Identification of a Cytoplasmic Peptidoglycan Precursor in Antibiotic

Chapter 5

Identification of a Cytoplasmic Peptidoglycan Precursor in Antibiotic-Resistant Bacteria 1

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Mike S. Lee , Kevin J. Volk , Jinping Liu , Michael J. Pucci , and Sandra Handwerger

Downloaded by UNIV MASSACHUSETTS AMHERST on October 14, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch005

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Analytical Research and Development, Pharmaceutical Research Institute, Bristol-Myers Squibb, Princeton, NJ 08543-4000 Analytical Research and Development and Anti-Infective Microbiology, Pharmaceutical Research Institute, Bristol-Myers Squibb, Wallingford, CT 06492 Laboratory of Microbiology, Rockefeller University, New York, NY 10021

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Research in the area of bacterial strains resistant to antibiotic therapies has gained much attention since the recent emergence of vancomycin resistant bacteria. Vancomycin is a glycopeptide antibiotic which functions by binding directly to the D-Ala-D-Ala terminus of peptidoglycan precursors thereby inhibiting crosslinking by the transpeptidase enzyme. We have developed an analytical approach which utilizes sensitive and selective electrospray LC/MS techniques to determine the molecular weight of cytoplasmic precursors present in samples harvested from vancomycin resistant cells. Substructure analysis strategies utilizing LC/MS/MS protocols provide on-line structure identification by comparison of the mass spectrometric characteristics of unknown precursors with the substructural "template" of the standard pentapeptide precursor. Differences between resulting product ion spectra are indicative of altered and/or modified substructures. Using a novel application of affinity capillary electrophoresis (ACE) techniques, we have also obtained binding information for these precursors. Rapid screening of a variety of peptidoglycan molecules has been performed and resulting binding constants were obtained from Scatchard plots. The affinity of peptidoglycan precursors to vancomycin can be related to structure and provide a basis for proposing possible mechanisms of action. The results of these findings are significant with respect to revealing the mechanism(s) of bacteria resistance as structural evidence was obtained which confirmed the presence of a peptidoglycan precursor terminating in lactate rather than alanine. This structural information serves as a valuable platform for understanding mechanisms of resistance, and hopefully, the design of novel and unique therapies for intervention.

0097-6156/95/0619-0106$13.00A) © 1996 American Chemical Society

In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; Snyder, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


5. LEE ET AL.

Identification of a Cytoplasmic Peptidoglycan Precursor

Vancomycin is used extensively throughout the world for treatment of infections due to methicillin-resistant Staphylococcus aureus (MRSA) and Grampositive organisms in patients allergic to β-lactam antibiotics (1). Treatments utilizing vancomycin have been quite attractive and successful. This is due, in part, to its selectivity for the D-Ala-D-Ala terminus of peptidoglycan precursors thought to be ubiquitous in many bacterial cell walls. However, these treatments often represent our last line of defense tofightinfectious disease. Research involving the glycopeptide family of antibiotics has gained much attention since the emergence of vancomycin resistant bacteria (2-4). Reports of transmissible high-level resistance to vancomycin in Enterococcus faecalis have been associated with the production of a 38 kDa membrane protein VanA (5-8). This protein is responsible for the synthesis of a number of mixed dipeptides, DAla-X, which could be incorporated into a peptidoglycan precursor resulting in structural alteration and possible reduction of its affinity to vancomycin (9). This situation serves to highlight the serious and potentially disastrous implications of a microbe's seemingly endless capability for adaptation and subsequent development of resistance. Vancomycins, like other bacterial agents such as penicillins and cephalosporins, target the bacterial cell wall and enzymes called transpeptidases which are involved with cell wall synthesis. The peptidoglycan molecule is the basic building block in the bacterial cell wall and consists of both carbohydrate and peptide substructures shown in Figure 1. The carbohydrate portion of the molecule is made up of the two sugar units N-acetyl-glucosamine and N-acetylmuramic acid while the peptide consists offiveamino acids L-Ala-D-Glu-(L-Lys or m-Dap)-D-Ala-D-Ala where m-Dap is meso-diaminopimelic acid. A requisite for cell wall synthesis involves the cross-linking of peptidyl substructures on adjacent glycan strands by transpeptidation between an amine group on one strand and the penultimate D-alanine of a D-Ala-D-Ala terminus on an adjacent strand (Figure 2). The β-lactam antibiotics (penicillins and cephalosporins) work by binding to a transpeptidase or an ensemble of transpeptidases which catalyze this cross-linking process (10). Vancomycin is a glycopeptide antibiotic and inhibits peptidoglycan synthesis by binding directly to the D-Ala-D-Ala terminus thereby inhibiting cross-linking by the transpeptidase (11-12). The structure of vancomycin features a modified heptapeptide with crosslinked tyrosine residues substituted with a sugar and aminosugar (Figure 3). The core structure containing the seven amino acids is biologically active while the sugar substituents do not affect antibiotic activity in vitro. It is the heptapeptide core which binds to stem pentapeptides terminating in D-Ala-D-Ala within the bacterial cell wall layer resulting in inhibition of peptidoglycan synthesis (13-17). Inhibition of this critical process during cell wall synthesis results in the accumulation of lipid intermediates and the peptidoglycan precursor uridine diphosphate-N-acetyl-muramyl-pentapeptide (UDP-N-acetyl-muramylpentapeptide) in the cytoplasm. Recent studies performed in our laboratory focus on the application of mass spectrometry (MS) based techniques for the structure profile analysis of vancomycin resistant bacteria. New advances in coupling liquid chromatography with electrospray/mass spectrometry have resulted in new areas of research and biological applications (18-19). Our analytical approach utilizes sensitive and selective micro-liquid chromatography/mass spectrometry (micro-LC/MS) profiling techniques to determine the molecular weight (MW) of cytoplasmic


