Delphine Patin

A WASp–VASP complex regulates actin polymerization at the plasma membrane

Proteins of the Wiskott–Aldrich syndrome and Ena/VASP families both play essential functions in the regulation of actin dynamics at the cell leading edge. However, possibilities of functional interplay between members of these two families have not been addressed. Here we show that, in hemopoietic cells, recruitment of the C‐terminal VCA (Verprolin homology, Cofilin homology, Acidic) domain of WASp at the plasma membrane by a ligand technique using rapamycin as an intermediate is not sufficient to elicit efficient Arp2/3 complex‐mediated actin polymerization. Other domains of WASp, in particular the proline‐rich domain, are required for the formation of actin‐rich structures. An in vitro analysis demonstrates that the proline‐rich domain of WASp binds VASP with an affinity of ∼106 M−1. In addition, WASp and VASP both accumulate in actin‐rich phagocytic cups. Finally, in a reconstituted motility medium, VASP enhances actin‐based propulsion of WASp‐coated beads in a fashion reminiscent of its effect on Listeria movement. We propose that VASP and WASp cooperation is essential in stimulating actin assembly and membrane protrusion at the leading edge.

Discovery of Novel 5-Benzylidenerhodanine and 5-Benzylidenethiazolidine-2,4-dione Inhibitors of MurD Ligase

We have designed, synthesized, and evaluated 5-benzylidenerhodanine- and 5-benzylidenethiazolidine-2,4-dione-based compounds as inhibitors of bacterial enzyme MurD with E. coli IC(50) in the range 45-206 μM. The high-resolution crystal structure of MurD in complex with (R,Z)-2-(3-[{4-([2,4-dioxothiazolidin-5-ylidene]methyl)phenylamino}methyl)benzamido)pentanedioic acid [(R)-32] revealed details of the binding mode of the inhibitor within the active site and provides a good foundation for structure-based design of a novel generation of MurD inhibitors.

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.

Activation of the l,d‐transpeptidation peptidoglycan cross‐linking pathway by a metallo‐d,d‐carboxypeptidase in Enterococcus faecium

Bypass of the penicillin‐binding proteins by an l,d‐transpeptidase (Ldtfm) confers cross‐resistance to β‐lactam and glycopeptide antibiotics in mutants of Enterococcus faecium selected in vitro. Ldtfm is produced by the parental strain D344S although it insignificantly contributes to peptidoglycan cross‐linking as pentapeptide stems cannot be used as acyl donors by this enzyme. Here we show that production of the tetrapeptide substrate of Ldtfm is controlled by a two‐component regulatory system (DdcRS) and a metallo‐d,d‐carboxypeptidase (DdcY). The locus was silent in D344S and its activation was due to amino acid substitutions in DdcS or DdcR that led to production of DdcY and hydrolysis of the C‐terminal d‐Ala residue of the cytoplasmic peptidoglycan precursor UDP‐MurNAc‐pentapeptide. The T161A and T161M substitutions affected a position of DdcS known to be essential for the phosphatase activity of related sensor kinases. Complete elimination of UDP‐MurNAc‐pentapeptide, which was required specifically for resistance to glycopeptides, involved substitutions in DdcY that increased the catalytic efficiency of the enzyme (E127K) and affected its interaction with the cell envelope (I14N). The ddc locus displays striking similarities with portions of the van vancomycin resistance gene clusters, suggesting possible routes of emergence of cross‐resistance to glycopeptides and β‐lactams in natural conditions.

New 5-benzylidenethiazolidin-4-one inhibitors of bacterial MurD ligase: Design, synthesis, crystal structures, and biological evaluation

Mur ligases (MurC-MurF), a group of bacterial enzymes that catalyze four consecutive steps in the formation of cytoplasmic peptidoglycan precursor, are becoming increasingly adopted as targets in antibacterial drug design. Based on the crystal structure of MurD cocrystallized with thiazolidine-2,4-dione inhibitor I, we have designed, synthesized, and evaluated a series of improved glutamic acid containing 5-benzylidenerhodanine and 5-benzylidenethiazolidine-2,4-dione inhibitors of MurD with IC(50) values up to 28 μM. Inhibitor 37, with an IC(50) of 34 μM, displays a weak antibacterial activity against S. aureus ATCC 29213 and E. faecalis ATCC 29212 with minimal inhibitory concentrations of 128 μg/mL. High-resolution crystal structures of MurD in complex with two new inhibitors (compounds 23 and 51) reveal details of their binding modes within the active site and provide valuable information for further structure-based optimization.

