Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem

Chlamydiaceae are obligate intracellular bacteria that do not synthesise detectable peptidoglycan although they possess an almost complete arsenal of genes encoding peptidoglycan biosynthetic activities. In this paper, the murF gene from Chlamydia trachomatis was shown to be capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase activity. Recombinant MurF from C. trachomatis was overproduced and purified from E. coli. It exhibited ATP-dependent UDP-MurNAc-X-γ-D-Glu-meso-A(2)pm:D-Ala-D-Ala ligase activity in vitro. No significant difference of kinetic parameters was seen when X was L-Ala, L-Ser or Gly. The L-Lys-containing UDP-MurNAc-tripeptide was a poorer substrate as compared to the meso-A(2)pm-containing one. Based on the respective substrate specificities of the chlamydial MurC, MurE, MurF and Ddl enzymes, a sequence L-Ala/L-Ser/Gly-γ-D-Glu-meso-A(2)pm-D-Ala-D-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.     

Functional and biochemical analysis of Chlamydia trachomatis MurC, an enzyme displaying UDP-N-acetylmuramate:amino acid ligase activity

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):L-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:L-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (L-alanine, L-serine, and glycine); comparable Vmax/Km values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide.     

Determinants for differential effects on D-Ala-D-lactate vs D-Ala-D-Ala formation by the VanA ligase from vancomycin-resistant enterococci.

Bacteria with either intrinsic or inducible resistance to vancomycin make peptidoglycan (PG) precursors of lowered affinity for the antibiotic by switching the PG-D-Ala-D-Ala termini that are the antibiotic-binding target to either PG-D-Ala-D-lactate or PG-D-Ala-D-Ser as a consequence of altered specificity of the D-Ala-D-X ligases in the cell wall biosynthetic pathway. The VanA ligase of vancomycin-resistant enterococci, a D-Ala-D-lactate depsipeptide ligase, has the ability to recognize and activate the weak nucleophile D-lactate selectively over D-Ala(2) to capture the D-Ala(1)-OPO(3)(2)(-) intermediate in the ligase active site. To ensure this selectivity in catalysis, VanA largely rejects the protonated (NH(3)(+)) form of D-Ala at subsite 2 (K(M2) of 210 mM at pH 7.5) but not at subsite 1. In contrast, the deprotonated (NH(2)) form of D-Ala (K(M2) of 0.66 mM, k(cat) of 550 min(-)(1)) is a 17-fold better substrate compared to D-lactate (K(M) of 0.69 mM, k(cat) of 32 min(-)(1)). The low concentration of the free amine form of D-Ala at physiological conditions (i.e., 0.1% at pH 7.0) explains the inefficiency of VanA in dipeptide synthesis. Mutational analysis revealed a residue in the putative omega-loop region, Arg242, which is partially responsible for electrostatically repelling the protonated form of D-Ala(2). The VanA enzyme represents a subfamily of D-Ala-D-X ligases in which two key active-site residues (Lys215 and Tyr216) in the active-site omega-loop of the Escherichia coli D-Ala-D-Ala ligase are absent. To look for functional complements in VanA, we have mutated 20 residues and evaluated effects on catalytic efficiency for both D-Ala-D-Ala dipeptide and D-Ala-D-lactate depsipeptide ligation. Mutation of Asp232 caused substantial defects in both dipeptide and depsipeptide ligase activity, suggesting a role in maintaining the loop position. In contrast, the H244A mutation caused an increase in K(M2) for D-lactate but not D-Ala, indicating a differential role for His244 in the recognition of the weaker nucleophile D-lactate. Replacement of the VanA omega-loop by that of VanC2, a D-Ala-D-Ser ligase, eliminated D-Ala-D-lactate activity while improving by 3-fold the catalytic efficacy of D-Ala-D-Ala and D-Ala-D-Ser activity.     

Selectivity for D-lactate incorporation into the peptidoglycan precursors of Lactobacillus plantarum: role of Aad, a VanX-like D-alanyl-D-alanine dipeptidase

Lactobacillus plantarum produces peptidoglycan precursors ending in D-lactate instead of D-alanine, making the bacterium intrinsically resistant to vancomycin. The ligase Ddl of L. plantarum plays a central role in this specificity by synthesizing D-alanyl-D-lactate depsipeptides that are added to the precursor peptide chain by the enzyme MurF. Here we show that L. plantarum also encodes a D-Ala-D-Ala dipeptidase, Aad, which eliminates D-alanyl-D-alanine dipeptides that are produced by the Ddl ligase, thereby preventing their incorporation into the precursors. Although D-alanine-ended precursors can be incorporated into the cell wall, inactivation of Aad failed to suppress growth defects of L. plantarum mutants deficient in d-lactate-ended precursor synthesis.     

Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori

D-Alanine-D-alanine ligase is the second enzyme in the D-Ala branch of bacterial cell wall peptidoglycan assembly, and recognized as an attractive antimicrobial target. In this work, the D-Ala-D-Ala ligase of Helicobacter pylori strain SS1 (HpDdl) was kinetically and structurally characterized. The determined apparent K(m) of ATP (0.87 microM), the K(m1) (1.89 mM) and K(m2) of D-Ala (627 mM), and the k(cat) (115 min(-1)) at pH 8.0 indicated its relatively weak binding affinity and poor catalytic activity against the substrate D-Ala in vitro. However, by complementary assay of expressing HpDdl in Escherichia coli Delta ddl mutant, HpDdl was confirmed to be capable of D-Ala-D-Ala ligating in vivo. Through sequence alignment with other members of the D-Ala-D-X ligase superfamily, HpDdl keeps two conservatively substituted residues (Ile16 and Leu241) and two nonconserved residues (Leu308 and Tyr311) broadly located in the active region of the enzyme. Kinetic analyses against the corresponding HpDdl mutants (I16V, L241Y, L241F, L308T, and Y311S) suggested that these residues, especially Leu308 and Tyr311, might partly contribute to the unique catalytic properties of the enzyme. This was fairly proved by the crystal structure of HpDdl, which revealed that there is a 3(10)-helix (including residues from Gly306 to Leu312) near the D-Ala binding region in the C-terminal domain, where HpDdl has two sequence deletions compared with other homologs. Such 3(10)-helix may participate in D-Ala binding and conformational change of the enzyme. Our present work hopefully provides useful information for understanding the D-Ala-D-Ala ligase of Helicobacter pylori.     

In vitro and in vivo functional activity of Chlamydia MurA,a UDP-N-acetylglucosamine enolpyruvyl transferase involved in peptidoglycan synthesis and fosfomycin resistance

Organisms of Chlamydia spp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal DeltamurA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA. In E. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.     

Substrate specificity of thermostable D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589

D-Alanine-D-alanine ligase (Ddl) and its mutants maintain the biosynthesis of peptidoglycan, and the substrate specificity of Ddls partially affects the resistance mechanism of vancomycin-resistant enterococci. Through investigation of Ddls, Ddl from Thermotoga maritima ATCC 43589 showed novel characteristics, vis. thermostability up to 90 degrees C and broad substrate specificity toward 15 D-amino acids, particularly D-alanine, D-cysteine, and D-serine, in that order.     

Staphylococcus aureus MurC participates in L-alanine recognition via histidine 343, a conserved motif in the shallow hydrophobic pocket

UDP-N-acetylmuramic acid:L-alanine ligase that is encoded by the murC gene, is indispensable for bacterial peptidoglycan biosynthesis and an important target for the development of antibacterial agents. Structure of MurC ligase with substrates has been described, however, little validation via studying the effects of mutations on the structure of MurC has been performed. In this study, we carried out a functional in vitro and in vivo characterization of Staphylococcus aureus MurCH343Y protein that has a temperature-sensitive mutation of a conserved residue in the predicted shallow hydrophobic pocket that holds a short L-alanine side chain. Purified H343Y and wild-type MurC had K(m) values for L-alanine of 3.2 and 0.44 mM, respectively, whereas there was no significant difference in their K(m) values for ATP and UDP-N-acetylmuramic acid, suggesting the specific alteration of L-alanine recognition in MurCH343Y protein. In a synthetic medium that excluded L-alanine, S. aureus murCH343Y mutant cells showed an allele-specific slow growth phenotype that was suppressed by addition of L-alanine. These results suggest that His343 of S. aureus MurC is essential for high-affinity binding to L-alanine both in vitro and in vivo and provide experimental evidence supporting the structural information of MurC ligase.     

Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium

Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P313 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.     

Localization and Characterization of GTP-Binding Protein CT703 in the <Emphasis Type="Italic">Chlamydia trachomatis</Emphasis>-Infected Cells

To localize and characterize the GTP-binding protein encoded by the chlamydial ORF CT703 in the Chlamydia trachomatis-infected cells, the gene coding for CT703 in the Chlamydia trachomatis serovar L2 genome was cloned into the prokaryotic expression vector pGEX and expressed as GST fusion protein in the E. coli BL21 strain. The GST-CT703 fusion protein was purified and used to raise antigen-specific antibodies. Using the anti-fusion protein antibodies, we localized the endogenous CT703 protein inside the chlamydial inclusion using an indirect immunofluorescence assay (IFA). We also detected a significantly decreased level of CT703 in cultures that were induced to undergo persistent infection. These observations suggest that CT703 may be an important regulator for promoting chlamydial productive infection.     

