1655, {Delta}tolC::kanr) were from our laboratory collection. Streptomyces sp. utilized for lipoglycopeptide production was deposited in ATCC (ATCC PTA-3546).

Purification of S. pneumoniae SPase I—Recombinant S. pneumoniae SPase I was purified as described previously (9).

Purification of E. coli SPase I—The gene encoding E. coli SPase I was cloned by PCR. Two primers, 5'-GATCGTTCATATGGTTCGTTCATTTCTTTATGAACCCTTTCAG-3' and 5'-GCCGCTAACTCGAGGGCAGCGTGAACGATCATTTCATCACAG-3', were synthesized according to the published sequence (10, 11). Expression vector pET15b-EcoliSPase was constructed by replacing the NdeI and XhoI fragments of pET15b with the PCR fragment. For expression of E. coli SPase I, E. coli strain BL21(DE3) was transformed with pET15b-EcoliSPase, grown, and induced with isopropyl-1-thio-{beta}-D-galactopyranoside as described (30). Because overexpressed E. coli SPase I was aggregated in the inclusion body, the purification went through a denaturing and refolding process. Typically, 1 liter of isopropyl-1-thio-{beta}-D-galactopyranoside-induced E. coli BL21(DE3) cells harboring pET15b-EcoliSPase were resuspended in 50 ml of lysis buffer containing 50 mM Na2HPO4, 300 mM NaCl, pH 8.0, and sonicated for 5 min on ice. The lysate was centrifuged at 50,000 x g for 1 h at 4 °C. The resultant pellet was then sonicated for 5 min in 20 ml of denaturing buffer containing 50 mM Na2HPO4, 300 mM NaCl, and 8 M urea. After centrifugation at 50,000 x g for 1 h at 4 °C, the supernatant was loaded onto a 5 ml of pre-equilibrated nickel-nitrilotriacetic acid column that was then washed with 120 ml of refolding buffer (50 mM NaH2PO4, 300 mM NaC1, 15 mM imidazole) containing a continuous urea gradient from 6 to 1 M with a flow rate of 1 ml/min. The protein was finally eluted with 30 ml of elution buffer (20 mM Tris-HCl, pH 8.0, 20% glycerol, 1 M urea, and 100 mM imidazole), and 1-ml fractions were collected and analyzed by SDS-PAGE. We found that 1 M urea in the elution buffer increased the recovery of active enzyme, and did not influence the enzymatic activity (data not shown).

HPLC Analysis of Peptide Substrate Cleavage by S. pneumoniae and E. coli SPase I—Previously described peptide substrates, KLTFGTVKPVQAIAGYEWL and KLTFGTVK(Abz)PVQAIAGY(NO2)EWL, were utilized for HPLC analysis (26). Typically, cleavage assay was performed in 50 µl of reaction mixture containing 20 mM Tris-HCl, pH 8.0, 50 µg of E. coli lipid extract, 0.05 µM SPase I, and 100 µM substrate. Reaction was incubated at 37 °C for 30 min and terminated by addition of an equal amount of 8 M urea. Cleavage of peptide substrate was determined by HPLC using a Hewlett Packard Series 1100 system equipped with an autosampler.

Fermentation—An aliquot (100 µl) of Streptomyces culture (ATCC PTA-3546) was inoculated into 20 ml of a vegetative medium (31) and incubated at 30 °C at 165 rpm. After 72 h, 2 ml of the vegetative culture was transferred to 800 ml of fermentation medium (31), incubated at 30 °C at 250 rpm, and harvested after 96 h.

Isolation of Lipoglycopeptides—The cell pellet from 32 liters of broth was extracted 2x with 5 liters of CH3OH. The combined extracts were concentrated to 500 ml and partitioned with 500 ml of CH3OH and 2x 1 liter of EtOAc. The combined EtOAc layers were evaporated (49 g), suspended in 300 ml of 3:1 (v/v) CH3OH/H2O, and filtered. The filtrate was diluted with 600 ml of H2O and applied onto a 500 ml of TosoHaas Amberchrom CG161m column equilibrated with 3:1 (v/v) H2O/CH3OH. The column was sequentially eluted with 4x 500 ml of 3:1 (v/v) H2O/CH3OH, 4x 500 ml of 1:1 (v/v) H2O/CH3OH, and 8x 500 ml of CH3OH. The first 2 liters of CH3OH effluent were concentrated (2 g) and chromatographed over a Sephadex LH-20 column (7.5 x 39 cm, Amersham Biosciences) with CH3OH as the solvent. The effluent containing the lipoglycopeptides was evaporated (960 mg) and further chromatographed over a Waters SymmetryPrep C18 column (50 x 250 mm, 7 µm, flow rate 45 ml/min, 35–70% CH3CN gradient buffered with 0.05% NH4OAc over 48 min and holding at 70% CH3CN for 24 min). 45 ml of fractions were collected and combined to yield three fractions, A (172 mg), B (370 mg), and C (128 mg).

