Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of penicillin binding protein 2A.

All clinical isolates of methicillin-resistant Staphylococcus aureus contain an extra penicillin binding protein (PBP) 2A in addition to four PBPs present in all staphylococcal strains. This extra PBP is thought to be a transpeptidase essential for the continued cell wall synthesis and growth in the presence of beta-lactam antibiotics. As an approach of testing this hypothesis we compared the muropeptide composition of cell walls of a highly methicillin-resistant S. aureus strain containing PBP2A and its isogenic Tn551 derivative with reduced methicillin resistance, which contained no PBP2A because of the insertional inactivation of the PBP2A gene. Purified cell walls were hydrolyzed into muropeptides which were subsequently resolved by reversed-phase high-performance liquid chromatography and identified by chemical and mass spectrometric analysis. The peptidoglycan composition of the two strains were identical. Both peptidoglycans were highly cross-linked mainly through pentaglycine cross-bridges, although other, chemically distinct peptide cross-bridges were also present including mono-, tri-, and tetraglycine; alanine; and alanyl-tetraglycine. Our experiments provided no experimental data for a unique transpeptidase activity associated with PBP2A.     

Role of penicillin-binding protein 2 (PBP2) in the antibiotic susceptibility and cell wall cross-linking of Staphylococcus aureus: evidence for the cooperative functioning of PBP2, PBP4, and PBP2A

Ceftizoxime, a beta-lactam antibiotic with high selective affinity for penicillin-binding protein 2 (PBP2) of Staphylococcus aureus, was used to select a spontaneous resistant mutant of S. aureus strain 27s. The stable resistant mutant ZOX3 had an increased ceftizoxime MIC and a decreased affinity of its PBP2 for ceftizoxime and produced peptidoglycan in which the proportion of highly cross-linked muropeptides was reduced. The pbpB gene of ZOX3 carried a single C-to-T nucleotide substitution at nucleotide 1373, causing replacement of a proline with a leucine at amino acid residue 458 of the transpeptidase domain of the protein, close to the SFN conserved motif. Experimental proof that this point mutation was responsible for the drug-resistant phenotype, and also for the decreased PBP2 affinity and reduced cell wall cross-linking, was provided by allelic replacement experiments and site-directed mutagenesis. Disruption of pbpD, the structural gene of PBP4, in either the parental strain or the mutant caused a large decrease in the highly cross-linked muropeptide components of the cell wall and in the mutant caused a massive accumulation of muropeptide monomers as well. Disruption of pbpD also caused increased sensitivity to ceftizoxime in both the parental cells and the ZOX3 mutant, while introduction of the plasmid-borne mecA gene, the genetic determinant of the beta-lactam resistance protein PBP2A, had the opposite effects. The findings provide evidence for the cooperative functioning of two native S. aureus transpeptidases (PBP2 and PBP4) and an acquired transpeptidase (PBP2A) in staphylococcal cell wall biosynthesis and susceptibility to antimicrobial agents.     

Inhibition of cell wall turnover and autolysis by vancomycin in a highly vancomycin-resistant mutant of Staphylococcus aureus.

A highly vancomycin-resistant mutant (MIC = 100 microg/ml) of Staphylococcus aureus, mutant VM, which was isolated in the laboratory by a step-pressure procedure, continued to grow and synthesize peptidoglycan in the presence of vancomycin (50 microg/ml) in the medium, but the antibiotic completely inhibited cell wall turnover and autolysis, resulting in the accumulation of cell wall material at the cell surface and inhibition of daughter cell separation. Cultures of mutant VM removed vancomycin from the growth medium through binding the antibiotic to the cell walls, from which the antibiotic could be quantitatively recovered in biologically active form. Vancomycin blocked the in vitro hydrolysis of cell walls by autolytic enzyme extracts, lysostaphin and mutanolysin. Analysis of UDP-linked peptidoglycan precursors showed no evidence for the presence of D-lactate-terminating muropeptides. While there was no significant difference in the composition of muropeptide units of mutant and parental cell walls, the peptidoglycan of VM had a significantly lower degree of cross-linkage. These observations and the results of vancomycin-binding studies suggest alterations in the structural organization of the mutant cell walls such that access of the vancomycin molecules to the sites of wall biosynthesis is blocked.     

Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation.

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilis vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in the dacA mutant. This major low-molecular-weight PBP (DD-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.     

