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Correlation between the structure of the bacterial peptidoglycan monomer unit, the specificity of transpeptidation, and susceptibility to β-lactams
Abstract
Peptidoglycan is a continuous covalent macromolecular structure found on the outside of the cytoplasmic membrane of almost all bacteria and exclusively in these organisms. As the main structural component of the bacterial cell wall, its function is to preserve cell integrity by withstanding the internal osmotic pressure. It is also responsible for the maintenance of a defined cell shape, and it is intimately involved in the cell division process (1). The two basic structural features of this giant macromolecule are linear glycan chains interlinked with short peptide bridges. The glycan chains are composed of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). The carboxyl group of each N-acetylmuramic acid residue is substituted by a short stem peptide subunit. The formation of the three-dimensional network structure of peptidoglycan is ensured by cross-linking between the peptide subunit of one chain with that of a neighboring chain (2).
... The reaction leads to an inversion of configuration at C1, from the R-configuration in the precursor to the -configuration in the glycosylated acceptor. Glycan chain elongation occurs either by transfer of the growing chain attached to the undecaprenyl-pyrophosphate, which is the donor, to C4 of N-acetylglucosamine of a lipid II precursor molecule (way 1, Figure 1) or by transfer of a lipid- linked disaccharide functioning as a glycosyl donor to C4 of the growing chain, which is the acceptor substrate and is not necessarily linked to the undecaprenyl (way 2, Figure 1) (1,21). To gain insight into the catalytic mechanism of the glycosyl transferase, the specificity profile of the enzyme for the lipid II substrate was determined by using substrate analogues. ...
... These experiments do not allow us to decide whether the growing saccharidic chain acts as a donor [as suggested in Gram-positive bacteria (7,21)] or as an acceptor in the transglycosylation reaction. However, it appears that PBP1b shows a strong preference for acceptor molecules containing a lipid moiety. ...
The glycosyl transferase of the Escherichia coli bifunctional penicillin-binding protein (PBP) 1b catalyzes the assembly of lipid-transported N-acetylglucosaminyl-beta-1,4-N-acetylmuramoyl-L-Ala-gamma-D-Glu-meso-A2pm-D-Ala-D-Ala units (lipid II) into linear peptidoglycan chains. These units are linked, at C1 of N-acetylmuramic acid (MurNAc), to a C55 undecaprenyl pyrophosphate. In an in vitro assay, lipid II functions both as a glycosyl donor and as a glycosyl acceptor substrate. Using substrate analogues, it is suggested that the specificity of the enzyme for the glycosyl donor substrate differs from that for the acceptor. The donor substrate requires the presence of both N-acetylglucosamine (GlcNAc) and MurNAc and a reactive group on C1 of the MurNAc and does not absolutely require the lipid chain which can be replaced by uridine. The enzyme appears to prefer an acceptor substrate containing a polyprenyl pyrophosphate on C1 of the MurNAc sugar. The problem of glycan chain elongation that presumably proceeds by the repetitive addition of disaccharide peptide units at their reducing end is discussed.
... The PBPs fall into two categories, high molecular mass (HMM) PBPs (1a, 1b, 2, 3, and 3a) and lower molecular mass (LMM) PBPs (4, 5, and 7) [86]. PBPs play a role in cellular division and controlling cellular morphology through the incorporation of NAG-NAM units into growing peptidoglycan chains via a glycosyltransferase activity, cross-linking of different NAG-NAM units through their peptide side chains, and modifying peptidoglycan peptide chains ( Figure 1) [87,88]. There is functional redundancy of PBPs, with only PBP3 being essential for growth [79]. ...
Pseudomonas aeruginosa is a major opportunistic pathogen, causing a wide range of acute and chronic infections. β-lactam antibiotics including penicillins, carbapenems, monobactams, and cephalosporins play a key role in the treatment of P. aeruginosa infections. However, a significant number of isolates of these bacteria are resistant to β-lactams, complicating treatment of infections and leading to worse outcomes for patients. In this review, we summarize studies demonstrating the health and economic impacts associated with β-lactam-resistant P. aeruginosa. We then describe how β-lactams bind to and inhibit P. aeruginosa penicillin-binding proteins that are required for synthesis and remodelling of peptidoglycan. Resistance to β-lactams is multifactorial and can involve changes to a key target protein, penicillin-binding protein 3, that is essential for cell division; reduced uptake or increased efflux of β-lactams; degradation of β-lactam antibiotics by increased expression or altered substrate specificity of an AmpC β-lactamase, or by the acquisition of β-lactamases through horizontal gene transfer; and changes to biofilm formation and metabolism. The current understanding of these mechanisms is discussed. Lastly, important knowledge gaps are identified, and possible strategies for enhancing the effectiveness of β-lactam antibiotics in treating P. aeruginosa infections are considered.
... 17), yielding lipid I (C 55 -P-MurNAc-pentapeptide) (162). The translocase MurG subsequently links UDPactivated N-acetyl-glucosamine (UDP-GlcNAc) to the muramoyl moiety of lipid I, thus yielding lipid II (C 55 -P-GlucNAc-MurNAc-pentapeptide)(388). ...
Das Gram-positive Pathogen Staphylococcus aureus ist zwingend auf die korrekte Synthese der Zellwand angewiesen. Die darin enthaltene Pentaglycinkette wird sequentiell durch die essentiellen Fem-Faktoren angehängt und ist obligat für Methicillin-Resistenz. Es wurde ein in vitro Assay entwickelt, welcher die Identifizierung der Substrate der einzelnen Fem-Faktoren erlaubte und die Untersuchung potentiell letaler Inhibitoren ermöglicht. Abwehrmechanismen des Wirts und die Wirkung von Antibiotika können in S. aureus durch die Bildung so genannter small colony variants (SCVs) umgangen werden. Es wurde eine stabile SCV konstruiert, welche signifikante Unterschiede in der Expression von Regulatoren und Virulenzfaktoren im Vergleich zum Wildtyp zeigte. In der SCV wurde die durchgehende Aktivität eines Stress-Faktors, Sigma B, nachgewiesen und somit einer der involvierten Regulatoren identifiziert. Accurate cell wall synthesis is crucial for the Gram-positive pathogen Staphylococcus aureus. The pentaglycine side chain of the peptidoglycan precursor is added in a sequential manner by the essential Fem factors and is a prerequisite for methicillin resistance. An in vitro assay was developed which led to the identification of the individual substrates of the Fem factors and which could be used for testing potentially lethal inhibitors. Defence mechanisms of the host and the effects of antibiotics can be bypassed in S. aureus via formation of so-called small colony variants (SCVs). A stable SCV was constructed which showed significant differences in regulator and virulence factor expression compared to the wild type. In the SCV, the general stress factor sigma B was found to be one of the regulators involved as it was active over an extended growth period.
... A short peptide of four amino acids is attached to the carboxyl group of NAM of mature peptidoglycan. Variability in the peptidoglycan structure is largely due to differences in the short peptide, although differences in the glycan backbone and nature of the crosslink have also been noted 22 . In Escherichia coli, for example, the mature stem peptide is composed of L-alanine, D-glutamic acid, meso-diaminopimelic acid and D-alanine, whereas in Staphylococcus aureus meso-diaminopimelic acid is replaced by L-lysine. ...
The introduction of antibiotic therapy for the treatment of bacterial infections has led to a greatly increased human life span compared to that in the pre-antibiotic era. However, a disturbing trend has also been noted in that, within a very short period of time following the introduction of a new antibiotic, resistance to that antibiotic begins to emerge, a factor that is becoming increasingly meaningful as the discovery of new antibiotics wanes [1–3]. There are a number of mechanisms by which a bacterium may become resistant to a particular antibiotic. Generally these include, but are not limited to, modification of the drug to render it inactive, modification of the drug target, such that it is incapable of interacting with the drug and decreased uptake of the antibiotic into the cell, due to reduced transport and/or increased efflux. Recent functional genomic studies have also implied that antibiotics may have more complex mechanisms of action than first thought and we are beginning to appreciate that in addition to the mutation of primary targets, subtle mutations in secondary targets are likely to be influential [4, 5]. Moreover, a growing body of evidence suggests that the temporary changes in susceptibility associated with the phenomenon of adaptive resistance may also be important for the global rise in bacterial resistance to antimicrobial compounds [6]. This chapter will focus on the contribution of a decreased antibiotic uptake to an increase in antibacterial resistance.
... The translo-case MurG subsequently links UDP-activated N-acetylglucosamine (UDP-GlcNAc) to the muramoyl moiety of lipid I, yielding lipid II. 88 MurM is able to use either lipid I or lipid II as a substrate in vivo, indicating that the N-acetylglucosaminyl group of lipid II is not necessary for MurM lipid precursor recognition. 117 Figure 2 illustrates the peptidogly-can formation and cross-linking portion of cell wall biogenesis including the known Fem enzyme family members and their precursor specificities. ...
Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families responsible for cell wall modifications, aminoacyl-phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids to phosphatidylglycerol (PG) by aaPGSs neutralizes the lipid bilayer making the bacteria less susceptible to positively charged antimicrobial agents. Fem transferases utilize aa-tRNA to form peptide bridges that link strands of peptidoglycan. These bridges vary among the bacterial species in which they are present and play a role in resistance to antibiotics that target the cell wall. Additionally, the formation of truncated peptides results in shorter peptide bridges and loss of branched linkages which makes bacteria more susceptible to antimicrobials. A greater understanding of the structure and substrate specificity of this diverse enzymatic family is necessary to aid current efforts in designing potential bactericidal agents. These two enzyme families are linked only by the substrate with which they modify the cell wall, aa-tRNA; their structure, cell wall modification processes and the physiological changes they impart on the bacterium differ greatly. WIREs RNA 2012, 3:247–264. doi: 10.1002/wrna.1108
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... The loss of this control is ultimately bactericidal. Moreover, the cell wall also contributes to infectivity and pathogenicity, and the glycopeptides (muropeptides) released from the cell wall during cell growth (and cell death) activate the human immune system (30,71,72,138,155,174). ...
The Gram-positive bacterium Staphylococcus aureus is a leading cause of hospital- and community-associated infections (16, 85, 108). ...
... The first membrane-associated step of cell wall synthesis is catalyzed by MraY, which transfers the soluble cell wall precursor UDP-MurNAc-pp to the membrane carrier undecaprenylphosphate (C 55 -P), yielding lipid I (undecaprenylphosphate-MurNAc-pp). The translocase MurG subsequently adds UDP-activated N-acetyl-glucosamine (UDP-GlcNAc) to the muramoyl moiety of lipid I, thus yielding lipid II (undecaprenylphosphate-GlcNAc-MurNAc-pp) (47). In staphylococci, the lipid II molecule is further modified by the attachment of a pentaglycine interpeptide bridge, catalyzed by the FemXAB peptidyltransferases (33), before the precursor is translocated across the cytoplasmic membrane where it is then assembled into the growing peptidoglycan network by the action of the PBPs. ...
