Cross-linking and O-acetylation of newly synthesized peptidoglycan in Staphylococcus aureus H

Staphylococcus aureus H growing exponentially was labelled with N-acetyl[14C]glucosamine, which became incorporated into the peptidoglycan. The portion of peptidoglycan not linked to teichoic acid (60-75% of the whole) was degraded with Chalaropsis muramidase to yield disaccharide-peptide monomers and dimers, trimers and oligomers formed by biosynthetic cross-linking of the monomers. The degree of O-acetylation of these fragments was also examined. Pulse-chase experiments showed that the proportion of label initially in the monomer fraction immediately after the 1 min pulse declined rapidly during a 3 min chase, while the oligomer fraction (fragments greater than trimer) gained the radioactivity proportionately. The radioactivity of the dimer and trimer fractions remained virtually unchanged. At 4 min after the commencement of labelling (i.e. approx. one-tenth of a generation time) final values had been reached. The O-acetylation of all fragments had achieved final values even at 1 min, except for the monomer fraction, which showed an increase from 40% to 60% during the first 3 min of chase. Although O-acetylation was clearly a very rapid process, no O-acetylated peptidoglycan lipid-intermediates could be detected.     

Gradual alterations in cell wall structure and metabolism in vancomycin-resistant mutants of Staphylococcus aureus

In five vancomycin-resistant laboratory step mutants selected from the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL (MIC of methicillin, 800 microg/ml; MIC of vancomycin, 1.5 microg/ml), the gradually increasing levels of resistance to vancomycin were accompanied by parallel decreases in the levels of methicillin resistance and abnormalities in cell wall metabolism. The latter included a gradual reduction in the proportion of highly cross-linked muropeptide species in peptidoglycan, down-regulation of the production of penicillin-binding protein 2A (PBP2A) and PBP4, and hypersensitivity to beta-lactam antibiotics each with a relatively selective affinity for the various staphylococcal PBPs; the PBP2-specific inhibitor ceftizoxime was particularly effective.     

Effect of oligomeric derivatives of prostaglandin B1 on oxidative phosphorylation and their Ca2+ ionophoretic activity

Dimers, trimers and tetramers of 15-dehydro-PGB1 and of 16,16'-dimethyl-15-dehydro-PGB1 have been synthesized and their effect on mitochondrial function evaluated. The trimers and tetramers, and to a lesser extent the dimers, of both series, protected isolated mitochondria from the loss of phosphorylating capacity during in vitro incubation. The monomers were inactive. The trimers and tetramers inhibited between 40 and 50% the F1F0-ATPase of submitochondrial particles. All of the oligomers, but not the monomers, had Ca2+ ionophoretic activity with isolated mitochondria. These activities are qualitatively similar to that reported for the oligomeric mixture of 15-dehydro-PGB1, termed PGBX.     

Antitumor activity and other biological actions of oligomers of ribonuclease A

Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.     

Characterization and isolation of intermediates in beta-lactoglobulin heat aggregation at high pH

The early stages of heat induced aggregation at 67.5 degrees C of beta-lactoglobulin were studied by combined static light scattering and size exclusion chromatography. At all conditions studied (pH 8.7 without salt and pH 6.7 with or without 60 mM NaCl) we observe metastable heat-modified dimers, trimers, and tetramers. These oligomers reach a maximum in concentration at about the time when large aggregates (1000-4000 kg/mol) appear, after which they decline in concentration. By isolating the oligomers it was demonstrated that they rapidly form aggregates upon heating in the absence of monomeric protein, showing that these species are central to the aggregation process. To our knowledge this is the first time that intermediates in protein aggregation have been isolated. At all stages of aggregation the dominant oligomer was the heat-modified dimer. Whereas the heat-modified oligomers are formed at a higher rate at pH 8.7 than at pH 6.7, the opposite is the case for the formation of aggregates from the metastable oligomers indicating cross-linking via disulfide bridges for the oligomers and noncovalent interaction in the formation of the aggregates. The data suggest that an aggregate nucleus is formed from four oligomers. For protein concentrations of 10 or 20 g/l a heat-modified monomer can be observed until about the time when the maximum in concentration appears of the heat-modified dimer. The disappearance of this heat-modified monomer correlates to the formation of dimers (trimers and tetramers).     

