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International audience; ObjectivesWe previously described extended-spectrum oxacillinase OXA-145 from Pseudomonas aeruginosa, which differs from narrow-spectrum OXA-35 by loss of Leu-155. The deletion results in loss of benzylpenicillin hydrolysis and acquisition of activity against ceftazidime. We report the crystal structure of OXA-145 and provide the basis of its switch in substrate specificity.MethodsOXA-145 variants were generated by site-directed mutagenesis and purified to homogeneity. The crystal structure of OXA-145 was determined and molecular dynamics simulations were performed. Kinetic parameters were investigated in the absence and in the presence of sodium hydrogen carbonate (NaHCO3) for representative substrates.ResultsThe structure of OXA-145 was obtained at a resolution of 2.3 Å and its superposition with that of OXA-10 showed that Trp-154 was shifted by 1.8 Å away from the catalytic Lys-70, which was not N-carboxylated. Addition of NaHCO3 significantly increased the catalytic efficiency against penicillins, but not against ceftazidime. The active-site cavity of OXA-145 was larger than that of OXA-10, which may favour the accommodation of large molecules such as ceftazidime. Molecular dynamics simulations of OXA-145 in complex with ceftazidime revealed two highly coordinated water molecules on the α- or β-face of the acyl ester bond, between Ser-67 and ceftazidime, which could be involved in the catalytic process.ConclusionsDeletion of Leu-155 resulted in inefficient positioning of Trp-154, leading to a non-carboxylated Lys-70 and thus to loss of hydrolysis of the penicillins. Ceftazidime hydrolysis could be attributed to enlargement of the active site and to a catalytic mechanism independent of the carboxylated Lys-70.

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.

International audience; An infant botulism clinical course was characterized by several relapses despite therapy with amoxicillin and metronidazole. Botulism was confirmed by identification of botulinum toxin and C. botulinum in stools. A C. botulinum A2 strain resistant to penicillins and with heterogeneous resistance to metronidazole was isolated from stool samples up to 110 days after onset. Antibiotic susceptibility was tested by disk agar diffusion and minimum inhibitory concentrations were determined by Etest. Whole genome sequencing allowed detection of a gene cluster composed of blaCBP for a novel penicillinase, blaI for a regulator, and blaR1 for a membrane bound penicillin receptor in the chromosome of the C. botulinum isolate. The purified recombinant penicillinase was assayed. Resistance to β-lactams was in agreement with the kinetic parameters of the enzyme. In addition, the β-lactamase gene cluster was found in three C. botulinum genomes in databanks and in two out of 62 genomes of our collection, all the strains belonging to group I C. botulinum. This is the first report of a C. botulinum isolate resistant to penicillins. This stresses the importance of antibiotic susceptibility testing for adequate therapy of botulism.

International audience; OBJECTIVES: To describe a novel extended-spectrum oxacillinase, named OXA-145, differing from narrow-spectrum OXA-35 (from the OXA-10 group) by deletion of residue Leu-165. The genetic environment of bla(OXA-145) and the biochemical properties of OXA-145 are reported. We also assessed the impact of the Leu-165 deletion on the hydrolysis spectrum of the ancestor OXA-10. METHODS: Extended-spectrum β-lactamase OXA-145 was identified in the multidrug-resistant clinical Pseudomonas aeruginosa 08-056, and characterized by isoelectric focusing, PCR and DNA sequencing. Antibiotic susceptibility tests were performed by agar dilution. The resistance profiles conferred by cloned bla(OXA-10), bla(OXA-35), bla(OXA-145) and a bla(OXA-10) derivative obtained by site-directed mutagenesis were determined in Escherichia coli. Kinetic parameters of OXA-35 and OXA-145 were established after purification of His-tagged proteins. RESULTS: The sequence of OXA-145, encoded by a class 1 integron-borne gene in strain 08-056, differed from that of narrow-spectrum penicillinase OXA-35 by a single amino acid deletion (Leu-165) located in the highly conserved omega loop. Deletion of Leu-165 from OXA-35 (yielding OXA-145) or OXA-10 (the progenitor of OXA-35) extended the hydrolysis spectrum to third-generation cephalosporins and to monobactams, while reducing that for penicillins. OXA-145 showed biphasic hydrolysis curves for all the substrates tested. Its activity against nitrocefin was 10-fold higher in the presence of sodium hydrogen carbonate. CONCLUSIONS: OXA-145 is a new extended-spectrum β-lactamase from the OXA-10 group. The deletion of Leu-165 is responsible for a shift in the hydrolysis spectrum from penicillins to third-generation cephalosporins, as well as monobactams. The loss of penicillin hydrolysis was due to a non-carboxylated Lys-73.