Two distinct transpeptidation reactions during murein synthesis in Escherichia coli.

Murein synthesized in ether-permeabilized cells of Escherichia coli deficient in individual penicillin-binding proteins (PBPs) and in the presence of certain beta-lactam antibiotics was analyzed by high-pressure liquid chromatography separation of the muramidase split products. PBP 1b was found to to be the major murein synthesizing activity that was poorly compensated for by PBP 1a. A PBP 2 mutant as well as mecillinam-inhibited cells showed increased activity in the formation of oligomeric muropeptides as well as UDP-muramylpeptidyl-linked muropeptides, the reaction products of transpeptidation, bypassing the lipid intermediate. In contrast, penicillin G and furazlocillin severely inhibited these reactions but stimulated normal dimer production. It is concluded that two distinct transpeptidases exist in E. coli: one, highly sensitive to penicillin G and furazlocillin, catalyzes the formation of hyper-cross-linked muropeptides, and a second one, quite resistant to these antibiotics, synthesizes muropeptide dimers.     

Streptococcus pneumoniae PBP2x mid‐cell localization requires the C‐terminal PASTA domains and is essential for cell shape maintenance

The transpeptidase activity of the essential penicillin‐binding protein 2x (PBP2x) of Streptococcus pneumoniae is believed to be important for murein biosynthesis required for cell division. To study the molecular mechanism driving localization of PBP2x in live cells, we constructed a set of N‐terminal GFP–PBP2x fusions under the control of a zinc‐inducible promoter. The ectopic fusion protein localized at mid‐cell. Cells showed no growth defects even in the absence of the genomic pbp2x, demonstrating that GFP–PBP2x is functional. Depletion of GFP–PBP2x resulted in severe morphological alterations, confirming the essentiality of PBP2x and demonstrating that PBP2x is required for cell division and not for cell elongation. A genetically or antibiotic inactivated GFP–PBP2x still localized at septal sites. Remarkably, the same was true for a GFP–PBP2x derivative containing a deletion of the central transpeptidase domain, although only in the absence of the protease/chaperone HtrA. Thus localization is independent of the catalytic transpeptidase domain but requires the C‐terminal PASTA domains, identifying HtrA as targeting GFP–PBP2x derivatives. Finally, PBP2x was positioned at the septum similar to PBP1a and the PASTA domain containing StkP protein, confirming that PBP2x is a key element of the divisome complex.     

Listeria monocytogenes EGD lacking penicillin-binding protein 5 (PBP5) produces a thicker cell wall

We report on the cloning of the structural gene for penicillin-binding protein 5 (PBP5), lmo2754. We also describe the enzymatic activity of PBP5 and characterize a mutant lacking this activity. Purified PBP5 has dd-carboxypeptidase activity, removing the terminal D-alanine residue from murein pentapeptide side chains. It shows higher activity against low molecular weight monomeric pentapeptide substrates compared to dimeric pentapeptide compound. Similarly, PBP5 preferentially cleaves monomeric pentapeptides present in high-molecular weight murein sacculi. A Listeria monocytogenes mutant lacking functional PBP5 was constructed. Cells of the mutant are viable, showing that the protein is dispensable for growth, but grow slower and have thickened cell walls.     

Hybrid proteins of the transglycosylase and the transpeptidase domains of PBP1B and PBP3 of Escherichia coli.

The construction of hybrid proteins of PBP1B and PBP3 has been described. One hybrid protein (PBP1B/3) contained the transglycosylase domain of PBP1B and the transpeptidase domain of PBP3. In the other hybrid protein, the putative transglycosylase domain of PBP3 was coupled to the transpeptidase domain of PBP1B (PBP3/1B). The hybrid proteins were localized in the cell envelope in a similar way as the wild-type PBP1B. In vitro isolates of the strains containing the hybrid proteins had a transglycosylase activity intermediate between that of wild-type PBP1B-producing strain and that of a PBP1B overproducer. Analysis with specific antibiotics against PBP1A/1B and PBP3 and mutant analysis in strains containing PBP3/1B revealed no detectable effects in vivo compared with wild-type strains. The same was shown for PBP1B/3 when the experiments were performed in a recA background. The data indicate that the hybrid proteins cannot replace native penicillin-binding proteins. This finding suggests that functional high-molecular-weight penicillin-binding protein specificity is at least in part determined by the unique combination of the two functional domains.     