Fraction C was further chromatographed over a PolyLC polyhydroxyethyl aspartamide column (25.4 x 250 mm, 12 µm, flow rate 25 ml/min, 95–70% CH3CN gradient buffered with 0.05% NH4OAc over 50 min and holding at 70% for 22 min) to yield two additional fractions, fraction D (40 mg) mostly containing lipoglycopeptides 1 and 2, and fraction E (12.5 mg) containing lipoglycopeptides 3, 4, and 5. Rechromatography of fraction D over a Waters SymmetryPrep C18 column (7.8 x 300 mm, 7 µm, flow rate 4.7 ml/min, 40–55% CH3CN gradient buffered with 0.05% NH4OAc over 60 min) produced 11.7 mg of lipoglycopeptide 1 and 4.1 mg of lipoglycopeptide 2. Repeated chromatography of fraction E twice as described above yielded 0.3 mg of lipoglycopeptide 3, 0.5 mg of lipoglycopeptide 4, and 3.2 mg of lipoglycopeptide 5. Fraction A was chromatographed over a PolyLC polyhydroxyethyl aspartamide column (95–65% CH3CN gradient over 48 min and holding at 65% CH3CN for 24 min) to furnish fraction F (90 mg) containing lipoglycopeptides 6 and 7, and fraction G (51 mg) containing lipoglycopeptide 8. Rechromatography of fraction F over a Waters SymmetryPrep C18 column (19 x 300 mm, 7 µm, flow rate 17 ml/min, 25–40% CH3CN gradient buffered with 0.05% NH4OAc over 48 min and holding at 40% CH3CN for 24 min) yielded 9 mg of enriched lipoglycopeptide 6 and 11 mg of enriched lipoglycopeptide 7. Each sample was re-purified as detailed above to yield 3.7 mg of lipoglycopeptide 6 and 2.3 mg of lipoglycopeptide 7. Similar chromatography of fraction G gave 6.4 mg of lipoglycopeptide 8. The purity of isolated peptides was determined by analytical chromatography over a Waters Symmetry C18 column (4.6 x 150 mm, 3.5 µm, flow rate 1 ml/min, 35–70% CH3CN gradient over 15 min).

Amino Acid Analysis—Amino acid analysis of lipoglycopeptide 1 was performed as described (32).

Deacylation of Lipoglycopeptides 7 and 8—A solution containing mostly lipoglycopeptides 7 and 8 (200 mg) in 20 ml of CH3CN/H2O/trifluororacetic acid (6:3:1) was stirred for 90 h at room temperature. The mixture was purified over a PolyLC polyhydroxyethyl aspartamide column (50.8 x 250 mm, 12 µm, flow rate 45 ml/min, 90–50% CH3CN gradient buffered with 0.05% NH4OAc over 72 min) to yield two fractions. These fractions were further purified independently over a CG161 column (85 ml) to yield 10.7 and 14 mg of glycopeptides 9 and 10, respectively.

LC-MS Study—LC-MS analysis was carried out on a Waters Alliance 2690 Separations Module coupled with a Platform LCZ mass spectrometer. ESI spectra were collected with capillary and sample cone potentials set at 3000 and 50 V, respectively. Accurate mass determination was performed using a Micromass Q-TOF 1 quadrupole/orthogonal time-of-flight mass spectrometer. Desvancosamine ion of vancomycin (m/z 1305.3434) was used as the lock mass in all accurate mass determinations.

NMR Study—NMR experiments were carried out on a Varian Inova spectrometer equipped with a pulse-field gradient module and a Nalorac Z-SPEC microdual 3-mm probe, operating at 500 MHz for 1H and 125.7 MHz for 13C. Proton and carbon chemical shifts were referenced to the residual solvent signal (CD3OH or CD3OD) at 3.30 and 49 ppm, respectively. Two-dimensional experiments including TOCSY, DQCOSY, HSQC, HMBC, and ROESY were performed using Varian standard pulse sequences.

IC50 Determination—IC50 was determined by fluorimetric assay as described (26). Standard reaction (50 µl) contained 50 nM of either E. coli or S. pneumoniae SPase I, 50 µM substrate and different concentrations of an inhibitor. Reactions were incubated at 37 °C for 1 h for E. coli SPase I and 2 h for S. pneumoniae SPase I. IC50 was calculated using nonlinear regression method with GraphPad Prism Software.

Kinetic Analysis—Kinetic analysis was performed with E. coli SPase I and the non-fluorescent peptide substrate, KLTFGTVKPVQAIAGYEWL by HPLC assay. Typically, a reaction (50 µl) containing 50 nM E. coli SPase I, different concentrations of the substrate and an inhibitor, was incubated at room temperature for 30 min, and terminated by the addition of an equal amount of 8 M urea. The substrate cleavage was analyzed by HPLC. The initial rate, Ki, and type of inhibition were calculated with Sigma Plot 2000, version 6.2 with enzyme kinetics module.

Determination of in Vitro Antibacterial Activity—In vitro antibacterial activity was determined by broth microdilution assay as recommended by National Committee for Clinical Laboratory Standards (33). The growth media utilized for MIC determination were cation-adjusted Mueller-Hinton broth for S. aureus and E. coli strains, Todd-Hewitt broth for S. pneumoniae,or Haemophilus test medium for H. influenzae (33). Bacterial strains used for antibacterial assay include both Gram-positive and Gram-negative bacteria (S. pneumoniae R6, S. aureus ATCC13709, H. influenzae ATCC 49247, and E. coli strains EL683, EL744, and K12). Typically, a 2-fold serial dilution of the test compound was performed in a sterile 96-well microplate. The microplate was then incubated for 23–24 h at 37 °C. The MIC was determined by visual examination of the microplate with the aid of a magnifying mirror apparatus. MIC is the lowest concentration of compound that showed no visible sign of bacterial growth.

