Analysis of Neisseria gonorrhoeae Peptidoglycan by Reverse-Phase, High-Pressure Liquid Chromatography

Transcription

1 JOURNAL OF BACTERIOLOGY, JUIY 1985, p Vol. 163, No /85/769-6$2./ Copyright 1985, American Society for Microbiology Analysis of Neisseria gonorrhoeae Peptidoglycan by Reverse-Phase, High-Pressure Liquid Chromatography THOMAS J. DOUGHERTY Laboratory of Microbiology, The Rockefeller University, New York, New York 121 Received 13 February 1985/Accepted 2 April 1985 The muramidase digest of peptidoglycan from Neisseria gonorrhoeae was isolated and analyzed by the use of a reverse-phase, high-pressure liquid chromatography system. As was found previously in the case of Escherichia coli, gonococci peptidoglycan is also composed of a greater number of muropeptides than can be resolved with thin-layer chromatography systems. Preliminary classification of the muropeptide components into subclasses based on O-acetyl modification and degree of cross-linkage was achieved. Examination of a penicillin-susceptible strain and a highly resistant strain with two penicillin-binding protein alterations synthesized distinctly different peptidoglycan structures, as revealed by this technique. All gram-negative bacteria studied possess one chemical type of peptidoglycan, Aly, and variations have been believed to be limited to degree of muropeptide cross-linkage, degree of amidation of the carboxyl groups of the peptide subunit, and O-acetylation of the disaccharide (15, 21). Recent investigations by Glauner and Schwarz, who used the extreme resolving power of reverse-phase, high-pressure liquid chromatography (HPLC), established that Escherichia coli peptidoglycan has a much greater heterogeneity in muropeptide structure than previously thought (5). Variations include substitution of amino acids, such as glycine for alanine, and direct cross-links between diaminopimelic acid residues at the site of lipoprotein attachment. These findings raise the possibility of specialized domains within the peptidoglycan structure and roles for the multiple penicillin-binding proteins (PBPs) in assembly of these regions. In another recent study, this technology was used to characterize several modifications that the peptidoglycan of E. coli undergoes during transitions in growth states (11). In the present study, the reverse-phase HPLC system was used for examination of the peptidoglycan structure of Neisseria gonorrhoeae. A complex, reproducible pattern of muramidase-digested peptidoglycan fragments was obtained. The fragments were classified into groups by the degree of peptide cross-linkage and O-acetylation, and preliminary identification of some species was assigned. Peptidoglycan from a highly penicillin-resistant strain with reduced binding affinity in two of its PBPs was examined. Several differences in the peptidoglycan digest from penicillin-susceptible and -resistant strains of gonococci were evident. MATERIALS AND METHODS Strains and growth conditions. N. gonorrhoeae FA19 was obtained from P. F. Sparling, University of North Carolina, Chapel Hill, N.C., and strain CDC was from the Centers for Disease Control, Atlanta, Ga. These strains have been extensively characterized in previous studies (2, 3). A series of non-beta-lactamase-producing, high-level penicillin-resistant strains was from J. Biddle, Centers for Disease Control. Several penicillin-susceptible isolates were obtained from the clinical laboratory at the New York Hospital- Cornell Medical Center, New York, N.Y. Strains were kept frozen in gonococcal broth with 15% glycerol at -7 C. 69 Stocks were thawed, and cultures were maintained by daily passage on GCBA agar plates with 1% IsoVitaleX supplement (BBL Microbiology Systems, Cockeysville, Md.). Incubation was at 37 C in 5% CO2. Colonies were p-, transparent by the criteria of Swanson (19). Peptidoglycan preparation. Gonococci were inoculated into 5-ml volumes of gonococcal broth with 1% IsoVitaleX and 42,ug of NaHCO3 per ml. These cultures, in 2-liter flasks, were grown at 37 C with agitation (14 rpm) in a New Brunswick G-25 shaker (New Brunswick Scientific Co., Inc., Edison, N.J.). Growth was monitored at frequent intervals with a Sequoia-Turner model 34 spectrophotometer at 61 