Molecular architecture of human properdin, a positive regulator of the alternative pathway of complement

The structure of the human properdin molecule was investigated by hydrodynamic, spectroscopic, and transmission electron microscope studies. Sucrose density gradient ultracentrifugation of purified, functionally active properdin showed a single component sedimenting at 5.5 S. Electron microscopic examination by two different methods, however, revealed polydispersity of the protein which consisted of cyclic dimers, trimers, tetramers, pentamers, and higher cyclic oligomers. Approximately 80% of the oligomers were dimers, trimers, and tetramers. Monomers could not be detected. These polymers could be partially separated by gel filtration on Sephacryl S-300 and all fractions were active in terms of binding to C3b. The specific activity increased with oligomer size. When reexamined after incubation at 37 degrees C for 4 h or at 4 degrees C for 2 weeks, the chromatographic behavior of the oligomers and their electron microscopic appearance were unchanged, suggesting that in vitro no rapid equilibration occurred. The protomer is clearly visualized within polymers as a flexible, rod-like structure 26.0 nm in length and 2.5 nm in diameter, with pronounced thickening at each end. The monomer is bivalent with respect to binding to other properdin monomers and the binding sites are localized to the ends of the structure. A model could be devised which is consistent with the distinct geometry of the intersubunit contacts observed in micrographs. The circular dichroism spectrum of properdin suggests the presence of little alpha helix or beta structure and shows positive ellipticity at 231 nm. In contrast to previous investigators, we conclude that isolated human properdin is polydisperse and consists of a set of cyclic polymers constructed from a single highly asymmetric and flexible protomer.     

Structural properties of trimers and tetramers of ribonuclease A

Ribonuclease A aggregates (dimers, trimers, tetramers, pentamers) can be obtained by lyophilization from 40% acetic acid solutions. Each aggregate forms two conformational isomers distinguishable by different basic net charge. The crystal structure of the two dimers has recently been determined; the structure of the higher oligomers is unknown. The results of the study of the two trimeric and tetrameric conformers can be summarized as follows: (1) RNase A trimers and tetramers form by a 3D domain-swapping mechanism. N-terminal and C-terminal types of domain swapping could coexist; (2) the secondary structures of the trimeric and tetrameric conformers do not show significant differences if compared with the secondary structure of monomeric RNase A or its two dimers; (3) a different exposure of tyrosine residues indicates that in the aggregates they have different microenvironments; (4) the two trimeric and tetrameric conformers show different susceptibility to digestion by subtilisin; (5) dimers, trimers, and tetramers of RNase A show unwinding activity on double-helical poly(dA-dT) x poly(dA-dT), that increases as a function of the size of the oligomers; (6) the less basic conformers are more stable than the more basic ones, and a low concentration in solution of trimers and tetramers favors their stability, which is definitely increased by the interaction of the aggregates with poly(dA-dT) x poly(dA-dT); (7) the products of thermal dissociation of the two trimers indicate that their structures could be remarkably different. The dissociation products of the two tetramers allow the proposal of two models for their putative structures.     

Peptidoglycan cross-linking in Staphylococcus aureus. An apparent random polymerisation process