{beta}-Lactamase Secretion Assay—S. aureus ATCC 33592, a gentamicin- and methicillin-resistant, and {beta}-lactamase-producing strain (29) was grown in a series dilution in brain heart infusion (from BD Biosciences, a semi-synthetic casein acid hydrolysate medium supplemented with 0.5% yeast extract) in a 37 °C incubator containing 5% CO2 overnight. The cultures with A620 less than 0.3 were centrifuged and resuspended with fresh brain heart infusion to make the initial A620 to 0.15–0.2. The selected inhibitor at different concentrations was added to the mixture, which was then incubated for 2 h. After incubation, the cells were centrifuged, and the supernatant was saved for determination of secreted {beta}-lactamase. The resultant pellet was washed once with fresh brain heart infusion medium and resuspended in same volume of fresh brain heart infusion medium. After 3 cycles of freeze-thaw, the mixture was used for measurement of retained {beta}-lactamase. For determination of {beta}-lactamase activity, typically, a 100-µl reaction containing 30 µl of supernatant or lysed cells was incubated with 250 µM nitrocefin in phosphate-buffered saline buffer, pH 6.8, at 37 °C for 30–60 min. The absorbance at 482 nm (A482) was measured at the beginning and end of the reaction. The secreted and the retained {beta}-lactamase activities were calculated based upon {Delta}A482.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Production and Purification of Lipoglycopeptides—To identify novel inhibitors of bacterial SPase I, 50,000 pre-fractionated natural product samples were screened in a high-throughput screen using the fluorimetric assay (26). This led to the identification of a single sample derived from a microbial source with reproducible activity. Further fractionation of limited amounts of this sample resulted in the identification of two isomeric components responsible for the activity. UV-visible and MS-based literature searches revealed that the active components might be novel. To fully characterize and study their antibiotic properties, we undertook a large scale fermentation. Fermentation followed by chromatographic purification resulted in the isolation of eight closely related lipoglycopeptides 1–8. The purity of compounds, except lipoglycopeptide 3 (92%), used for structural and biochemical studies exceeded 98% as determined by HPLC. The purity was estimated based on evaporative light scattering detector response (data not shown).

Structure Determination of Lipoglycopeptides—The two most abundant new isomeric lipoglycopeptides 1 and 2 showed identical molecular composition (C52H78N6O16) as determined by high resolution ESIMS (calculated 1043.5553 (M + H), observed 1043.5531 and 1043.5551, respectively). The structures were primarily deduced by NMR spectroscopy (Fig. 1). After trial and error, the optimum resolution of the amide proton signals of lipoglycopeptide 1 was observed at 10 °C in CD3OH solution. The NMR results of lipoglycopeptide 1 reported in Table I were obtained after a detailed analysis of 1H, 13C, DQCOSY, TOCSY, HSQC, and HMBC, which revealed the presence of four common amino acid residues, i.e. glycine, two alanines, and N-methylserine, in addition to two uncommon aromatic amino acid residues, i.e. a 3-substituted tyrosine and a 3,4,5-trisubstituted N-methylphenylglycine. Consistent with this observation, the amino acid analysis revealed the presence of glycine and alanine in a ratio of 1:2.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 1.
Structures of lipoglycopeptides 1–8 and glycopeptides 9 and 10.

 


View this table:
[in this window]
[in a new window]
  		TABLE I
1H and 13C NMR chemical shift assignments of lipoglycopeptide 1 in CD3OH

Chemical shifts referenced with respect to residual solvent signal at 3.3 ppm (1H) and 49.0 ppm (13C). Assignments were made by the analysis of 1H, DQCOSY, TOCSY, HSQC, and HMBC data. The abbreviations used are: s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; m, multiplet; dd, double doublet; dq, double quartet; br, broad.

 
The other prominent features in the NMR spectra of lipoglycopeptide 1 included resonances for a 6-deoxy sugar and a fatty acyl chain. In accordance with the proposed molecular formula and MS/MS, the number of carbons in the acyl chain was determined to be 16 (Figs. 1 and 2). Furthermore, the NMR spectrum suggested that the acyl chain terminated with an isopropyl group in lipoglycopeptide 1 ({delta}H 0.86, d, J = 6 Hz, 6 protons) and with a normal methyl group in lipoglycopeptide 2 ({delta}H 0.89, t, J = 7 Hz, 3 protons), which, in fact, is the only structural difference between 1 and 2. The HMBC cross-peaks observed from the amide carbonyl carbon ({delta} 176.9) of N-methylserine to the CH2 protons ({delta} 2.44) of the acyl chain, N-CH3 protons ({delta} 3.09) of N-methylserine, and {alpha} proton ({delta} 4.97) of the N-methylserine strongly suggested attachment of the long fatty acyl chain on the N terminus of N-methylserine (Fig. 3A). Likewise, the HMBC correlations observed from the sugar anomeric proton ({delta} 5.36) and the mutually coupled phenylglycine protons ({delta} 6.47 and 6.77) to the same carbon ({delta} 143.8, C-4 of N-methylphenylglycine) clearly indicated that the 6-deoxysugar unit was attached to the 4 position of the phenylglycine through a glycosidic linkage (anomeric carbon resonance at {delta} 103.9). The HMBC data (13C–1H correlations between the carbonyl and the adjacent amino acid amide proton and/or proton attached to the {alpha} carbon) established the linear amino acid sequence as N-methylserine-alanine-glycine-(3,4,5-trisubstituted N-methylphenylglycine)-alanine-(3-substituted tyrosine) (Fig. 3A) and was confirmed by ROESY (correlations from the amide proton to the adjacent amino acid {alpha}-proton) data (Fig. 3B).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 2.
MS/MS spectrum and diagnostic ESI mass spectral fragmentation pattern of lipoglycopeptide. A, MS/MS spectrum of lipoglycopeptide 1. B, diagnostic ESI mass spectral fragmentation pattern of lipoglycopeptides.

 



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 3.
Spectroscopic analysis of lipoglycopeptide. A, selected 1H–13C HMBC correlations of lipoglycopeptide 1. HMBC data were acquired as 2048 x 512 data points with 52 transients per t1 increment. The J-filter was optimized for 1JC-H = 140 Hz and N bond delay was set to 0.063 s corresponding to NJC-H = 8 Hz. B, selected ROESY correlations of lipoglycopeptide 1. ROESY data were acquired as 2048 x 128 data points with 32 transients per t1 increment. A mixing time of 0.2 s was used. C, proton coupling constants and ROESY correlations of deoxy-{alpha}-mannose. ROESY data were acquired as 2048 x 128 data points with 32 transients per t1 increment. A mixing time of 0.2 s was used. Proton coupling constants 3J1,2 = 2 Hz, 3J2,3 = 3 Hz, 3J3,4 = 9 Hz, 3J4,5 = 9 Hz, and 3J5,6 = 6 Hz were measured directly from the one-dimensional spectra of lipoglycopeptides 1, 5, 9, and 10.