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BIOLOGICAL AND BIOTECHNOLOGICAL APPLICATIONS OF ESI-MS


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5. LEE ET AL.


109


110


Ο

Figure 3. Illustration of the binding between vancomycin and a normal peptidoglycan precursor.



5. LEE ET AL.


11

peptidoglycan precursors present in samples harvested from vancomycin resistant cells. Substructure analysis strategies utilizing tandem mass spectrometry (MS/MS) protocols provide on-line structure identification by comparison of the mass spectrometry characteristics of unknown precursors with the substructural "template" of the standard pentapeptide precursor present in non-resistant bacteria. This approach is based on the premise that the precursors of interest would be expected to retain much of the substructural characteristics of the standard peptidoglycan precursor and would therefore be expected to undergo similar MS/MS fragmentations. Identical MS/MS product ions and neutral losses are correlated and provide direct evidence for common substructures. Differences between resulting MS/MS product ion spectra are indicative of altered and/or modified substructures. This MS/MS substructure analysis strategy has been delineated in previous studies involving the rapid identification of drug metabolites (20) Recently, integrated LC/MS and LC/MS/MS profiling approaches have been utilized in our laboratories with studies involving the structure profile analysis of natural products (21-23) contained in complex matrices. Here we describe details of our recentfindings(24,25) which reveal the presence of a peptidoglycan precursor terminating in D-Ala-lactate rather than DAla-D-Ala. We also feature our subsequent work dealing with the novel application of affinity capillary electrophoresis (ACE) techniques for the rapid generation of binding or molecular recognition profiles (26). Rapid screening of a variety of peptidoglycan molecules are performed and binding constants are obtained from Scatchard plots (29). The affinity of peptidoglycan precursors to vancomycin is related to structure and provides a basis for proposing possible mechanisms of action. The analytical methodology described in this chapter highlights the powerful capabilities of nebulizer assisted electrospray ionization (ESI) techniques, particularly for the sensitive analysis of challenging and difficult to analyze samples. When incorporated within a micro-LC/MS environment, this arrangement affords excellent opportunities for application with sample limited conditions such as the structure profile analysis of peptidoglycan precursors. Sensitivity and speed of analysis are other factors worthwhile noting since peptidoglycan precursors are transient intermediates present in very low amounts. The results obtained from these micro-LC/MS structure profiles are fed into subsequent ACE binding assays. The purpose of such an integrated effort is to provide a rapid assessment of structure and binding. This combined information can serve as a valuable platform for revealing, predicting or postulating mechanisms of resistance, and hopefully, the design of novel and unique therapies for intervention. Experimental

