Analysis of Neisseria gonorrhoeae peptidoglycan by reverse-phase, high-pressure liquid chromatography

Abstract

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. N.Y. Strains were kept frozen in gonococcal broth with 15% glycerol at -70°C. 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 500-ml volumes of gonococcal broth with 1% IsoVitaleX and 420 ,ug of NaHCO3 per ml. These cultures, in 2-liter flasks, were grown at 37°C with agitation (140 rpm) in a New Brunswick G-25 shaker (New Brunswick Scienti...

Major fragment of soluble peptidoglycan released from growing Bordetella pertussis is tracheal cytotoxin

“…The homogeneity of soluble PG (peak i) detected in B. pertussis supernatants, although not predicted on the basis of structural heterogeneity of PG subunits found in other gram-negative PGs (1,4,8,11), is consistent with the marked homogeneity of monomer subunits in intact B. pertussis PG (3). However, the principal monomeric fragment making up intact B. pertussis PG was identified unambiguously by tandem mass spectrometry (3) as N-acetylglucosaminyl-N-acetylmuramyl-alanyl-glutamyl-DAP-alanine (NAG-NAM-Ala-Glu-DAP-Ala; 939 daltons), whereas TCT might be a somewhat larger fragment (about 1,400 daltons) containing an additional residue of the muramyl dipeptide N-acetylmuramyl-alanyl-glutamic acid (5).…”

Biological activities and chemical composition of purified tracheal cytotoxin of Bordetella pertussis

“…Since TCT is undetectable by pulse-chase-type experiments designed to look at turnover of macromolecular peptidoglycan (25,28), detecting soluble fragments released by bacterial microorganisms may require more direct methods (like those described here). The use of HPLC to separate fragments of purified peptidoglycan has been reported by workers in several laboratories (8,11,20), but its use has been limited to the analysis of enzymatic digestions of high-molecularweight cell wall material precipitated by hot sodium dodecyl sulfate. TCT, for instance, is refactory to commonly used protein precipitation procedures (unpublished observations).…”

Two lytic transglycosylases inNeisseria gonorrhoeaeimpart resistance to killing by lysozyme and human neutrophils

“…Insoluble peptidoglycan and proteins were removed using an Amico‐Ultra 10 kDa filter. Soluble peptidoglycan fragments were separated by HPLC as described (Dougherty, ).…”