 
The molecular formula of lipoglycopeptides 1 and 2, C52H78N6O16, requires 17 degrees of unsaturation. The six amino acid carbonyls, one acyl group, one sugar unit, and two aromatic rings account for 16 of the 17 degrees of unsaturation. This suggested that the peptide is monocyclic. The HMBC correlations observed from H-2 of tyrosine to C-5 of N-methylphenylglycine and H-6 of N-methylphenylglycine to C-3 of tyrosine indicated that the two aromatic amino acids were linked via a carbon-carbon bond resulting in the 14-membered cyclic structure as shown in Fig. 1. This was further confirmed by a strong ROESY correlation observed between the H-2 of tyrosine and H-6 of N-methylphenylglycine (Fig. 3B). Significant fragments observed in the quadrupole/orthogonal time-of-flight mass spectrometry corroborated the gross structure assignment of lipoglycopeptide 1 by NMR spectroscopy (Fig. 2).

In regard to stereochemistry, amino acid analysis of lipoglycopeptide 1 revealed L- and D-configuration for the two alanines (34). The coupling constant analysis of the sugar protons and ROESY data established the identity of the deoxy sugar as deoxy-{alpha}-mannose (Table I, Fig. 3C). Thus, small coupling constants observed between H-1 and H-2, and H-2 and H-3 were consistent with equatorial/axial (H-1), equatorial (H-2), and axial (H-3) orientations for these protons. The large coupling constants observed between H-3 and H-4, and H-4 and H-5 were consistent with axial orientations for H-3, H-4, and H-5. Accordingly, in the ROESY spectrum, a strong correlation was observed between the 1,3-diaxially oriented H-3 and H-5, and no correlation was observed between H-1 (equatorial) and H-3 (axial). The absence of ROESY correlation between H-3 and H-1 clearly demonstrated equatorial orientation for H-1.

The related lipoglycopeptide 5 had the molecular formula C52H78N6O15 as deduced by the high resolution ESIMS (calculated for 1027.5603 (M + H), observed 1027.5637), which differed from lipoglycopeptide 1 by one less oxygen. The 1H NMR spectrum of lipoglycopeptide 5 overall contained resonances reminiscent of a lipoglycopeptide and was very similar to lipoglycopeptide 1, except that the aromatic ring of the N-methylphenylglycine residue was tri-substituted rather than tetra-substituted, indicating that lipoglycopeptide 5 is a deshydroxy analog of lipoglycopeptide 1 (Table II). Notably, the lack of the phenolic group ortho to the sugar substituent in lipoglycopeptide 5 resulted in the restoration of the sugar methyl to its normal frequency ({delta}H 0.64 in 1 versus 1.12 ppm in 5).


View this table:
[in this window]
[in a new window]
  		TABLE II
1H and 13C NMR chemical shift assignments of lipoglycopeptide 5 in CD3OD

Chemical shifts referenced with respect to residual solvent signal at 3.3 ppm (1H) and 49.0 ppm (13C). Assignments were made by the analysis of 1H, DQCOSY, TOCSY, HSQC, and HMBC data. The abbreviations used are: s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; m, multiplet; dd, double doublet; dq, double quartet; br, broad; obsc, obscured.

 
The structure of the remaining lipoglycopeptides 3, 4, 6, 7, and 8 was suggested by the high resolution ESIMS data shown in Table III and confirmed by 1H NMR data (data not shown). Thus, the family of lipoglycopeptides obtained from the Streptomyces sp. can, in general, be classified into two major cores differing only in the oxidation state of the N-methylphenylglycine residue. The rest of the diversity stems from the nature of the acyl chain that differs in chain length and degree of branching (Fig. 1). The presence of two different peptide cores and the attachment of the fatty acyl chain on the N terminus of N-methylserine were further confirmed by the trifluoroacetic acid-mediated hydrolysis of a mixture of lipoglycopeptides 7 and 8 to yield glycopeptides 9 and 10 (Fig. 1). The structures of glycopeptides 9 and 10 were fully supported by high-resolution mass measurement (Table III) and NMR data (Table IV). Specifically, in NMR spectra of glycopeptides 9 and 10, the N-methyl resonance of N-methylserine exhibited a significant up-field shift (~0.7 ppm) when compared with their acylated counterparts.


View this table:
[in this window]
[in a new window]
  		TABLE III
High Resolution electrospray ionization mass spectral data of lipoglycopeptides 1-8 and glycopeptides 9 and 10

 


View this table:
[in this window]
[in a new window]
  		TABLE IV
1H and 13C NMR chemical shift assignments of lipoglycopeptides 9 and 10 in CD3OD

Chemical shifts referenced with respect to residual solvent signal at 3.3 ppm (1H). Assignments were made by the analysis of 1H, DQCOSY, and TOCSY data. The abbreviations used are: s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; m, multiplet; dd, double doublet; dq, double quartet; br, broad; obsc, obscured.

 
At the time of the completion of this work, a literature search revealed that this family of lipoglycopeptides is novel. Subsequently, we learned that structures of a similar family of lipopeptides, called arylomycins, have been disclosed in a conference proceeding (35) followed by a publication (36). The major structural difference is the absence of the sugar unit in arylomycins.