Cytoplasmic pools of UDP-linked peptidoglycan precursors were extracted as previously described (24,25). Briefly, Enterococcus faecalis 221 was obtained by introduction of the glycopeptide resistance plasmid phKKlOO (27) into the susceptible Enterococcus faecalis JH2-2. A l l bacterial cultures including Leuconostoc mesenteroides VR1 and Lactobacillus casei ATCC 7469 were grow to midlogarithmic phase and bacitracin was added to a final concentration of 100 μg/mL to accumulate precursors. After 1 h, cultures were chilled rapidly, and cells were harvested by centrifugation and extracted with cold trichloroacetic acid (final concentration 5%) for 30 min. The supernatant fluid was separated by gel



Transfer line

Nebulizing gas

lonspray interface

UV detector

Figure 4. Schematic diagram of the microcolumn-LC//MS system.

Waste

Restrictor

lOOOOD 1OGOO0

Capillary column (250 μπι i.d.)

Injector

Splitter

Micromixer

In line filter

HPLC pump



5. LEE ET AL.


filtration (Sephadex G-25) with water elution. Hexosamine-containing fractions were identified by the assay of Ghuysen et al. (28), and they were pooled and lyophilized. Precursor extracts were chromatographically separated on-line with a Beckman System Gold high-performance liquid chromatography (Fullerton, CA, U.S.A.) and a Sciex API triple quadrupole mass spectrometer (Sciex, Thorahill, Ontario, Canada) equipped with a nebulizer assisted electrospray LC/MS interface (Figure 4). The conventional HPLC system was modified for performing microLC at low flow rates. Solvent gradients were directly delivered into a micromixer obtained from Upchurch Scientific, Inc. (Oak Harbor, WA, U.S.A.) at flow rates of 0.2-0.4 mL/min. A precolumn splitting device was used to obtain appropriate output flow rates (approximately 3 \\Umm) for packed capillary columns. The split ratio was easily regulated by adjusting the length of restriction line where a fused silica capillary with 50 μτη i.d. was used. The capillary columns (36 cm χ 250 μτη i.d., 375 μτη o.d.) were packed in-house with C18,5-μτη particle of 300 A pore size, from Vydac (Hesperia, CA, U.S.A.) using an ISCO mLC-500 pump (Lincoln, Nebraska, U.S.A). The columns were directly connected into a Valco microinjector with 100 nL and 500 nL internal loops. The transfer line from the column outlet consisted of fused silica capillary with 50 μτη i.d. and 190 μτη o.d. on-line connected to a UV detector and mass spectrometer. An ABI Model 785A UV detector (Applied Biosystem Inc., Foster City, CA, U.S.A.) equipped with Zshape capillary flow cell obtained from LCpacking (San Francisco, CA, U.S.A.) was used in this study. The mobile phase used in this system consisted of 50 mM ammonium formate at pH=6.75 (solvent A) and 30% solvent A/70% acetonitrile (solvent B). Solvent gradient (0% - 20% solvent Β over 20 minutes) was performed for all separations. The low eluent flow from the micro-LC system was coupled directly to the mass spectrometer equipped with an articulated nebulizer assisted electrospray interface with a voltage of +5300V. The mass spectrometer was scanned from m/z 400-1500 with a step size of 0.4 u and an acquisition time of 2.6s per scan. MS/MS experiments were performed on the doubly charged M H 2 ions of the precursors with a collision energy of 45 eV and an argon collision gas thickness of 400 X 1 0 atomsfcm . Experiments involving ACE were performed with a Beckman P/ACE Model 2100 CE system (Fullerton, CA, U.S.A.). An untreated fused silica capillary (Polymicro Technology, Phoenix, AZ, U.S.A.), 57.3 cm X 50 μτη i.d., was used within a capillary chamber maintained at 25°C. The resulting components were monitored using the UV detector which was set at 254 nm. All binding assays were performed using an open-tubular ACE system described previously (26). The running buffer consisted of 0.2 M glycine and 0.03 M Tris at pH=8.30. A 25 μΜ vancomycin stock solution was used as ligand substrate added into the running buffer. A neutral marker, mesityl oxide (0.2 mg/mL), was utilized as a reference for the measurement of relative migration times reported (29). A 10 μ ί aliquot of each peptidoglycan precursor was mixed with 10 |iL of mesityl oxide and used directly for injection without further sample purification. Pressure injection was utilized with a duration time of 20 seconds, which represents about 20 nL of sample volume injected. A series of running buffers containing appropriate concentrations of vancomycin ligand was used to measure migration shifting with precursors. The relationship between ligand concentration and migration time can be illustrated using Scatchard analysis affording the determination of binding constants or dissociation constants. 2+