The peptidoglycan of Staphylococcus aureus contains relatively short glycan chains and is highly cross-linked via its peptide chains. The material from wild-type (strain H) and mutants H28, H4B and MR-1 was freed from the teichoic-acid-linked component and then hydrolysed by Chalaropsis muramidase to yield disaccharide-repeating units of the glycan with attached peptides either non-cross-linked (monomer) or joined to similar units by one (dimer), two (trimer) or more (oligomer) peptide cross links. The resulting fragments were separated by high-resolution HPLC so that distinguishable components as large as nonamer could be identified. Extrapolation showed that, in S. aureus H, H28 and MR-1, oligomers at least as large as eicosamer formed part of the smooth distribution of oligomer fragments, whereas in strain H4B (PBP4-) the maximum size was around dodecamer. The oligomer distribution profile was related to the polymerization theories of Flory, which allow a distinction to be made between a monomer addition model, whereby each oligomer can only be synthesized by the addition of a single monomer unit to its next lower homologue, and a random addition model, in which an oligomer can be formed by linkage of any combination of its constituent smaller units. In S. aureus close approximation to the random addition model for oligomer synthesis and hence for peptidoglycan cross-linking was observed, both in PBP4+ and PBP4- mutants. The implications for secondary cross-linking in S. aureus cell wall formation are inescapable, although the possibility of an endopeptidase/transpeptidase providing later modification of the peptidoglycan is not completely ruled out.     

Inactivated pbp4 in highly glycopeptide-resistant laboratory mutants of Staphylococcus aureus.

Both vancomycin- and teicoplanin-resistant laboratory mutants of Staphylococcus aureus produce peptidoglycans of altered composition in which the proportion of highly cross-linked muropeptide species is drastically reduced with a parallel increase in the representation of muropeptide monomers and dimers (Sieradzki, K., and Tomasz, A. (1997) J. Bacteriol. 179, 2557-2566; and Sieradzki, K. , and Tomasz, A. (1998) Microb. Drug Resist. 4, 159-168). We now report that the distorted peptidoglycan composition is related to defects in penicillin-binding protein 4 (PBP4); no PBP4 was detectable by the fluorographic assay in membrane preparations from the mutants, and comparison of the sequence of pbp4 amplified from the mutants indicated disruption of the gene by two types of abnormalities, a 17-amino acid long duplication starting at position 305 of the pbp4 gene was detected in the vancomycin-resistant mutant, and a stop codon was found to be introduced into the pbp4 KTG motif at position 261 in the mutant selected for teicoplanin resistance. Additional common patterns of disturbances in the peptidoglycan metabolism of the mutants are indicated by the increased sensitivity of mutant cell walls to the M1 muramidase and decreased sensitivity to lysostaphin, which is a reversal of the susceptibility pattern of the parental cell walls. Furthermore, the results of high performance liquid chromatography analysis of lysostaphin digests of peptidoglycan suggest an increase in the average chain length of the glycan strands in the peptidoglycan of the glycopeptide-resistant mutants. The increased molar proportion of muropeptide monomers in the cell wall of the glycopeptide-resistant mutants should provide binding sites for the "capture" of vancomycin and teicoplanin molecules, which may be part of the mechanism of glycopeptide resistance in S. aureus.     

Colchicine photosensitizes covalent tubulin dimerization.

Pure rat brain tubulin can be cross-linked by ultraviolet irradiation of tubulin-colchicine complexes at the high-wavelength maximum of colchicine to form covalent dimers greater than trimers greater than tetramers. With colchicine concentrations approximately 3 x 10(-4) M (mole ratio to tubulin 3-12) and irradiation for 5-10 min at 95-109 mW/cm2, the yield of dimers is 11-17% and of trimers is 4-6% of the total tubulin. The oligomers show polydispersity and anomalously high apparent molecular masses that converge toward expected values in low-density gels. Maximal dimer yields are obtained with MTC and the decreasing photosensitizing potency is MTC greater than colchicine greater than colchicide greater than isocolchicine greater than thiocolchicine. Single-ring troponoids also promote dimerization. Evidence is presented suggesting that the initial, low-affinity, binding step of colchicine and its analogues is sufficient to photosensitize tubulin dimerization.     

Dimeric, trimeric and tetrameric complexes of immunoglobulin G fix complement.