Friulimicin B is a naturally occurring cyclic lipopeptide, produced by the actinomycete Actinoplanes friuliensis, with excellent activity against gram-positive pathogens, including multidrug-resistant strains. It consists of a macrocyclic
decapeptide core and a lipid tail, interlinked by an exocyclic amino acid. Friulimicin is water soluble and amphiphilic, with
an overall negative charge. Amphiphilicity is enhanced in the presence of Ca2+, which is also indispensable for antimicrobial activity. Friulimicin shares these physicochemical properties with daptomycin,
which is suggested to kill gram-positive bacteria through the formation of pores in the cytoplasmic membrane. In spite of
the fact that friulimicin shares features of structure and potency with daptomycin, we found that friulimicin has a unique
mode of action and severely affects the cell envelope of gram-positive bacteria, acting via a defined target. We found friulimicin
to interrupt the cell wall precursor cycle through the formation of a Ca2+-dependent complex with the bactoprenol phosphate carrier C55-P, which is not targeted by any other antibiotic in use. Since C55-P also serves as a carrier in teichoic acid biosynthesis and capsule formation, it is likely that friulimicin blocks multiple
pathways that are essential for a functional gram-positive cell envelope.
... These sites could be related to the binding sites of the peptidoglycan N-acetylmuramidases, which have several subsites each for one sugar unit of peptidoglycan (Thunnissen et al., 1995). The peptide moiety of the monomer unit plays a minor role in the recognition process as exemplified with shorter or modified peptide subunits (see references in van Heijenoort and Gutmann, 2000). ...
The main structural features of bacterial peptidoglycan are linear glycan chains interlinked by short peptides. The glycan chains are composed of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), all linkages between sugars being beta,1-->4. On the outside of the cytoplasmic membrane, two types of activities are involved in the polymerization of the peptidoglycan monomer unit: glycosyltransferases that catalyze the formation of the linear glycan chains and transpeptidases that catalyze the formation of the peptide cross-bridges. Contrary to the transpeptidation step, for which there is an abundant literature that has been regularly reviewed, the transglycosylation step has been studied to a far lesser extent. The aim of the present review is to summarize and evaluate the molecular and cellullar data concerning the formation of the glycan chains in the synthesis of peptidoglycan. Early work concerned the use of various in vivo and in vitro systems for the study of the polymerization steps, the attachment of newly made material to preexisting peptidoglycan, and the mechanism of action of antibiotics. The synthesis of the glycan chains is catalyzed by the N-terminal glycosyltransferase module of class A high-molecular-mass penicillin-binding proteins and by nonpenicillin-binding monofunctional glycosyltransferases. The multiplicity of these activities in a given organism presumably reflects a variety of in vivo functions. The topological localization of the incorporation of nascent peptidoglycan into the cell wall has revealed that bacteria have at least two peptidoglycan-synthesizing systems: one for septation, the other one for elongation or cell wall thickening. Owing to its location on the outside of the cytoplasmic membrane and its specificity, the transglycosylation step is an interesting target for antibacterials. Glycopeptides and moenomycins are the best studied antibiotics known to interfere with this step. Their mode of action and structure-activity relationships have been extensively studied. Attempts to synthesize other specific transglycosylation inhibitors have recently been made.
... Although this is the basic design of PG, a great variety of PG structures have been observed in different bacterial species, and modifications can occur, depending on growth conditions. 20 From an immunologic point of view, PG can be regarded as a functional analog of lipopolysaccharide, because it also binds the CD14 molecule expressed by macrophages and neutrophils, and induces a similar pattern of proinflammatory cytokines in vitro. However, lipopolysaccharide and PG are distinct in their differential co-engagement of TLR for cellular signaling, employing TLR-4 and TLR-2, respectively. ...
Peptidoglycan (PG) is a major component of the cell wall of gram-positive bacteria that is abundantly present in all human mucosa. PG is a functional lipopolysaccharide analog that binds to CD14 on macrophages and induces proinflammatory cytokine production and metalloproteinases. We investigated the hypothesis that bacterial PG is present in atherosclerotic tissue. In addition, plaque phenotypes were characterized in relation to presence of PG. Immunohistology of carotid (n = 15) and femoral (n = 6) endarterectomy specimens revealed the presence of PG in the cytoplasm of cells located in plaques. PG was detected in 14 of 15 carotid arteries and 5 of 6 femoral arteries. From the 14 coronary arteries, 31 atherosclerotic segments were selected. PG was detected within 19 of 31 of these coronary segments. Western blot demonstrated the presence of the toll-like receptor (TLR-2), the co-receptor for PG, in coronary artery tissue. The number of PG-containing cells in coronary arteries was significantly higher when the histologic features of plaque vulnerability were evident. Inflammation of the cap or shoulder was observed in 11 of 19 PG-positive versus 2 of 12 PG-negative segments (p = 0.023). More than 50% of the plaque area consisted of atheroma in 7 of 19 PG-positive segments and 0 of 12 PG-negative segments (p = 0.025). Heavy smooth muscle cell staining occurred in the plaque cap and shoulder in 3 of 19 PG-positive segments versus 9 of 12 PG-negative segments. Proinflammatory bacterial PG and its co-receptor have been observed in atherosclerotic arteries, in association with the vulnerable plaque phenotype.
... Lipid I (undecaprenylphosphate-MurNAcpentapeptide) is formed in the first lipid-linked step of cell wall synthesis by MraY, which transfers the soluble UDP-MurNAc-pentapeptide to the lipid carrier undecaprenylphosphate (C55-P). The translocase MurG subsequently links UDP-activated N -acetyl-glucosamine (UDP-GlucNAc) to the muramoyl moiety of lipid I, thus yielding lipid II (undecaprenylphosphate-GlucNAc-MurNAc-pentapeptide) (van Heijenoort and Gutmann, 2000). ...
Staphylococcus aureus peptidoglycan is cross-linked via a characteristic pentaglycine interpeptide bridge. Genetic analysis had identified three peptidyltransferases, FemA, FemB and FemX, to catalyse the formation of the interpeptide bridge, using glycyl t-RNA as Gly donor. To analyse the pentaglycine bridge formation in vitro, we purified the potential substrates for FemA, FemB and FemX, UDP-MurNAc-pentapeptide, lipid I and lipid II and the staphylococcal t-RNA pool, as well as His-tagged Gly-tRNA-synthetase and His-tagged FemA, FemB and FemX. We found that FemX used lipid II exclusively as acceptor for the first Gly residue. Addition of Gly 2,3 and of Gly 4,5 was catalysed by FemA and FemB, respectively, and both enzymes were specific for lipid II-Gly1 and lipid II-Gly3 as acceptors. None of the FemABX enzymes required the presence of one or two of the other Fem proteins for activity; rather, bridge formation was delayed in the in vitro system when all three enzymes were present. The in vitro assembly system described here will enable detailed analysis of late, membrane-associated steps of S. aureus peptidoglycan biosynthesis.
... Staphylococcus aureus is an important human pathogen that causes life-threatening diseases including septicemia, endocarditis, toxic shock syndrome, and abscesses in organ tissues (15,25). The cell wall of the microorganism plays an important role in infectivity and pathogenicity (40). Over several decades of research, extensive knowledge has accumulated concerning epidemiology (9), virulence (25,28), genetics (3), genomic evolution (14), the biochemistry of cell wall assembly (31), the crystal structures of -lactam-resistant enzymes (24), the ultrastructure of the cell wall (4,18), and the muropeptide composition of wild-type, methicillin-resistant (7), and vancomycinresistant strains (34). ...
The recently described scaffold model of murein architecture depicts the gram-negative bacterial cell wall as a gel-like matrix
composed of cross-linked glycan strands oriented perpendicularly to the plasma membrane while peptide bridges adopt a parallel
orientation (B. A. Dmitriev, F. V. Toukach, K. J. Schaper, O. Holst, E. T. Rietschel, and S. Ehlers, J. Bacteriol. 185:3458-3468,
2003). Based on the scaffold model, we now present computer simulation studies on the peptidoglycan arrangement of the gram-positive
organism Staphylococcus aureus, which show that the orientation of peptide bridges is critical for the highly cross-linked murein architecture of this microorganism.
According to the proposed refined model, staphylococcal murein is composed of glycan and oligopeptide chains, both running
in a plane that is perpendicular to the plasma membrane, with oligopeptide chains adopting a zigzag conformation and zippering
adjacent glycan strands along their lengths. In contrast to previous models of murein in gram-positive bacteria, this model
reflects the high degree of cross-linking that is the hallmark of the staphylococcal cell wall and is compatible with distinguishing
features of S. aureus cytokinesis such as the triple consecutive alteration of the division plane orientation and the strictly centripetal mode
of septum closure.
... Mutations resulting in a modification of the composition of the interpeptide bridges or in a by-pass of the essential PBPs by L,D-transpeptidation in Gram-positive species were also shown to modify the susceptibility of strains to β-lactams. 23,[32][33][34] Morphological transition of Helicobacter pylori from spiral to coccoid was also correlated with a modification of the structure of its cell wall. 35 Also, O-acetylation of MurNAc or N-deacetylation of GlcNAc were shown to increase resistance of pathogenic bacteria to lysozyme, an important host defense component. ...
Peptidoglycan (murein) is a major essential and specific constituent of the bacterial cell wall. Its main function is to protect cells against the internal osmotic pressure and to maintain the characteristic cell shape. It also serves as a platform for the anchoring of specific proteins and other cell wall components. This giant macromolecule is composed of long glycan chains cross-linked by short peptides. Any alteration of the disaccharide-peptide basic unit results in a global change of peptidoglycan structure and properties. Such global variations are encountered in nature as conserved variations along phyletic lines but have sometimes been acquired as a result of mutations or as a mechanism of resistance against cell-wall targeted antibiotics. During bacterial cell growth and division, the peptidoglycan mesh is constantly broken down by a set of highly specific hydrolases in a maturation process allowing insertion of newly synthesized units in the pre-existing polymerized material. Depending on the bacterial species considered, degradation fragments are either released in the growth medium or efficiently re-utilized for synthesis of new murein in a sequence of events termed the recycling pathway. Peptidoglycan is one of the main pathogen-associated molecular patterns recognized by the host innate immune system. Variations of the structure and metabolism of this cell wall component have been exploited by host defense mechanisms for detection/identification of invading bacterial species. Modification of the peptidoglycan structure could also represent a mechanism allowing bacteria to escape these host defense systems.
