Structural insight into YcbB-mediated beta-lactam resistance in Escherichia coli.

The bacterial cell wall plays a crucial role in viability and is an important drug target. In Escherichia coli, the peptidoglycan crosslinking reaction to form the cell wall is primarily carried out by penicillin-binding proteins that catalyse D,D-transpeptidase activity. However, an alternate crosslinking mechanism involving the L,D-transpeptidase YcbB can lead to bypass of D,D-transpeptidation and beta-lactam resistance. Here, we show that the crystallographic structure of YcbB consists of a conserved L,D-transpeptidase catalytic domain decorated with a subdomain on the dynamic substrate capping loop, peptidoglycan-binding and large scaffolding domains. Meropenem acylation of YcbB gives insight into the mode of inhibition by carbapenems, the singular antibiotic class with significant activity against L,D-transpeptidases. We also report the structure of PBP5-meropenem to compare interactions mediating inhibition. Additionally, we probe the interaction network of this pathway and assay beta-lactam resistance in vivo. Our results provide structural insights into the mechanism of action and the inhibition of L,D-transpeptidation, and into YcbB-mediated antibiotic resistance.

Purification and partial characterization of LdtP, a cell envelope modifying enzyme in Liberibacter asiaticus.

BACKGROUND: The aggressive spread of Liberibacter asiaticus, a bacterium closely associated with citrus greening, has given rise to an acute crisis in the citrus industry, making it imperative to expand the scientific knowledge base regarding L. asiaticus. Despite several endeavors to culture L. asiaticus, this bacterium has yet to be maintained in axenic culture, rendering identification and analysis of potential treatment targets challenging. Accordingly, a thorough understanding of biological mechanisms involved in the citrus host-microbe relationship is critical as a means of directing the search for future treatment targets. In this study, we evaluate the biochemical characteristics of CLIBASIA_01175, renamed LdtP (L,D-transpeptidase). Surrogate strains were used to evaluate its potential biological significance in gram-negative bacteria. A strain of E. coli carrying quintuple knock-outs of all genes encoding L,D-transpeptidases was utilized to demonstrate the activity of L. asiaticus LdtP. RESULTS: This complementation study demonstrated the periplasmic localization of mature LdtP and provided evidence for the biological role of LdtP in peptidoglycan modification. Further investigation highlighted the role of LdtP as a periplasmic esterase involved in modification of the lipid A moiety of the lipopolysaccharide. This work described, for the first time, an enzyme of the L,D-transpeptidase family with moonlighting enzyme activity directed to the modification of the bacterial cell wall and LPS. CONCLUSIONS: Taken together, the data indicates that LdtP is a novel protein involved in an alternative pathway for modification of the bacterial cell, potentially affording L. asiaticus a means to survive within the host.

Structure and Inhibitor Specificity of L,D-Transpeptidase (LdtMt2) from Mycobacterium tuberculosis and Antibiotic Resistance: Calcium Binding Promotes Dimer Formation.

The final step of peptidoglycan (PG) synthesis in all bacteria is the formation of cross-linkage between PG-stems. The cross-linking between amino acids in different PG chains gives the peptidoglycan cell wall a 3-dimensional structure and adds strength and rigidity to it. There are two distinct types of cross-linkages in bacterial cell walls. D,D-transpeptidase (D,D-TPs) generate the classical 4➔3 cross-linkages and the L,D-transpeptidase (L,D-TPs) generate the 3➔3 non-classical peptide cross-linkages. The present study is aimed at understanding the nature of drug resistance associated with L,D-TP and gaining insights for designing novel antibiotics against multi-drug resistant bacteria. Penicillin and cephalosporin classes of ß-lactams cannot inhibit L,D-TP function; however, carbapenems inactivate its function. We analyzed the structure of L,D-TP of Mycobacterium tuberculosis in the apo form and in complex with meropenem and imipenem. The periplasmic region of L,D-TP folds into three domains. The catalytic residues are situated in the C-terminal domain. The acylation reaction occurs between carbapenem antibiotics and the catalytic Cys-354 forming a covalent complex. This adduct formation mimics the acylation of L,D-TP with the donor PG-stem. A novel aspect of this study is that in the crystal structures of the apo and the carbapenem complexes, the N-terminal domain has a muropeptide unit non-covalently bound to it. Another interesting observation is that the calcium complex crystallized as a dimer through head and tail interactions between the monomers.