The binding of pure dimers, trimers and tetramers of randomly cross-linked non-immune rabbit immunoglobulin G to the first component and subcomponent of the complement system, C1 and C1q respectively, was studied. These oligomers possessed open linear structures. All three oligomers fixed complement with decreasing affinity in the order: tetramer, trimer, dimer. Complement fixation by dimeric immunoglobulin exhibited the strongest concentration-dependence. No clear distinction between a non-co-operative and a co-operative binding mechanism could be achieved, although the steepness of the complement-fixation curves for dimers and trimers was better reflected by the co-operative mechanism. Intrinsic binding constants were about 10(6)M-1 for dimers, 10(7)M-1 for trimers and 3 X 10(9)M-1 for tetramers, assuming non-co-operative binding. The data are consistent with a maximum valency of complement component C1 for immunoglobulin G protomers in the range 6-18. The binding of dimers to purified complement subcomponent C1q was demonstrated by sedimentation-velocity ultracentrifugation. Mild reduction of the complexes by dithioerythritol caused the immunoglobulin to revert to the monomeric state (S20,w = 6.2-6.5S) with concomitant loss of complement-fixing ability.     

Dimers initiate and propagate serine protease inhibitor polymerisation

The serine protease inhibitor (serpin) family can readily form long-chain polymers by a process that underlies a variety of diseases. We show here that monomers of plasma serpins α₁-antitrypsin and antithrombin are stable on incubation with the rate-limiting step in their polymerisation being the formation of the initial dimer. Once formed, the dimers readily interlink to form tetramers and can bind monomers to form trimers and longer oligomers. Cleavage of the only exposed reactive loop, in unit I of the dimers, prevents their interlinkage, but these cleaved dimers can still link to monomers. The rapid binding by the cleaved dimers of a peptide specific to the lower half of β-sheet A of the molecule indicates the ready opening of this β-sheet in unit II of the dimers. The failure of the cleaved dimers to bind peptide-complexed monomers, together with the relative inaccessibility of the P14 hinge residue in the oligomers, is evidence that partial insertion of the reactive loop into its own A-sheet is required for polymer formation. We propose that serpin dimers initiate and propagate polymerisation by having one exposed loop with an optimal conformation as a β-strand donor and a readily opened β-sheet as an acceptor. The sequential reformation of these activated β-interfaces as the oligomer extends, molecule by molecule, provides a model for the fibril and amyloid formation of conformational diseases in general as well as for the infectivity of prion encephalopathies.     

Factors affecting gamma-chain multimer formation in cross-linked fibrin.

The major covalently linked multimolecular D fragments found in plasmic digests of factor XIIIa cross-linked fibrin formed under physiological pH and ionic strength conditions consist of D dimers, D trimers, and D tetramers. These fragments are linked by epsilon-amino-gamma-glutamyllysine bonds in the carboxy-terminal regions of their gamma chains, which had originated in the cross-linked fibrin as gamma dimers, gamma trimers, and gamma tetramers, respectively. In this study, factors affecting the degree and rate of formation of these three classes of cross-linked gamma chains were determined by analyzing the D-fragment content of plasmic digests of cross-linked fibrin that had been sampled after all gamma-chain monomers had been consumed in the cross-linking process. D trimers and D tetramers, expressed as a proportion of the total D-fragment content, both increased at the expense of the D-dimer population as a function of increasing factor XIII concentration, the time of cross-linking, or the CaCl2 concentration. Their levels decreased as the ionic strength was raised by NaCl addition. However, the ionic strength effect could be reversed by concomitantly raising the CaCl2 concentration. Digests of clots prepared from recalcified fresh citrated plasma also contained each type of cross-linked D fragment, and the proportion of D trimers and D tetramers in the digest increased with increasing clot incubation time. These results indicate that gamma-trimer and gamma-tetramer formation is a dynamic physiological process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiation of methicillin activity against methicillin-resistant Staphylococcus aureus by diterpenes

Totarol is a diterpene compound extracted from the totara tree. Totarol and eight other diterpenes were found to potentiate methicillin, one reducing the minimum inhibitory concentration of methicillin against resistant Staphylococcus aureus 256-fold. Totarol did not inhibit the synthesis of DNA or peptidoglycan in S. aureus, but reduced the respiration rate by 70%. Under potentiation conditions, diterpenes had only a slight effect on the respiration rate, but had a significant effect on expression of PBP 2a. We conclude that the primary staphylococcal target for totarol is the respiratory chain, but that potentiation of methicillin by diterpenes is by interference with PBP 2a expression.     