Conservation of the biochemical properties of IncA from Chlamydia trachomatis and Chlamydia caviae: oligomerization of IncA mediates interaction between facing membranes

The developmental cycle of Chlamydiaceae occurs in a membrane compartment called an inclusion. IncA is a member of a family of proteins synthesized and secreted onto the inclusion membrane by bacteria. IncA proteins from different species of Chlamydiaceae show little sequence similarity. We report that the biochemical properties of Chlamydia trachomatis and Chlamydia caviae are conserved. Both proteins self-associate to form multimers. When artificially expressed by the host cell, they localize to the endoplasmic reticulum. Strikingly, heterologous expression of IncA in the endoplasmic reticulum completely inhibits concomitant inclusion development. Using truncated forms of IncA from C. caviae, we show that expression of the C-terminal cytoplasmic domain of the protein at the surface of the endoplasmic reticulum is sufficient to disrupt the bacterial developmental cycle. On the other hand, development of a C. trachomatis strain that does not express IncA is not inhibited by artificial IncA expression, showing that the disruptive effect observed with the wild-type strain requires direct interactions between IncA molecules at the inclusion and on the endoplasmic reticulum. Finally, we modeled IncA tetramers in parallel four helix bundles based on the structure of the SNARE complex, a conserved structure involved in membrane fusion in eukaryotic cells. Both C. trachomatis and C. caviae IncA tetramers were highly stable in this model. In conclusion, we show that the property of IncA proteins to assemble into multimeric structures is conserved between chlamydial species, and we propose that these proteins may have co-evolved with the SNARE machinery for a role in membrane fusion.     

The MurC ligase essential for peptidoglycan biosynthesis is regulated by the serine/threonine protein kinase PknA in Corynebacterium glutamicum

The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (L-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis.     

Requirement of the VanY and VanX D,D-peptidases for glycopeptide resistance in enterococci

Transposon Tn 1546 confers resistance to glycopeptide antibiotics in enterococci and encodes two D,D-peptidases (VanX and VanY) in addition to the enzymes for the synthesis of D-alanyl-D-lactate (D-Ala-D-Lac). VanY was produced in the baculovirus expression system and purified as a proteolytic fragment that lacked the putative N-terminal membrane anchor of the protein. The enzyme was a Zn2+-dependent D,D-carboxypeptidase that cleaved the C-terminal residue of peptidoglycan precursors ending in R-D-Ala-D-Ala or R-D-Ala-D-Lac but not the dipeptide D-Ala-D-Ala. The specificity constants kcat/Km were 17- to 67-fold higher for substrates ending in the R-D-Ala-D-Ala target of glycopeptides. In Enterococcus faecalis, VanY was present in membrane and cytoplasmic fractions, produced UDP-MurNAc-tetrapeptide from cytoplasmic peptidoglycan precursors and was required for high-level glycopeptide resistance in a medium supplemented with D-Ala. The enzyme could not replace the VanX D,D-dipeptidase for the expression of glycopeptide resistance but a G237D substitution in the host D-Ala:D-Ala ligase restored resistance in a vanX null mutant. Deletion of the membrane anchor of VanY led to an active D,D-carboxypeptidase exclusively located in the cytoplasmic fraction that did not contribute to glycopeptide resistance in a D-Ala-containing medium. Thus, VanX and VanY had non-overlapping functions involving the hydrolysis of D-Ala-D-Ala and the removal of D-Ala from membrane-bound lipid intermediates respectively.     

Detection of sequence variation in PCR-amplified fragments of omp2 gene from three species of the family Chlamydiaceae using agarose gel electrophoresis containing bisbenzimide-PEG

A simple technique providing a means for rapid genetic differentiation of chlamydial strains is described. The technique is based on a single-step sequence-specific separation of PCR-amplified DNA fragments by electrophoresis in an agarose gel containing a DNA ligand - bisbenzimide-PEG. A hypervariable region at the 5' end of the omp2 gene of Chlamydiaceae species encoding the 60-kDa cysteine-rich outer membrane protein was selected as a target for PCR. The appropriate fragments were amplified from strains of Chlamydia trachomatis, Chlamydophila pneumoniae, and Chlamydophila psittaci, and the PCR products originating from different species were electrophoretically separated in the presence of the DNA ligand. We therefore demonstrated that PCR with a single pair of primers followed by simple agarose gel electrophoresis with bisbenzimide-PEG can be applied to the differentiation of three members of the family Chlamydiaceae which are commonly recognized as human pathogens.     