1 2

2


113



J

0.0

10.0

20.0

Retention Time (min)

Figure 5. Microcolumn-LC/UV chromatogram of the precursor extract obtained from Enterococcus faecalis. The asterisk denotes the peak corresponding to the modified peptidoglycan precursor.


5. LEE ET AL.


115


Structure Analysis Mixtures of peptidoglycan precursors extracted from vancomycin resistant bacteria were characterized using micro-LC/MS profiling techniques. As discussed previously, vancomycin acts by binding to the D-Ala-D-Ala terminus of the stem pentapeptides present in bacterial peptidoglycan. Although it was considered highly unlikely that vancomycin resistance would develop since the DAla-D-Ala terminus is ubiquitous among bacterial species, vancomycin resistant Enterococcus faecalis strains have been identified. In addition, other species such as Leuconostoc mesenteroides and Lactobacillus casei are known to be intrinsically resistant to high levels of glycopeptide antibiotics such as vancomycin. Based on the development of resistant strains, it was postulated that vancomycin resistant bacteria could produce a stem pentapeptide terminating in something other than D- Alanine. The initial goal of these studies was to develop analytical methodologies to profile and characterize precursor extracts from various types of bacteria. To determine whether the mechanism of acquired resistance in specific strains of Enterococcus faecalis, and intrinsically resistant Leuconostoc mesenteroides and Lactobacillus casei was similar, cytoplasmic precursors from resistant and non-resistant strains of Enterococcus faecalis were obtained and profiled on-line using micro-LC/MS and compared to those of Leuconostoc mesenteroides and Lactobacillus casei. A tripeptide precursor, UDPN-acetyl-muramyl-L-Ala-D-Glu-L-Lys (MW 1107), isolated by inhibition of murein synthesis in the presence of D-cycloserine and the normal UDP-linked pentapeptide precursor, UDP-N-acetyl-muramyl-L-Ala-D-Glu-L-Lys-D-Ala-DAla (MW 1149), isolated from Staphylococcus aureus, were used as reference materials and substructural "templates". The micro-LC separation of precursor extracts from Enterococcus faecalis, Leuconostoc mesenteroides, and Lactobacillus casei each demonstrated a peak which eluted later than the normal pentapeptide precursor from Staphylococcus aureus. Figure 5 illustrates the micro-LC/UV chromatogram of the precursor extract from Enterococcus faecalis and indicates the modified precursor is a component in the mixture. Full scan mass spectra containing both singly charged and doubly charged ions were generated from the on-line analysis of the standard and precursors. The standard pentapeptide precursor isolated from Staphylococcus aureus has a molecular weight of 1149 Da. The corresponding full scan mass spectrum of the standard pentapeptide precursor is shown in Figure 6 and illustrates the ions corresponding to the protonated (MH+) and doubly charged (MH2 ) molecular ions accompanied by a fragment ion at m/z 746 corresponding to a neutral loss of the uridine diphosphate (UDP) substructure (404 Da) from the MH+ species. This full scan fragmentation information is specific and can be used diagnostically to identify peptidoglycan precursors in complex mixtures and indicate modifications of the N-acetyl-muramyl-peptide substructure. The molecular weight analysis performed on extracts from Enterococcus faecalis and Lactobacillus casei indicated a modified precursor containing the UDP substructure with a MW of 1150 Da. Micro-LC/MS analysis of the extract obtained from Leuconostoc mesenteroides indicated a precursor with a MW of 1221 Da. Substructural "templates" were generated for the tripeptide and normal pentapeptide precursors using micro-LC/MS/MS techniques. The association of specific MS/MS product ions with unique substructures or fragment ions of known precursors provides a basis for the interpretation of the MS/MS substructural data 2+



116


100-

576.0

243,000

+ 2

Standard Pentapeptide (MW1149)

75

MH+

50-

1150.4

25

.UDP 746.4

400

L

600

1000

800

1200

m/z

Figure 6. A typical full scan mass spectrum of the standard pentapeptide precursor isolated from Staphylococcus aureus. This spectrum was obtained from a standard averaged over 8 seconds.


