Peptidoglycan of Legionella pneumophila: apparent resistance to lysozyme hydrolysis correlates with a high degree of peptide cross-linking

Abstract

Peptidoglycan (PG) from Legionella pneumophila was composed of muramic acid, glucosamine, glutamic acid, alanine, and meso-diaminopimelic acid in a molar ratio of 0.8:0.8:1.1:1.7:1. Partially purified PG contained trypsin-insensitive proteins which were extracted by 1 N NaOH hydrolysis without apparent dissolution of the PG. Lysozyme hydrolysis of purified PG or cell walls caused an increase in reducing groups which correlated with roughly 70 to 100% digestion of disaccharides. However, there was no significant decrease in turbidity during lysozyme hydrolysis of purified PG or cell wall. Additionally, 80 to 90% of the meso-diaminopimelic acid e-amino groups were not susceptible to dinitrophenylation. Collectively, the PG of L. pneumophila was sensitive to lysozyme hydrolysis and insensitive to alkali dissolution, and 80 to 90% of the NH2 groups of mesodiaminopimelic acid were apparently involved in cross-linkages between peptides. The structural features of the cell envelope of Legionella pneumophila have been studied by several investigators (8, 14, 21). The cell envelope consists of three layers: the cytoplasmic membrane, a peptidoglycan (PG) layer or cell wall, and the outer membrane (11). However, the chemical composition of the cell envelope and sacculus (32) of L. pneumophila has not been extensively investigated. Keel et al. (14) reported that the PG constituents, alanine, glutamic acid, muramic acid, and glucosamine, were present in molar ratios of 2:1.14:0.51:0.44 but that diaminopimelic acid (DAP) was undetectable. In contrast, Guerrant et al. (13) found DAP in the cell walls or PG of seven species of L. pneumophila. These results led us to examine the chemical composition and structure of the PG of L. pneumophila Philadelphia 2. The PG of this organism contains chemical components common to most gram-negative stained bacteria. Moreover, the PG peptide subunits may be 80 to 90% cross-linked with meso-DAP to produce a highly stable PG complex.

Biochemical and immunological properties of Coxiella burnetii cell wall and peptidoglycan-protein complex fractions

“…Purification of PG-PC from SCVs was easily accomplished. Proteins associated with the PG were resistant to proteolysis by trypsin, protease VI, and proteinease K. These results were similar to the resistance properties of proteins associated with the PG of Legionella pneumophila (1,2). Chemical analysis of the PG-PC indicated that a KDOlike component present in phase I lipopolysaccharide (3a, 20) copurified with this fraction, thus suggesting that LPS and protein were associated with the PG.…”

“…Proteolytic enzyme treatments of PG-PC. Samples (0.4 mg) were incubated at 370C with 50 Fg of trypsin, protease VI, and proteinase K (E. Merck AG, Darmstadt, West Germany) as previously described (1,2).…”

Structural Studies of Peptidoglycans in Campylobacter Species

“…The bacterial cells were harvested by centrifugation at 100,000 x g for 30 min, washed twice with distilled water and lyophilized . Two hundred mg of dried cells were suspended in 50 ml of 4% sodium dodecyl sulfate (SDS), boiled for 1 hr, and cooled at room temperature as described before (1). After centrifugation, the residue was washed three times with distilled water .…”

Legionella pneumophila surface antigens cloned and expressed in Escherichia coli are translocated to the host cell surface and interact with specific anti-Legionella antibodies

“…Certain foreign surface antigens that have been cloned from closely related donor species have been shown to localize to the E. coli cell surface (13); however, molecules from distantly related species, such as streptococcal M protein, are not surface localized in E. coli (5). It is therefore surprising that L. pneumophila surface antigens are properly sorted in E. coli, as these two species are phylogenetically distant, having only 1 to 3% DNA homology (3), and as they possess cell envelopes with distinctly different structures (1,16).…”