... The MurM protein catalyses the first step in the addition of short dipeptide branches to the muropeptide units of the pneumococcal peptidoglycan . PBPs are biosynthetic enzymes with transpeptidase (TPase) and/or transglycosylase (TGase) activity that catalyse terminal stages in the synthesis of bacterial peptidoglycan (Goffin and Ghuysen, 1998) and it has been proposed that the reduced reactivity of these proteins for the antibiotic molecule in resistant mutants may also bring along changes in their catalytic efficiency with respect to their physiological substrates (Garcia-Bustos and Tomasz, 1990;van Heijenoort and Gutmann, 2000). ...
The level of penicillin resistance in clinical isolates of Streptococcus pneumoniae depends not only on the reduced affinity of penicillin binding proteins (PBPs) but also on the functioning of enzymes that modify the stem peptide structure of cell wall precursors. We used mariner mutagenesis in search of additional genetic determinants that may further attenuate the level of penicillin resistance in the bacteria. A mariner mutant of the highly penicillin-resistant S. pneumoniae strain Pen6 showed reduction of the penicillin minimum inhibitory concentration (MIC) from 6 to 0.75 microg ml(-1). Decrease in penicillin MIC was also observed upon introduction of the mutation (named provisionally adr, for attenuator of drug resistance) into representatives of major epidemic clones of penicillin-resistant pneumococci. Attenuation of resistance levels was specific for beta-lactams. The adr mutant has retained unchanged (low affinity) PBPs, unaltered murM gene and unchanged cell wall stem peptide composition, but the mutant became hypersensitive to exogenous lysozyme and complementation experiments showed that both phenotypes--reduced resistance and lysozyme sensitivity--were linked to the defective adr gene. DNA sequence comparison and chemical analysis of the cell wall identified adr as the structural gene of the pneumococcal peptidoglycan O-acetylase.
Since Fleming's discovery of penicillin nearly a century ago, a bounty of natural product antibiotics have been discovered, many of which continue to be of clinical importance today. The structural diversity encountered among nature's repertoire of antibiotics is mirrored by the varying mechanisms of action by which they selectively target and kill bacterial cells. The ability for bacteria to construct and maintain a strong cell wall is essential for their robust growth and survival under a range of conditions. However, the need to maintain the cell wall also presents a vulnerability that is exploited by many natural antibiotics. Bacterial cell wall biosynthesis involves both the construction of complex membrane-bound precursor molecules and their subsequent crosslinking by dedicated enzymes. Interestingly, many naturally occurring antibiotics function not by directly inhibiting the enzymes associated with cell wall biosynthesis, but rather by binding tightly to their membrane-bound substrates. Such substrate sequestration mechanisms are comparatively rare outside of the antibiotics space with most small-molecule drug discovery programs instead aimed at developing inhibitors of target enzymes. In this feature article we provide the reader with an overview of the unique and ever increasing family of natural product antibiotics known to specifically function by binding to membrane-anchored bacterial cell wall precursors. In doing so, we highlight both our own contributions to the field as well as those made by other researchers engaged in exploring the potential offered by antibiotics that target bacterial cell wall precursors.
Developing molecular models to capture the complex physicochemical architecture of the bacterial cell wall and to study the interaction with antibacterial molecules is an important aspect of assessing and developing novel antimicrobial molecules. We carried out molecular dynamics simulations using an atomistic model of peptidoglycan to represent the architecture for Gram-positive S. aureus. The model is developed to capture various structural features of the Staphylococcal cell wall, such as the peptide orientation, area per disaccharide, glycan length distribution, cross-linking, and pore size. A comparison of the cell wall density and electrostatic potentials is made with a previously developed cell wall model of Gram-negative bacteria, E. coli, and properties for both single and multilayered structures of the Staphylococcal cell wall are studied. We investigated the interactions of the antimicrobial peptide melittin with peptidoglycan structures. The depth of melittin binding to peptidoglycan is more pronounced in E. coli than in S. aureus, and consequently, melittin has greater contacts with glycan units of E. coli. Contacts of melittin with the amino acids of peptidoglycan are comparable across both the strains, and the D-Ala residues, which are sites for transpeptidation, show enhanced interactions with melittin. A low energetic barrier is observed for translocation of a naturally occurring antimicrobial thymol with the four-layered peptidoglycan model. The molecular model developed for Gram-positive peptidoglycan allows us to compare and contrast the cell wall penetrating properties with Gram-negative strains and assess for the first time binding and translocation of antimicrobial molecules for Gram-positive cell walls.
This chapter covers five main elements which are: the ions including the divalent cations, the antibiotics particularly those affected by the presence of the divalent cations and the ionophor antibiotics, the enzymes involved in the antibiotic resistance, and the macromolecules and their interaction with the ions. It describes the relationship between the divalent cation and the antibiotic resistance through different related mechanisms. The chapter addresses some case studies concerning the role of the divalent cations in the resistance and the sensitivity of the bacteria during the treatment with the antibiotics. The biological system is sensitive to the chemical structure, particularly the metals. P. aeruginosa is always reported as a multidrug resistant microbe. Divalent cations have an important role in prokaryote cells especially in cell protection or in infection adaptation. A better understanding of the role of the divalent cations in antibiotic resistance will enable better pathogen control.
Beta‐lactam antibiotics are the most popular antibacterial agents used for treating bacterial infections, due to their great activity, broad spectrum, and safety profile. All of them share the beta‐lactam ring and because of the particular chemical structure, they are classified into five groups: Penicillins (natural and semisynthetic), cephalosporins (first‐, second‐, third‐, fourth‐, and fifth‐generation), monobactams, carbapenems and the association of a beta‐lactam with a beta‐lactamase inhibitor (Beta‐lactam and non‐beta‐lactam inhibitors). Carbapenems have a carbon atom instead of a sulfur or an oxygen atom in the bicyclic nucleus and a hydroxyethyl side chain in trans configuration at position 6 and are the widest spectrum antibiotics available among the beta‐lactams. Beta‐lactams are bactericidal agents that kill bacteria by inhibiting the synthesis of the cell wall in both Gram‐negative and Gram‐positive bacteria, interacting with PBPs, so the transpeptidation reaction is blockaded, leading to the cell lysis and death. As a consequence of the widespread use of this antibiotics, bacterial resistance is a growing problem in both community and hospital settings. Emergence and dissemination of beta‐lactam resistance have renewed interest in the development of novel beta‐lactam antibiotics or beta‐lactamase inhibitors, and some of them are currently available for multi‐resistant bacteria treatment.
The bacterial cell wall is composed of membrane layers and a rigid yet flexible scaffold called peptidoglycan (PG). PG provides mechanical strength to enable bacteria to resist damage from the environment and lysis due to high internal turgor. PG also has a critical role in dictating bacterial cell morphology. The essential nature of PG for bacterial propagation, as well as its value as an antibiotic target, has led to renewed interest in the study of peptidoglycan biosynthesis. However, significant knowledge gaps remain that must be addressed before a clear understanding of peptidoglycan synthesis and dynamics is realized. For example, the enzymes involved in the PG biosynthesis pathway have not been fully characterized. Our understanding of PG biosynthesis has been frequently revamped by the discovery of novel enzymes or newly characterized functions of known enzymes. In addition, we do not clearly know how the respective activities of these enzymes are coordinated with each other and how they control the spatial and temporal dynamics of PG synthesis. The emergence of molecular probes and imaging techniques has significantly advanced the study PG synthesis and modification. Prior efforts utilized the specificity of PG-targeting antibiotics and proteins to develop PG-specific probes, such as fluorescent vancomycin and fluorescent wheat germ agglutinin. However, these probes suffer from limitations due to toxic effects toward bacterial cells and poor membrane permeability. To address these issues, we designed and introduced a family of novel molecular probes, fluorescent d-amino acids (FDAAs), which are covalently incorporated into PG through the activities of endogenous bacterial transpeptidases. Their high biocompatibility and PG specificity have made them powerful tools for labeling peptidoglycan. In addition, their enzyme-mediated incorporation faithfully reflects the activity of PG synthases, providing a direct in situ method for studying PG formation during the bacterial life cycle. In this Account, we describe our efforts directed at the development of FDAAs and their derivatives. These probes have enabled for the first time the ability to visualize PG synthesis in live bacterial cells and in real time. We summarize experimental evidence for FDAA incorporation into PG and the enzyme-mediated incorporation pathway. We demonstrate various applications of FDAAs, including bacterial morphology analyses, PG growth model studies, investigation of PG-enzyme correlation, in vitro PG synthase activity assays, and antibiotic inhibition tests. Finally, we discuss the current limitations of the probes and our ongoing efforts to improve them. We are confident that these probes will prove to be valuable tools that will enable the discovery of new antibiotic targets and expand the available arsenal directed at the public health threat posed by antibiotic resistance.
The increased antibiotic pollutants in aquatic environments pose severe threats on microbial ecology due to their extensive distribution and antibacterial properties. A total of 16 antibiotics including fluoroquinolones (FQs) (ofloxacin (OFX), ciprofloxacin (CFX), norfloxacin (NFX)), Sulfonamides (SAs) (sulfamonomethoxine (SMM), sulfadiazine (SDZ), sulfaquinoxaline (SQX)), Tetracyclines (TCs) (tetracycline (TC), doxycycline (DC)), β-lactams (penicillin G (PEN G), penicillin V (PEN V), cefalexin (LEX)), Macrolides (MLs) (erythromycin-H2O (ETM), tylosin (TYL)) and other antibiotics (Polymix-B (POL), Vancomycin (VAN), Lincomycin (LIN)) were detected in the surface water of the Qingcaosha Reservoir. Multivariate statistical analysis indicated that both water quality and physicochemical indexes have less contributions on variations of these antibiotics, suggesting the concentrations of antibiotics inside the reservoir are mainly affected by upstream runoff and anthropic activity along the river. Antibiotics including TYL, PEN G and ETM showed significant correlations with variations of bacterial community composition, and closely connected with various gram-negative bacteria in co-occurrence/exclusion patterns of the network, suggesting these bacterial taxa play important roles in the course of migration and transformation of related antibiotics. In conclusion, further research is required to evaluate the potential risk of genetic transfer of resistance to related bacteria induced by long-term exposure to low levels of antibiotics in the environment.
Research into antibacterial agents has recently gathered pace in light of the disturbing crisis of antimicrobial resistance. The development of modern tools offers the opportunity of reviving the fallen era of antibacterial discovery through uncovering novel lead compounds that target vital bacterial cell components, such as lipid II. This paper provides a summary of the role of lipid II as well as an overview and insight into the structural features of macrocyclic peptides that inhibit this bacterial cell wall component. The recent discovery of teixobactin, a new class of lipid II inhibitor has generated substantial research interests. As such, the significant progress that has been achieved towards its development as a promising antibacterial agent is discussed.
