Anchor structure of staphylococcal surface proteins. III. Role of the FemA, FemB, and FemX factors in anchoring surface proteins to the bacterial cell wall

Surface proteins of Staphylococcus aureus are covalently linked to the bacterial cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. Cleavage between the threonine and the glycine of the LPXTG motif liberates the carboxyl of threonine to form an amide bond with the pentaglycyl cross-bridge in the staphylococcal peptidoglycan. Here, we asked whether altered peptidoglycan cross-bridges interfere with the sorting reaction and investigated surface protein anchoring in staphylococcal fem mutants. S. aureus strains carrying mutations in the femA, femB, femAB, or the femAX genes synthesize altered cross-bridges, and each of these strains displayed decreased sorting activity. Characterization of cell wall anchor structures purified from the fem mutants revealed that surface proteins were linked to cross-bridges containing one, three, or five glycyl residues, but not to the epsilon-amino of lysyl in muropeptides without glycine. When tested in a femAB strain synthesizing cross-bridges with mono-, tri-, and pentaglycyl as well as tetraglycyl-monoseryl, surface proteins were found anchored mostly to the five-residue cross-bridges (pentaglycyl or tetraglycyl-monoseryl). Thus, although wild-type peptidoglycan appears to be the preferred substrate for the sorting reaction, altered cell wall cross-bridges can be linked to the COOH-terminal end of surface proteins.     

Anchor structure of staphylococcal surface proteins. A branched peptide that links the carboxyl terminus of proteins to the cell wall

Surface proteins of Staphylococcus aureus are anchored to the cell wall by a mechanism requiring a COOH-terminal sorting signal. Previous work demonstrated that the sorting signal is cleaved at the conserved LPXTG motif and that the carboxyl of threonine (T) is linked to the staphylococcal cell wall. By employing different cell wall lytic enzymes, surface proteins were released from the staphylococcal peptidoglycan and their COOH-terminal anchor structure was revealed by a combination of mass spectrometry and chemical analysis. The results demonstrate that surface proteins are linked to a branched peptide (NH2-Ala-gamma-Gln-Lys-(NH2-Gly5)-Ala-COOH) by an amide bond between the carboxyl of threonine and the amino of the pentaglycine cross-bridge that is attached to the epsilon-amino of lysyl. This branched anchor peptide is amide-linked to the carboxyl of N-acetylmuramic acid, thereby tethering the COOH-terminal end of surface proteins to the staphylococcal peptidoglycan.     

Effects of no precursor and donor on neuronal activity of rat hippocampal slices

Human N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28) degrades peptidoglycan, a major component of bacterial cell walls with potent pro-inflammatory cytokine-inducing properties. We postulate that degradation of peptidoglycan by N-acetylmuramyl-L-alanine amidase is important for the inactivation of inflammatory peptidoglycan products in human tissues. The inflammatory activities of peptidoglycan digested by lysozyme and/or amidase were investigated using two properties of peptidoglycan: its capacity to induce the release of the inflammatory cytokines IL-1, IL-6 and TNF-alpha in vivo and in vitro and its capacity to induce arthritis in Lewis rats. The results show that after subsequent treatment with both lysozyme and amidase, the peptidoglycan products were unable to induce arthritis in Lewis rats. The production of pro-inflammatory cytokines in mice after intravenous injection of cell wall fragments was lower after in vitro degradation of the cell wall fragments by amidase. These in vivo results were confirmed with whole blood assays in which the production of pro-inflammatory cytokines was measured after stimulation with lysozyme- and amidase-treated peptidoglycan. The results show that human N-acetylmuramyl-L-alanine amidase possesses an enzymatic activity capable of inactivating inflammatory peptidoglycan by lowering its cytokine-inducing properties.     

Anesthetics modulate phospholipase C hydrolysis of monolayer phospholipids by surface pressure

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.     