Rahnella aquatilis is an environmental enterobacterial species with a chromosomal bla(RAHN-1) gene encoding extended-spectrum class A beta-lactamase RAHN-1. We describe the diversity of bla(RAHN) genes from two groups of strains, G1 and G2, isolated from raw fruits and vegetables, and the new class A beta-lactamase RAHN-2.MICs were determined by Etest. bla(RAHN) genes were amplified by PCR, sequenced, and cloned to produce RAHN-1 and RAHN-2 proteins whose kinetic parameters were determined.All strains had similar beta-lactam resistance patterns. However, isolates of G1 were at least 2-fold more susceptible to piperacillin, amoxicillin, piperacillin/clavulanic acid, piperacillin/tazobactam and cefotaxime. Sequences of bla(RAHN) from G1 had82.9% identity with that of bla(RAHN-1), whereas those of G2 were92% identical. The RAHN-2 beta-lactamase was 89.8% identical to RAHN-1, 5-fold more efficient than RAHN-1 in hydrolysing ticarcillin and 2.5-fold more efficient in cefotaxime and cefuroxime hydrolysis. However, the specific activity of RAHN-1 was 2-fold higher than that of RAHN-2 suggesting that the bla(RAHN) genes are regulated differently.The new class A beta-lactamase RAHN-2 is phenotypically difficult to detect and requires MIC determination.

VanA-type Staphylococcus aureus strain VRSA-7 was partially dependent on glycopeptides for growth. The vanA gene cluster, together with the erm (A) and the ant(9)-Ia resistance genes, was carried by the ca. 35- to 40-kb conjugative plasmid pIP848 present at five copies per cell. The chromosomal ddl gene had a mutation that led to a N 308 K substitution in the d -Ala: d -Ala ligase that resulted in a 1,000-fold decrease in activity relative to that of strain VRSA-6. Strain VRSA-7 grown in the absence or in the presence of vancomycin mainly synthesized precursors ending in d -Ala- d -Lac, indicating that the strain relied on the vancomycin resistance pathway for peptidoglycan synthesis. Greatly enhanced growth in the presence of glycopeptides and the absence of mutations in the genes for VanR and VanS indicated the inducible expression of resistance. Thus, a combination of loose regulation of the vanA operon by the two-component system and a gene dosage effect accounts for the partial glycopeptide dependence of VRSA-7. Since peptidoglycan precursors ending in d -Ala- d -Lac are not processed by PBP 2′, the strain was fully susceptible to oxacillin, despite the production of a wild-type PBP 2′.

Brachyspira pilosicoliBM4442, isolated from the feces of a patient with diarrhea at the Hospital Saint-Michel in Paris, was resistant to oxacillin (MIC > 256 μg/ml) but remained susceptible to cephalosporins and to the combination of amoxicillin and clavulanic acid. Cloning and sequencing of the corresponding resistance determinant revealed a coding sequence of 807 bp encoding a new class D β-lactamase named OXA-63. TheblaOXA-63gene was chromosomally located and not part of a transposon or of an integron. OXA-63 shared 54% identity with FUS-1 (OXA-85), an oxacillinase fromFusobacterium nucleatumsubsp.polymorphum, and 25 to 44% identity with other class D β-lactamases (DBLs) and contained all the conserved structural motifs of DBLs.Escherichia colicarrying theblaOXA-63gene exhibited resistance to benzylpenicillin and amoxicillin but remained susceptible to amoxicillin in combination with clavulanic acid. Mature OXA-63 consisted of a 31.5-kDa polypeptide and appeared to be dimeric. Kinetic analysis revealed that OXA-63 exhibited a narrow substrate profile, hydrolyzing oxacillin, benzylpenicillin, and ampicillin with catalytic efficiencies of 980, 250, and 150 mM−1s−1, respectively. The enzyme did not apparently interact with oxyimino-cephalosporins, imipenem, or aztreonam. Unlike FUS-1 and other DBLs, OXA-63 was strongly inhibited by clavulanic acid (50% inhibitory concentration [IC50] of 2 μM) and tazobactam (IC50of 0.16 μM) and exhibited low susceptibility to NaCl (IC50of >2 M). OXA-63 is the first DBL described for the anaerobic spirocheteB. pilosicoli.