5. LEE ET AL.


for the modified precursors. The MS/MS product ion spectrum corresponding to the MH2 * species of the standard pentapeptide precursor is shown in Figure 7. Neutral loss of the UDP substructure is a facile fragmentation route resulting in the singly and doubly charged product ions at m/z 746 and m/z 374, respectively. Cleavage of the muramyl-peptide ether linkage also yields a product ion which provides MW information for the peptide portion of the peptidoglycan precursor. Several important fragmentations occur in die peptidic backbone which helps to delineate the portion of the precursor which is modified. One common pathway involves cleavage of the Glu-Lys amide bond to yield a Y series product ion at m/z 289 (Lys-Ala-Ala substructure) and cleavage of the Ala-Glu amide bond to yield a Y series product ion at m/z 418 (Glu-Lys-Ala-Ala substructure). A second important fragmentation pathway involves the neutral loss of the C-terminal AlaAla substructure from the product ion at m/z 418 to produce a product ion at m/z 258. The neutral loss of the C-terminal and penultimate amino acids, such as AlaAla in the standard pentapeptide precursor, is a common fragmentation theme for all of the peptidoglycan precursors studied. Previous studies in other laboratories have indicated key proteins (VanH and VanA) involved in peptidoglycan synthesis which are modified in resistant bacteria and appear to have altered substrate specificity (9). Hydroxy acids such as D-2-hydroxybutyrate were proposed as potential substrates in these studies. In studies performed in our laboratories, we have utilized LC/MS/MS methodologies to provide detailed information about modified precursors which indicate hydroxybutyrate is not incorporated into the modified peptidoglycan precursors we have characterized. The MW (1150 Da) and product ion spectra of die precursors from Enterococcus faecalis and Leuconostoc mesenteroides were identical. The MS/MS product ion spectra of the doubly charged MH2 * species of the precursors from Leuconostoc mesenteroides, Lactobacillus casei, and Enterococcus faecalis were consistent with substructures of modified pentapeptide and modified tetrapeptide precursors. Comparison of the MS/MS product ion spectra of the precursors from Enterococcus faecalis, Lactobacillus casei (Figure 8) and Leuconostoc mesenteroides (Figure 9) to the fragmentation "template" of the normal pentapeptide obtained from Staphylococcus aureus (Figure 7) indicated an alteration of an amino acid near the C-terminus for each precursor. As discussed previously, a key fragmentation route common to each precursor involved cleavage of the Glu-Lys amide bond. Y-series fragment ions were observed consistent with Lys-Ala-Ala (m/z 289), Lys-Ala-Lactate (m/z 290), and Lys-(Ala)Ala-Lactate (m/z 361) substructures for the standard pentapeptide precursor {Staphylococcus aureus), modified tetrapeptide precursor {Lactobacillus casei and Enterococcus faecalis), and modified pentapeptide precursor {Leuconostoc mesenteroides), respectively. Cytoplasmic hexapeptide precursors in bacterial species with interpeptide bridges are present in some Leuconostoc mesenteroides and Lactobacillus casei species. In these organisms, the first amino acid of the interpeptide bridge is added to the e-amino group of the L-Lys at the level of the UDP-linked cytoplasmic precursor radier than at the lipid intermediate level (30). Consecutive MS/MS fragmentation resulting in the neutral loss of the Ala-Lactate substructure from the Y-series product ions (Glu-Lys-Ala-Lactate and Glu-Lys(Ala)-Ala-Lactate) provide additional complementary evidence for the proposed structural modification. Although not fully utilized in these studies, MS/MS screening techniques such as constant neutral loss or precursor scans may be employed on a wider scale to evaluate other species of bacteria suspected of producing modified precursors. For 2


117

2



25Θ 289

,

Ull

200

m/z

MH

600

576

561

2

2 +

746

800

•Ala-Ala

I

NH

I

Q=Q

2+

COOH

CH-CH

NH

I

CH-CH

2

2

3

3

129*

H Ç—(CH )T-NH

289* 2H

Figure 7. The MS/MS product ion spectrum corresponding to the MH2 species of the standard pentapeptide precursor isolated from Staphylococcus aureus.