The introduction of antibiotic therapy for the treatment of bacterial infections has led to a greatly increased human lifespan
compared to that in the pre-antibiotic era. However, a disturbing trend has also been noted in that, within a very short period
of time following the introduction of a new antibiotic, resistance to that antibiotic begins to emerge, a factor that is becoming
increasingly meaningful as the discovery of new antibiotics wanes (1-3). There are a number of mechanisms by which a bacterium
may become resistant to a particular antibiotic. Generally these include, but are not limited to, modifi cation of the drug
to render it inactive, modifi cation of the drug target, such that it is incapable of interacting with the drug and decreased
uptake of the antibiotic into the cell, due to reduced transport and/or increased effl ux. Recent functional genomic studies
have also implied that antibiotics may have more complex mechanisms of action than fi rst thought and we are beginning to
appreciate that in addition to the mutation of primary targets, subtle mutations in secondary targets are likely to be infl
uential (4, 5). This chapter will focus on the contribution of a decreased antibiotic uptake to an increase in antibacterial
resistance.
The pyrolysis–gas chromatography (Py–GC) module of a fielded, briefcase Py–GC–ion mobility spectrometry (Py–GC–IMS) biodetector is shown to generate a set of specific products that reflect the biochemical composition of bacterial cell envelopes. A prominent pyrolysis biomarker derived from the cell walls of Gram-positive bacteria has been identified as pyridine-2-carboxamide (2-picolinamide). Experiments performed using model polymers strongly suggest its origin from the thermal rearrangements of the meso-diaminopimelic acid and/or l-lysine peptide residue cross-linker species in peptidoglycan. Biomarkers produced by pyrolysis from Gram-negative organisms were found to originate from the lipid A portion of the lipopolysaccharide (LPS) cell surface. These biomarkers include pyrolysis products of the 3-hydroxymyristate fatty acid residues such as 1-tridecene, dodecanal and methylundecylketone. A nitrile derivative from amide bound fatty acids and free fatty acids originally present as ester bound moieties in lipid A are also observed upon bacterial pyrolysis. The presence of these biomarkers directly reflect similar information content provided by a classical Gram staining procedure and make them suitable for direct Gram-type differentiation of micro-organisms.
Regardless of the existence of antibiotics, infectious diseases are the leading causes of death in the world. Staphylococci cause many infections of varying severity, although they can also exist peacefully in many parts of the human body. Most often Staphylococcus aureus colonises the nose, and that colonisation is considered to be a risk factor for spread of this bacterium. S. aureus is considered to be the most important Staphylococcus species. It poses a challenge to the field of medicine, and one of the most problematic aspects is the drastic increase of the methicillin-resistant S. aureus (MRSA) strains in hospitals and community world-wide, including Finland. In addition, most of the clinical coagulase-negative staphylococcus (CNS) isolates express resistance to methicillin. Methicillin-resistance in S. aureus is caused by the mecA gene that encodes an extra penicillin-binding protein (PBP) 2a. The mecA gene is found in a mobile genomic island called staphylococcal chromosome cassette mec (SCCmec). The SCCmec consists of the mec gene and cassette chromosome recombinase (ccr)gene complexes. The areas of the SCCmec element outside the ccr and mec complex are known as the junkyard J regions. So far, eight types of SCCmec(SCCmec I- SCCmec VIII) and a number of variants have been described. The SCCmec island is an acquired element in S. aureus. Lately, it appears that CNS might be the storage place of the SCCmec that aid the S. aureus by providing it with the resistant elements. The SCCmec is known to exist only in the staphylococci. The aim of the present study was to investigate the horizontal transfer of SCCmec between the S. aureus and CNS. One specific aim was to study whether or not some methicillin-sensitive S. aureus (MSSA) strains are more inclined to receive the SCCmec than others. This was done by comparing the genetic background of clinical MSSA isolates in the health care facilities of the Helsinki and Uusimaa Hospital District in 2001 to the representatives of the epidemic MRSA (EMRSA) genotypes, which have been encountered in Finland during 1992-2004. Majority of the clinical MSSA strains were related to the EMRSA strains. This finding suggests that horizontal transfer of SCCmec from unknown donor(s) to several MSSA background genotypes has occurred in Finland. The molecular characteristics of representative clinical methicillin-resistant S. epidermidis (MRSE) isolates recovered in Finnish hospitals between 1990 and 1998 were also studied, examining their genetic relation to each other and to the internationally recognised MRSE clones as well, so as to ascertain the common traits between the SCCmec elements in MRSE and MRSA. The clinical MRSE strains were genetically related to each other; eleven PFGE types were associated with sequence type ST2 that has been identified world-wide. A single MRSE strain may possess two SCCmec types III and IV, which were recognised among the MRSA strains. Moreover, six months after the onset of an outbreak of MRSA possessing a SCCmec type V in a long-term care facility in Northern Finland (LTCF) in 2003, the SCCmec element of nasally carried methicillin-resistant staphylococci was studied. Among the residents of a LTCF, nasal carriage of MR-CNS was common with extreme diversity of SCCmec types. MRSE was the most prevalent CNS species. Horizontal transfer of SCCmec elements is speculated to be based on the sharing of SCCmec type V between MRSA and MRSE in the same person. Additionally, the SCCmec element of the clinical human S. sciuri isolates was studied. Some of the SCCmec regions were present in S. sciuri and the pls gene was common in it. This finding supports the hypothesis of genetic exchange happening between staphylococcal species. Evaluation of the epidemiology of methicillin-resistant staphylococcal colonisation is necessary in order to understand the apparent emergence of these strains and to develop appropriate control strategies. SCCmec typing is essential for understanding the emergence of MRSA strains from CNS, considering that the MR-CNS may represent the gene pool for the continuous creation of new SCCmec types from which MRSA might originate. Antibioottien olemassaolosta huolimatta tartuntataudit ovat yksi maailman yleisimpiä kuolinsyitä. Vaikka stafylokokit aiheuttavat monia vakavuudeltaan eriasteisia infektioita, voivat ne esiintyä myös harmittomina ihmisen elimistössä. Staphylococcus aureus löytyy yleisimmin nenän limakalvolta, mitä pidetään riskitekijänä kyseisen bakteerin leviämiselle. S. aureusta pidetään tärkeimpänä stafylokokkilajina ja se asettaa haasteita lääketieteelle. Yksi sen ongelmallisimmista piirteistä on metisil-liiniresistenttien S. aureus (MRSA) bakteerikantojen jyrkkä lisääntyminen sairaaloissa ja avohoidossa, niin Suomessa kuin maailmallakin. Lisäksi kliinisistä koagulaasinegatiivisista stafylokokki-kannoista (KNS) suurin osa on vastustuskykyisiä metisilliinille. Metisilliiniresistenssi S. aureus -bakteerissa johtuu mecA geenistä. Kyseinen geeni koodaa penisilliiniä sitovaa proteiinia (PBP) 2a, ja se löytyy liikkuvasta yksiköstä, jota kutsutaan stafylokokkaaliseksi kromosomikasetti mec:ksi (SCCmec). SCCmec koostuu mec-geenistä ja rekombinanteista kasettikromosomi geenikomplekseissta (ccr). Ne SCCmec elementtien alueet, jotka sijaitsevat ccr ja mec -kompleksien ulkopuolella, tunnetaan nimellä junkyard J alueet. Tähän mennessä kahdeksan SCCmec (SCCmec I- SCCmec VIII) lajia, sekä useita eri variantteja on kuvattu, ja on saatu selville, että SCCmec -kasetti on hankittu elementti S. aureuksessa. Uusimpien tutkimustulosten valossa näyttää siltä, että KNS saattaisi olla vastustuskykyisiä SCCmec elementtejä S. aureukselle tarjoava reservuaari. SCCmec:n tiedetään löytyvän vain stafylokokeissa. Tämän tutkimuksen tavoitteena oli selvittää SCCmec:n horisontaalinen siirtyminen S. aureuksen ja KNS:n välillä. Erityisesti tavoitteena oli selvittää, ovatko jotkut metisilliinille herkät S. aureus (MSSA) bakteerikannat taipuvaisempia vastaanottamaan SCCmec:n kuin muut. Tämä tehtiin vertaamalla Helsingin ja Uudenmaan sairaanhoitopiirin terveydenhuollon laitoksista vuonna 2001 otettujen kliinisten MSSA -näytteiden geneettisiä piirteitä Suomessa vuosina 1992 2004 esiintyneisiin epideemisiin MRSA (EMRSA)-genotyyppeihin. Suurin osa kliinisistä MSSA -kannoista oli sukua EMRSA-kannoille. Tämä havainto viittaa siihen, että SCCmec:n horisontaalinen siirtyminen tuntemattomalta luovuttajalta (/luovuttajilta) useille MSSA genotyypeille on tapahtunut Suomessa. Suomen sairaaloista vuosina 1990 1998 saatujen kliinisten metisilliiniresistenttien S. epidermidis (MRSE) -kantojen molekyylitason ominaisuuksia tarkasteltiin tutkimalla niiden geneettistä suhdetta sekä toisiinsa että kansainvälisesti tunnettuihin MRSE -klooneihin, jotta SCCmec elementtien yhteiset piirteet MRSE:ssa ja MRSA:ssa saataisiin selvitettyä. Kliiniset MRSE -kannat olivat geneettisesti toisilleen sukua; yksitoista PFGE -tyyppiä yhdistettiin maailmanlaajuisesti esiintyvään sekvenssi ST2:een. Yksi MRSE -kanta saattaa omata kaksi SCCmec -tyyppiä (III ja IV), jotka ovat tunnettuja MRSA-kantojen keskuudessa. Kuusi kuukautta SCCmec tyyppi V:n omaavan MRSA tautiryppään puhkeamisen jälkeen pitkäaikaishoidon laitoksessa Pohjois-Suomessa (LTCF) vuonna 2003, tutkittiin nenässä esiintyvien metisilliiniresistenttien stafylokokkien SCCmec elementtien kantajuutta. LTFC:n asukkaiden keskuudessa nenässä kulkeutuva MR-KNS oli yleinen, joskin SCCmec tyypeissä oli valtavaa vaihtelua. MRSE oli yleisin KNS -laji. SCCmec elementtien horisontaalisen siirtymisen on arveltu perustuvan SCCmec tyyppi V:n siirtymisen MRSA:n ja MRSE:n välillä samassa henkilössä. Tämän lisäksi tutkittiin myös ihmisen kliinisten S. sciuri kantojen SCCmec -elementtiä. Useat SCCmec alueet olivat läsnä S. sciurissa, ja niissä pls -geeni oli yleinen. Tämä havainto tukee olettamusta geneettisen vaihdon tapahtumisesta stafylokokkilajien välillä. Metisilliiniresistentin stafylokokkikolonisaation epidemiologian arviointi on tarpeen, jotta voidaan ymmärtää näiden kantojen ilmaantuminen ja kehittää asianmukaisia seurantastrategioita. SCCmec geenikasetin tyypittäminen on olennaista KNS:stä kehittyvien MRSA -kantojen ymmärtämiselle ottaen huomioon sen, että MR-KNS saattaa edustaa geenipoolia, jossa syntyy jatkuvasti uusia SCCmec tyyppejä, joista MRSA saattaa mahdollisesti olla peräisin.
Human beta-defensin 3 (hBD3) is a highly charged (+11) cationic host defense peptide, produced by epithelial cells and neutrophils. hBD3 retains antimicrobial activity against a broad range of pathogens, including multiresistant Staphylococcus aureus, even under high-salt conditions. Whereas antimicrobial host defense peptides are assumed to act by permeabilizing cell membranes, the transcriptional response pattern of hBD3-treated staphylococcal cells resembled that of vancomycin-treated cells (V. Sass, U. Pag, A. Tossi, G. Bierbaum, and H. G. Sahl, Int. J. Med. Microbiol. 298:619-633, 2008) and suggested that inhibition of cell wall biosynthesis is a major component of the killing process. hBD3-treated cells, inspected by transmission electron microscopy, showed localized protrusions of cytoplasmic contents, and analysis of the intracellular pool of nucleotide-activated cell wall precursors demonstrated accumulation of the final soluble precursor, UDP-MurNAc-pentapeptide. Accumulation is typically induced by antibiotics that inhibit membrane-bound steps of cell wall biosynthesis and also demonstrates that hBD3 does not impair the biosynthetic capacity of cells and does not cause gross leakage of small cytoplasmic compounds. In in vitro assays of individual membrane-associated cell wall biosynthesis reactions (MraY, MurG, FemX, and penicillin-binding protein 2 [PBP2]), hBD3 inhibited those enzymes which use the bactoprenol-bound cell wall building block lipid II as a substrate; quantitative analysis suggested that hBD3 may stoichiometrically bind to lipid II. We report that binding of hBD3 to defined, lipid II-rich sites of cell wall biosynthesis may lead to perturbation of the biosynthesis machinery, resulting in localized lesions in the cell wall as demonstrated by electron microscopy. The lesions may then allow for osmotic rupture of cells when defensins are tested under low-salt conditions.
Bacterial cell wall transglycosylases (TGs) and transpeptidases (TPs) are ideal drug targets due to their essentiality, accessibility and lack of mammalian homologs. Although antibacterial therapy using the beta-lactam family of TP inhibitors has been successful for decades, potent TG inhibitors which are suitable for development into antibiotics for human use have yet to be identified. We sought to further understand the molecular interactions required to inhibit bacterial transglycosylation by characterizing the active site of Staphylococcus aureus (Sa) monofunctional transglycosylase (Mtg). Ten mutants were tested for their ability to polymerize Lipid II and to crystallize in the presence of moenomycin. Five of six putative active site mutants (E100Q, D101N, Q136E, E156T, and Y176F) were found to be catalytically inactive whereas a F104Y mutation did not affect activity. Four mutants generated to enhance crystal formation (F143T, V154T, L157T, and F158T) also retained activity. Here we also report the crystal structure of Sa Mtg E100Q mutant in complex with the inhibitor moenomycin to 2.1A resolution. The co-crystal structure revealed detailed interactions between the protein and inhibitor including portions of the polycarbon tail of moenomycin. The structure also contained an ordered phosphate ion which helped to identify the Lipid II binding site.
In staphylococci, crosslinking of the peptide moiety of peptidoglycan is mediated via an additional spacer, the interpeptide bridge, consisting of five glycine residues. The femAB operon, coding for two approximately 50-kDa proteins is known to be involved in pentaglycine bridge formation. Using chemical mutagenesis of the beta-lactam-resistant strain BB270 and genetic, biochemical, and biophysical characterization of mutants selected for loss of beta-lactam resistance and reduced lysostaphin sensitivity it is shown that peptide bridge formation proceeds via three intermediate bridge lengths (cell wall peptides with no, one, three, and five glycine units). To proceed from one intermediate to the next, three genes appear necessary: femX, femA, and femB. The drastic loss of beta-lactam resistance after inactivation of FemA or partial impairment of FemX even beyond the level of the sensitive wild-type strains renders these proteins attractive antistaphylococcal targets.
The pneumococcus is one of the longest-known pathogens. It has been instrumental to our understanding of biology in many ways, such as in the discovery of the Gram strain and the identification of nucleic acid as the hereditary material. Despite major advances in our understanding of pneumococcal pathogenesis, the need for vaccines and antibiotics to combat this pathogen is still vital. Genomics is beginning to uncover new virulence factors to advance this process, and it is enabling the development of DNA chip technology, which will permit the analysis of gene expression in specific tissues and in virulence regulatory circuits.
The bacterial acyltransferases of the SxxK superfamily vary enormously in sequence and function, with conservation of particular amino acid groups and all-alpha and alpha/beta folds. They occur as independent entities (free-standing polypeptides) and as modules linked to other polypeptides (protein fusions). They can be classified into three groups. The group I SxxK D,D-acyltransferases are ubiquitous in the bacterial world. They invariably bear the motifs SxxK, SxN(D), and KT(S)G. Anchored in the plasma membrane with the bulk of the polypeptide chain exposed on the outer face of it, they are implicated in the synthesis of wall peptidoglycans of the most frequently encountered (4-->3) type. They are inactivated by penicillin and other beta-lactam antibiotics acting as suicide carbonyl donors in the form of penicillin-binding proteins (PBPs). They are components of a morphogenetic apparatus which, as a whole, controls multiple parameters such as shape and size and allows the bacterial cells to enlarge and duplicate their particular pattern. Class A PBP fusions comprise a glycosyltransferase module fused to an SxxK acyltransferase of class A. Class B PBP fusions comprise a linker, i.e., protein recognition, module fused to an SxxK acyltransferase of class B. They ensure the remodeling of the (4-->3) peptidoglycans in a cell cycle-dependent manner. The free-standing PBPs hydrolyze D,D peptide bonds. The group II SxxK acyltransferases frequently have a partially modified bar code, but the SxxK motif is invariant. They react with penicillin in various ways and illustrate the great plasticity of the catalytic centers. The secreted free-standing PBPs, the serine beta-lactamases, and the penicillin sensors of several penicillin sensory transducers help the D,D-acyltransferases of group I escape penicillin action. The group III SxxK acyltransferases are indistinguishable from the PBP fusion proteins of group I in motifs and membrane topology, but they resist penicillin. They are referred to as Pen(r) protein fusions. Plausible hypotheses are put forward on the roles that the Pen(r) protein fusions, acting as L,D-acyltransferases, may play in the (3-->3) peptidoglycan-synthesizing molecular machines. Shifting the wall peptidoglycan from the (4-->3) type to the (3-->3) type could help Mycobacterium tuberculosis and Mycobacterium leprae survive by making them penicillin resistant.
Reduced susceptibility to penicillin G in Neisseria meningitidis is directly correlated with alterations in the penA gene, which encodes the penicillin-binding protein 2 (PBP2). Using purified PBP2s from different backgrounds, we confirmed that the reduced susceptibility to penicillin G is associated with a decreased affinity of altered PBP2s for penicillin G. Infrared spectroscopy analysis using isogenic penicillin-susceptible strains and strains with reduced susceptibility to penicillin G suggested that the meningococcal cell wall is also modified in a penA-dependent manner. Moreover, reverse-phase high pressure liquid chromatography and mass spectrometry analysis of these meningococcal strains confirmed the modifications of peptidoglycan components and showed an increase in the peaks corresponding to pentapeptide-containing muropeptides. These results suggest that the D,D-transpeptidase and/or D,D-carboxypeptidase activities of PBP2 are modified by the changes in penA gene.
Staphylococci have two mechanisms for resistance to beta-lactam antibiotics. One is the production of beta-lactamases, enzymes that hydrolytically destroy beta-lactams. The other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by beta-lactam antibiotics. Strains of S. aureus exhibiting either beta-lactamase or PBP 2a-directed resistance (or both) have established a considerable ecological niche among human pathogens. The emergence and subsequent spread of bacterial strains designated as methicillin-resistant S. aureus (MRSA), from the 1960s to the present, has created clinical difficulties for nosocomial treatment on a global scale. The recent variants of MRSA that are resistant to glycopeptide antibiotics (such as vancomycin) have ushered in a new and disconcerting chapter in the evolution of this organism.
We analyzed the mode of action of the lantibiotic plantaricin C (PlnC), produced by Lactobacillus plantarum LL441. Compared to the well-characterized type A lantibiotic nisin and type B lantibiotic mersacidin, which are both able
to interact with the cell wall precursor lipid II, PlnC displays structural features of both prototypes. In this regard, we
found that lipid II plays a key role in the antimicrobial activity of PlnC besides that of pore formation. The pore forming
activity of PlnC in whole cells was prevented by shielding lipid II on the cell surface. However, in contrast to nisin, PlnC
was not able to permeabilize Lactococcus lactis cells or to form pores in 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes supplemented with 0.1 mol% purified lipid II. This emphasized the different requirements
of these lantibiotics for pore formation. Using cell wall synthesis assays, we identified PlnC as a potent inhibitor of (i)
lipid II synthesis and (ii) the FemX reaction, i.e., the addition of the first Gly to the pentapeptide side chain of lipid
II. As revealed by thin-layer chromatography, both reactions were clearly blocked by the formation of a PlnC-lipid I and/or
PlnC-lipid II complex. On the basis of the in vivo and in vitro activities of PlnC shown in this study and the structural
lipid II binding motifs described for other lantibiotics, the specific interaction of PlnC with lipid II is discussed.
This review is an attempt to bring together and critically evaluate the now-abundant but dispersed data concerning the lipid intermediates of the biosynthesis of bacterial peptidoglycan. Lipid I, lipid II, and their modified forms play a key role not only as the specific link between the intracellular synthesis of the peptidoglycan monomer unit and the extracytoplasmic polymerization reactions but also in the attachment of proteins to the bacterial cell wall and in the mechanisms of action of antibiotics with which they form specific complexes. The survey deals first with their detection, purification, structure, and preparation by chemical and enzymatic methods. The recent important advances in the study of transferases MraY and MurG, responsible for the formation of lipids I and II, are reported. Various modifications undergone by lipids I and II are described, especially those occurring in gram-positive organisms. The following section concerns the cellular location of the lipid intermediates and the translocation of lipid II across the cytoplasmic membrane. The great efforts made since 2000 in the study of the glycosyltransferases catalyzing the glycan chain formation with lipid II or analogues are analyzed in detail. Finally, examples of antibiotics forming complexes with the lipid intermediates are presented.
The mechanism of addition of glycine during synthesis of the peptidoglycan of cell walls of Staphylococcus aureus has been investigated. Glycine was activated as glycyl soluble ribonucleic acid (sRNA) for this reaction. The initial acceptor for glycine was disaccharide(-pentapeptide)-P-P-phospholipid, an intermediate in peptidoglycan synthesis. Acetylmuramyl(-pentapeptide)-P-P-phospholipid also acted as a glycine acceptor, but less effectively. The glycyl-sRNA of S. aureus has been fractionated into two glycyl-sRNAs, only one of which was active in peptidoglycan synthesis. The product of this sequence, disaccharide(-pentapeptide-pentaglycine)-P-P-phospholipid, then transferred disaccharide-decapeptide to an endogenous cell wall acceptor. Analyses of the lipid intermediate and of the peptidoglycan product indicated that a chain of about 5 glycine residues was substituted on the ε-amino group of lysine both in the lipid intermediate and in the peptidoglycan. These pentaglycine chains had free amino ends, and the closure of the pentaglycine bridges was therefore not catalyzed by the enzyme preparations employed. The inhibition of this system by various solvents, detergents, and antibiotics is reported. Two antibiotics, ristocetin and vancomycin, were specific inhibitors of the reaction sequence. Of the three reactions involved in the sequence (attachment of disaccharide-pentapeptide to the lipid, transfer of glycine to the lipid intermediates, and utilization of the lipid intermediates for peptidoglycan synthesis), the first was the least sensitive and the last was the most sensitive to these agents.
The synthesis of UDP-MurNAc-Ala-DGlu-Lys[Nepsilon-dimethylaminonaphthalene sulfonyl (Dns)]-DAla-DAla provides a method for the specific introduction of a fluorescent reporter group into the membrane environment of nascent peptidoglycan synthesis. To assess the degree of perturbation of this environment caused by the introduction of the dansyl substituent, this nucleotide was compared with UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla in the reaction catalyzed by phospho-MurNAc-pentapeptide translocase (UDP-MurNAc-Ala-gammaDGlu-Lys-DAla-DAla:undecaprenyl phosphate phospho-MurNA-C-pentapeptide transferase) and in the membrane-associated synthesis of nascent peptidoglycan. Phospho-MurNAc-pentapeptide translocase in membrane fragments from Staphylococcus aureus Copenhagen catalyzed the transfer of phospho-MurNAc-Ala-DGlu-Lys(Nepsilon-Dns)-DAla-DAla to undecaprenyl phosphate with a Vmax/Km of 3.8 and a Vmax of 1.6 times the values for UDP-MurNAc-pentapeptide. In the exchange reaction catalyzed by the translocase, the Rmax/Km and Rmax for the dansylated substrate were 1.8 and 0.78 times the respective values for the reference nucleotide. The equilibrium constant for the transfer reaction utilizing UDP-MurNAc-(Nepsilon-Dns)pentapeptide was 5.9 +/- 0.13 compared to 1.1 +/- 0.02 for UDP-MurNAc-pentapeptide. With respect to the proposed reaction model (Pless, D. D., and Neuhaus, F. C. (1973) J. Biol. Chem. 248, 1568-1576), the increase in Keq is consistent with a decrease in the affinity of undecaprenyl diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Dns)-DAla-DAla for the translocase. The fluorescence emission maximum of the phospho-MurNAc-(Nepsilon-Dns)pentapeptide moiety of UDP-MurNAc-(Nepsilon-Dns)pentapeptide was blue-shifted from 525 to 495 nm upon transfer from UMP to undecaprenyl phosphate with a 6-fold increase in quantum yield. These spectral changes provided a sensitive and continuous assay for the formation of undecaprenyl diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Dans)-DAla-DAla. The nascent peptidoglycan synthesizing system from Gaffkya homari utilized the dansylated nucleotide with a Vmax/Km of 0.05 and a Vmax of 0.10 times the values for UDP-MurNAc-pentapeptide. These results demonstrate that phospho-MurNAc-Ala-DGlu-Lys(Nepsilon-Dns)-DAla-DAla linked to the undecaprenyl phosphate will serve as a precursor for the synthesis of nascent peptidoglycan and that the dansyl moiety will report on the membrane environment it experiences during this synthesis.
The peptide network of Streptococcus pneumoniae cell walls was solubilized using the pneumococcal autolytic amidase (N-acetylmuramoyl-L-alanine amidase, EC 3.5.1.28). The peptide material was fractionated into size classes by gel filtration followed by reverse-phase high-performance liquid chromatography which resolved the peptide population into over 40 fractions. About 40% of the lysines present participate in cross-links between stem peptides. The main components (3 monomers, 5 dimers, and 2 trimers), accounting for 77% of all the wall peptides, were purified. Their structures were determined using a combination of amino acid and end-group analysis, mass spectrometry, and gas-phase sequencing. Two different types of cross-links between stem peptides were found. In the most abundant type there is an alanylserine cross-bridge between the alanine in position 4 of the donor stem peptide and the lysine at position 3 of the acceptor peptide, as in type A3 peptidoglycan. In the second type of cross-link there is no intervening cross-bridge, as in the type A1 peptidoglycan of Gram-negative bacteria. The data indicate that pneumococcal peptidoglycan has a structural complexity comparable to that recently shown in some Gram-negative species.
The activity of penicillin-binding protein 3 of Escherichia coli has been studied both in vivo and in ether-permeabilized cells. The peptidoglycan transpeptidase activity of penicillin-binding protein 3 appears to use either nascent or exogenously added UDP-N-acetylmuramyl tripeptide-derived substrates as acceptors. By means of a defilamentation system which elicited the activity of penicillin-binding protein 3 in vivo, the structure of peptidoglycan made by this enzyme has been elucidated. This peptidoglycan, very probably of septal location, contained increased amounts of cross-linked peptidoglycan as well as a higher ratio of tripeptide-containing cross-linked subunits.
Enzyme preparations from Streptococcus faecalis and Lactobacillus casei catalyzed the incorporation of d-aspartic acid into a peptidoglycan in the presence of uridine diphosphate acetylmuramylpentapeptide and uridine diphosphate acetylglucosamine. The reaction required ATP but was independent of either supernatant enzymes or tRNA. A d-aspartic acid-activating enzyme present in the membrane fraction has been solubilized with 2 m LiCl and purified approximately 100-fold. The preparation so obtained catalyzed the formation of equivalent amounts of β-d-aspartylhydroxamate, ADP, and inorganic phosphate in the presence of d-aspartic acid, ATP, and hydroxylamine. The intermediate formed is presumably β-d-aspartylphosphate.
A new transposon library constructed in the background of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL yielded 70 independent insertional mutants with reduced levels of antibiotic resistance. Restriction analysis with HindIII, EcoRV, EcoRI, and PstI and then Southern hybridization with probes for the transposon and for the femA-femB gene demonstrated that 41 of the 70 Tn551 mutants carried distinct and novel, as yet undescribed insertion sites, all of which were outside of the mecA gene and were also outside the already-characterized auxiliary genes femA, femB, femC, and femD. All previously described Tn551 mutations of this type were in genes located either on SmaI fragment A or SmaI fragment I. In contrast, inserts of the new library were located in 7 of the 16 SmaI chromosomal fragments, fragments A, B, C, D, E, F, and I. In all of the mutants, expression of methicillin resistance became heterogeneous, and the MIC for the majority of cells was reduced (1.5 to 200 micrograms ml-1) from the homogeneous methicillin MIC (1,600 micrograms ml-1) of the parental cells. Although identification of the exact number of genes inactivated through the new set of transposon inserts will require cloning and sequencing, a rough estimate of this number from mapping data suggests a minimum of at least 10 to 12 new genetic determinants, all of which are needed together with femA, femB, femC, and femD for the optimal expression of methicillin resistance.
Screening of a new Tn551 library constructed in the background of a highly methicillin-resistant Staphylococcus aureus strain identified a new insertion site located on the SmaI B-fragment of the chromosome that reduced the minimal inhibitory concentration of the parent (1600 micrograms/ml) to 25-50 micrograms/ml in the mutant and caused heterogeneous expression of resistance and abnormality in peptidoglycan composition (absence of the unsubstituted pentapeptide and incorporation of alanylglutamate- and alanylisoglutamate-containing muropeptides). There was an accumulation of large amounts of the UDP-linked muramyl dipeptide in the cytoplasmic wall precursor pool of the mutant. Reduced (heterogeneous) antibiotic resistance and all the biochemical abnormalities were reproduced in genetic backcrosses by transduction with phage 80 alpha. Mutant RUSA235 appears to be impaired in the biosynthesis of the staphylococcal cell wall precursor muropeptide before the lysine addition step. We propose to provisionally call the gene inactivated in this mutant femF.
The biochemical basis for the acquired or natural resistance of various gram-positive organisms to glycopeptides was studied by high-pressure liquid chromatographic analysis of their peptidoglycan UDP-MurNAc-peptide precursors. In all cases, resistance was correlated with partial or complete replacement of the C-terminal D-Ala-D-Ala-containing UDP-MurNAc-pentapeptide by a new precursor with a modified C terminus. Nuclear magnetic resonance analysis by sequential assignment showed that the new precursor encountered in Enterococcus faecium D366, a strain belonging to the VANB class, which expresses low-level resistance to vancomycin, was UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-lactate, identical to that previously found in the VANA class, which expresses high-level resistance to vancomycin. High-pressure liquid chromatographic analyses, composition determinations, and digestion by R39 D,D-carboxypeptidase demonstrated the exclusive presence of the new precursor in Lactobacillus casei and Pediococcus pentosaceus, which are naturally highly resistant to glycopeptides. The low-level natural resistance of Enterococcus gallinarum to vancomycin was found to be associated with the synthesis of a new precursor identified as a UDP-MurNAc-pentapeptide containing a C-terminal D-serine. The distinction between low and high levels of resistance to glycopeptides appeared also to depend on the presence or absence of a substantial residual pool of a D-Ala-D-Ala-containing UDP-MurNAc-pentapeptide.
Tn551 inactivation of femA, a factor involved in methicillin resistance of Staphylococcus aureus, caused the production of peptidoglycan in which the fraction of monoglycyl- and serine-containing muropeptides was increased at the expense of pentaglycyl muropeptides. femA mutants have a specific block in the biosynthesis of pentaglycine cross bridges after the addition of the first glycine residue.