Carnobacterium divergens clinical isolates BM4489 and BM4490 were resistant to penicillins but remained susceptible to combinations of amoxicillin-clavulanic acid and piperacillin-tazobactam. Cloning and sequencing of the responsible determinant from BM4489 revealed a coding sequence of 912 bp encoding a class A β-lactamase named CAD-1. The bla CAD-1 gene was assigned to a chromosomal location in the two strains that had distinct pulsed-field gel electrophoresis patterns. CAD-1 shared 53% and 42% identity with β-lactamases from Bacillus cereus and Staphylococcus aureus , respectively. Alignment of CAD-1 with other class A β-lactamases indicated the presence of 25 out of the 26 isofunctional amino acids in class A β-lactamases. Escherichia coli harboring bla CAD-1 exhibited resistance to penams (benzylpenicillin and amoxicillin) and remained susceptible to amoxicillin in combination with clavulanic acid. Mature CAD-1 consisted of a 34.4-kDa polypeptide. Kinetic analysis indicated that CAD-1 exhibited a narrow substrate profile, hydrolyzing benzylpenicillin, ampicillin, and piperacillin with catalytic efficiencies of 6,600, 3,200, and 2,900 mM −1 s −1 , respectively. The enzyme did not interact with oxyiminocephalosporins, imipenem, or aztreonam. CAD-1 was inhibited by tazobactam (50% inhibitory concentration [IC 50 ] = 0.27 μM), clavulanic acid (IC 50 = 4.7 μM), and sulbactam (IC 50 = 43.5 μM). The bla CAD-1 gene is likely to have been acquired by BM4489 and BM4490 as part of a mobile genetic element, since it was not found in the susceptible type strain CIP 101029 and was adjacent to a gene for a resolvase.

Vancomycin resistance in Gram-positive bacteria results from the replacement of the d-alanyl–d-alanine target of peptidoglycan precursors with d-alanyl–d-lactate or d-alanyl–d-serine (d-Ala-d-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of d-Ala-d-Ser-terminating precursors by converting l-Ser to d-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in l-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 was determined. The structure showed significant similarity to type III pyridoxal 5′-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for the l-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against l-Ser versus l-Ala implied that this enzyme relies on its membrane-bound domain for l-Ser transport to increase the overall rate of d-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria., IMPORTANCE Vancomycin is one of the drugs of last resort against Gram-positive antibiotic-resistant pathogens. However, bacteria have evolved a sophisticated mechanism which remodels the drug target, the d-alanine ending precursors in cell wall synthesis, into precursors terminating with d-lactate or d-serine, to which vancomycin has less affinity. d-Ser is synthesized by VanT serine racemase, which has two unusual characteristics: (i) it is one of the few serine racemases identified in bacteria and (ii) it contains a membrane-bound domain involved in l-Ser uptake. The structure of the catalytic domain of VanTG showed high similarity to alanine racemases, and we identified three specific active site substitutions responsible for l-Ser specificity. The data provide the molecular basis for VanT evolution to a bifunctional enzyme coordinating both transport and racemization. Our findings also illustrate the evolution of the essential alanine racemase into a vancomycin resistance enzyme in response to antibiotic pressure.

Antimicrobial peptide plectasin targeting bacterial cell wall precursor Lipid II has been reported to be active against benzylpenicillin-resistant Streptococcus pneumoniae but less potent against vancomycin-resistant enterococci than their susceptible counterparts. The aim of this work was to test plectasin NZ2114 in combination with cell wall targeting antibiotics on vancomycin-resistant Enterococcus faecalis. The activity of antibiotic combinations was evaluated against VanA-type vancomycin-resistant E. faecalis strain BM4110/pIP816-1 by disk agar-induction, double-disk assay, determination of fractional inhibitory concentration (FIC) index, and time-kill curve. The results indicated that plectasin NZ2114 was synergistic in combination with teicoplanin, moenomycin, and dalbavancin but not with vancomycin, telavancin, penicillin G, bacitracin, ramoplanin, daptomycin, and fosfomycin. To gain an insight into the synergism, we tested other cell wall antibiotic combinations. Interestingly, synergy was observed between teicoplanin or moenomycin and the majority of the antibiotics tested; however, vancomycin was only synergistic with penicillin G. Other cell wall active antibiotics such as ramoplanin, bacitracin, and fosfomycin did not synergize. It appeared that most of the synergies observed involved inhibition of the transglycosylation step in peptidoglycan synthesis. These results suggest that teicoplanin, dalbavancin, vancomycin, and telavancin, although they all bind to the C-terminal D-Ala-D-Ala of Lipid II, might act on different stages of cell wall synthesis.