Targeting of muralytic enzymes to the cell division site of Gram-positive bacteria: repeat domains direct autolysin to the equatorial surface ring of Staphylococcus aureus

Staphylococcus aureus secretes autolysin (Atl) to complete cell division by hydrolyzing its thick cell wall layer at a designated site, known as the equatorial surface ring. Secreted pro-Atl (1256 amino acids) is cleaved at residues 198 and 775 to generate a pro-peptide, amidase and glucosaminidase, respectively. Here we examined the mechanism that directs amidase and glucosaminidase to the cell division site on the staphylococcal surface. Targeting of pro-Atl to the cell surface occurred prior to its proteolytic processing. Three repeat domains (R1, R2 and R3) located at the center of pro-Atl are necessary and sufficient for the targeting of reporter proteins to the equatorial surface ring. Pro-Atl cleavage at residue 775 separates the polypeptide such that R1 and R2 are linked to the C-terminus of amidase, whereas R3 is located at the N-terminus of glucosaminidase. Thus, it appears that the repeat domains direct pro-Atl, amidase and glucosaminidase to a specific receptor at the equatorial surface ring of staphylococci, thereby allowing localized peptidoglycan hydrolysis and separation of the dividing cells.     

Pth1/Vam3p is the syntaxin homolog at the vacuolar membrane of Saccharomyces cerevisiae required for the delivery of vacuolar hydrolases

We constructed hybrid proteins containing a plant alpha-galactosidase fused to various C-terminal moieties of the hypoxic Srp1p; this allowed us to identify a cell wall-bound form of Srp1p. We showed that the last 30 amino acids of Srp1p, but not the last 16, contain sufficient information to signal glycosyl-phosphatidylinositol anchor attachment and subsequent cell wall anchorage. The cell wall-bound form was shown to be linked by means of a beta1,6-glucose-containing side-chain. Pmt1p enzyme is known as a protein-O-mannosyltransferase that initiates the O-glycosidic chains on proteins. We found that a pmt1 deletion mutant was highly sensitive to zymolyase and that in this strain the alpha-galactosidase-Srp1 fusion proteins, an alpha-galactosidase-Sed1 hybrid protein and an alpha-galactosidase-alpha-agglutinin hybrid protein were absent from both the membrane and the cell wall fractions. However, the plasma membrane protein Gas1p still receives its glycosyl-phosphatidylinositol anchor in pmt1 cells, and in this mutant strain an alpha-galactosidase-Cwp2 fusion protein was found linked to the cell wall but devoid of beta1,6-glucan side-chain, indicating an alternative mechanism of cell wall anchorage.     

Architecture of the yeast cell wall. Beta(1-->6)-glucan interconnects mannoprotein, beta(1-->)3-glucan, and chitin

In a previous study (Kollár, R., Petráková, E., Ashwell, G., Robbins, P. W., and Cabib, E. (1995) J. Biol. Chem. 270, 1170-1178), the linkage region between chitin and beta(1-->3)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with beta(1-->3)-glucanase, reduction with borotritide, and subsequent incubation with chitinase. In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material. We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, beta(1-->3)-glucan, beta(1-->6)-glucan, chitin, and mannoprotein are linked together. Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to beta(1-->6)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five alpha-linked mannosyl residues. The beta(1-->6)-glucan has some beta(1-->3)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a beta(1-->4) or beta(1-->2) linkage. Finally, the reducing end of beta(1-->6)-glucan is connected to the nonreducing terminal glucose of beta(1-->3)-glucan through a linkage that remains to be established. A fraction of the isolated material has three of the main components but lacks mannoprotein. From these results and previous findings on the linkage between mannoproteins and beta(1-->6)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall. The possible mechanism of synthesis and physiological significance of the cross-links is discussed.     

Functional display of a heterologous protein on the surface of Lactococcus lactis by means of the cell wall anchor of Staphylococcus aureus protein A

In this study, we showed that the cell wall anchor of protein A from Staphylococcus aureus is functional in the food-grade organism Lactococcus lactis. A fusion protein composed of the lactococcal Usp45 secretion signal peptide, streptavidin monomer, and the S. aureus protein A anchor became covalently attached to the peptidoglycan when expressed in L. lactis. The streptavidin moiety of the fusion protein was functionally exposed at the cellular surface. L. lactis cells expressing the anchored fusion polypeptide could be specifically immobilized on a biotinylated alkaline phosphatase-coated polystyrene support.     