nm. At absorbance values of.6 to.7 (ca. 246 mg/liter [dry weight], 5 x 18 CFU/ml), culture vessels were swirled in ice-alcohol baths with temperature monitoring. Culture temperatures dropped from 37 to 2 C in less than 5 min. This was essential to avoid autolysis artifacts. Cells were collected by centrifugation (Sorvall RC-2B, DuPont-Sorvall, Wilmington, Del.) at 8, x g for 1 min at 2 C. The cells were washed once with ice-cold 2 mm sodium acetate (ph 5.) and then were resuspended in 15 ml of the same buffer on ice. The cells were added dropwise to 15 ml of 1% sodium dodecyl sulfate (SDS) buffered with 2 mm acetate (ph 5.) at 1 C. After all of the cells were added (ca. 5 min), the SDS extraction at 1 C was continued for 2 min. After cooling, the SDS-insoluble material was collected by centrifugation (Beckman L5-5, Beckman Instruments, Inc., Palo Alto, Calif.) at 8, x g for 3 min. The material was then washed by centrifugation three times with glassdistilled water. The SDS-insoluble material was treated for 1 h with 2,ug of a-amylase (Sigma Chemical Co., St. Louis, Mo.) and then with 2,ug of pronase (Pronase-CB, Calbiochem-Behring, La Jolla, Calif., preheated for 1 h at 6 C). Both incubations were in 2 mm Tris-chloride (ph 7.4) and 1 mm NaCl at 24 C. After 2 h of pronase treatment, the SDS-insoluble fraction was reextracted with.1% SDS at 1 C for 5 min. The material was collected and washed by centrifugation, as described above, four times. After all washes, residual fluid was carefully removed with a Pasteur pipette, leaving a clear, gelatinous pellet. The peptidoglycan pellet was resuspended in distilled water by brief (5-s) treatment with a low-power sonicator (Kontes Micro Disruptor, Kontes Co., Vineland, N.J.). The final pellet was

2 7 DOUGHERTY J. BACTERIOL. An Be salt ni I origin 1 I \ t i.. IK I H G..!.-A - T -----I , 1 12 MIN FIG. 1. (A) Separation of radiolabeled gonococcal peptidoglycan digest by thin-layer chromatography in isobutyric acid-ammonia system (2). Detection was by fluorography. Components: 1, mono-o-acetylated disaccharide monomer; 2, disaccharide monomer; 3, di-o-acetylated bis-disaccharide dimer; 4, mono-o-acetylated bis-disaccharide dimer; 5, bis-disaccharide dimer; 6, oligomers. (B) Reverse-phase HPLC pattern of strain FA19 gonococcal peptidoglycan. The salt peak contained both the borate buffer and the muramidase used in the digestion. suspended in 2,ul of 25 mm sodium phosphate buffer (ph 6.5) with.1 mm MgCl2. Streptomyces globisporus muramidase (Miles Scientific, Naperville, Ill.) was added to a concentration of 25,ug/2 RI1 (ca. 55 U). Sodium azide was added (.5%), and the material was incubated at 37 C for 18 h. Before analysis, the digested peptidoglycan was reduced with sodium borohydride (5) to prevent anomerization at the Cl position. The digested peptidoglycan was mixed with an equal volume of.5 M borate buffer (ph 8.) in which NaBH4 (1 mg/ml) had been freshly dissolved. After exactly 2 min, the reaction was terminated by the addition of phosphoric acid, and the reaction mixture was brought to a final ph of ca. 4. and filtered (.2-,um pore size filter). Because of the effect of high ph on O-acetyl groups, the O-acetyl data were not considered quantitative. Reverse-phase HPLC analysis. The HPLC system consisted of two Waters 51 pumps (Waters Associates, Inc., Milford, Mass.), a Waters 721 system controller, a Waters U6K injector, and a Waters 73 Data Module that integrated the areas under the curves and performed other documentation functions. Detection of the peptide side chains on the peptidoglycan was at 25 nm in an Isco V4 detector (Isco, Lincoln, Nebr.) equipped with a heat exchanger flow cell with a 1-mm path length and 7-,u volume. The column was a Shandon ODS-Hypersil (4.6 by 25 mm; 5 p.m particles; Shandon Southern Instruments Inc., Sewickley, Pa.). The analysis consisted of a linear gradient from 5 mm phosphate (ph 4.33) to 5 mm phosphate buffer (ph 5.1) with 15% methanol over 12 min at a flow rate of.5 ml/min. Final conditions were held for an additional S min. HPLC grade reagents were from Burdick & Jackson Laboratories Inc. (Muskegon, Mich.). Sodium hydroxide (solid) and orthophosphoric acid were analar grade from BDH