Determination of Bacterial SPases I Activity—As illustrated in Table V, all 8 compounds showed potent inhibitory activity against E. coli SPase I with IC50 ranging from 0.11 to 0.19 µM. They also inhibited S. pneumoniae SPase I with IC50 of 2.4–24.9 µM. The activity was further confirmed by the HPLC assay. As revealed by HPLC profiles, the intact peptide substrate has one peak with retention time of 5.45 min (Fig. 4A). After incubation with SPase I, the substrate was specifically cleaved, two products with retention times of 4.1 and 4.9 min, respectively, were generated (Fig. 4B). When a lipoglycopeptide was included in the reaction mixture, the cleavage of the peptide substrate was inhibited (Fig. 4C), thus confirming the inhibitory activity.


View this table:
[in this window]
[in a new window]
  		TABLE V
Enzymatic inhibition of E. coli and S. pneumoniae SPase I by lipoglycopeptides 1–8

 



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 4.
HPLC analysis of peptide substrate cleavage by SPase I and its inhibition by lipoglycopeptide. The peptide substrate (A) was incubated at 37 °C for 2 h with S. pneumoniae SPase I in the absence (B) or presence of a lipoglycopeptide (C). The cleavage of the peptide substrate was determined by HPLC as described under the "Experimental Procedures." The peaks labeled 1, 2, and 3 correspond to the substrate, the C-terminal cleavage product, and the N-terminal cleavage product, respectively.

 
Determination of Mechanism of Inhibition—Kinetic analysis with lipoglycopeptides 2, 5, 7, and 8 by HPLC assay revealed competitive inhibition with Ki ranging from 50 to 158 nM (Table V). Fig. 5 is an example of the Lineweaver-Burk plot for lipoglycopeptide 5. Analysis demonstrated that increasing substrate concentration proportionally reduced the inhibition of SPase I activity by lipoglycopeptide 5 as determined by velocity changes. Therefore, the mechanism of action of this compound is competitive with respect to the substrate. Similar kinetic analysis was also performed for compounds 2, 7, and 8, which also showed competitive mechanism (data not shown).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 5.
Lineweaver-Burk plot of SPase I inhibition by lipoglycopeptide 5. The kinetic analysis was performed with E. coli SPase I and the non-fluorogenic peptide substrate by HPLC assay. Reactions contained 50 nM E. coli SPase I, 100, 200, 300, 400, or 500 µM substrate, and different concentrations of inhibitor as indicated. The initial rate, Ki, and inhibition type of the inhibitor were calculated with Sigma Plot 2000, version 6.2 with enzyme kinetics module.

 
Determination of Antibacterial Activity—Table VI summarizes the antibacterial activity of these novel lipoglycopeptides against a panel of major human bacterial pathogens, including Gram-positive S. pneumoniae and S. aureus, and Gram-negative H. influenzae and E. coli. They exhibited moderate antibacterial activity with MIC of 4–8 µM against E. coli strain EL683, 8–32 µM against E. coli strain EL744, 8–64 µM against S. pneumoniae, 32–64 µM against S. aureus, and 64–64 µM against H. influenzae.


View this table:
[in this window]
[in a new window]
  		TABLE VI
Antibacterial activity of lipoglycopeptides 1, 2 and 5–8

MIC was determined by broth micro-dilution assay using 2xsTH (for S. pneumoniae), MHII (for S. aureus and E. coli strains) or HTM medium (for H. inflienzae). E. coli EL683 and EL744 are isogenic strains of K12. EL683 is an isogenic envA mutation of wild type E. coli strain K12. envA is a component involved in the biosynthesis of lipid A, the hydrophobic anchor of lipopolysaccharide, which makes up the outer monolayer of outer membrane. E. coli EL744 is a K12 isogenic strain in which tolC, a component of an outer membrane efflux pump was deleted.

 
Inhibition of {beta}-Lactamase Secretion in S. aureus—SPase I is responsible for processing many secreted proteins in both Gram-positive and Gram-negative bacteria and is essential for effective protein secretion. {beta}-Lactamase is a well characterized secretory protein that is processed by SPase I. The uncleaved leader sequence of {beta}-lactamase acts as a membrane anchor and prevents release of the {beta}-lactamase from membrane. After cleavage by SPase I, the mature {beta}-lactamase is released and secreted to the growth medium. Before performing the {beta}-lactamase secretion experiment, we screened a few S. aureus strains for {beta}-lactamase production. We found that S. aureus ATCC33592 produced maximum {beta}-lactamase and was most suitable for protein secretion study (data not shown). To test if these lipoglycopeptides mechanistically inhibit protein secretion within bacterial cells, lipoglycopeptides 1, 2, and 5 were tested in S. aureus, ATCC33592. The results demonstrated that the cells treated with lipoglycopeptides secreted less {beta}-lactamase in the medium in a dose-dependent manner when compared with the control cells, and conversely, the activity of {beta}-lactamase retained within the cells treated with lipopeptide was slightly higher than the control (Fig. 6), suggesting that these SPase I inhibitors indeed inhibit protein secretion within the bacterial cells.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 6.
Effects of lipoglycopeptide 5 on {beta}-lactamase secretion from S. aureus. The selected inhibitor with the indicated concentrations was added to S. aureus cells, and incubated at 37 °C for 2 h. The A620 was measured at the end of the incubation (connected line). The cells were then centrifuged, and the supernatant was saved for determination of the secreted {beta}-lactamase activities (white bars). The pellet was used for determination of retained {beta}-lactamase activities (black bars) as described under "Experimental Procedures."