Functional and Biochemical Analysis of the Chlamydia trachomatis Ligase MurE

Chlamydiae are unusual obligately intracellular bacteria that do not synthesize detectable peptidoglycan. However, they possess genes that appear to encode products with peptidoglycan biosynthetic activity. Bioinformatic analysis predicts that chlamydial MurE possesses UDP-MurNAc-L-Ala-D-Glu:meso-diaminopimelic acid (UDP-MurNAc-L-Ala-D-Glu:meso-A(2)pm) ligase activity. Nevertheless, there are no experimental data to confirm this hypothesis. In this paper we demonstrate that the murE gene from Chlamydia trachomatis is capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-L-Ala-D-Glu:meso-A(2)pm ligase activity. Recombinant MurE from C. trachomatis (MurE(Ct)) was overproduced in and purified from E. coli in order to investigate its kinetic parameters in vitro. By use of UDP-MurNAc-L-Ala-D-Glu as the nucleotide substrate, MurE(Ct) demonstrated ATP-dependent meso-A(2)pm ligase activity with pH and magnesium ion optima of 8.6 and 30 mM, respectively. Other amino acids (meso-lanthionine, the ll and dd isomers of A(2)pm, D-lysine) were also recognized by MurE(Ct.) However, the activities for these amino acid substrates were weaker than that for meso-A(2)pm. The specificity of MurE(Ct) for three possible C. trachomatis peptidoglycan nucleotide substrates was also determined in order to deduce which amino acid might be present at the first position of the UDP-MurNAc-pentapeptide. Relative k(cat)/K(m) ratios for UDP-MurNAc-L-Ala-D-Glu, UDP-MurNAc-L-Ser-D-Glu, and UDP-MurNAc-Gly-D-Glu were 100, 115, and 27, respectively. Our results are consistent with the synthesis in chlamydiae of a UDP-MurNAc-pentapeptide in which the third amino acid is meso-A(2)pm. However, due to the lack of specificity of MurE(Ct) for nucleotide substrates in vitro, it is not obvious which amino acid is present at the first position of the pentapeptide.

Purification and biochemical characterization of Mur ligases from Staphylococcus aureus.

The Mur ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the UDP-N-acetylmuramoyl-pentapeptide precursor of peptidoglycan. The murC, murD, murE and murF genes from Staphylococcus aureus, a major pathogen, were cloned and the corresponding proteins were overproduced in Escherichia coli and purified as His(6)-tagged forms. Their biochemical properties were investigated and compared to those of the E. coli enzymes. Staphylococcal MurC accepted L-Ala, L-Ser and Gly as substrates, as the E. coli enzyme does, with a strong preference for L-Ala. S. aureus MurE was very specific for L-lysine and in particular did not accept meso-diaminopimelic acid as a substrate. This mirrors the E. coli MurE specificity, for which meso-diaminopimelic acid is the preferred substrate and L-lysine a very poor one. S. aureus MurF appeared less specific and accepted both forms (L-lysine and meso-diaminopimelic acid) of UDP-MurNAc-tripeptide, as the E. coli MurF does. The inverse and strict substrate specificities of the two MurE orthologues is thus responsible for the presence of exclusively meso-diaminopimelic acid and L-lysine at the third position of the peptide in the peptidoglycans of E. coli and S. aureus, respectively. The specific activities of the four Mur ligases were also determined in crude extracts of S. aureus and compared to cell requirements for peptidoglycan biosynthesis.

The functional vanGCd cluster of Clostridium difficile does not confer vancomycin resistance

vanGCd, a cryptic gene cluster highly homologous to the vanG gene cluster of Enterococcus faecalis is largely spread in Clostridium difficile. Since emergence of vancomycin resistance would have dramatic clinical consequences, we have evaluated the capacity of the vanGCd cluster to confer resistance. We showed that expression of vanGCd is inducible by vancomycin and that VanGCd, VanXYCd and VanTCd are functional, exhibiting D‐Ala : D‐Ser ligase, D,D‐dipeptidase and D‐Ser racemase activities respectively. In other bacteria, these enzymes are sufficient to promote vancomycin resistance. Trans‐complementation of C. difficile with the vanC resistance operon of Enterococcus gallinarum faintly impacted the MIC of vancomycin, but did not promote vancomycin resistance in C. difficile. Sublethal concentration of vancomycin led to production of UDP‐MurNAc‐pentapeptide[D‐Ser], suggesting that the vanGCd gene cluster is able to modify the peptidoglycan precursors. Our results indicated amidation of UDP‐MurNAc‐tetrapeptide, UDP‐MurNAc‐pentapeptide[D‐Ala] and UDP‐MurNAc‐pentapeptide[D‐Ser]. This modification is passed on the mature peptidoglycan where a muropeptide Tetra‐Tetra is amidated on the meso‐diaminopimelic acid. Taken together, our results suggest that the vanGCd gene cluster is functional and is prevented from promoting vancomycin resistance in C. difficile.

Functional and Structural Characterization of PaeM, a Colicin M-like Bacteriocin Produced by Pseudomonas aeruginosa

Background: Pathogenic Pseudomonas aeruginosa strains produce a colicin M-like bacteriocin exhibiting peptidoglycan lipid II-degrading activity. Results: We have determined the crystal structure of the Pseudomonas aeruginosa PaeM bacteriocin and functionally characterized its C-terminal activity domain. Conclusion: This study highlights structural plasticity of the active site of this enzyme family. Significance: The PaeM pyocin could potentially be exploited as antibacterial agent. Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg2+ ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria.

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-A2pm: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-A2pm-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-A2pm-d-Ala-d-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.