Chemicals, Ltd. (Poole, England). Determinations of buffer ph values, which were critical, were made on an Orion Research model 71 A digital ionalyzer with an Orion Ii i i1# research grade electrode (Orion Research Inc., Cambridge, Mass.). Buffers for ph standardization were from Corning (Corning Science Products, Medfield, Mass.) and were referenced to National Bureau of Standards standards at +.1 ph unit. Peptidoglycan fractionation. Peptidoglycan prepared from a 5-ml culture of FA19S was mixed with 3H-labeled peptidoglycan (3, cpm) and digested with muramidase as described above. The material was loaded onto coupled Sephadex G-5-G-25 columns (1.8 by 7 cm each; Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) and eluted with.1 M LiCl (4). Fractions of 1.2 ml volume were collected, and 1-,ul samples were counted in Ultrafluor scintillation fluid (National Diagnostics, Inc., Somerville, N.J.). Each set of peak fractions was pooled, taken to dryness on a rotary evaporator, and suspended in 1 ml of water. The individual peaks were desalted on a Sephadex G-1 column (1.8 by 1 cm) eluted with distilled water. The material was concentrated again, reduced with borohydride as described above, and injected into the HPLC system. The 1-6 anhydro muramyl peptide was prepared by R. Rosenthal, Indiana University School of Medicine, Indianapolis, Ind. An E. coli anhydromuramidase preparation was used to digest the gonococcal peptidoglycan, and the anhydro muramyl monomer was isolated. Amino acid analysis. Peptidoglycan was hydrolyzed for 4 and 18 h in 5.8 N HCl at 11 C in vacuo. The use of two hydrolysis times allowed estimation of the degree of muramic acid and glucosamine breakdown under these conditions. Samples were analyzed on a Durrum analyzer, model D-5. RESULTS Basic characteristics of gonococcal peptidoglycan. Gonococcal peptidoglycan from strain FA19 was prepared as described above, and a portion was subjected to amino acid analysis after acid hydrolysis. A composition identical to

3 VOL. 163, 1985 HPLC ANALYSIS OF GONOCOCCAL PEPTIDOGLYCAN 71 a > * 4 * E~ 1 ) FRACT ION (1.2 ml) FIG. 2. Separation of muramidase-digested gonococcal peptidoglycan on coupled Sephadex G-5 and G-25 columns. Peak fractions: 1, tetramer; 2, trimer; 3, dimer; and 4, monomer. For the subsequent analysis by HPLC, tetramer and trimer peaks were combined before desalting on Sephadex G-1. Peak identities were confirmed by thin-layer chromatography with known standards. Void and total column volumes for the coupled columns are indicated. that previously reported by others was obtained, including small amounts of glycine and aspartic acid present (9, 14, 22). The remaining portion was digested with S. globisporus muramidase. After reduction with borohydride, the material was adjusted to ph 4., and 5 to 1,ug of peptidoglycan fragments was injected into the reverse-phase HPLC system. The resulting chromatograph, and an analysis of peptidoglycan by thin-layer chromatography (2), are shown in Fig. 1. Several notable features are evident. The thin-layer system sorts components solely on the basis of cross-linkage and O-acetylation. There are by reverse-phase HPLC analysis ca. 13 major species of muropeptide that comprise about 7% of the total peptidoglycan. The 4 to 45 minor peaks make up the remaining 3% of the material. During the early stages of this work, several peaks were noted to be metastable. This instability could be attributed to two sources, namely, the borohydride reduction at high ph, at which the O-acetyl groups can be removed from the disaccharide units (1), and the highly active system of peptidoglycan hydrolases present in the gonococcus (6, 12, 16). Standardization of the reduction time to exactly 2 min for each sample greatly improved the reproducibility of the system. Care must also be exercised during the initial cell harvesting, when rapid cooling is essential. For a preliminary assessment of the structural features of the muropeptides, a preparation of strain FA19 peptidoglycan was mixed with.2,uci of [3HJglucosamine-labeled FA19 peptidoglycan (2) and digested with muramidase. The digest was chromatographed on coupled Sephadex G-5-G- 25 columns in.1 M LiCl. The peaks, detected by radioactivity, were identified