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Proteases in general are divided into four classes according to their mechanism of action, and they are serine, cysteine, metallo-, and aspartyl proteases. However, recent investigations have unambiguously demonstrated that SPase I is not a member of any of these four traditional classes. For example, it is not sensitive to any of the standard protease inhibitors (2, 3). The catalytic mechanism of the bacterial SPase I has been studied by site-directed mutagenesis using E. coli enzyme (15, 16), Bacillus subtilis SipS (37), and S. pneumoniae enzyme (9). In all these cases, a conserved serine and a conserved lysine were identified to be critical for enzymatic activity. These results suggest that SPases belong to a novel class of serine proteases that utilize a serine and a lysine to form a catalytic dyad. This serine/lysine catalytic dyad structure has been recently confirmed by the structure of E. coli SPase I in complex with 5S,6S-penem (38). This unique catalytic mechanism may explain the difficulty to identify effective SPase inhibitors and thus, to date, only {beta}-lactams with moderate potency have been reported (19, 24, 25). In the present study, we introduce a novel class of lipoglycopeptides as potent inhibitors of SPase I. These peptides have shown competitive inhibition of SPase I, with Ki ranging from 50 to 158 nM. Mechanistically, they blocked the bacterial protein secretion and inhibited the growth of both Gram-positive and Gram-negative bacteria. These favorable biochemical and physiological properties place this class of compounds as potential candidates for further development as effective novel antibiotics.

Our work has shown that these lipoglycopeptides inhibit SPase I from both E. coli and S. pneumoniae, but with different potency. In general, they are more potent against Gram-negative E. coli SPase I than Gram-positive S. pneumoniae enzyme (Table V). A similar difference in potency was also observed with a known {beta}-lactam inhibitor, 5S,6S-penem, which had IC50 of 4.67 and 733.4 µM against E. coli and S. pneumoniae enzymes, respectively, in our assay conditions. This difference in sensitivity of SPases to inhibitors may reflect some subtle structural variations between them. In fact, it is documented that SPases from Gram-positive bacteria are generally onethird smaller than those from Gram-negative species (1). In future, detailed structural studies of SPases from Gram-positive bacteria may offer a better explanation why these enzymes are more difficult to inhibit.

Gene knock-out and other genetic experiments have shown that SPase I is an essential component for bacterial viability and growth in both Gram-positive and Gram-negative bacteria (5–8). Hence, inhibitors of SPase I should have antibacterial activity. Overall, the MIC values observed for the lipoglycopeptides against Gram-positive species, S. pneumoniae and S. aureus (8–64 µM), are in good correlation with the inhibition of S. pneumoniae SPase I (IC50 2.4–24.9 µM). However, the MIC values observed against Gram-negative species, E. coli and H. influenzae (4–64 µM), are significantly higher than the enzyme inhibition of E. coli SPase I (IC50 0.11–0.19 µM). The possible reason for this difference in potency is likely because of the outer membrane barrier that exists in Gram-negative bacteria. As we know, SPase I is located in the outside of the inner membrane of Gram-negative bacteria, and any potential SPase I inhibitor has to cross the outer membrane to access the active site of the SPase I. In our studies, we have demonstrated that modifying the permeability of E. coli outer membranes could indeed result in the improvement of antibacterial activity of these novel lipoglycopeptides. E. coli strain EL683 utilized in this study is an isogenic envA mutation of wild type E. coli K12. envA is a component involved in the biosynthesis of lipid A, the hydrophobic anchor of lipopolysaccharide, which makes up the outer monolayer of outer membrane. envA mutation lowers the lipopolysaccharide content of the outer membrane by 25–30%, and allows passage of large hydrophobic and hydrophilic molecules through the outer membrane (39). As shown in Table VI, the lipoglycopeptides showed MIC of 4–8 µM against E. coli EL683 strain, whereas these compounds had MIC >64 µM against the wild type E. coli K12 strain. Apparently, changing the outer membrane permeability of Gram-negative E. coli improved the antibacterial activity of these lipoglycopeptides. Therefore, the structure of these lipoglycopeptides needs to be optimized for better permeability to inhibit the growth of Gram-negative bacteria. In addition, these lipoglycopeptides also showed MIC of 8–32 µM against another E. coli strain, EL744. EL744 is an E. coli K12 isogenic strain in which tolC, a component of an outer membrane efflux pump, was deleted (40). This implies that an intrinsic resistance to these lipoglycopeptides may exist via a multidrug efflux system in E. coli. Further investigation to confirm this mechanism is needed.

From mechanism of action standpoint, the currently marketed antibiotics inhibit either bacterial cell wall formation or synthesis of essential macromolecules, such as DNA, RNA, proteins, or lipids. Use of these antibiotics over the past three decades has resulted in bacterial resistance and limited the effectiveness of these drugs to treat bacterial infections. Hence, there is an urgent need for new antibiotics with novel mechanisms of action to combat bacterial resistance and infection. In this study, we have demonstrated that these novel lipoglycopeptides are able to inhibit activity of bacterial SPase I, a key enzyme essential for bacterial growth. They also mechanistically block bacterial protein secretion in vivo and cause bacterial death. This consequence suggests that the killing mechanism of these lipoglycopeptides is highly likely by inhibiting SPase I activity and blocking protein secretion within bacterial cells. SPase I has long been considered as an attractive target for antibiotic development, and the data presented in this study has proved this concept by a SPase I specific inhibitor. Therefore, inhibition of bacterial SPase I and the protein secretion machinery indeed represents a new approach to develop novel antibacterial agents with previously unexploited mechanism of action.