LdtMav2, a nonclassical transpeptidase and susceptibility of Mycobacterium avium to carbapenems.

AIM: Mycobacterium avium infections, especially in immune-compromised individuals, present a significant challenge as therapeutic options are limited. In this study, we investigated if M. avium genome encodes nonclassical transpeptidases and if newer carbapenems are effective against this mycobacteria. MATERIALS & METHODS: Biochemical and microbiological approaches were used to identify and characterize a nonclassical transpeptidase, namely L,D-transpeptidase, in M. avium. RESULTS & CONCLUSION: We describe the biochemical and physiological attributes of a L,D-transpeptidase in M. avium, LdtMav2. Suggestive of a constitutive requirement, levels of LdtMav2, a L,D-transpeptidase in M. avium, remain constant during exponential and stationary phases of growth. Among ß-lactam antibacterials, only a subset of carbapenems inhibit LdtMav2 and tebipenem, a new oral carbapenem, inhibits growth of M. avium.

Inhibition of sortase, a bacterial surface protein anchoring transpeptidase, by beta-sitosterol-3-O-glucopyranoside from Fritillaria verticillata.

A glucosylsterol, beta-sitosterol-3-O-glucopyranoside, has been isolated as an active principle with sortase inhibitory effect from the bulbs of Fritillaria verticillata by bioassay-guided chromatographic fractionation. The isolate was a potent inhibitor of sortase, with an IC(50) value of 18.3 microg/ml and had antibacterial activity against Bacillus subtilis, Staphylococcus aureus, and Micrococcus leuteus with MIC values of 50, 200, and 400 microg/ml, respectively, indicating that this compound is a possible candidate for the development of a bacterial sortase inhibitor. In addition, sitosterol was found to be inactive upon sortase and bacterial cell growth. These results suggest that the inhibitory potency of beta-sitosterol-3-O-glucopyranoside is sensitively dependent upon the glucopyranoside side chain moiety.

Structural studies of the transpeptidase domain of PBP1a from Streptococcus pneumoniae.

The synthesis of the bacterial cell wall requires enzymes which are localized both in the cytoplasm and in the periplasm. Penicillin-binding proteins (PBPs) catalyze the last, crucial steps in peptidoglycan biosynthesis and several of them are essential for bacterial survival. High-molecular-mass PBPs can be bifunctional (class A) or monofunctional (class B) and to date no structural information on any class A PBP is available. To initiate the determination of the three-dimensional structure of a class A PBP, crystals of the transpeptidase domain of PBP1a from Streptococcus pneumoniae were prepared by limited proteolysis of the full-length molecule and purification by anion-exchange chromatography and gel filtration. The samples crystallize in space group C222(1), contain one molecule per asymmetric unit and diffract X-rays to 2.7 A. Selenomethionine-labelled crystals have been prepared and structure solution is under way.

Moenomycin-mediated affinity purification of penicillin-binding protein 1b.

The antibiotic moenomycin A inhibits the biosynthesis of peptidoglycan, the main structural polymer of the bacterial cell wall. The inhibition is based on a reversible binding of the antibiotic to one of the substrate binding sites at enzymes such as the penicillin binding protein 1b (PBP 1b). This binding has been employed to isolate PBP 1b by affinity chromatography. Suitable ligands have been prepared from moenomycin A and coupled both to affinity supports and to surface plasmon resonance sensor surfaces. The reactions that take place upon immobilization of the ligands to the affinity support and the sensor surface, respectively, have been studied in detail. With the help of surface plasmon resonance the optimal conditions for binding of PBP 1b to moenomycin-derivated ligands have been established. For the first time the selective binding of the moenomycin sugar moiety to the enzyme has been demonstrated.

Two-step procedure for purification and separation of the essential penicillin-binding proteins PBP 1A and 1Bs of Escherichia coli.