400

18472

«>5

• i . J U .. 1.4- ι JUl

6

186

a . JJ

374

47T

2

CH OH


2

2

2

138*

CH 0

168*

CH

3

I -H 0

NH

?-°

186*

Ο"

2

CH OH^.


Figure 8. The MS/MS product ion spectrum corresponding to the MH2 species of the precursors from Lactobacillus casei.

m/z 2+


OZl Downloaded by UNIV MASSACHUSETTS AMHERST on October 14, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch005

SIM-ISS ΛΟ SMOlLVOndJV ΤνθΙΟΟΊΟΜΗ33ΧΟΙβ QMV TVDIOOTOia Relative Intensity (%)


5. LEE ET AL.


example, a constant neutral loss scan corresponding to the UDP substructure may be used to screen complex samples for the presence of peptidoglycan precursors.


Binding Studies The identification of a peptidoglycan precursor terminating in Ala-Lactate resulted in efforts directed toward establishing a general binding assay to further confirm these findings. A novel ACE approach was developed and incorporated into our investigations to provide a rapid assessment of the binding affinity of vancomycin to these precursors and provide additional evidence for the identification of altered peptidoglycan precursors. The normal pentapeptide precursor isolated from Staphylococcus aureus (UDP-N-acetyl-muramyl-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala) was used as a model system in this portion of our studies. The proposed structure of this precursor together with its binding to vancomycin is displayed in Figure 3. Based on the proposed hydrogen bond interaction of vancomycin and the terminus of the precursor, a modification of the amide linkage to an ester linkage as indicated by the arrow in Figure 3 could disrupt the binding process (9). Affinity interaction between the precursor and vancomycin is mediated by a series of five hydrogen bonds between the dipeptide backbone and the glycopeptide. It is known that the amide linkage and the free terminal carboxyl group are essential for vancomycin binding (13-17). Modification or replacement of these functional groups (by other structural moieties) may significantly reduce their interaction with vancomycin (30). The binding behavior of this normal precursor with vancomycin serves as a binding profile "template" for subsequent studies involving other precursors. Vancomycin was selected as the substrate ligand (added directly into the operating buffer) while peptidoglycan precursors were used as the probe for monitoring migration shifting. A commonly used biological buffer consisting of 0.2 M glycine/0.03 M Tris (pH=8.30) was selected. Two typical electropherograms obtained under conditions with and without vancomycin in the buffer is shown in Figure 10. The neutral marker, mesityl oxide, has the same migration time (2.9 min) under both conditions. Upon die addition of 25 μΜ vancomycin to the buffer, the precursor peak shifts towards the reference peak corresponding to a shift in migration time from 4.3 to 3.9 min. The complexation between the precursor and vancomycin results in a reduction of net charge and the complex is thus "dragged" towards the cathode (detection window) somewhat faster than the unbound component due to the electroosmotic flow generated under present experimental conditions. The observed change in mobility of the complex indicates that the normal pentapeptide precursor (UDP-N-acetyl-muramyl-L-AlaD-Glu-L-Lys-D-Ala-D-Ala) from Staphylococcus aureus, is bound to vancomycin. As expected, the formed complex has a different mobility from the original free precursor. A single component appears in both the micro-LC chromatogram and the CE electropherogram for the normal peptidoglycan precursor from Staphylococcus aureus. The major components present in the extracts correspond to precursors, and similarities in structure are inferred from migration times. It is important to note that the precursors studied here are prepared from cytoplasmic extracts and pre-purified by gelfiltrationprior to other analyses. A series of buffers containing various concentrations of vancomycin ranging from 0 to 200 μΜ were prepared for binding constant determination. The corresponding series of ACE binding profiles as a function of ligand concentration are shown in Figure 11. An increase of vancomycin concentration results in gradual migration shifting of the precursor towards the reference peak. Binding