International audience; The VanA D-Ala:D-Lac ligase is a key enzyme in the emergence of high level resistance to vancomycin in Enterococcus species and methicillin-resistant Staphylococcus aureus. It catalyzes the formation of D-Ala-D-Lac instead of the vancomycin target, D-Ala-D-Ala, leading to the production of modified, low vancomycin binding affinity peptidoglycan precursors. Therefore, VanA appears as an attractive target for the design of new antibacterials to overcome resistance. The catalytic site of VanA is delimited by three domains and closed by an ω-loop upon enzymatic reaction. The aim of the present work was (i) to investigate the conformational transition of VanA associated with the opening of its ω-loop and of a part of its central domain and (ii) to relate this transition with the substrate or product binding propensities. Molecular dynamics trajectories of the VanA ligase of Enterococcus faecium with or without a disulfide bridge distant from the catalytic site revealed differences in the catalytic site conformations with a slight opening. Conformations were clustered with an original machine learning method, based on self-organizing maps (SOM), which revealed four distinct conformational basins. Several ligands related to substrates, intermediates, or products were docked to SOM representative conformations with the DOCK 6.5 program. Classification of ligand docking poses, also performed with SOM, clearly distinguished ligand functional classes: substrates, reaction intermediates, and product. This result illustrates the acuity of the SOM classification and supports the quality of the DOCK program poses. The protein-ligand interaction features for the different classes of poses will guide the search and design of novel inhibitors.

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.

The synthesis of the title compound is described as well as a series of derivatives with wide biological applications

International audience; Although trypanothione [T(S)2] is the major thiol component in trypanosomatidae, significant amounts of glutathione are present in Trypanosoma cruzi. This could be explained by the existence of enzymes using glutathione or both glutathione and T(S)2 as cofactors. To assess these hypotheses, a cytosolic fraction of T. cruzi epimastigotes was subjected to affinity chromatography columns using as ligands either S-hexylglutathione or a non-reducible analogue of trypanothione disulphide. A similar protein of 52 kDa was eluted in both cases. Its partial amino acid sequence indicated that it was identical with the protein encoded by the TcAc2 cDNA previously described [Schoneck, Plumas-Marty, Taibi et al. (1994) Biol. Cell 80, 1-10]. This protein showed no significant glutathione transferase activity but surprisingly catalysed the thiol-disulphide exchange between dihydrotrypanothione and glutathione disulphide. The kinetic parameters were in the same range as those determined for trypanothione reductase toward its natural substrate. This trypanothione-glutathione thioltransferase provides a new target for a specific chemotherapy against Chagas' disease and may constitute a link between the glutathione-based metabolism of the host and the trypanothione-based metabolism of the parasite.

International audience; d-Alanyl:d-lactate (d-Ala:d-Lac) and d-alanyl:d-serine ligases are key enzymes in vancomycin resistance of Gram-positive cocci. They catalyze a critical step in the synthesis of modified peptidoglycan precursors that are low binding affinity targets for vancomycin. The structure of the d-Ala:d-Lac ligase VanA led to the understanding of the molecular basis for its specificity, but that of d-Ala:d-Ser ligases had not been determined. We have investigated the enzymatic kinetics of the d-Ala:d-Ser ligase VanG from Enterococcus faecalis and solved its crystal structure in complex with ADP. The overall structure of VanG is similar to that of VanA but has significant differences mainly in the N-terminal and central domains. Based on reported mutagenesis data and comparison of the VanG and VanA structures, we show that residues Asp-243, Phe-252, and Arg-324 are molecular determinants for d-Ser selectivity. These residues are conserved in both enzymes and explain why VanA also displays d-Ala:d-Ser ligase activity, albeit with low catalytic efficiency in comparison with VanG. These observations suggest that d-Ala:d-Lac and d-Ala:d-Ser enzymes have evolved from a common ancestral d-Ala:d-X ligase. The crystal structure of VanG showed an unusual interaction between two dimers involving residues of the omega loop that are deeply anchored in the active site. We constructed an octapeptide mimicking the omega loop and found that it selectively inhibits VanG and VanA but not Staphylococcus aureus d-Ala:d-Ala ligase. This study provides additional insight into the molecular evolution of d-Ala:d-X ligases and could contribute to the development of new structure-based inhibitors of vancomycin resistance enzymes.

Bioorg. Med. Chem. Lett. ISI Document Delivery No.: MH050 Times Cited: 26 Cited Reference Count: 16 Tromelin, a moutiez, m mezianecherif, d aumercier, m tartar, a sergheraert, c; International audience; A good alternative substrate for the trypanothione reductase from Trypanosoma cruzi N,N'-bis(benzyloxycarbonyl)-L-cysteinylglycine 3-dimethylaminopropylamide disulfide, has been modified at the disulfide bridge to obtain non reducible derivatives. Cystine was replaced by three natural diamino diacids (djenkolic acid, lanthionine and cystathionine). The compounds were selective linear competitive inhibitors for trypanothione reductase versus trypanothione, its physiological substrate.

A naturally occurring fungal compound has been found to restore the susceptibility of bacteria to a class of antibiotic that is currently considered to be our last defence against serious infections. See Article p.503 Infection with Gram-negative pathogens bearing metallo-β-lactamases such as NDM-1 and VIM is a growing public health problem and threatens the use of penicillin, cephalosporin and carbapenem antibiotics to treat infections. Here, Gerard Wright and colleagues report a screen for naturally produced inhibitors of NDM-1 in an extensive collection of DMSO-dissolved natural product extracts derived from environmental microorganisms. One extract (from Aspergillus versicolor) exhibited a particularly potent anti-NDM-1 activity and was identified as aspergillomarasmine A (AMA), a natural product first reported some 50 years ago associated with leaf wilting. AMA is a rapid and potent inhibitor of both NDM-1 and VIM-2, and the authors find that AMA fully restores antibiotic efficacy in vitro and in vivo against bacterial pathogens possessing either VIM- or NDM-type resistance genes. AMA is non-toxic and well tolerated, making it a realistic prospect as an antibiotic adjuvant.

The vancomycin-resistant Staphylococcus aureus VRSA-9 clinical isolate was partially dependent on glycopeptide for growth. The responsible vanA operon had the same organization as that of Tn 1546 and was located on a plasmid. The chromosomal d -Ala: d -Ala ligase ( ddl ) gene had two point mutations that led to Q260K and A283E substitutions, resulting in a 200-fold decrease in enzymatic activity compared to that of the wild-type strain VRSA-6. To gain insight into the mechanism of enzyme impairment, we determined the crystal structure of VRSA-9 Ddl and showed that the A283E mutation induces new ion pair/hydrogen bond interactions, leading to an asymmetric rearrangement of side chains in the dimer interface. The Q260K substitution is located in an exposed external loop and did not induce any significant conformational change. The VRSA-9 strain was susceptible to oxacillin due to synthesis of pentadepsipeptide precursors ending in d -alanyl- d -lactate which are not substrates for the β-lactam-resistant penicillin binding protein PBP2′. Comparison with the partially vancomycin-dependent VRSA-7, whose Ddl is 5-fold less efficient than that of VRSA-9, indicated that the levels of vancomycin dependence and susceptibility to β-lactams correlate with the degree of Ddl impairment. Ddl drug targeting could therefore be an effective strategy against vancomycin-resistant S. aureus .

International audience; Acquired VanG-type resistance to vancomycin in Enterococcus faecalis BM4518 arises from inducible synthesis of peptidoglycan precursors ending in d-alanyld-serine, to which vancomycin exhibits low binding affinity. VanG, a d-alanine: d-serine ligase, catalyzes the ATP-dependent synthesis of the d-Ala-d-Ser dipeptide, which is incorporated into the peptidoglycan synthesis of VanG-type vancomycin-resistant strains. Here, the purification, crystallization and preliminary crystallographic analysis of VanG in complex with ADP are reported. The crystal belonged to space group P3 1 21, with unit-cell parameters a = b = 116.1, c = 177.2 Å , and contained two molecules in the asymmetric unit. A complete data set has been collected to 2.35 Å resolution from a single crystal under cryogenic conditions using synchrotron radiation.

The use of date juice as a substrate for lactic acid production was investigated. Various nitrogen sources were compared with yeast extract for efficient lactic acid production by Lactobacillus casei subsp. rhamnosus. Among different nitrogen sources added to date juice (yeast extract, ammonium sulfate, tryptic soy, urea, peptone, and casein hydrolysate), yeast extract was the most efficient. The effect of yeast extract could have been due to its B vitamin content. The addition of five B vitamins at less than 25 mg/l to date juice with any nitrogen source enhanced lactic acid production to some extent, except for date juice with yeast extract or urea or peptone. The most significant increase was obtained with ammonium sulfate. Half of the yeast extract content (10 g/l) in a supplemented date juice could be replaced by a mixture of B vitamins at less than 25 mg/l, and ammonium sulfate at 2.6 g/l with no significant decrease in lactic acid production.

International audience; Acquired resistance to glycopeptides in enterococci is associated with the production of D‐Alanine: D‐Alanine ligase‐related proteins. The VanA protein associated with high‐level vancomycin and teicoplanin resistance (VanA phenotype) synthesizes a new peptidoglycan precursor, D‐alanine‐D‐lactate, that has reduced glycopeptide affinity. Production of a similar protein, VanB, is induced in strains that display variable levels of vancomycin resistance but remain susceptible to teicoplanin (VanB phenotype). This paper describes the over‐production, purification and characterization of VanB. Comparison of kinetic parameters of the two Van enzymes suggests that differences in catalytic efficiency could account, at least in part, for the various levels of vancomycin resistance.

Emergence of high-level resistance to the last resort glycopeptide antibiotic vancomycin in Enterococcus spp. and its spread to methicillin-resistant Staphylococcus aureus is a public health threat. Resistance to vancomycin is due to substitution of the D-Ala-D-Ala terminus of cell wall precursors, which forms the antibiotic target, by D-Ala-D-Lac or D-Ala-D-Ser of low binding affinities. Resistance also requires depletion of the normal precursors catalyzed by the zinc-dependent D,D-peptidases VanX and VanY acting on dipeptide (D-Ala-D-Ala) or pentapeptide (UDP-MurNac-L-Ala-D-γ-Glu-L-Lys-D-Ala-D-Ala), respectively. Some resistance operons encode VanXY D,D-peptidase acting on both substrates. Van D,D-peptidases represent attractive targets for combinational antimicrobial therapies to curb resistance; however, the molecular basis of their specificity remains poorly understood, hindering development of potent inhibitors. Therefore we undertook detailed structure-function analysis of VanXY and VanY enzymes. Obtained structural information revealed the substrate-binding site of VanXYC as an extended cavity suitable for binding of di- or pentapeptides, contrasting with previous results showing that VanX contains a small, shallow active site. Biochemical and mutagenesis analysis identified a mobile cap over the catalytic site of VanXYC as the key structural element involved in a switch between di- and pentapeptide hydrolysis. The structures also provided the molecular basis for selectivity towards Van-susceptible peptidoglycan precursors. Overall, this study illustrates the adaptability of the D,D-peptidase fold in response to antibiotic pressure via evolution of particular structural elements that modulate substrate specificity. The results open new opportunities for structure-guided development of Van D,D-peptidases specific inhibitors as glycopeptides adjuvants. This project has been funded by NIAID under Contracts No. HHSN272200700058C and HHSN272201200026C.

International audience

In order to compare the non-enzymatic capacities of the xenobiotic conjugation of trypanothione (a spermidine-glutathione conjugate unique to kinetoplastidae) and glutathione, the reactivity of their respective thiols was investigated. The acido-basic properties of both compounds and their nucleophilicity toward Elliman's reagent and 1-chloro-2, 4-dinitrobenzene were studied. Our results show that although glutathione is a better nucleophile than trypanothione, the latter is more reactive because it is more ionized in a large pH range. This pH range likely includes the pH to which such conjugation reactions are expected to happen in vivo. Thus, the better conjugation capacity of trypanothione could make it the cornerstone for the xenobiotic detoxication of trypanosomatidae.