Active oligomeric ATP synthases in mammalian mitochondria

Recently, by analysis of mildly solubilized mitochondrial membranes new biochemical evidences were obtained for the occurrence of ATP synthase dimers in mitochondria of different eukaryotes from yeast to mammals. In the case of yeast even higher ATP synthase oligomers could be found. Here, we analysed by BN- and CN-PAGE mammalian (bovine and rat) mitochondria from five different tissues, which were efficiently but very mildly solubilized with digitonin. In mitochondria from all investigated tissues besides ATP synthase monomers (V(1)) not only dimeric ATP synthase (V(2)) but for the first time also higher oligomers, at least trimers (V(3)) and tetramers (V(4)), were separated. Compared with BN-PAGE, by CN-PAGE analysis the yields of preserved respiratory supercomplexes as well as of oligomeric ATP synthases (V(2-4)) were significantly increased. The latter represent the majority of total ATP synthases in all cases. Importantly, all different ATP synthase species from the five tissues displayed in-gel ATP hydrolase activity, suggesting that homooligomeric ATP synthases are the constitutive, enzymatically competent organization of mammalian ATP synthases in the inner mitochondrial membrane.     

Primary structure of cyanelle peptidoglycan of Cyanophora paradoxa: a prokaryotic cell wall as part of an organelle envelope.

The peptidoglycan layer surrounding the photosynthetic organelles (cyanelles) of the protist Cyanophora paradoxa is thought to be a relic of their cyanobacterial ancestors. The separation of muropeptides by gel filtration and reverse-phase high-performance liquid chromatography revealed four different muropeptide monomers. A number of muropeptides were identical in retention behavior to muropeptides of Escherichia coli, while others had remarkably long retention times with respect to their sizes, as indicated by gel filtration. Molecular mass determination by plasma desorption and matrix-assisted laser desorption ionization mass spectrometry showed that these unusual muropeptides had molecular masses greater by 112 Da or a multiple thereof than those of ones common to both species. Fast atom bombardment-tandem mass spectrometry of these reduced muropeptide monomers allowed the localization of the modification to D-glutamic acid. High-resolution fast atom bombardment-mass spectrometry and amino acid analysis revealed N-acetylputrescine to be the substituent (E. Pittenauer, E. R. Schmid, G. Allmaier, B. Pfanzagl, W. Löffelhardt, C. Quintela, M. A. de Pedro, and W. Stanek, Biol. Mass Spectrom. 22:524-536, 1993). In addition to the 4 monomers already known, 8 dimers, 11 trimers, and 6 tetramers were characterized. An average glycan chain length of 51 disaccharide units was determined by the transfer of [U-14C]galactose to the terminal N-acetylglucosamine residues of cyanelle peptidoglycan. The muropeptide pattern is discussed with respect to peptidoglycan biosynthesis and processing.     

Mapping Conformational Ensembles of Aβ Oligomers in Molecular Dynamics Simulations

Although the oligomers formed by Aβ peptides appear to be the primary cytotoxic species in Alzheimer's disease, detailed information about their structures appears to be lacking. In this article, we use exhaustive replica exchange molecular dynamics and an implicit solvent united-atom model to study the structural properties of Aβ monomers, dimers, and tetramers. Our analysis suggests that the conformational ensembles of Aβ dimers and tetramers are very similar, but sharply distinct from those sampled by the monomers. The key conformational difference between monomers and oligomers is the formation of β-structure in the oligomers occurring together with the loss of intrapeptide interactions and helix structure. Our simulations indicate that, independent of oligomer order, the Aβ aggregation interface is largely confined to the sequence region 10-23, which forms the bulk of interpeptide interactions. We show that the fractions of β structure computed in our simulations and measured experimentally are in good agreement.     