121


122


0.0

2.0

4.0

6.0

Time (mln)

Figure 10. Evaluation of vancomycin binding to pentapeptide precursors from Staphylococcus aureus by affinity capillary electrophoresis. 1 - neutral marker; 2 - UDP-N-acetyl-muramyl-L-ala-D-glu-L-lys-D-ala-D-ala. Buffer in upper trace (A): 0.2 M glycine/0.03 M Tris (pH=8.30) and additional 25 μΜ vancomycin, and in lower trace (B): 0.2 M glycine/0.03 M Tris (pH=8.30) without vancomycin. Sample injection: 20 s; Operating voltage: 25 kV (7 μΑ).


5. LEE ET AL.


Strain 209P


neutral marker Vancomycin (μπι)

0.0

5.0

15.0

Φ

υ c

25.0

50.0

ZD

100.0

200.0

0.0

2.0

4.0

6.0

Time (min)

Figure 11. Affinity capillary electrophoresis of pentapeptide precursors from Staphylococcus aureus using 0.2 M glycine/0.03 M Tris (pH=8.30) containing vancomycin with concentrations ranging from 0 to 200 μΜ. Instrumental conditions are the same as in Figure 10.


123


124


0.00

-0.08 H 1.0

1

•

1

1.1

•

1

1

1.2

•

•

1

1.3

·

•

1.4

Rf

Figure 12. Scatchard plot from data obtained in Figure 11 for the determination of the binding constant for the peptidoglycan precursor from Staphylococcus aureus. Rf = dDt/ dDt , dDt is the relative migration shift of receptor at saturating concentration of ligand, [L] is the concentration of ligand. s

s



5. LEE ET AL.


saturation was observed at a concentration of about 200 μΜ vancomycin. Peak broadening was observed at some intermediate concentrations. This condition is generally caused by the retardation of migrating molecules due to their frequent interactions with the ligand. A receptor-ligand binding interaction in biological studies is generally a thermodynamic equilibrium process. Relaxation times between free receptor and its saturated complex with the ligand appear to take longer in the range of intermediate substrate concentrations, reflecting the slow process of equilibration between species with different migration times, which results in slightly broader peaks. The precursor peak becomes sharper at the saturating concentrations of the ligand. Similar results were also reported by simulation studies of this phenomenon (29). A possible explanation for decreased peak height of the neutral marker may be due to the evaporation of mesityl oxide which is miscible with common organic solvent and evaporates slowly. It should be emphasized that this change has no effect on the measurement of the relative migration time as long as the migration time of the neutral marker remains unchanged and measurable. Based on some assumptions dealing with interaction, equilibrium and surface absorption, equations relating the migration time to binding constant were proposed (29,32). The general equation for Scatchard analysis in ACE studies was derived as Rf/[L] = K - Ai>Rf, where Rf = dDt/ dDt , dDts is the relative migration shift of receptor at saturating concentration of ligand, [L] is the concentration of ligand, and K is the binding constant. A Scatchard plot derived from these experiments (Figure 12) displays linearity with a correlation coefficient of 0.97. The binding constant, K measured is 1.6x10 M ' and the dissociation constant, K was found to be 6.25 μΜ, which compares well with those obtained from other assays for structurally similar compounds (9,11). Binding profiles were also obtained for precursors isolated from Leuconostoc mesenteroides and Lactobacillus casei using the method established for vancomycin-peptidoglycan precursor binding discussed above. Figure 13 displays two electropherograms obtained from Leuconostoc mesenteroides under the same conditions as previously described. The cell extract was directly injected without further purification. No apparent migration shifting occurs under these conditions which indicates the bioaffinity of this precursor to vancomycin has been reduced to a great extent, suggesting a structural modification at the terminal amino acid. These findings appear to be consistent with our earlier micro-LC/MS/MS studies which revealed a modified terminus corresponding to Ala-Lactate. A similar lack of migration shift was also observed for the precursor from Lactobacillus casei, as shown in Figure 14, supporting an altered C-terminus structure. The amide hydrogen of the C-terminal D-Ala-D-Ala has been reported to be a crucial binding site involved in the formation of hydrogen bonds, which binds specifically to the carbonyl oxygen of the (p-hydroxyphenyl) glycine residue located at the "core" center of vancomycin (17). The modified structure resulted from the incorporation of a depsipeptide terminating in D-lactate which no longer possesses this amide linkage, and therefore, loses its capacity to hydrogen-bond and its affinity to vancomycin. This substitution of the amide NH for oxygen has been reported to result in at least 1000-fold lower binding (9,11), which is not detectable by this assay. h

s

h

5

1

bf

d

Conclusions and Future Prospects Our structural studies dealing with abnormal precursors provide useful information aimed at developing a better understanding of molecular recognition and potential mechanisms of resistance. These findings along with previously