Analysis by high-performance liquid chromatography of the stem peptide composition of cell walls purified from a large number of pneumococcal strains indicates that these bacteria produce a highly conserved species-specific peptidoglycan independent of serotype, isolation date, and geographic origin. Characteristic features of this highly reproducible peptide pattern are the dominance of linear stem peptides with a monomeric tripeptide, a tri-tetra linear dimer, and two indirectly cross-linked tri-tetra dimers being the most abundant components. Screening of strains with the high-performance liquid chromatography technique has identified two naturally occurring peptidoglycan variants in which the species-specific stem peptide composition was replaced by two drastically different and distinct stem peptide patterns, each unique to the particular clone of pneumococci producing it. Both isolates were multidrug resistant, including resistance to penicillin. In one of these clones--defined by multilocus enzyme analysis and pulsed-field gel electrophoresis of the chromosomal DNAs--the linear stem peptides were replaced by branched peptides that most frequently carried an alanyl-alanine substituent on the epsilon amino group of the diamino acid residue. In the second clone, the predominant stem peptide species replacing the linear stem peptides carried a seryl-alanine substituent. The abnormal peptidoglycans may be related to the altered substrate preference of transpeptidases (penicillin-binding proteins) in the pneumococcal variants.
Compared with most penicillin-susceptible isolates of Streptococcus pneumoniae, penicillin-resistant clinical isolate Hun 663 contains mosaic penicillin-binding protein (PBP) genes encoding PBPs with reduced penicillin affinities, anomalous molecular sizes, and also cell walls of unusual chemical composition. Chromosomal DNA prepared from Hun 663 was used to transform susceptible recipient cells to donor level penicillin resistance, and a resistant transformant was used next as the source of DNA in the construction of a second round of penicillin-resistant transformants. The greatly reduced penicillin affinity of the high-molecular-weight PBPs was retained in all transformants through both genetic crosses. On the other hand, PBP pattern and abnormal cell wall composition, both of which are stable, clone-specific properties of strain Hun 663, were changed: individual transformants showed a variety of new, abnormal PBP patterns. Furthermore, while the composition of cell walls resembled that of the DNA donor in the first-round transformants, it became virtually identical to that of susceptible pneumococci in the second-round transformants. The findings indicate that genetic elements encoding the low affinity of PBPs and the penicillin resistance of the bacteria are separable from determinants that are responsible for the abnormal cell wall composition that often accompanies penicillin resistance in clinical strains of pneumococci.
The muropeptide compositions of isogenic vancomycin-resistant and -susceptible Enterococcus faecalis strains were analyzed by reverse-phase high-performance liquid chromatography combined with amino acid analysis and fast atom bombardment mass spectrometry. Peptidoglycan of the susceptible strain contained pentapeptides as stem peptides, whereas peptidoglycan of the isogenic resistant strain was composed of muropeptides with tetrapeptide stem peptides. Despite the synthesis of lactate-terminating peptidoglycan precursors, no lactate-containing muropeptides were detected in peptidoglycan of the resistant strain. These findings indicate that either lactate-terminating precursors are not incorporated into peptidoglycan of the resistant strain or that the lactate residues are removed from peptidoglycan during synthesis.
The influence of NaCl on the susceptibility of Enterococcus faecalis to cefotaxime was tested with JH2-2, a laboratory strain, and 20 clinical strains grown on tryptic soy agar supplemented with 5% horse blood. Growth with 3% NaCl in the medium resulted in an increase in cefotaxime resistance and the appearance of a heterogeneous resistance phenotype: for the majority of the strains, the MICs of cefotaxime increased from 4 to 512 micrograms ml-1. By a competition assay using cefotaxime and [3H]benzylpenicillin, it was shown for strain JH2-2 that at the MIC penicillin-binding protein (PBP) 2 and PBP3 were the apparent essential PBPs in medium without NaCl, whilst the low-affinity PBPs 4 and 1 were the apparent essential PBPs for cell growth in medium containing 3% NaCl. Analysis of JH2-2 peptidoglycan by HPLC and MS after growth in the presence of 3% NaCl showed a relative increase in unsubstituted monomers and a relative decrease in alanine- and dialanine-substituted monomers. It is therefore hypothesized that modification of the number of alanine-substituted precursors in the presence of NaCl could interfere with the functions of the different PBPs and thus play a role in cefotaxime resistance in E. faecalis.
Penicillin-resistant strains of Streptococcus pneumoniae contain low affinity penicillin-binding proteins and often also produce abnormal indirectly crosslinked cell walls. However the relationship between cell wall abnormality and penicillin resistance has remained obscure. We now show that the genome of S. pneumoniae contains an operon composed of two genes (murM and murN) that encode enzymes involved with the biosynthesis of branched structured cell wall muropeptides. The sequences of murMN were compared in two strains: the penicillin-susceptible strain R36A producing the species-specific pneumococcal cell wall peptidoglycan in which branched stem peptides are rare, and the highly penicillin-resistant transformant strain Pen6, the cell wall of which is enriched for branched-structured stem peptides. The two strains carried different murM alleles: murM of the penicillin-resistant strain Pen6 had a "mosaic" structure encoding a protein that was only 86.5% identical to the product of murM identified in the isogenic penicillin-susceptible strain R36A. Mutants of R36A and Pen6 in which the murMN operon was interrupted by insertion-duplication mutagenesis produced peptidoglycan from which all branched muropeptide components were missing. The insertional mutant of Pen6 carried a pbp2x gene with the same "mosaic" sequence found in Pen6. On the other hand, inactivation of murMN in strain Pen6 and other resistant strains caused a virtually complete loss of penicillin resistance. Our observations indicate that the capacity to produce branched cell wall precursors plays a critical role in the expression of penicillin resistance in S. pneumoniae.
The fundamental polymer that is a common component of the cell walls of Grampositive and Gram-negative bacteria, Rickettsiae and blue-green bacteria is called peptidoglycan (formerly mucopeptide or murein). As its name implies, it consists of glycan chains with peptide substituents, and in all examples that have been studied the peptide subunits are cross-linked so that the overall structure is a network that surrounds the cell. This network seems responsible for the integrity of the shape of Gram-positive bacteria, and at least partially of Gram-negative bacteria as well. Certainly when the peptidoglycan is degraded, as for instance by lysozyme, the bacterium tends to lose its characteristic shape and to form a spherical body known as a spheroplast, which usually needs to be maintained in a hypertonic medium if it is not to burst because of the high osmotic pressure within it and the lack of external support. The chemical composition of peptidoglycan has been established over the period since the early 1950s, when Salton [40] first showed that the cell walls prepared from Gram-positive organisms were of a comparatively simple amino acid composition, although both he and Weidel [56] found that the walls of Gramnegative species were more complex.
Publisher Summary This chapter discusses the analysis of the chemical composition and primary structure of the murein. Murein (peptidoglycan, mucopeptide) is the main cell wall polymer of eubacteria and is common to both Gram-negative and Gram-positive bacteria. There are only a few prokaryotic organisms, such as mycoplasmas and archaebacteria, which lack murein. The glycan moiety is rather uniform and shows only a few variations such as O-acetylation or O-phosphorylation or the exceptional absence of peptide substituents. The peptide moiety of the murein shows, in contrast to the glycan part, a considerable variation. Extensive investigation of the chemical structure of murein has demonstrated the existence of almost 100 different variations of the peptide moiety. Depending on the mode of cross-linking two main groups of murein, named A and B, have been distinguished. The different structure of cell walls of Gram-positive and Gram-negative bacteria necessitates discrete methods for preparing cell walls. The cell walls of Gram-positive bacteria reveal in profile one thick and more or less homogeneous layer, whereas Gram-negative bacteria have thinner, but distinctly layered cell walls with an outer membrane resembling the cytoplasmic membrane in profile.
This chapter discusses the biosynthesis of the bacterial peptidoglycan unit. The biosynthesis of bacterial peptidoglycan is a complex two stage process. The first stage is concerned with the formation of the disaccharide peptide monomer unit, and the second the polymerization reactions accompanied by the insertion of the newly made peptidoglycan material into the cell wall. The assembly of the peptidoglycan unit proceeds by a series of cytoplasmic and membrane reactions. This implies a passage through the hydrophobic environment of the membrane. Lipid intermediates are involved in this process. The various steps of the pathway have been identified in one bacteria or another, and a general scheme established that is valid for both Gram-positive and negative eubacteria. The elucidation of the pathway leading to the complete peptidoglycan unit was established by isolating and characterizing the muramic acid containing precursors, and by developing a specific in vitro assay for the enzymatic activity catalyzing each step. Assays involving more than one step have also been developed.
The shape of Escherichia coli is strikingly simple compared to those of higher eukaryotes. In fact, the end result of E. coli morphogenesis is a cylindrical tube with hemispherical caps. It is argued that physical principles affect biological forms. In this view, genes code for products that contribute to the production of suitable structures for physical factors to act upon. After introduction of a physical model, the discussion is focused on the shape-maintaining (peptidoglycan) layer of E. coli. This is followed by a detailed analysis of the structural relationship of the cellular interior to the cytoplasmic membrane. A basic theme of this review is that the transcriptionally active nucleoid and the cytoplasmic translation machinery form a structural continuity with the growing cellular envelope. An attempt has been made to show how this dynamic relationship during the cell cycle affects cell polarity and how it leads to cell division.
The two membrane precursors (pentapeptide lipids I and II) of peptidoglycan are present in Escherichia coli at cell copy numbers no higher than 700 and 2,000 respectively. Conditions were determined for an optimal accumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for its isolation and purification. When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptide lipid II was formed, and it was isolated and purified. Both lipids II were compared as substrates in the in vitro polymerization by transglycosylation assayed with PBP 1b or PBP 3. With PBP 1b, tripeptide lipid II was used as efficiently as pentapeptide lipid II. It should be stressed that the in vitro PBP 1b activity accounts for at best to 2 to 3% of the in vivo synthesis. With PBP 3, no polymerization was observed with either substrate. Furthermore, tripeptide lipid II was detected in D-cycloserine-treated cells, and its possible in vivo use in peptidoglycan formation is discussed. In particular, it is speculated that the transglycosylase activity of PBP 1b could be coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor.
About 80 different muropeptides, the subunits which comprise the polymer murein of Escherichia coli, were resolved by high-performance liquid chromatography. The muropeptides were released from isolated murein by complete digestion with muramidase from Chalaropsis spec. The separation method is based on reversed phase chromatography of the sodium borohydride-reduced compounds using ODS (C18) columns and a linear gradient elution with sodium phosphate buffer and methanol as organic modifier. The effect of temperature, pH, ionic strength, and the steepness of the gradient and of different support materials on the separation of the muropeptides was investigated. The new method represents a major improvement over previous methods with respect to resolution, sensitivity, and speed. Analytical as well as preparative separations can be realized. Quantitative analysis of murein composition is achieved by a linear gradient from 50 mM sodium phosphate, pH 4.31, to 75 mM sodium phosphate, pH 4.95, containing 15% methanol for 135 min on a 250 X 4.6 mm 3-micron Hypersil ODS column at 55 degrees C using a flow rate of 0.5 ml/min. With uv detection at 205 nm about 20 micrograms of murein per analysis is sufficient. The detection limit per compound is about 5 ng. A method for the evaluation of the analytical data allowing a convenient comparison of different muropeptide pattern is described.