Recruitment of penicillin-binding protein PBP2 to the division site of Staphylococcus aureus is dependent on its transpeptidation substrates

Staphylococcus aureus penicillin-binding protein PBP2 is an enzyme involved in the last stages of peptidoglycan assembly and is an important player in the mechanism of methicillin resistance of this pathogen. PBP2 localized to the division site but its recruitment to the forming division septum was prevented after acylation by oxacillin. The presence of the antibiotic did not affect FtsZ ring maintenance nor the localization of externalized peptidoglycan precursors. Delocalization of PBP2 was also observed when its pentapeptide substrate was eliminated by addition of d-cycloserine or blocked by addition of vancomycin. Taken together these observations suggest that PBP2 is recruited to the division site by binding to its substrate, which is localized at that place. In methicillin-resistant S. aureus, addition of oxacillin does not result in delocalization of PBP2 indicating that acylated PBP2 can be maintained in place by functional PBP2A, the central element of this resistance mechanism.     

Oligomerization of ribonuclease A under reducing conditions

By lyophilization from 40% acetic acid solutions, bovine ribonuclease A forms well characterized, three-dimensional domain-swapped oligomers: dimers, trimers, tetramers, and higher order multimers. Each oligomeric species consists of at least two conformers. Identical oligomers also form by thermally-inducing the oligomerization of highly concentrated RNase A dissolved in fluids endowed with various denaturing power. Now, our question is: which might the influence of a reducing agent be on RNase A oligomerization, i.e., of conditions that decrease the stability of the protein and increase the mobility of its swapping domains? To address this question, we carried out experiments of RNase A oligomerization in the presence of increasing concentrations of dithiothreitol (DTT) under the two experimental conditions mentioned above. Results indicate that RNase A oligomers similar to those previously known form anyhow, but with a change of their relative proportions. The amounts of dimers and trimers decrease by increasing the concentration of DTT, while the yields of two tetramers remarkably increase. Moreover, in the presence of DTT RNase A forms labile and probably unstructured aggregates that can possibly drive the protein towards precipitation when the reducing agent's concentration increases. Taken together, these results point out once again (i) the important role of the 3D domain swapping mechanism in protein oligomerization, and (ii) the importance of the native structure of RNase A (and of proteins in general) in preventing an uncontrolled aggregation and precipitation in a reducing and highly crowded environment like that existing in a living cell.     

Peptidoglycan synthesis by Enterococcus faecalis penicillin binding protein 5

Low-affinity penicillin binding proteins (PBPs) are a particular class of proteins involved in β-lactam antibiotic resistance of enterococci. The activity of these PBPs is just sufficient to allow the cells to survive in the presence of high concentrations of β-lactams that cause saturation (and inhibition) of the other PBPs. For this reason, the low-affinity PBPs are thought to be multifunctional enzymes capable of catalyzing the entire peptidoglycan synthesis. To test the validity of this claim, we analyzed the muropeptide composition by reversed-phase high-performance liquid chromatography of the peptidoglycan synthesized by PBP5 (the low-affinity PBP) of Enterococcus faecalis, in comparison with the peptidoglycan produced normally by the concerted action of the usual PBPs (namely PBPs 1, 2, and 3). Cross-linked peptidoglycan was produced. The main difference consisted in the lack of oligomers higher than trimers, thus suggesting that this oligomer cannot be used as an acceptor/donor by the transpeptidase component of PBP5. The lack of higher oligomers had little impact on total cross-linking because of the increase observed in the dimer family. This increase was distributed among the various members of the dimer family with the result that minor dimer components figured among the prevalent ones in cells in which peptidoglycan was synthesized by PBP5. This also suggests that E. faecalis PBP5 is capable of catalyzing the synthesis of a peptidoglycan that is less precise and refined than usual, and for this reason PBP5 can be considered an enzyme endowed with poor specificity for substrates, as may be expected on the basis of its survival function. Received: 18 March 1998 / Accepted: 26 May 1998