Structural characterization of an abnormally cross-linked muropeptide dimer that is accumulated in the peptidoglycan of methicillin- and cefotaxime-resistant mutants of Staphylococcus aureus

Laboratory mutants of Staphylococcus aureus strain ATCC 8325 (27S) selected for increased minimal inhibitory concentration (MIC) values to methicillin and cefotaxime showed increased rates of cell wall turnover and detergent-induced autolysis in virtual parallel with the increasing MIC for the antibiotic. Also in parallel with the increasing MICs for the particular antibiotic used in the selection was the gradual accumulation of an unusual muropeptide in the peptidoglycan of the mutants, muropeptide 12, which is a minor component of the cell wall of the parental strain. Analysis of muropeptide 12, its peptide derivative, and its lysostaphin degradation products by high pressure liquid chromatography, Edman degradation, and mass spectrometry suggests that muropeptide 12 is a dimer in which the two monomeric components are interlinked by two pentaglycyl cross-bridges, thus generating a 14-member macrocyclic ring structure. This unusual cross-linked structure may be the product of the abnormal activity of penicillin-binding protein 2 which has grossly reduced antibiotic binding capacity in the mutant staphylococci.     

New information on the structure of mycobacteria

Recent progress about the mycobacterial structures have been realized and two major structures have been concerned: the genome and the cell wall. From these acquired new knowledge several lines of clinical research and diagnosis application emerged. Cloning and sequencing of several mycobacterial genes led to the development of diagnostic tools (DNA probes, PCR, finger printings of indated mycobacterial strains) and the potential detection of multiding resistant strains of M. tuberculosis. Genetic manipulations involving various mycobacterial genes do open the way for more precise molecular approaches concerning virulence factors involved in the pathophysiological understanding of mycobacterial diseases. Comparative physico-chemical and ultra-structural analysis of the mycobacterial cell wall evoked a highly complexed cell wall structure, constituted of a double lipidic layer linked to the peptidoglycan (PG). The first layer is constituted of mycolic acids that are linked to the PG by arabinogalactan, and to the superficial layer by hydrophobic interactions of glycolipids. The superficial layer is constituted of amphiphatic glycolipids, having a lipidic banal pole and a polysaccharidic apical pole. The knowledge of the mycobacterial cell wall structure opened the way of: the development of immunological diagnostic tools, being now days in clinical evaluation phase, a better approach for host-bacteria relationship study at the cellular level (macrophage, lymphocytes), and the understanding of the mode of action of antimycobacterial drugs such as isoniazid and ethambutol.     

Expression and intracellular localization of the human N-acetylmuramyl-L-alanine amidase, a bacterial cell wall-degrading enzyme

N-acetylmuramyl-L-alanine amidase (NAMLAA) specifically degrades peptidoglycan, which is a major component of bacterial cell walls with strong inflammatory properties. For instance, peptidoglycan is capable of stimulating peripheral blood cells to release pro-inflammatory cytokines and is capable of inducing chronic arthritis in an animal model. In a previous study we found that degradation of peptidoglycan by purified NAMLAA reduced its inflammatory effects. To determine where NAMLAA is located in tissues, monoclonal antibodies against purified NAMLAA were produced for use in immunohistochemistry, immunoelectron microscopy, flow cytometric analysis, and Western blotting. The immunohistochemical studies showed NAMLAA-positive cells in human spleen, liver, arthritic synovial tissues, and lymph nodes. In flow cytometric studies of blood and bone marrow, neutrophilic and eosinophilic granulocytes proved to be positive. Monocytes were negative, although they do contain lysozyme, the other important peptidoglycan-degrading enzyme. However, mature macrophages obtained by bronchoalveolar lavage and subsequent selection based on autofluorescence did possess NAMLAA. In immunocytochemical staining of blood smears, thrombocytes were also positive for NAMLAA. Western blot analysis and immunoelectron microscopy of neutrophils and eosinophils showed that NAMLAA is located in azurophilic granules of neutrophils and in secretory vesicles and crystalloid-containing granules of eosinophils. Flow cytometric analysis of blood and bone marrow from different French-American-British-classified acute myeloid leukemia (AML) patients showed that AML-M2 myeloblasts were the first in the granulocyte maturation lineage that were positive for NAMLAA. The more immature AML, such as AML-M0 and AML-M1, did not express NAMLAA. CD15- and CD13-negative megakaryoblasts, corresponding to AML-M7, were also positive for NAMLAA. The expression pattern of NAMLAA in the myeloid lineage suggests that the monoclonal antibody AAA4, recognizing NAMLAA, is useful for discrimination between AML in the monocyte lineage and in the granulocyte lineage.     