Several naturally occurring glycopeptides and lipopeptides and their analogs have been successfully marketed or are in late phase clinical development. Among them, vancomycin, teicoplanin, ramoplanin, and oritavancin act by inhibiting bacterial cell wall biosynthesis (41–43). The recently marketed daptomycin and related cyclic peptides are promising new antibiotics that act on the cytoplasmic membrane by inhibiting lipoteicholic acid biosynthesis (44, 45). Polymyxin, octapeptin, and related peptides are also membrane-acting antibiotics by interacting with lipopolysaccharide on the outer membrane of Gram-negative bacteria (46, 47). The present research encountered yet another class of lipoglycopeptides with distinct structural features and unique mechanisms of action from any of the previously known peptide antibiotics. Considering the success of peptides in antibiotic development, we feel that the present discovery offers hope for the development of a new class of antibiotics to combat bacterial resistance and infections.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285. Tel.: 317-433-4549; Fax: 317-276-1414; E-mail: Peng_Sheng-Bin{at}Lilly.com.

1 The abbreviations used are: SPase, signal peptidase; MIC, minimal inhibitory concentration; ESIMS, electrospray ionization mass spectrometry; DQCOSY, double-quantum filtered correlation spectroscopy; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single-quantum coherence; HMBC, heteronuclear multiple bond correlation; ROESY, rotating frame nuclear Overhauser effect spectroscopy; HPLC, high performance liquid chromatography; 5S,6S-penem, (5S,6S)-6-((R)-acetoxyethyl)-penem-3-carboxylate. Back