The penicillin-binding proteins PBP 1A and 1Bs are the essential murein polymerases of Escherichia coli. Purification of these membrane-bound bifunctional transglycosylase-transpeptidases was a major obstacle in studying the details of both enzymatic reactions. Here we describe a simple, highly specific affinity chromatography method that takes advantage of the availability of the specific inhibitor of the transglycosylase site moenomycin A in order to enrich PBP 1A and 1Bs in one step from crude membrane preparations. Separation of PBP 1A from PBP 1Bs is achieved in a second step employing cation exchange chromatography yielding enzymatically active native murein polymerases.

Identification, purification, and characterization of transpeptidase and glycosyltransferase domains of Streptococcus pneumoniae penicillin-binding protein 1a.

Resistance to beta-lactam antibiotics in Streptococcus pneumoniae is due to alteration of penicillin-binding proteins (PBPs). S. pneumoniae PBP 1a belongs to the class A high-molecular-mass PBPs, which harbor transpeptidase (TP) and glycosyltransferase (GT) activities. The GT active site represents a new potential target for the generation of novel nonpenicillin antibiotics. The 683-amino-acid extracellular region of PBP 1a (PBP 1a*) was expressed in Escherichia coli as a GST fusion protein. The GST-PBP 1a* soluble protein was purified, and its domain organization was revealed by limited proteolysis. A protease-resistant fragment spanning Ser 264 to Arg 653 exhibited a reactivity profile against both beta-lactams and substrate analogues similar to that of the parent protein. This protein fragment represents the TP domain. The GT domain (Ser 37 to Lys 263) was expressed as a recombinant GST fusion protein. Protection by moenomycin of the GT domain against trypsin degradation was interpreted as an interaction between the GT domain and the moenomycin.

Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics.

To understand the biochemical basis of resistance of bacteria to beta-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k(cat)/Km values for the two purified enzymes (3,400 M(-1) s(-1) for S-PBP2x and 11.2 M(-1) s(-1) for R-PBP2x) suggests that they are significantly different kinetically. Implications of this finding are discussed. We also found that the two purified enzymes did not possess a detectable level of beta-lactam hydrolytic activity. Finally, we show that the expression levels of both PBP2x enzymes were similar during different growth phases.

Cloning and characterization of the ponA gene encoding penicillin-binding protein 1 from Neisseria gonorrhoeae and Neisseria meningitidis.

The ponA gene encoding penicillin-binding protein 1 (PBP 1) from Neisseria gonorrhoeae was cloned by a reverse genetic approach. PBP 1 was purified from solubilized membranes of penicillin-susceptible strain FA19 by covalent ampicillin affinity chromatography and used to obtain an NH2-terminal amino acid sequence. A degenerate oligonucleotide based on this protein sequence and a highly degenerate oligonucleotide based on a conserved amino acid motif found in all class A high-molecular-mass PBPs were used to isolate the PBP 1 gene (ponA). The ponA gene encodes a protein containing all of the conserved sequence motifs found in class A PBPs, and expression of the gene in Escherichia coli resulted in the appearance of a new PBP that comigrated with PBP 1 purified from N. gonorrhoeae. A comparison of the gonococcal ponA gene to its homolog isolated from Neisseria meningitidis revealed a high degree of identity between the two gene products, with the greatest variability found at the carboxy terminus of the two deduced PBP 1 protein sequences.

Localization of a putative second membrane association site in penicillin-binding protein 1B of Escherichia coli.

We have shown previously that the periplasmic domain of penicillin-binding protein 1B (PBP 1Bper; residues 90-844) from Escherichia coli is insoluble in the absence of detergents, and can be reconstituted into liposomes [Nicholas, Lamson and Schultz (1993) J. Biol. Chem. 268, 5632-5641]. These data suggested that native PBP 1B contains a membrane association site in addition to its N-terminal transmembrane anchor. We have studied the membrane topology of PBP 1B in greater detail by assessing detergent binding and solubility in the absence of detergents for PBP 1Bper and a set of proteolytic fragments of PBP 1B. PBP 1Bper was shown by three independent methods to bind to detergent micelles, which strongly suggests that the periplasmic domain interacts with the hydrophobic milieu of membrane bilayers. Digestion with high weight ratios of thrombin of purified PBP 1B containing an engineered thrombin cleavage site on the periplasmic side of the transmembrane anchor generated four fragments in addition to PBP 1Bper that varied in size from 71 to 48 kDa. In contrast to PBP 1Bper, all fragments of 67 kDa and smaller were eluted from a gel-filtration column in the absence of detergents and did not bind to detergent micelles. The N-terminal sequences of the four fragments were determined, allowing the cleavage sites to be located in the primary sequence of PBP 1B. These data localize the membrane association site of PBP 1B to a region comprising the first 163 amino acids of the periplasmic domain, which falls within the putative transglycosylase domain. Lipid modification does not appear to be the mechanism by which PBP 1Bper associates with membranes.