A simple method for electrophoretic analysis of cell surface glycoproteins based on concanavalin A binding

A simple and sensitive method is described for the detection of cell surface proteins. This method consists of reacting concanavalin A (Con A) with 35S-labeled methionine, detergent solubilized whole cell extracts and immunoprecipitating the Con A-protein complexes with anti-Con A antibody bound to Staphylococcus aureus Cowan I strain (SACI). These reagents are commercially available, stable and easy to standardize. Mouse lymphocytes cell surface proteins purified by this procedure show great similarities to those purified with goat anti-cell surface antibodies.     

Bacillus subtilis N-acetylmuramic acid L-alanine amidase

The N-acetymuramic acid L-alanine amidase from Bacillus subtilis (ATCC 6051) has been purified to homogeneity. It is a monomeric protein of molecular weight 50,000. The enzyme has a high affinity for homologous cell walls and once attached to a cell wall will hydrolyze the wall completely before initiating the hydrolysis of a new cell wall. The affinity of the enzyme for cell walls devoid of teichoic acid or for cell walls of Bacillus megaterium is much lower than that for B. subtilis cell walls. A second homogenous protein has been isolated from B. subtilis which specifically combines with the amidase in a 1:1 molar ratio and stimulates enzyme activity. This modifier protein has no intrinsic cell wall lytic activity. The binding of enzyme and modifier protein has a dissociation constant of 8.5 times 10-9 M in 0.1 M LiCl, pH 8.0, but the two proteins can be completely dissociated in 3 M LiCl at pH 8.0.     

The biologic activities of peptidoglycan in experimental Haemophilus influenzae meningitis

While gram-positive bacterial cell walls are known to incite inflammation, the contribution of gram-negative peptidoglycan to disease has not been characterized. The ability of cell wall, purified peptidoglycan, and soluble peptidoglycan subcomponents from Haemophilus influenzae to provoke inflammation was determined in a rabbit model of meningitis. Haemophilus peptidoglycan, with or without associated proteins, produced brain edema at > or = 0.1 micrograms/mL of cerebrospinal fluid (CSF); leukocytosis and protein accumulation in CSF occurred only at > or = 10.0 micrograms/mL of CSF. Solubilized peptidoglycan was 10-fold more active than intact cell wall. The bioactivity of peptidoglycan from ampicillin-resistant H. influenzae was at least twofold greater than that of ampicillin-sensitive strains. Consistent with these pathologic effects of purified peptidoglycan, ampicillin-induced bacterial lysates in which endotoxin was neutralized induced brain edema and protein influx but little leukocytosis. Thus, peptidoglycan seems to contribute to the pathology of gram-negative meningitis, particularly brain edema.     

Glucose-induced sequential processing of a glycosyl-phosphatidylinositol-anchored ectoprotein in Saccharomyces cerevisiae

Transfer of spheroplasts from the yeast Saccharomyces cerevisiae to glucose leads to the activation of an endogenous (glycosyl)-phosphatidylinositol-specific phospholipase C ([G]PI-PLC), which cleaves the anchor of at least one glycosyl-phosphatidylinositol (GPI)-anchored protein, the cyclic AMP (cAMP)-binding ectoprotein Gce1p (G. Müller and W. Bandlow, J. Cell Biol. 122:325-336, 1993). Analyses of the turnover of two constituents of the anchor, myo-inositol and ethanolamine, relative to the protein label as well as separation of the two differently processed versions of Gce1p by isoelectric focusing in spheroplasts demonstrate the glucose-induced conversion of amphiphilic Gce1p first into a lipolytically cleaved hydrophilic intermediate, which is then processed into another hydrophilic version lacking both myo-inositol and ethanolamine. When incubated with unlabeled spheroplasts, the lipolytically cleaved intermediate prepared in vitro is converted into the version lacking all anchor constituents, whereby the anchor glycan is apparently removed as a whole. The secondary cleavage ensues independently of the carbon source, attributing the key role in glucose-induced anchor processing to the endogenous (G)PI-PLC. The secondary processing of the lipolytically cleaved intermediate of Gce1p at the plasma membrane is correlated with the emergence of a covalently linked high-molecular-weight form of a cAMP-binding protein at the cell wall. This protein lacks anchor components, and its protein moiety appears to be identical with double-processed Gce1p detectable at the plasma membrane in spheroplasts. The data suggest that glucose-induced double processing of GPI anchors represents part of a mechanism of regulated cell wall expression of proteins in yeast cells.     