125

126



Strain VR1

neutral marker

Β

ο

Buffer with 25 μπι vancomycin

_

δ c

I Buffer

Γ 0.0

2.0

- ι

1

r-

4.0

—r6.0

8.0

Time (mln)

Figure 13. Evaluation of vancomycin binding to pentapeptide precursors from Leuconostoc mesenteroides by affinity capillary electrophoresis. 1 - neutral marker; 2 - UDP-N-acetyl-muramyl-L-ala-D-glu-L-lys-(L-ala)-D-ala-Dlactate. Instrumental conditions are the same as in Figure 10.


LEE ET AL.



Strain 7469

—·

0.0

1

1

1

2.0

1

i

1

1 4.0

1

1

>

1

6.0

'

«

«

9.0

Time (min)

Figure 14. Evaluation of vancomycin binding to pentapeptide precursors from Lactobacillus casei by affinity capillary electrophoresis. 1 - neutral marker; 2 - UDP-N-acetyl-muramyl-L-ala-D-glu-L-lys-D-ala-D-lactate. Instrumental conditions are the same as in Figure 10.


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128


described biological data (9,24-26,33) demonstrate that the mechanism of glycopeptide resistance found in Enterococcus faecalis, Leuconostoc mesenteroides, and Lactobacillus casei involves utilization of a peptidoglycan precursor terminating in lactate rather than alanine. The micro-LC and CE based techniques described in this chapter represent extremely powerful approaches for the separation, analysis and characterization of complex biological mixtures. The high separation efficiency of micro-LC in combination with MS/MS provides a rapid and systematic assessment of MW and structure profile. Subsequent ACE assays generate binding profiles for a variety of precursors. Certainly, the strategies described here are suitable for a wide variety of applications. Micro-LC/MS methods offer distinct advantages over conventional LC/MS systems. First, reduced column diameters result in enhanced mass sensitivity. Also, high linear mobile phase velocities can be generated facilitating fast separations. These analytical features are quite useful with sample-limited applications. The novel ACE approach affords many useful advantages dealing with high separation efficiency, high sensitivity and an extremely rapid analysis time. Similar to other CE based techniques, A C E also offers advantages of reproducibility and ease of automation. Perhaps the most attractive feature of ACE is its unique ability to profile receptor-ligand interactions with only small amounts of non-radiolabeled sample. High protein purity or accurate values of concentration are not requisites for ACE binding analyses. These factors are quite attractive for the profile analysis of simultaneous binding events (receptor-ligands) occurring in the same solution. The advantages of modern LC/MS techniques for the profile analysis of complex mixtures are evident in mis work as well as other fundamental studies and applications. To continue and further delineate these advantages would likely be redundant and unnecessary for the purposes of this book However, we should pause and recognize the significance of the current quality of LC/MS technology and instrumentation available today. Continued exploitation of the analytical advantages offered by the LC/MS interface (nebulizer assisted electrospray) will result in numerous and significant advances across scientific disciplines. As in the past, we should follow these advances closely while "stopping" along the way to participate, share and learn. We realize that application of tins valuable technology has led our investigations of today to places, destinations and collaborations that were simply visions 5-10 years ago. The extension of analytical methodologies involving the LC/MS interface will likely continue beyond micro-LC or CE. The anticipation of newer and improved technologies will no doubt culminate in novel and unique applications. The future appears bright, signaling a new beginning for LC/MS based techniques which appear ready to assume expanding roles in science and play an integral part in providing a faster rate of discovery than ever experienced. References 1. 2. 3.

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Identification of a Cytoplasmic Peptidoglycan Precursor in Antibiotic

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