The "DEAD box" protein DbpA interacts specifically with the peptidyltransferase center in 23S rRNA.

The Escherichia coli DEAD (Asp-Glu-Ala-Asp) box protein DbpA is a putative RNA helicase and established RNA-dependent ATPase and is the only member of the DEAD box protein family for which a specific RNA substrate, bacterial 23S rRNA, has been identified. We have investigated the nature of this specificity in depth and have localized by deletion mutagenesis and PCR a single region of 93 bases (bases 2496-2588) in 23S rRNA that is both necessary and sufficient for complete activation of ATPase activity of DbpA. This target region forms part of the peptidyltransferase center and includes many bases involved in interaction with the 3' terminal adenosines of both A- and P-site tRNAs. Deletion of stem loops within the 93-base segment abolished ATPase activation. Similarly, point mutations that disrupt base pairing within stem structures ablated stimulation of ATPase activity. These data are consistent with roles for DbpA either in establishing and/or maintaining the correct three-dimensional structure of the peptidyltransferase center in 23S rRNA during ribosome assembly or in the peptidyltransferase reaction.

Specific interaction of penicillin-binding proteins 3 and 7/8 with soluble lytic transglycosylase in Escherichia coli.

Soluble lytic transglycosylase 70 (Slt70), one of the better characterized murein hydrolases of Escherichia coli, was covalently bound to CNBr-activated Sepharose and used as a specific tool to screen for proteins showing an affinity for Slt70. Several proteins were specifically enriched by Slt-Sepharose affinity chromatography. Two of them were identified as the penicillin-binding proteins (PBP)3 and PBP7/8. Thus, the bifunctional synthase PBP3, specifically involved in septum formation, and PBP7/8, recently shown to be a DD-endopeptidase, bind to Slt70 in vitro. In addition, PBP7/8 was found not only to stabilize but also to stimulate the enzymatic activity of Slt70 by a protein-protein interaction. It is concluded that Slt70, PBP7/8, and PBP3 may form a multienzyme complex in vivo.

Penicillin-binding protein 2x of Streptococcus pneumoniae: enzymic activities and interactions with beta-lactams.

The high-molecular-mass penicillin-binding protein (PBP) 2x, one of the primary targets of beta-lactam antibiotics in Streptococcus pneumoniae, has been produced as a soluble form and purified in large amounts. It has been shown to catalyse hydrolysis and transfer reactions with different ester and thiolester substrates and its catalytic behaviour was often similar to that of the soluble DD-peptidase from Streptomyces R61. This provided an easy method to monitor the activity of the PBP. For the first time, a reliable kinetic study of the interaction between a lethal target and beta-lactam antibiotics has been performed. Characteristic kinetic parameters were obtained with different beta-lactam compounds. These results not only validated the mechanism established with non-essential extracellular enzymes, but will also constitute the basis for comparative studies of the low-affinity variants from penicillin-resistant strains.

Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3.

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.

Unusual resistance of peptidyl transferase to protein extraction procedures.

Peptidyl transferase, the ribosomal activity responsible for catalysis of peptide bond formation, is resistant to vigorous procedures that are conventionally employed to remove proteins from protein-nucleic acid complexes. When the "fragment reaction" was used as a model assay for peptide bond formation, Escherichia coli ribosomes or 50S subunits retained 20 to 40 percent activity after extensive treatment with proteinase K and SDS, but lost activity after extraction with phenol or exposure to EDTA. Ribosomes from the thermophilic eubacterium Thermus aquaticus remained more than 80 percent active after treatment with proteinase K and SDS, which was followed by vigorous extraction with phenol. This activity is attributable to peptidyl transferase, as judged by specific inhibition by the peptidyl transferase-specific antibiotics chloramphenicol and carbomycin. In contrast, activity is abolished by treatment with ribonuclease T1. These findings support the possibility that 23S ribosomal RNA participates in the peptidyl transferase function.