   ACKNOWLEDGMENTS
 
We thank Paushika S. Shah for amino acid analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Paetzel, M., Karla, A., Strynadka, N. C., and Dalbey, R. E. (2002) Chem. Rev. 102, 4549–4580[CrossRef][Medline] [Order article via Infotrieve]
  2. Dalbey, R. E., Lively, M. O., Bron, S., and van Dijl, J. M. (1997) Protein Sci. 6, 1129–1138[Medline] [Order article via Infotrieve]
  3. Tschantz, W. R., and Dalbey, R. E. (1994) Methods Enzymol. 224, 285–301
  4. Innis, M. A., Tokunaga, M., Williams, M. E., Loranger, J. M., Chang, S. Y., and Wu, H. C. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3708–3712[Abstract/Free Full Text]
  5. Dalbey, R. E., and Wickner, W. (1985) J. Biol. Chem. 260, 15925–15931[Abstract/Free Full Text]
  6. van Dijl, J. M., Smith, H., Bron, S., and Venema, G. (1988) Mol. Gen. Genet. 214, 55–61[CrossRef][Medline] [Order article via Infotrieve]
  7. Date, T. (1983) J. Bacteriol. 154, 76–83[Abstract/Free Full Text]
  8. Cregg, K. M., Wilding, E. I., and Black, M. T. (1996) J. Bacteriol. 178, 5712–5718[Abstract/Free Full Text]
  9. Peng, S. B., Wang, L., Moomaw, J., Peery, R. B., Sun, P. M., Johnson, R. B., Lu, J., Treadway, P., Skatrud, P. L., and Wang Q. M. (2001) J. Bacterol. 183, 621–627[Abstract/Free Full Text]
  10. Wolfe, P. B., Wickner, W., and Goodman, J. M. (1983) J. Biol. Chem. 258, 12073–12080[Abstract/Free Full Text]
  11. Moore, K. E., and Miura, S. (1987) J. Biol. Chem. 262, 8806–8813[Abstract/Free Full Text]
  12. Lee, J. I., Kuhn, A., and Dalbey, R. E. (1992) J. Biol. Chem. 267, 938–943[Abstract/Free Full Text]
  13. Evans, E. A., Gilmore, R., and Blobel, G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 581–585[Abstract/Free Full Text]
  14. Sung, M., and Dalbey, R. E. (1992) J. Biol. Chem. 267, 13154–13159[Abstract/Free Full Text]
  15. Black, M. T. (1993) J. Bacteriol. 175, 4957–4961[Abstract/Free Full Text]
  16. Tschantz, W. R., Sung, M., Delgado-Partin, V. M., and Dalbey, R. E. (1993) J. Biol. Chem. 268, 27349–27354[Abstract/Free Full Text]
  17. Valkenburg, C. V., Chen, X., Mullins, C., Fang, H., and Green, N. (1999) J. Biol. Chem. 274, 11519–11525[Abstract/Free Full Text]
  18. Kuo, D. W., Chan, H. K., Wilson, C. J., Griffin, P. R., Williams, H., and Knight, W. B. (1993) Arch. Biochem. Biophys. 303, 274–280[CrossRef][Medline] [Order article via Infotrieve]
  19. Kuo, D., Weidner, J., Griffin, P., Shah, S. K., and Knight, W. B. (1994) Biochemistry 33, 8347–8354[CrossRef][Medline] [Order article via Infotrieve]
  20. Wilmouth, R. C., Kassamally, S., Westwood, N. J., Sheppard, R. J., Claridge, T. D., Aplin, R. T., Wright, P. A., Pritchard, G. J., and Schofield, C. J. (1999) Biochemistry 38, 7989–7998[Medline] [Order article via Infotrieve]
  21. Taylor, P., Anderson, V., Dowden, J., Flitsch, S. L., Turner, N. J., Loughran, K., and Walkinshaw, M. D. (1999) J. Biol. Chem. 274, 24901–24905[Abstract/Free Full Text]
  22. Tew, D. G., Boyd, H. F., Ashman, S., Theobald, C., and Leach, C. A. (1998) Biochemistry 37, 10087–10093[CrossRef][Medline] [Order article via Infotrieve]
  23. Strynadka, N. C., Adachi, H., Jensen, S. E., Johns, K., Sielecki, A., Betzel, C., Sutoh, K., and James, M. N. (1992) Nature 359, 700–705[CrossRef][Medline] [Order article via Infotrieve]
  24. Allsop, A. E., Brook, G., Bruton, G., Coulton, S., Edwards P. D., Hatton, I. K., Kaura, A. C. McLean, S. D., Pearson, N. D., Smale, T. C., and Southgate, R. (1995) Bioorg. Med. Chem. Lett. 5, 443–448
  25. Allsop, A., Brooks, G., Edwards, P. D., Kaura, A. C., and Southgate, R. (1996) J. Antibiot. 49, 921–928[Medline] [Order article via Infotrieve]
  26. Peng, S. B., Zheng, F., Angleton, E. L., Smiley, D., Carpenter, J., and Scott, J. E. (2001) Anal. Biochem. 293, 88–95[Medline] [Order article via Infotrieve]
  27. Hoskins, J., Alborn, W. E., Jr., Arnold, J., Blaszczak, L. C., Burgett, S., DeHoff, B. S., Estrem, S. T., Fritz, L., Fu, D. J., Fuller, W., Geringer, C., Gilmour, R., Glass, J. S., Khoja, H., Kraft, A. R., Lagace, R. E., LeBlanc, D. J., Lee, L. N., Lefkowitz, E. J., Lu, J., Matsushima, P., McAhren, S. M., McHenney, M., McLeaster, K., Mundy, C. W., Nicas, T. I., Norris, F. H., O'Gara, M., Peery, R. B., Robertson, G. T., Rockey, P., Sun, P. M., Winkler, M. E., Yang, Y., Young-Bellido, M., Zhao, G., Zook, C. A., Baltz, R. H., Jaskunas, S. R., Rosteck, P. R., Jr., Skatrud, P. L., and Glass, J. I. (2001) J. Bacteriol. 183, 5709–5717[Abstract/Free Full Text]
  28. Smith, M. D., and Guild, W. R. (1979) J. Bacteriol. 137, 735–739[Abstract/Free Full Text]
  29. Schaefler, S., Jones, D., Perry, W., Ruvinskaya, L., Baradet, T., Mayr, E., and Wilson, M. E. (1981) J. Clin. Microbiol. 13, 754–759[Abstract/Free Full Text]
  30. Studier, F. W., Rosenburg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60–89[Medline] [Order article via Infotrieve]
  31. Zahn, J. A., Higgs, R. E., and Hilton, M. D. (2001) Appl. Environ. Microbiol. 67, 377–386[Abstract/Free Full Text]
  32. Irvine, G. B. (1994) Methods Mol. Biol. 32, 257–265[Medline] [Order article via Infotrieve]
  33. National Committee for Clinical Laboratory Standards (1990) Methods for Dilution Antimicrobial Susceptibility Testing for Bacteria That Grow Aerobically: Standard M7-A2, 2nd Ed., National Committee for Clinical Laboratory Standards, Villanova, PA
  34. Aswad, D. W. (1984) Anal. Biochem. 137, 405–409[CrossRef][Medline] [Order article via Infotrieve]
  35. Schmid, D. G., Holtzel, A., Nicholson, G. J., Gebhardt, K., Fiedler, H., and Jung, G. (2001) in Peptides: The Wave of the Future, Proceedings of the 2nd International Symposium/17th American Peptide Symposium, San Diego, CA, June 9–14, 2001 (Lebl, M., and Houghten, R. A., eds) pp. 768–769, Kluwer Academic Publishers, Norwell, MA
  36. Holtzel, A., Schmid, D. G., Nicholson, G. J., Stevanovic, S., Schimana, J., Gebhardt, K., and Fiedler, H. (2002) J. Antibiot. 55, 571–577[Medline] [Order article via Infotrieve]
  37. van Dijl, J. M., de Long, A., Venema, G., and Bron, S. (1995) J. Biol. Chem. 270, 3611–3618[Abstract/Free Full Text]
  38. Paetzel, M., Dalbey, R. E., and Strynadka, N. C. (1998) Nature 396, 186–190[CrossRef][Medline] [Order article via Infotrieve]
  39. Normark, S., Boman, H. G., and Matsson, E. (1969) J. Bacteriol. 97, 1334–1342[Abstract/Free Full Text]
  40. Hikaido, H. (1996) J. Bacteriol. 178, 5853–5859[Free Full Text]
  41. Boger, D. L. (2001) Med. Res. Rev. 21, 356–381[CrossRef][Medline] [Order article via Infotrieve]
  42. Allen, N. E., and Nicas, T. I. (2003) FEMS Microbiol. Rev. 26, 511–532[CrossRef][Medline] [Order article via Infotrieve]
  43. Ge, M., Chen, Z., Onishi, H. R., Kohler, J., Silver, L. L., Kerns, R., Fukuzama, S., Thompson, C., and Kahne, D. (1999) Science 284, 507–511[Abstract/Free Full Text]
  44. Tally, F. P., and Debruin, M. F. (2000) J. Antimicrob. Chemother. 46, 523–526[Free Full Text]
  45. Laganas, V., Alder, J., and Silverman, J. A. (2003) Antimicrob. Agents Chemother. 47, 2682–2684[Abstract/Free Full Text]
  46. Storm, D. R., Rosenthal, K. S., and Swanson, P. (1977) Annu. Rev. Biochem. 46, 723–763[CrossRef][Medline] [Order article via Infotrieve]
  47. Tsubery, H., Ofek, I., Cohen, S., and Fridkin, M. (2000) J. Med. Chem. 43, 3085–3092[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Bacteriol.Home page
R. Gilmour, J. E. Foster, Q. Sheng, J. R. McClain, A. Riley, P.-M. Sun, W.-L. Ng, D. Yan, T. I. Nicas, K. Henry, et al.
New Class of Competitive Inhibitor of Bacterial Histidine Kinases
J. Bacteriol., December 1, 2005; 187(23): 8196 - 8200.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/35/36250    most recent
M405884200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kulanthaivel, P.
Right arrow Articles by Peng, S.-B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kulanthaivel, P.
Right arrow Articles by Peng, S.-B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement