Quantitative Proteomics of the Endothelial Secretome Identifies RC0497 as Diagnostic of Acute Rickettsial Spotted Fever Infections.

Mediterranean spotted fever is a reemerging acute tick-borne infection produced by the α-proteobacterium, Rickettsia conorii. Rickettsia conorii infects vascular endothelial cells producing disseminated plasma leakage, manifesting as nonspecific fever, headache, and maculopapular rash. Because there are no available tests of early infection, Mediterranean spotted fever is often undiagnosed and untreated, resulting in significant mortality. To address this critical need, we have applied a quantitative proteomics pipeline for analyzing the secretome of primary human umbilical vein endothelial cells. Of the 104 proteins whose abundance changed significantly in the R. conorii-infected human umbilical vein endothelial cells' secretome, 46 proteins were up-regulated: 45 were host secreted proteins (including cytokines), and 1 was a rickettsial protein, the putative N-acetylmuramoyl-l-alanine amidase RC0497. Proteins with sequence highly homologous to RC0497 were found to be shared by many species of the spotted fever group rickettsiae, but not typhus group rickettsiae. Quantitative targeted proteomics studies of plasma from a mouse model of sublethal and lethal R. conorii identified RC0497 in the blood, and its circulating levels were proportionally associated with infection outcome. Finally, the presence of RC0497 in the serum samples from a cohort of humans presenting with acute rickettsioses was confirmed. The detection of RC0497 has the potential to be a sensitive and specific marker for acute rickettsial spotted rickettsioses.

Proteomic profiling identifies N-acetylmuramoyl-l-alanine amidase as a novel biomarker of sepsis.

AIM: Sepsis is a critical condition that leads to high mortality and is the most common cause of death in intensive care units. Despite exhaustive efforts by the scientific community, a reliable biomarker for diagnosis, evolution and prognosis of sepsis is still lacking. Results & methodology: Here, using high-throughput proteomics, we describe N-acetylmuramoyl-l-alanine amidase as a novel candidate for differentiating infectious and noninfectious inflammatory syndromes. DISCUSSION & CONCLUSION: This is the first description of N-acetylmuramoyl-l-alanine amidase as a biomarker that can be used alone or in conjunction with other biomarkers to facilitate the diagnosis of sepsis in the critically ill.

Development of amperometric magnetogenosensors coupled to asymmetric PCR for the specific detection of Streptococcus pneumoniae.

A disposable magnetogenosensor for the rapid, specific and sensitive detection of Streptococcus pneumoniae is reported. The developed procedure involves the use of streptavidin-modified magnetic beads, a specific biotinylated capture probe that hybridizes with a specific region of lytA, the gene encoding the pneumococcal major autolysin, and appropriate primers for asymmetric polymerase chain reaction (PCR) amplification. Capture probes and amplicons specific for S. pneumoniae were selected by a careful analysis of all lytA alleles available. The selected primers amplify a 235-bp fragment of pneumococcal lytA. A detection limit (LOD) of 5.1 nM was obtained for a 20-mer synthetic target DNA without any amplification protocol, while the LOD for the asymmetric PCR amplicon was 1.1 nM. A RSD value of 6.9% was obtained for measurements carried out with seven different genosensors for 1.1-nM aPCR product. The strict specificity of the designed primers was demonstrated by aPCR amplification of genomic DNA prepared from different bacteria, including some closely related streptococci. Direct asymmetric PCR (daPCR), using cells directly from broth cultures of S. pneumoniae, showed that daPCR products could be prepared with as few as 2 colony-forming units (CFU). Furthermore, this methodology did not show any cross-reaction with closely related streptococci such as Streptococcus mitis (or Streptococcus pseudopneumoniae) even when present in the culture at concentrations up to 10(5) times higher than that of S. pneumoniae. Preliminary data for rapid detection of pneumococcus directly in clinical samples has shown that it is possible to discriminate between non-inoculated blood and urine samples and samples inoculated with only 10(3) CFU mL(-1) S. pneumoniae.

Identification of serum N-acetylmuramoyl-l-alanine amidase as liver peptidoglycan recognition protein 2.

N-acetylmuramoyl-l-alanine amidase (NAMLAA) hydrolyzes bacterial peptidoglycan and is present in human serum. A peptidoglycan-recognition protein 2 (PGLYRP2) is expressed in human liver and has N-acetylmuramoyl-l-alanine amidase activity. Here, we determined the amino acid sequences of human serum NAMLAA and liver PGLYRP2 and tested the hypothesis that serum NAMLAA and PGLYRP2 are the same protein. Liver PGLYRP2 and serum NAMLAA had the same mass determined by mass spectrometry and polyacrylamide gel electrophoresis, and both proteins and recombinant PGLYRP2 reacted with polyclonal anti-NAMLAA and anti-PGLYRP2 antibodies, and with monoclonal anti-NAMLAA antibodies. Digestion of serum NAMLAA with trypsin, chymotrypsin, or trypsin plus V8 protease, or with CNBr yielded, respectively, 37, 40, and 3 overlapping peptides that matched 100% and covered 81% of the deduced amino acid sequence of mature PGLYRP2. These peptides overlapped all exon-intron junctions indicating no alternative splice forms. Digestion of liver PGLYRP2 with trypsin yielded 23 peptides that matched 100% and covered 44% of the deduced amino acid sequence of mature PGLYRP2. Serum NAMLAA had a C398-C404 disulfide, partial phosphorylation of S218, and deamidation of N253 and N301. These results indicate that serum NAMLAA and liver PGLYRP2 are the same protein encoded by the pglyrp2 gene.

Purification and characterization of N-acetylmuramyl-L-alanine amidase from human plasma using monoclonal antibodies.

N-Acetylmuramyl-L-alanine amidase (EC 3.5.1.28) cleaves the amide bond between N-acetyl muramic acid and L-alanine in the peptide side chain of different peptidoglycan products. The enzyme was purified from human plasma using a three-step column chromatography procedure. Monoclonal antibodies were produced against the purified human enzyme. By coupling of a high affinity monoclonal antibody to sepharose beads an immunoadsorbent column was prepared. Using this second purification method it was possible to purify large amounts of the amidase from human plasma in a single step. SDS-PAGE showed one single band of 70 kDa and two-dimensional electrophoresis showed the presence of multiple isomeric forms of the protein with pI between 6.5 and 7.9. Two different methods were used for determination of substrate specificity, a HPLC method separating peptidoglycan monomers from the reaction products after incubation with amidase and a colorimetric method when high molecular weight peptidoglycan was used as a substrate for amidase. It is shown that the disaccharide tetra peptide, disaccharide penta peptide and the anhydro disaccharide tetrapeptide are good substrates for the amidase and that muramyl dipeptide and disaccharide dipeptide are not a substrate for the amidase. Using one of the monoclonal antibodies against the amidase it was shown in FACScan analysis that N-acetylmuramyl-L-alanine amidase is present in granulocytes but not in monocytes from unstimulated peripheral blood of a healthy donor. The presence of N-acetylmuramyl-L-alanine amidase in granulocytes is a novel finding and perhaps important for the inactivation of biologically active peptidoglycan products still present after hydrolysis by lysozyme.

Characterization of human serum N-acetylmuramyl-L-alanine amidase purified by affinity chromatography.

Human serum N-acetylmuramyl-L-alanine amidase was purified to homogeneity by a relatively short procedure including affinity chromatography. For this purpose, a specific adsorbent was prepared by coupling the main substrate of the enzyme, N-acetyl-muramyl-L-alanine-gamma-D-glutamyl-L-meso-2,6[3,4,5-3H] diaminopimelic acid, to a divinylsulfone agarose gel. The enzyme is unable to hydrolyze this muramylpeptide when it is attached as a ligand to the gel, whereas a high affinity is conserved. In addition to affinity chromatography, the presented purification scheme includes ion exchange chromatography on DEAE-Sepharose and molecular sieving on Superdex 200. The enzyme was purified 739-fold with a yield of 22.5%. One single band at 135 kDa was obtained on native gradient PAGE. Gradient PAGE in denaturing conditions gave one single band at 74 kDa, which was lowered to 64 kDa when the enzyme was denatured in nonreducing conditions. This suggests that the native enzyme is a dimer consisting of two subunits of identical molecular weight with only intramolecular disulfide bonds. Isoelectric focusing gave one single band at pI 5.0. Glycan detection before and after treatment with N-glycosidase F showed that the enzyme is a glycoprotein. Further analysis by lectin immuno detection on dot blots confirmed that the enzyme is an N-glycosylated protein of complex type with sialic acid, terminally linked alpha (2-->6) to galactose or N-acetylgalactosamine. The 15 amino acid N-terminal sequence was determined by microsequence analysis.

The human and mammalian N-acetylmuramyl-L-alanine amidase: distribution, action on different bacterial peptidoglycans, and comparison with the human lysozyme activities.

N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28) specifically hydrolyzes the bacterial cell wall peptidoglycans (or mureins) and the muropeptides. The enzyme splits these molecules into two parts: the peptide subunits and the glycan strands or moieties. The bacterial peptidoglycans and their derived muropeptides display a number of biological properties. Removal of the glycosidic part of these molecules abolishes their beneficial as well as their detrimental properties. We report the high level of enzymatic activity found in all mammalian (including human) sera tested. The enzyme also occurred in human saliva, milk, cerebrospinal fluid, and synovial liquid. Mucosal tissue from different parts of the mammalian digestive tract exhibited enzymatic activity, but the enzyme was not detectable in the lumen content. The range of substrate specificity of the human enzyme was evaluated by measuring its action on the peptidoglycans extracted from several bacterial strains and representing different chemotypes and structures. Time course of the muramylalanine amidase and of the lysozyme (both of human origin) activities on some of these peptidoglycans are also reported, with the enzymes acting separately or together. From these data, we would speculate that a probable physiological role of the muramylalanine amidase is the maintenance of adequate ratios between the biologically active muropeptides and their inactive derivatives in the organism, the amidase activity antagonizing the production of biologically active molecules by lysozyme.

Assay for N-acetylmuramyl-L-alanine amidase in serum by determination of muramic acid released from the peptidoglycan of Brevibacterium divaricatum.

A method is reported for the determination of N-acetylmuramyl-L-alanine amidase in serum. Muramic acid, released from the interpeptide bridges of Brevibacterium divaricatum peptidoglycan, is measured by a modified colorimetric method. Using this procedure, it was possible to determine N-acetylmuramyl-L-alanine amidase in aliquots of less than 10 microliters human serum with an incubation time of 10 min. Amidase activity was found in all the sera tested (n = 11). The relevance of this simple and fast assay is discussed.

Purification and characterization of N-acetylmuramoyl-L-alanine amidase from human serum.

Purification to homogeneity of the N-acetylmuramoyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) from human serum has been achieved with a high yield. By molecular sieving chromatography, a molecular weight of 120,000-130,000 has been found for the native enzyme. Polyacrylamide gel electrophoresis under native conditions gave a unique band of Mr = 125,000. The same technique performed under denaturing conditions revealed that the protein is a dimer composed of one subunit of Mr = 57,000 and another of Mr = 70,000. In isoelectrofocalization assays, the amidase behaved as an acidic protein. Ethylenediaminetetraacetate inhibited the enzyme activity; the Mg2+ requirement was confirmed. The simultaneous presence of sulfhydryl groups and disulfide bonds in the protein was evidenced by the inhibitions produced by different thiol-blocking reagents and by several thiol-bearing substances. Direct measurements established the presence of two accessible thiol groups and the occurrence of nine disulfide bonds per protein molecule. Studies of substrate hydrolyzing capacities showed a marked preference for the muramoyl tripeptide derived from the Escherichia coli or Bacillus cereus mureins, the disaccharide tetrapeptide and the bis disaccharide tetra-tetrapeptide from E. coli were also good substrates. Activities on small muropeptides of other composition are also reported. Whole (insoluble) peptidoglycans representing the main bacterial chemotypes were submitted to the enzyme action; although with weak specific activities, the human amidase was nevertheless able to release soluble peptides from some of them. A bacteriolytic capacity on some microorganisms cannot be excluded. Results are discussed and the human enzyme is compared to presently known microbial muramoyl amidases.

Murein hydrolase (N-acetyl-muramyl-L-alanine amidase) in human serum.

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and L-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetyl-muramyl-L-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed. The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.

Partial purification and characterization of N-acetylmuramyl-L-alanine amidase from human and mouse serum.

The enzyme N-acetylmuramyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) has been detected in human, mouse, rabbit, bovine and sheep sera. A method for detection of amidase activity using [14C]peptidoglycan monomer as the substrate has been developed. Partial purification of human and mouse amidase was achieved by gel chromatography on Bio-Gel A-1.5 m, DEAE-Sephadex A-50 and Sephadex G-100. Both amidase preparations exhibited maximal activity at pH 9.0 in Tris-HCl buffer and required Mg2+ for maximal activity. Following digestion of peptidoglycan monomer, GlcNAc-MurNAc-L-Ala-D-isoglutaminyl-meso-diaminopimelyl-D-Ala-D-Ala, the disaccharide GlcNAc-MurNAc and the corresponding pentapeptide L-Ala-D-isoglutaminyl-meso-diaminopimelyl-D-Ala-D-Ala were formed and subsequently isolated and chemically characterized. The enzyme therefore acts as an N-acetylmuramyl-L-alanine amidase by cleaving the bond between N-acetylmuramic acid and L-alanine. The glycan linked, peptide-not-cross-linked peptidoglycan dimer was also shown to be a substrate for human and mouse amidase.

The metabolic fate of 14C-labeled immunoadjuvant peptidoglycan monomer. II. In vitro studies.

Peptidoglycan monomer (GlcNAc-MurNAc-L-Ala-D-isoglutamine-meso-diaminopimelic acid-D-Ala-D-Ala), labeled with 14C both in the disaccharide and pentapeptide portions, was incubated with slices of mouse liver, kidney or spleen as well as with mouse and human blood, blood cells plasma and serum. Peptidoglycan monomer was isolated unchanged after incubations with mouse organs and blood cells. However, upon incubation with mouse or human blood, 10-50% of the peptidoglycan monomer underwent hydrolysis to the corresponding disaccharide and pentapeptide. After incubations with plasma and serum more than 90% of the [14C]peptidoglycan monomer was metabolized: about 50% of the administered radioactive dose was recovered in the disaccharide unit and about 35% in the pentapeptide part. These results suggest that in blood, plasma and serum of mouse and man, an N-acetylmuramoyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) exists which splits the amide bond between the lactyl carboxyl group of the muramyl residue and the amino group of the peptide moiety in the peptidoglycan molecule.