Construction of a water-soluble form of penicillin-binding protein 2a from a methicillin-resistant Staphylococcus aureus isolate.

The mecA gene from methicillin-resistant Staphylococcus aureus 27r, which encodes the membrane-bound penicillin-binding protein 2a (PBP 2a), was cloned, sequenced, and expressed in Escherichia coli. PBP 2a is the major factor that mediates methicillin resistance in staphylococci. The DNA sequence of the mecA gene from strain 27r was greater than 99% identical to the DNA sequence of other S. aureus mecA genes and the mecA gene from Staphylococcus epidermidis. Analysis of the deduced amino acid sequence of PBP 2a from strain 27r revealed a hydrophobic region at the amino terminus that possessed characteristics of an uncleaved signal peptide such as those found in type II integral membrane proteins. Site-specific mutagenesis was used to modify the strain 27r mecA gene to permit removal of the region encoding the putative transmembrane region (amino acids 2 to 22). When it was expressed in E. coli, the modified mecA gene from strain 27r encoded a water-soluble form of PBP 2a that was detectable in the cytoplasm of transformants. The water-soluble form of PBP 2a protein from S. aureus 27r retained the same binding efficiency for beta-lactam antibiotics as the unmodified membrane-bound PBP 2a from S. aureus 27r.

Characterization of penicillin-binding protein 2 of Staphylococcus aureus: deacylation reaction and identification of two penicillin-binding peptides.

Penicillin-binding protein (PBP) 2 is the major PBP of five that have been identified in susceptible strains of Staphylococcus aureus. Beta-lactam antibiotic binding to PBP 2 is important for the antibacterial effect. Antibiotic binding to PBP 2 in strain 209P was examined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in competition assays using [3H]penicillin as the radiolabel. Clavulanic acid, which is specifically bound by PBP 2, and cefaclor, which is specific for PBP 3, were studied. Cefaclor, which alone appeared not to bind PBP 2, in combination inhibited PBP 2 binding of clavulanic acid. By varying the temperature during radiolabeling with [3H]penicillin in cefaclor competition assays and in direct radiolabeling assays with [3H]cefaclor, it was shown that cefaclor was bound by PBP 2 with high affinity (50% inhibitory concentration, less than or equal to 0.1 microgram/ml) and that the apparent low-affinity binding (50% inhibitory concentration, greater than 10 micrograms/ml) in competition assays performed at 37 degrees C was due to rapid deacylation. Two penicillin-binding peptides of PBP 2 also were identified in fluorographs of PBPs separated by nonequilibrium pH gradient gel and two-dimensional electrophoresis. Rapid deacylation for some antibiotics and the presence of two penicillin-binding peptides are two properties of PBP 2 that should be considered when correlating results of binding assays with effects of beta-lactam antibiotics on S. aureus.

Comparison of conventional susceptibility tests with direct detection of penicillin-binding protein 2a in borderline oxacillin-resistant strains of Staphylococcus aureus.

Six selected strains of Staphylococcus aureus classified as borderline oxacillin-resistant, according to standard disk diffusion and microdilution susceptibility test methods, and seven methicillin-resistant and seven methicillin-susceptible control strains were examined for the presence of penicillin-binding protein 2a (PBP 2a) by fluorography and immunoblotting and for DNA hybridization with a mec-specific probe in a dot blot assay. Oxacillin agar screen tests with and without NaCl supplementation were also performed with all strains. PBP 2a was detected both by fluorography and by immunoblotting in all seven methicillin-resistant control strains and in none of the susceptible controls. PBP 2a was detected in two borderline strains. Results of agar screen tests performed without NaCl supplementation were completely concordant with susceptibility determined by PBP 2a and mec detection methods. Agar screening with NaCl supplementation was less accurate. These findings were confirmed with 20 additional borderline strains. Direct detection methods for the presence of PBP 2a or mec, the gene encoding it, allow accurate and definitive classification of borderline strains. Further efforts to develop a rapid, clinically useful, antibody detection system for PBP 2a are warranted.