Characterization of a lipoprotein common to Legionella species as a urinary broad-spectrum antigen for diagnosis of Legionnaires' disease.

We have previously identified the Legionella 19-kDa peptidoglycan-associated lipoprotein (PAL) as a species-common immunodominant antigen. We describe here for the first time the excretion and detection of the PAL antigen in infected urine specimens, which is useful for the diagnosis of Legionnaires' disease. Rabbit anti-PAL immunoglobulin G (IgG) antibody was produced by immunization with the purified, recombinant PAL of Legionella pneumophila serogroup 1 and used in the PAL antigen capture enzyme-linked immunosorbent assay (ELISA) to detect urinary PAL antigen. A soluble-antigen capture ELISA using rabbit IgG antibodies against Legionella soluble antigens was prepared independently and used as a broad-spectrum standard test to detect soluble antigens of several Legionella species. Urine samples were obtained from guinea pigs experimentally infected with each of L. pneumophila serogroups 1, 3, and 6, and other Legionella species. The absorbance values of the PAL antigen ELISA highly correlated with those of the soluble-antigen ELISA in infected urine samples, with a correlation coefficient of 0.84 (P < 0.01). When applied to 17 infected urine samples and 67 negative controls from guinea pigs, the sensitivity and specificity of the PAL antigen capture ELISA were 88.2 and 95.5%, respectively. Compared to the commercial Biotest enzyme immunoassay, the PAL antigen ELISA was more efficient for detecting pneumophila non-serogroup 1 and nonpneumophila species. None of the 161 control human urine specimens obtained from healthy adults and patients with either non-Legionella pneumonia or urinary tract infections tested positive in the PAL antigen ELISA. The present study shows that the Legionella PAL is a very useful broad-spectrum antigen for urinary diagnostic testing. Moreover, since recombinant PAL antigen can be produced more efficiently than the soluble antigens, the development of a broad-spectrum diagnostic immunoassay based on the detection of the PAL antigen appears to be warranted.

Bacterial peptidoglycans as modulators of sleep. I. Anhydro forms of muramyl peptides enhance somnogenic potency.

Chemically defined muramyl peptides (MPs), derived primarily from enzymatic digests of Neisseria gonorrhoeae peptidoglycan, were used to define the structural determinants of MP-mediated somnogenic activity. One of these, i.e. N-acetylglucosaminyl-N-acetyl-1,6-anhydro-N-acetylmuramyl-alanyl-glutamy l- diaminopimelyl-alanine, was structurally identical to the major naturally occurring MP previously detected in mammalian brain and urine. The somnogenic potency of this MP was similar to that of the corresponding disaccharide pentapeptide containing an additional alanine at the C-terminus and the analogous anhydro-muramic acid-containing monosaccharide tetrapeptide lacking the glucosamine moiety. Infusion of as little as 1 pmol of these highly active MPs increased significantly the percentage of slow-wave sleep in experimental animals. In fact, each of 5 anhydro-muramyl disaccharide peptides tested was somnogenic at a dose of 10 pmol or less and, as far as tested, the activity was affected only slightly by the length or composition of the peptide side chain. However, none of a matched set of analogous MPs, differing only in replacement of the anhydro-muramyl end by a hydrated muramic acid residue, was somnogenic at this dose. A modified form of the hydrated muramyl tripeptide containing a free amide on the diaminopimelic acid residue was completely inactive in amounts up to 1000 pmol. Together, the current data suggested: that the anhydro-muramic acid end (but not the glucosamine moiety) is essential for maximal somnogenic potency; and that amidation of carboxyl groups on the peptide-side chain may block MP-mediated somnogenic activity.

Peptidoglycans as promoters of slow-wave sleep. I. Structure of the sleep-promoting factor isolated from human urine.

Fast atom bombardment-mass spectrometry (FABMS) has been used to determine the structure of the urinary sleep-promoting factor (FSu), the nature of whose components had been reported earlier. Less than 1 nmol of the underivatized substance sufficed for the FABMS experiments. The major somnogenic constituent of the purified preparation was a peptidoglycan of Mr = 921 with the structure N-acetylglucosaminyl-N -acetylanhydromuramylalanylglutamyldiaminopimelylalanine. The anhydro linkage is between C-1 and C-6 of the muramyl entity. Two additional substances accompanied the above compound. These were the hydrated form (i.e. in which the muramyl entity had a free reducing end, and a free hydroxyl on C-6), and an anhydro analogue lacking the terminal alanine. The Mr values were 939 and 850, respectively. Methyl esters were prepared, and these were also acetylated. The mass spectra of the methyl ester of Mr = 921 displayed an increase in Mr of 42 (i.e. 3 X 14), indicating the presence, originally, of three free carboxyls. Acetylation increased Mr by a further 168 units (i.e. 4 X 42), indicating 4 hydroxyl or amino groups. These data are consistent with the structure cited above for the main entity of FSu. Similar confirmatory results were obtained for the two minor constituents described above. These operations were worked out on natural muramyl peptides of known structure, obtained from other sources, and the data are given for comparison.

Detection of soluble peptidoglycan in urine after penicillin administration.

A unique enzyme-linked immunosorbent assay was developed to detect soluble peptidoglycans in biological fluids. It makes use of the similar affinities of vancomycin and purified rabbit antibodies to peptidoglycan precursor sequences found in soluble peptidoglycans. This assay has been used to detect as little as 500 pg of soluble peptidoglycan per ml of serum and 5 pg/ml of urine. Studies of normal individuals and Staphylococcus aureus-infected patients revealed only a few sera with detectable levels of soluble peptidoglycans. Studies of normal volunteers who were given a single oral dose of 250 mg of penicillin VK showed that about half had detectable levels of soluble peptidoglycans in their urine up to 6 h after ingestion. This suggests that soluble peptidoglycans can be released by indigenous bacteria in detectable amounts. In one volunteer, a detectable level of soluble peptidoglycan in the urine at 6 h decreased to an undetectable level at 12 h. Such an ephemeral appearance of soluble peptidoglycan in the urine could account for the small number of human sera that had detectable levels of soluble peptidoglycan.

The metabolic fate of 14C-labeled peptidoglycan monomer in mice. I. Identification of the monomer and the corresponding pentapeptide in urine.

The distribution of radioactive products in mouse urine following intravenous administration of the 14C-labeled peptidoglycan monomer, GlcNAc-MurNaC-L-Ala-D-isoglutamine-meso-diaminopimelic acid-D-Ala-D-Ala, has been studied. 60--80% of radioactivity was recovered within first 3 h, 4--8% in the next 3 h and 2--7% in the following 18 h. The majority of the label was associated with the unchanged peptidoglycan monomer (42--56% of the dose). 14--21% of the label was incorporated into a compound which was isolated and tentatively identified as L-Ala-D-isoglutamine-meso-diaminopimelic acid-D-Ala-D-Ala. The kinetics of excretion and the distribution of radioactivity did not differ in immunized, when compared to non-immunized, mice.