as disaccharide peptide monomers, bis-disaccharide peptide dimers, and higher oligomers (Fig. 2). After concentration and desalting, these peaks were injected separately into the reverse-phase HPLC system (Fig. 3). The elution order was disaccharide monomers (15 to 65 min), bis-disaccharide dimers (55 to 95 min), and the higher oligomers (8 to 12 min). Two of the disaccharide monomers had retention times identical to those of E. coli disaccharide tripeptide (22 min) and disaccharide tetrapeptide (35 min). Interestingly, the distribution of these two components was ca. 25% tripeptide and 75% tetrapeptide, which agrees well with previously reported values for gonococci (14). In the bis-disaccharide class, only E. coli bis-disaccharide connected by two tetrapeptide side chains was unambiguously identified with a gonococcal component (82 min) of identical retention time. In the oligomers, the Tris-disaccharide with three tetrapeptides was tentatively found at 11 min. Treatment of O-acetylated peptidoglycan under basic conditions (NH4H, ph 1 for 6 h) removes the majority of the O-acetyl groups from the disaccharide residues (1). This allowed assignment of the O-acetylated residues in the monomer, dimer, and oligomer classes (Fig. 3). A small number of additional minor peaks that eluted after the salt peak were noted after the treatment with base; these are the beta-elimination products (2). Gonococcal peptidoglycan was treated with 1-6 anhydro muramidase isolated from E. coli and fractionated to obtain the 1-6 anhydro peptidoglycan monomer. The main product peak (Fig. 3) eluted much later than the other monomeric products. Analysis of peptidoglycan from a gonococcal strain with high-level penicillin resistance. Peptidoglycan was prepared from FA19, a penicillin-susceptible strain (MIC,.1,ug/ml), and CDC , a highly resistant (MIC, 2.,ug/ml) gonococcus. These two strains differ in that two of the three PBPs in the resistant strain have a much-reduced affinity for

4 72 DOUGHERTY + J. BACTERIOL. Downloaded from MIN FIG. 3. Analysis of peptidoglycan digest fractions from Sephadex columns by HPLC. (A) HPLC pattern of disaccharide monomers; (B) HPLC pattern of bis-disaccharide dimers; (C) HPLC pattern of combined trimers and tetramers. Tentative identifications of the O-acetylated muropeptides, made in a separate experiment by treatment with base, are indicated by solid arrows. Open arrow indicates the position of the 1-6 anhydro monomer generated by treatment of gonococcal peptidoglycan with the E. coli anhydromuramidase. Tentative identifications of disaccharide tripeptide (a), disaccharide tetrapeptide (b), bis-disaccharide tetrapeptide, tetrapeptide (c), and Tris-disaccharide tetrapeptide, tetrapeptide, tetrapeptide (d) are indicated. on September 5, 218 by guest penicillin (3). The PBPs are involved in the terminal stages of peptidoglycan assembly, so it was of interest to examine the structural details of peptidoglycan from these organisms. Figure 4 shows the peptidoglycan digest reverse-phase HPLC patterns of these two gonococcal strains. A number of differences, indicated in the figure by arrows, are evident. Most of these changes appear to be either amplification or diminution of preexisting muropeptides. DISCUSSION Analysis of the peptidoglycan structure of N. gonorrhoeae indicated that, similar to the situation in E. coli (5), the

5 VOL. 163, 1985 HPLC ANALYSIS OF GONOCOCCAL PEPTIDOGLYCAN 73 A U) (4c B Downloaded from I I I I I MIN FIG. 4. HPLC pattern of peptidoglycan digest from a penicillin-susceptible strain and highly penicillin-resistant strains of N. gonorrhoeae. Peptidoglycan was prepared and muramidase digested from (A) FA19 (MIC,.1,ug of penicillin per ml) and (B) CDC (MIC, 2.,ug of penicillin per ml). Areas of major difference between the strains are indicated by arrows. muramidase digestion products present in gonococci are more numerous than previously believed (Fig. 1). In the present study, several techniques were used to obtain a preliminary classification of the muramidase-treated peptidoglycan products into subgroups. In general terms, disaccharide peptide monomers have shorter retention times than bis-disaccharide peptide dimers, which in turn are less retarded