Isolation, cloning, and sequencing of the Salmonella typhimurium ddlA gene with purification and characterization of its product, D-alanine:D-alanine ligase (ADP forming)

A gene coding for D-alanine:D-alanine (D-Ala-D-Ala) ligase (ADP forming) (EC 6.3.2.4) activity has been isolated from a lambda library of Salmonella typhimurium DNA. Insertion mutations in the gene indicate that the gene is not essential for growth of the bacterium. The encoded enzyme was purified from an overproducing strain of S. typhimurium. D-Ala-D-Ala ligase is a protein of 39,271 molecular weight and has a kcat of 644 min-1 at pH 7.2. A 2.4-kilobase SalI-SphI fragment containing the gene was sequenced, and the ddlA gene consists of 1092 nucleotides. The gene sequence was compared to the sequence of the ddl gene of Escherichia coli [Robinson, A. C., Kenan, D. J., Sweeney, J., & Donachie, W. D. (1986) J. Bacteriol. 167, 809-817]. Because of differences between the S. typhimurium gene and the E. coli ddl gene, the S. typhimurium gene has been named ddlA.     

Molecular characterization of atypical Chlamydia and evidence of their dissemination in different European and Asian chicken flocks by specific real-time PCR

Chlamydia psittaci is a zoonotic pathogen associated primarily with avian chlamydiosis. New chlamydial agents with suspected zoonotic potential were recently detected from domestic poultry in Germany and France indicating that the spectrum of Chlamydiaceae encountered in birds is not confined to a single chlamydial species. For further characterization, a specific real-time PCR targeting the conserved 16S rRNA gene was developed and validated for a specific detection of these atypical Chlamydiaceae. In order to address the epidemiological importance of the new chlamydial agents and their distribution, Chlamydiaceae-positive chicken samples collected from flocks from five different countries were examined. The results confirmed that C.psittaci is not the predominant chlamydial species among chickens examined and suggested that the new chlamydial agents could putatively be widespread in poultry flocks (France, Greece, Croatia, Slovenia and China at least) justifying their systematic investigation when poultry samples are submitted to laboratories for avian chlamydiosis diagnosis. Besides, 16S rRNA-based dendrogram, including sequences from both isolates of the new chlamydial agents or positive samples as well as representative sequences from species belonging to the order Chlamydiales, showed the new chlamydial agents to form a distinct line of descent separated from those of other chlamydial species, but clearly grouped within the family Chlamydiaceae. Finally, the phylogenetic tree inferred from the multi-locus sequence typing based on four housekeeping fragments (gatA, gidA, enoA and hflX) and the ompA-based dendrogram showed an almost identical topology of the new chlamydial agents with that recovered by 16S rRNA-based dendrogram. Interestingly, partial ompA gene sequences displayed considerable diversity among isolates.     

Comparative proteomic analysis of Staphylococcus aureus strains with differences in resistance to the cell wall-targeting antibiotic vancomycin

Three isogenic strains derived from a clinical vancomycin-intermediate Staphylococcus aureus isolate were examined by comparative protein abundance analysis. Subcellular fractionation was followed by protein separation in 2-DE gels and spot identification by MALDI-TOFTOF-MS and LC-MS/MS. Sixty-five significant protein abundance changes were determined. Numerous enzymes participating in the purine biosynthesis pathway were dramatically increased in abundance in strain VP32, which featured the highest minimal inhibitory concentration for vancomycin, compared to strains P100 and HIP5827. Peptidoglycan hydrolase LytM (LytM) and the SceD protein, a putative transglycosylase, were increased in abundance in the cell envelope fraction of strain VP32, whereas the enzyme D-Ala-D-Ala ligase was decreased in its cytosol fraction. Furthermore, penicillin-binding protein 2 (PBP2) had substantially higher activity in strain VP32 compared to that in strain HIP5827. LytM, PBP2 and D-Ala-D-Ala ligase catalyze reactions in the biosynthesis or the metabolism of cell wall peptidoglycan. It is plausible that expression and activity changes of these enzymes in strain VP32 are responsible for an altered cell wall turnover rate, which has been observed, and an altered peptidoglycan structure, which has yet to be elucidated for this highly vancomycin-resistant strain.