Recently a dapF mutant of Escherichia coli lacking the diaminopimelate epimerase was found to have an unusual large LL-diaminopimelic acid (LL-DAP) pool as compared with that of meso-DAP (C. Richaud, W. Higgins, D. Mengin-Lecreulx, and P. Stragier, J. Bacteriol. 169:1454-1459, 1987). In this report, the consequences of high cellular LL-DAP/meso-DAP ratios on the structure and metabolism of peptidoglycan were investigated. For this purpose new efficient high-pressure liquid chromatography techniques for the separation of the DAP isomers were developed. Sacculi from dapF mutants contained a high proportion of LL-DAP that varied greatly with growth conditions. The same was observed with the two DAP-containing precursors, UDP-N-acetylmuramyl-tripeptide and UDP-N-acetylmuramyl-pentapeptide. The limiting steps for the incorporation of LL-DAP into peptidoglycan were found to be its addition to UDP-N-acetylmuramyl-L-alanyl-D-glutamate and the formation of the D-alanyl-DAP cross-bridges. The Km value of the DAP-adding enzyme for LL-DAP was 3.6 x 10(-2) M as compared with 1.1 x 10(-5) M for meso-DAP. When isolated sacculi were treated with Chalaropsis N-acetylmuramidase and the resulting soluble products were analyzed by high-pressure liquid chromatography, the proportion of the main peptidoglycan dimer was lower in the dapF mutant than in the parental strain. Moreover, the proportion of LL-DAP was higher in the main monomer than in the main dimer, where it was almost exclusively located in the donor unit. There are thus very few D-alanyl-LL-DAP cross-bridges, if any. We also observed that large amounts of LL-DAP and N-succinyl-LL-DAP were excreted in the growth medium by the dapF mutant.
A wall-plus-membrane preparation from a Bacillus licheniformis mutant incorporated radioactivity from a peptidoglycan precursor in which the free amino group of diaminopimelic acid was blocked by (14)C-labelled acetyl group. This incorporation was penicillin-sensitive. The enzymically degraded product contained cross-linked dimers, showing that newly synthesized peptidoglycan chains had been cross-linked to the pre-existing cell wall.
Resistance to glycopeptide antibiotics in enterococci is due to the synthesis of UDP-MurNAc-tetrapeptide-D-lactate (where Mur is muramic acid) replacing the normal UDP-MurNAc-pentapeptide precursor. The peptidoglycan structures of an inducible VanB-type glycopeptide-resistant Enterococcus faecium, D366, and its constitutively resistant derivative, MT9, were determined. Using HPLC, 17 muropeptides were identified and were present regardless of whether resistance was expressed or not. The structures of 15 muropeptides were determined using MS and amino acid analysis. The cross-bridge between D-alanine and L-lysine consisted of one asparagine. No monomer pentapeptide or tetrapeptide-D-lactate could be identified. These results obtained with D366 (non-induced) and MT9 indicate that, in the absence of vancomycin, the cell wall synthetic machinery of E. faecium can process the lactate-containing precursor as efficiently as the normal pentapeptide. In contrast, the presence of subinhibitory inducing concentrations of vancomycin interfered with the synthesis of oligomers.
The structures of cytoplasmic peptidoglycan precursor and mature peptidoglycan of an isogenic series of Staphylococcus haemolyticus strains expressing increasing levels of resistance to the glycopeptide antibiotics teicoplanin and vancomycin (MICs, 8 to 32 and 4 to 16 microg/ml, respectively) were determined. High-performance liquid chromatography, mass spectrometry, amino acid analysis, digestion by R39 D,D-carboxypeptidase, and N-terminal amino acid sequencing were utilized. UDP-muramyl-tetrapeptide-D-lactate constituted 1.7% of total cytoplasmic peptidoglycan precursors in the most resistant strain. It is not clear if this amount of depsipeptide precursor can account for the levels of resistance achieved by this strain. Detailed structural analysis of mature peptidoglycan, examined for the first time for this species, revealed that the peptidoglycan of these strains, like that of other staphylococci, is highly cross-linked and is composed of a lysine muropeptide acceptor containing a substitution at its epsilon-amino position of a glycine-containing cross bridge to the D-Ala 4 of the donor, with disaccharide-pentapeptide frequently serving as an acceptor for transpeptidation. The predominant cross bridges were found to be COOH-Gly-Gly-Ser-Gly-Gly-NH2 and COOH-Ala-Gly-Ser-Gly-Gly-NH2. Liquid chromatography-mass spectrometry analysis of the peptidoglycan of resistant strains revealed polymeric muropeptides bearing cross bridges containing an additional serine in place of glycine (probable structures, COOH-Gly-Ser-Ser-Gly-Gly-NH2 and COOH-Ala-Gly-Ser-Ser-Gly-NH2). Muropeptides bearing an additional serine in their cross bridges are estimated to account for 13.6% of peptidoglycan analyzed from resistant strains of S. haemolyticus. A soluble glycopeptide target (L-Ala-gamma-D-iso-glutamyl-L-Lys-D-Ala-D-Ala) was able to more effectively compete for vancomycin when assayed in the presence of resistant cells than when assayed in the presence of susceptible cells, suggesting that some of the resistance was directed towards the cooperativity of glycopeptide binding to its target. These results are consistent with a hypothesis that alterations at the level of the cross bridge might interfere with the binding of glycopeptide dimers and therefore with the cooperative binding of the antibiotic to its target in situ. Glycopeptide resistance in S. haemolyticus may be multifactorial.
Glycopeptide resistance in enterococci results from the production of peptidoglycan precursors with low affinity for these antibiotics. The mobility of the resistance genes by transposition and conjugation and the ability of the resistance proteins to interfere with synthesis of normal precursors in different hosts indicate that dissemination into other bacterial species should be anticipated.
Muropeptide composition of peptidoglycan isolated from isogenic vancomycin-resistant and sensitive Enterococcus faecium strains was analyzed by reverse-phase high-performance liquid chromatography combined with amino acid and fast atom bombardment mass spectrometric analyses. Peptidoglycan of the sensitive and resistant strains was the same and was composed of tri- and tetrapeptides stem peptide subunits with or without aspartate or asparagine substitutions on the epsilon-amino group of the lysine residue. Thus, the synthesis of lactate-terminating peptidoglycan precursors in vancomycin-resistant E. faecium did not affect the chemical composition of peptidoglycan.
In staphylococci, crosslinking of the peptide moiety of peptidoglycan is mediated via an additional spacer, the interpeptide bridge, consisting of five glycine residues. The femAB operon, coding for two approximately 50-kDa proteins is known to be involved in pentaglycine bridge formation. Using chemical mutagenesis of the beta-lactam-resistant strain BB270 and genetic, biochemical, and biophysical characterization of mutants selected for loss of beta-lactam resistance and reduced lysostaphin sensitivity it is shown that peptide bridge formation proceeds via three intermediate bridge lengths (cell wall peptides with no, one, three, and five glycine units). To proceed from one intermediate to the next, three genes appear necessary: femX, femA, and femB. The drastic loss of beta-lactam resistance after inactivation of FemA or partial impairment of FemX even beyond the level of the sensitive wild-type strains renders these proteins attractive antistaphylococcal targets.
Low-affinity penicillin-binding proteins (PBPs), which participate in the beta-lactam resistance of several pathogenic bacteria, have different origins. Natural transformation and recombination events with DNA acquired from neighbouring intrinsically resistant organisms are responsible for the appearance of mosaic genes encoding two or three low-affinity PBPs in highly resistant strains of transformable microorganisms such as Neisseria and Streptococcus pneumoniae. Methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococcal strains possess the mecA determinant gene, which probably evolved within the Staphylococcus genus from a closely related and physiologically functional gene that was modified by point mutations. The expression of mecA is either inducible or constitutive. A stable high-level resistant phenotype requires the synthesis of a normally constituted peptidoglycan. Enterococci have a natural low susceptibility to beta-lactams related to the presence of an intrinsic low-affinity PBP. Highly resistant enterococcal strains overexpress this PBP and/or reduce its affinity.
The monomer units in the Escherichia coli and Staphylococcus aureus cell wall peptidoglycans differ in the nature of the third amino acid in the L-alanyl-gamma-D-glutamyl-X-D-alanyl-D-alanine side chain, where X is meso-diaminopimelic acid or L-lysine, respectively. The murE gene from S. aureus encoding the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: L-lysine ligase was identified and cloned into plasmid vectors. Induction of its overexpression in E. coli rapidly results in abnormal morphological changes and subsequent cell lysis. A reduction of 28% in the peptidoglycan content was observed in induced cells, and analysis of the peptidoglycan composition and structure showed that ca. 50% of the meso-diaminopimelic acid residues were replaced by L-lysine. Lysine was detected in both monomer and dimer fragments, but the acceptor units from the latter contained exclusively meso-diaminopimelic acid, suggesting that no transpeptidation could occur between the epsilon-amino group of L-lysine and the alpha-carboxyl group of D-alanine. The overall cross-linking of the macromolecule was only slightly decreased. Detection and analysis of meso-diaminopimelic acid- and L-lysine-containing peptidoglycan precursors confirmed the presence of L-lysine in precursors containing amino acids added after the reaction catalyzed by the MurE ligase and provided additional information about the specificity of the enzymes involved in these latter processes.
The two-dimensional membrane topology of the Escherichia coli and Staphylococcus aureus MraY transferases, which catalyse the formation of the first lipid intermediate of peptidoglycan synthesis, was established using the beta-lactamase fusion system. All 28 constructed mraY-blaM fusions produced hybrid proteins. Analysis of the ampicillin resistance of the strains with hybrids led to a common topological model possessing 10 transmembrane segments, five cytoplasmic domains and six periplasmic domains including the N- and C-terminal ends. The agreement between the topologies of E. coli and S. aureus, their agreement to a fair extent with predicted models and a number of features arising from the comparative analysis of 25 orthologue sequences strongly suggested the validity of the model for all eubacterial MraYs. The primary structure of the 10 transmembrane segments diverged among orthologues, but they retained their hydrophobicity, number and size. The similarity of the sequences and distribution of the five cytoplasmic domains in both models, as well as their conservation among the MraY orthologues, clearly suggested their possible involvement in substrate recognition and in the catalytic process. Complementation tests showed that only fusions with untruncated mraY restored growth. It was noteworthy that S. aureus MraY was functional in E. coli. An increased MraY transferase activity was observed only with the untruncated hybrids from both organisms.
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