than the oligomers. There is considerable overlap among these classes (Fig. 3). Another point evident in Fig. 3 is that the number of major monomeric species is very high compared with that of the dimer and oligomeric digestion products. One might expect that if bis-disaccharide peptide dimers were composed of any possible combination of disaccharide monomers, then there should be a much larger repertoire of the dimer form. The fact that this is not evident (nor is it found in the trimer case) and only a few major species predominate may indicate that the transpeptidase system responsible for cross-linking may have tight restraints on donor-acceptor structure. It remains to be seen whether the large number of minor peaks in Fig. 3B may represent rare cases of these possible combinations. Isolation of the component peaks in quantities sufficient for further chemical characterization is under way. It is anticipated from data in the literature that some of the peaks may be due to variations in the amino acid composition of the peptide side chains. There are several reports from different groups of the presence of glycine and aspartic acid in gonococcal peptidoglycan (9, 14, 22). In addition, in the better-characterized E. coli system, amino acid substitutions in the side chain account for a proportion of the species observed (5). In E. coli, the diaminopimelyl-diaminopimelic acid cross-links serve as sites for lipoprotein attachment (5). No protein with a structure analogous to that of lipoprotein has been reported in gonococci; however, growth of several gonococcal strains at low ph causes increased amounts of a protein that is apparently covalently attached to the pepti- on September 5, 218 by guest

6 74 DOUGHERTY doglycan (7). There is a strain, CS-7, that produces large amounts of this peptidoglycan-associated protein (8). Studies on this strain are currently in progress. The observations with the penicillin-susceptible and -resistant strains of N. gonorrhoeae are of great interest. These two strains have been extensively characterized, and the decreased penicillin-binding affinity of two of the three PBPs in the resistant strain has been documented (3). Penicillin binds to the active site of the PBPs, and the change in penicillin affinity presumably reflects a mutational modification of these enzymes (17, 18). The notable differences in the peptidoglycan synthesized, particularly in the crosslinked muropeptide species, suggest that the PBP alterations affect the assembly of this macromolecule. Most of the changes appear to be in the relative concentrations of preexisting muropeptide peaks, and this may reflect a shift in precursor utilization by the modified PBPs. It should be emphasized that this peptidoglycan is the product of logarithmically growing cultures of these two strains, neither of which had penicillin present during growth. An additional pair of susceptible and resistant strains with virtually identical penicillin MICs yielded the same reverse-phase HPLC patterns (not shown). With the high-resolution system developed by Glauner and Schwarz (5) in E. coli, gonococci have been shown to possess remarkable heterogeneity in their peptidoglycan constituents. There are several points of significance to these findings. The change in peptidoglycan structure observed in highly penicillin-resistant strains indicates that a change in the penicillin target enzymes is not without consequence to the structure of the gonococcal cell envelope. Another aspect of gonococcal biology in which the diverse peptidoglycan structures may be of importance is host interactions. Gonococci exhibit substantial cell wall turnover, and release of gonococcal peptidoglycan fragments has been implicated in several pathological host responses (1, 13). The possibility of variations in host effects by different peptidoglycan subunits must be entertained. In addition to chemical characterization of the peptidoglycan subunits, studies of the effects of beta-lactam antibiotics on the sacculus structure are planned. These investigations are expected to further delineate the process of cell surface assembly in gonococci. ACKNOWLEDGMENTS I thank Bernd Glauner, Joachim-V. Holtje, and Uli Schwarz for their generosity