耐甲氧西林金黄色葡萄球菌PBP2a转肽酶区的克隆表达及纯化鉴定

目的:对编码耐甲氧西林金黄色葡萄球菌(MRSA)青霉素结合蛋白2a(PBP2a)转肽酶区的mecA基因片段进行克隆、表达、纯化及鉴定。方法:根据基因文库登录的mecA基因的编码序列,设计合成了一对寡核苷酸引物,应用PCR技术从MRSA基因组DNA中扩增获得编码PBP2a转肽酶区的DNA片段,将此目的基因片段克隆至pET-His载体,经酶切鉴定、测序正确后,转化E.coliBL21(DE3)plysS;用IPTG进行诱导表达后,利用Ni2 亲和层析技术从表达蛋白中纯化目的蛋白;对表达的蛋白以MRSA胶乳凝集试剂盒进行鉴定。结果:成功构建了PBP2a转肽酶区原核表达载体,并获得了高效表达,制备了高纯度的目的蛋白。结论:获得了高纯度的PBP2a转肽酶区蛋白,为其进一步研究奠定了基础。     

Cloning, characterization, and inactivation of the gene pbpC, encoding penicillin-binding protein 3 of Staphylococcus aureus

The gene pbpC from Staphylococcus aureus was sequenced: it encodes a 691-amino-acid protein with all of the conserved motifs of a class B high-molecular-weight penicillin-binding protein (PBP), including the transpeptidase conserved motifs SXXK, SXN, and KTG. Insertional inactivation of pbpC and introduction of the intact gene in a laboratory mutant missing PBP 3 showed that the pbpC gene encodes the staphylococcal PBP 3. Inactivation of pbpC caused no detectable change in the muropeptide composition of cell wall peptidoglycan and had only minimum, if any, effect on growth rates, but caused a small but significant decrease in rates of autolysis. Cells of abnormal size and shape and disoriented septa were produced when bacteria with inactivated pbpC were grown in the presence of a sub-MIC of methicillin.     

Characterization of the bifunctional glycosyltransferase/acyltransferase penicillin-binding protein 4 of Listeria monocytogenes

Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M(-1) s(-1) from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to beta-lactam antibiotics but did increase the resistance of the mutant to moenomycin.     

The balance between different peptidoglycan precursors determines whether Escherichia coli cells will elongate or divide.

The rodA(Sui) mutation allows cell division to take place at 42 degrees C in ftsI23 mutant cells, which produce a thermolabile penicillin-binding protein 3 (PBP3, the septation-specific peptidoglycan transpeptidase). We show here that the mutation in rodA is a single-base change from a glutamine to a chain termination (amber) codon, and that an amber suppressor (supE) present in the strain restores the ability to produce a reduced level of normal RodA protein. The reduced level of RodA is accompanied by an increase in the levels of two other proteins (PBP2 and PBP5) encoded by genes in the rodA operon. We show that an increased level of PBP5 is by itself sufficient to restore cell division to ftsI23 cells at 42 degrees C. Two other treatments were found to restore division capacity to the mutant: an increase in PBP6 (which is a D-alanine carboxypeptidase like PBP5) or suitable concentrations of D-cycloserine. All of the above treatments have the effect of reducing the number of pentapeptide side chains in peptidoglycan and increasing the number of tripeptides. We conclude that the effect of the rodA(Sui) mutation is to indirectly increase the availability of tripeptide side chains, which are used preferentially by PBP3 as acceptors in transpeptidation. A change in the proportions of different kinds of peptide side chain in the peptidoglycan can therefore determine whether cells will divide.     

Acquisition of Five High-Mr Penicillin-Binding Protein Variants during Transfer of High-Level β-Lactam Resistance from Streptococcus mitis to Streptococcus pneumoniae

Penicillin-resistant isolates of Streptococcus pneumoniae generally contain mosaic genes encoding the low-affinity penicillin-binding proteins (PBPs) PBP2x, PBP2b, and PBP1a. We now present evidence that PBP2a and PBP1b also appear to be low-affinity variants and are encoded by distinct alleles in β-lactam-resistant transformants of S. pneumoniae obtained with chromosomal donor DNA from a Streptococcus mitis isolate. Different lineages of β-lactam-resistant pneumococcal transformants were analyzed, and transformants with low-affinity variants of all high-molecular-mass PBPs, PBP2x, -2a, -2b, -1a, and -1b, were isolated. The MICs of benzylpenicillin, oxacillin, and cefotaxime for these transformants were up to 40, 100, and 50 μg/ml, respectively, close to the MICs for the S. mitis donor strain. Recruitment of low-affinity PBPs was accompanied by a decrease in cross-linked muropeptides as revealed by high-performance liquid chromatography of muramidase-digested cell walls, but no qualitative changes in muropeptide chemistry were detected. The growth rates of all transformants were identical to that of the parental S. pneumoniae strain. The results stress the potential for the acquisition by S. pneumoniae of high-level β-lactam resistance by interspecies gene transfer.     

Discrete and overlapping functions of peptidoglycan synthases in growth,cell division and virulence of Listeria monocytogenes

Upon ingestion of contaminated food, Listeria monocytogenes can cause serious infections in humans that are normally treated with β‐lactam antibiotics. These target Listeria's five high molecular weight penicillin‐binding proteins (HMW PBPs), which are required for peptidoglycan biosynthesis. The two bi‐functional class A HMW PBPs PBP A1 and PBP A2 have transglycosylase and transpeptidase domains catalyzing glycan chain polymerization and peptide cross‐linking, respectively, whereas the three class B HMW PBPs B1, B2 and B3 are monofunctional transpeptidases. The precise roles of these PBPs in the cell cycle are unknown. Here we show that green fluorescent protein (GFP)‐PBP fusions localized either at the septum, the lateral wall or both, suggesting distinct and overlapping functions. Genetic data confirmed this view: PBP A1 and PBP A2 could not be inactivated simultaneously, and a conditional double mutant strain is largely inducer dependent. PBP B1 is required for rod‐shape and PBP B2 for cross‐wall biosynthesis and viability, whereas PBP B3 is dispensable for growth and cell division. PBP B1 depletion dramatically increased β‐lactam susceptibilities and stimulated spontaneous autolysis but had no effect on peptidoglycan cross‐linkage. Our in vitro virulence assays indicated that the complete set of all HMW PBPs is required for maximal virulence.     

Overproduction of inactive variants of the murein synthase PBP1B causes lysis in Escherichia coli

Penicillin-binding protein 1B (PBP1B) of Escherichia coli is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms (alpha, beta, and gamma) which differ in the length of their N-terminal cytoplasmic region. Expression plasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lytic transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a multienzyme complex is discussed.     

dacD, an Escherichia coli gene encoding a novel penicillin-binding protein (PBP6b) with DD-carboxypeptidase activity.

In the course of a study of genes located at min 44 of the Escherichia coli genome, we identified an open reading frame with the capacity to encode a 43-kDa polypeptide whose predicted amino acid sequence is strikingly similar to those of the well-known DD-carboxipeptidases penicillin-binding proteins PBP5 and PBP6. The gene product was shown to bind [3H]benzylpenicillin and to have DD-carboxypeptidase activity on pentapeptide muropeptides in vivo. Therefore, we called the protein PBP6b and the gene dacD. As with other E. coli DD-carboxypeptidases, PBP6b is not essential for cell growth. A quadruple dacA dacB dacC dacD mutant was constructed and shown to grow as well as its isogenic wild-type strain, indicating that the loss of any known PBP-associated DD-carboxypeptidase activity is not deleterious for E. coli. We also identified the homologous gene of dacD in Salmonella typhimurium as one of the components of the previously described phsBCDEF gene cluster.     

Structural basis for the beta lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus

The multiple antibiotic resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) has become a major clinical problem worldwide. The key determinant of the broad-spectrum beta-lactam resistance in MRSA strains is the penicillin-binding protein 2a (PBP2a). Because of its low affinity for beta-lactams, PBP2a provides transpeptidase activity to allow cell wall synthesis at beta-lactam concentrations that inhibit the beta-lactam-sensitive PBPs normally produced by S. aureus. The crystal structure of a soluble derivative of PBP2a has been determined to 1.8 A resolution and provides the highest resolution structure for a high molecular mass PBP. Additionally, structures of the acyl-PBP complexes of PBP2a with nitrocefin, penicillin G and methicillin allow, for the first time, a comparison of an apo and acylated resistant PBP. An analysis of the PBP2a active site in these forms reveals the structural basis of its resistance and identifies features in newly developed beta-lactams that are likely important for high affinity binding.