Structure and assembly of hemidesmosomes

The hemidesmosome is a complex junction containing many proteins. The keratin cytoskeleton attaches to its cytoplasmic plaque, while its transmembrane elements interact with components of the extracellular matrix. Hemidesmosome assembly involves recruitment of alpha 6 beta 4 integrin heterodimers, as well as cytoskeletal elements and cytoskeleton-associated proteins to the cell surface. In our cell culture models, these phenomena appear to be triggered by laminin-5 in the extracellular matrix. Cell interaction with laminin-5 apparently induces both phosphorylation and dephosphorylation of subunits of alpha 6 beta 4 integrin. There is emerging evidence that such events are necessary for subsequent cytoskeleton anchorage to the hemidesmosome cytoplasmic plaque. Once assembled, the hemidesmosome plays an essential role in maintaining firm epithelial adhesion to the basement membrane, with hemidesmosome disruption being a hallmark of certain devastating blistering diseases. However, the hemidesmosome is more than just a stable anchor, as it may also be the site of signal transduction, mediated by its alpha 6 beta 4 integrin component. This review discusses our current knowledge of the structure and assembly of the hemidesmosome.     

Site-directed mutagenesis of the arginine-glycine-aspartic acid in vitronectin abolishes cell adhesion

Vitronectin (VN), a major cell adhesion protein, is found in plasma and in the extracellular matrix. At least three distinct cell surface receptors for vitronectin belonging to the integrin superfamily have been identified in normal and neoplastic cells. Many cell adhesion ligands, including vitronectin, contain an Arg-Gly-Asp (RGD) sequence mediating, in part, the ligand-receptor interaction. These ligands bind different integrins with varying specificity and affinity. The mechanism of receptor specificity remains controversial. To determine the role of the RGD sequence in receptor specificity, we amplified the cDNA for human vitronectin from a liver cDNA library and generated two separate mutants by utilizing site-directed mutagenesis resulting in aspartic acid (Asp47) to glutamic acid (Glu47) substitution and glycine (Gly46) to alanine (Ala46) substitution. The mammalian expression vector, pZEM229R, was used to transfect baby hamster kidney cells which secreted recombinant proteins into the supernatant. All recombinant proteins were isolated by heparin-agarose chromatography and tested for interaction with three known vitronectin receptors, namely, alpha IIIb beta 3 on thrombin-activated platelets, alpha v beta 3 on human umbilical vein endothelial cells and alpha v beta 5 on Panc-1 cells. Recombinant wild-type vitronectin behaved in a fashion similar to plasma-derived vitronectin. Both the RGE-VN and RAD-VN recombinant mutant proteins showed complete loss of cell adhesion activity, regardless of the receptor. These results confirm the essential and central role of the RGD sequence in vitronectin for cell adhesion. This expression system allows further structure/function analysis of vitronectin.     

Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae

Three glucanase-extractable cell wall proteins from Saccharomyces cerevisiae were purified, and their N-terminal amino acid sequences were determined. With this information, we were able to assign gene products to three known open reading frames (ORFs). The N-terminal sequence of a 55-kDa mannoprotein corresponded with the product of ORF YKL096w, which we named CWP1 (cell wall protein 1). A 80-kDa mannoprotein was identified as the product of the TIP1 gene, and a 180-kDa mannoprotein corresponded to the product of the ORF YKL444, which we named CWP2. CWP1, TIP1, and CWP2 encode proteins of 239, 210, and 92 amino acids, respectively. The C-terminal regions of these proteins all consist for more than 40% of serine/threonine and contain putative glycosylphosphatidylinositol attachment signals. Furthermore, Cwp1p and Tip1p were shown to carry a beta 1,6-glucose-containing side chain. The cwp2 deletion mutant displayed an increased sensitivity to Congo red, calcofluor white, and Zymolyase. Electron microscopic analysis of the cwp2 deletion mutant showed a strongly reduced electron-dense layer on the outside of the cell wall. These results indicate that Cwp2p is a major constituent of the cell wall and plays an important role in stabilizing the cell wall. Depletion of Cwp1p or Tip1p also caused increased sensitivities to Congo red and calcofluor white, but the effects were less pronounced than for cwp2 delta. All three cell wall proteins show a substantial homology with Srp1p, which also appears to be localized in the cell wall. We conclude that these four proteins are small structurally related cell wall proteins.