and hospitality during my visit to the Max-Planck- Institut fur Virusforschung, Tubingen, Federal Republic of Germany. I also thank R. Rosenthal for the anhydro muramyl peptide monomer compound, and Margaret Geller for help in manuscript preparation. This work was supported by Public Health Service grants Al 22 from the National Institute of Allergy and Infectious Diseases and BSRG S7 RR 765 from the National Institutes of Health. LITERATURE CITEI) 1. Blundell, J. K., G. J. Smith, and R. H. Perkins The peptidoglycan of Neisseria gonorrhoeae: O-acetyl groups and lysozyme sensitivity. FEMS Microbiol. Lett. 9: Dougherty, T. J Peptidoglycan biosynthesis in Neisseria gonorrhoeae strains sensitive and intrinsically resistant to 13- lactam antibiotics. J. Bacteriol. 153: Dougherty, T. J., A. E. Koller, and A. Tomasz Penicillinbinding proteins of penicillin-susceptible and intrinsically resistant Neisseria gonorrhoeae. Antimicrob. Agents Chemother. J. BACTERIOL. 18: Ghuysen, J. M., E. Bricas, M. Lache, and M. Leyh-Bouille Structure of the cell walls of Micrococcus lysodeikticus. III. Isolation of a new peptide dimer, N-(L-alanyl-y-(cx-D-glutamylglycine)) - L - lysyl - D - alanyl - N(L - alanyl - 'y - (a - D - glutamylglycine))l-jysyl-d-alanine. Biochemistry 7: Glauner, B., and U. Schwarz The analysis of murein composition with high pressure liquid chromatography, p In R. Hakenbeck, J.-V. Holtje, and H. Labischinski (ed.), The target of penicillin. W. de Gruyter, Berlin. 6. Goodell, E. W., M. Fazio, and A. Tomasz Effect of benzylpenicillin on the synthesis and structure of the cell envelope of Neisseria gonorrhoeae. Antimicrob. Agents Chemother. 13: Hebeler, B. H., S. A. Morse, W. Wong, and F. E. Young Effect of ph on the chemical and biological properties of the cell envelope of Neisseria gonorrhoeae: isolation of a proteinpeptidoglycan complex, p In G. F. Brooks, E. C. Gotschlich, K. K. Holmes, W. D. Sawyer, and F. E. Young (ed.), Immunobiology of Neisseria gonorrhoeae. American Society for Microbiology, Washington, D.C. 8. Hebeler, B. H., W. Wong, S. A. Morse, and F. E. Young Cell envelope of Neisseria gonorrhoeae CS7: peptidoglycanprotein complex. Infect. Immun. 23: Hebeler, B. H., and F. E. Young Chemical composition and turnover of peptidoglycan in Neisseria gonorrhoeae. J. Bacteriol. 126: Petersen, B. H., and R. S. Rosenthal Complement consumption by gonococcal peptidoglycan. Infect. Immun. 35: Pisabarro, A. G., M. A. de Pedro, and D. Vazque'z Structural modifications in the peptidoglycan of Escherichia coli associated with changes in the state of growth of the culture. J. Bacteriol. 161: Rosenthal, R. S Release of soluble peptidoglycan from growing gonococci: hexaminidase and amidase activities. Infect. Immun. 24: Rosenthal, R. S., W. J. Folkening, D. R. Miller, and S. C. Swim Resistance of O-acetylated gonococcal peptidoglycan to human peptidoglycan-degrading enzymes. Infect. Immun. 4: Rosenthal, R. S., R. M. Wright, and R. K. Sinha Extent of peptide cross-linking in the peptidoglycan of Neisseria gonorrhoeae. Infect. Immun. 28: Schleifer, K. H., and. Kandler Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36: Sinha, R. K., and R. S. Rosenthal Release of soluble peptidoglycan from growing gonococci: demonstration of anhydro-muramyl-containing fragments. Infect. Immun. 29: Spratt, B. G Escherichia coli resistance to beta-lactam antibiotics through a decrease in the affinity of a target for lethality. Nature (London) 274: Spratt, B. G Penicillin binding proteins and the future of beta-lactam antibiotics. J. Gen. Microbiol. 129: SWanson, J Studies on gonococcus infection. XII. Colony color and opacity variants of gonococci. Infect. Immun. 19: Tipper, D. J Alkali-catalyzed elimination of D-lactic acid from muramic acid and its derivatives and the determination of muramic acid. Biochemistry 7: Tipper, D. J., and A. Wright The structure and biosynthesis of bacterial cell walls, p In J. R. Sokatch and L. N. Ornston (ed.), The bacteria, vol. 7. Academic Press, Inc., New York. 22. Wolf-Watz, H., T. Elmros, S. Normark, and G. D. Bloom Cell envelope of Neisseria gonorrhoeae: outer membrane and peptidoglycan composition of penicillin-sensitive and -resistant strains. Infect. Immun. 11: