Sensitivity of Coxiella burnetii peptidoglycan to lysozyme hydrolysis and correlation of sacculus rigidity with peptidoglycan-associated proteins

Abstract

The protease-resistant proteins associated with the peptidoglycan (PG) of the phase I small-cell variant Coxiella burnetii were either partially released from the PG by boiling the PG-protein complex (PG-PC) in sodium dodecyl sulfate containing 2-mercaptoethanol and EDTA or totally released by 1 N NaOH hydrolysis at 23°C. An 18,300-dalton protein was released from the PG-PC under reducing conditions, whereas 1 N NaOH treatment extracted PG-associated proteins without apparent dissolution of the PG. Purified PG was composed of muramic acid, glucosamine, glutamic acid, alanine, and meso-diaminopimelic acid in a molar ratio of 0.9:0.9:1.0:1.4:1.0. Lysozyme hydrolysis of cell walls, PG-PC, and purified PG caused an increase in reducing groups which correlated with roughly 60 to 100% digestion of disaccharides. There was no significant decrease in turbidity during lysozyme hydrolysis of cell walls and PG-PC; however, hydrolysis of purified PG caused about 90% decrease in turbidity. Approximately 60% of the meso-diaminopimelic acid groups of PG were not susceptible to dinitrophenylation, thus, demonstrating an apparent contribution of PG-associated proteins, rather than cross-linkage between peptides, to sacculus rigidity of cell wall and PG-PC. This association of PG and protease-resistant covalently bound proteins may be important structural and functional determiners of resistance to both environmental conditions and intracellular digestion of C. burnetii by eucaryotic cells.

Transcriptional Profiling of Coxiella burnetii Reveals Extensive Cell Wall Remodeling in the Small Cell Variant Developmental Form

“…Cross-linking results contrast with an early study of the C . burnetii Ohio strain where chemical analysis of bacteria purified from embryonated eggs suggested 40% muropeptide cross-linking [ 117 ]. Of total cross-linked muropeptides, 46% of SCV were 3–3 cross-linked whereas only 16% of LCV were 3–3 cross-linked ( Fig 6 ).…”

Lounging in a lysosome: the intracellular lifestyle of Coxiella burnetii

“…The Coxiella surface must be intrinsically resistant to these insults as non-metabolizing organisms retain viability for several days in a lysosome-like, tight-fitting vacuole (Howe et al, 2003). A biochemically unusual peptidoglycan with associated protease-resistant proteins may provide protection from lysosomal digestion (Amano and Williams, 1984). In professional phagocytes, noxious reactive oxygen and nitrogen species are also produced that aid in disposal of invading pathogens.…”

Use of Monoclonal Antibodies for Analyses of Coxiella burnetii Major Antigens

“…Recently, we produced several MAbs to C. burnetii LPS and analyzed the antigenic change during phase variation [7] and the antigenic difference among strains [8]. In this study, we produced MAbs to OMPs [11], 62-kDa protein antigen and peptidoglycan protein complex (PG-PC) [1]. With the MAbs, we obtained invaluable data to elucidate the antigenic properties of C. burnetii.…”

“…Our results suggest that the MAbs of groups IV (K75), V (H106, K59 and K82) and VI (K43) recognize OMPs. Since the 29-to 31-kDa OMP aggregate contains 18.3-kDa PG-PC that is resistant to proteinase K as well [1,3], the MAb of group VII (K64) may recognize PG-PC. The MAbs of group VIII (K7, K24 and K57) appear to recognize a 62-kDa protein antigen.…”