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Infection due to multi-resistant bacteria is a public health concern. Unfortunately, the prospect of developing new antibiotics seem not to be on the horizon. Faced with this impasse, medicinal plants could be an alternative for the development of new molecules. The aim of this study was to determine the inhibitory effect of H. madagascariensis extracts on β-lactamases. Extraction of bioactive compounds from trunk barks and leaves of the plant was done in an ethanol-water mixture (70:30). Anti-β-lactamase activity was evaluated by spectrophotometry after the removal of tannins and a phytochemical screening was used to identify the groups of compounds. Concentrations inhibiting 50% of enzyme activity were 0.005±0.001 mg/mL (CTX-M-15), 0.01±0.001 mg/mL (P99) and 0.027±0.009 mg/mL (NDM-1) for bark extracts and 0.704 mg/mL for leaves extracts. Phytochemical screening revealed the presence of flavonoids in bark extracts. The ethanolic extracts of the trunk bark exert a good inhibitory activity on CTX-M-15, P99 and NDM-1 β-lactamases and this activity could be attributed to the presence of the flavonoids. Further studies by bio-guided fractionation of the ethanolic extracts of the bark could yield fractions with high inhibitory potential of β-lactamases.

International audience; Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-β-lactamases (MBLs), which inactivate virtually all β-lactams, increasingly contribute to this phenomenon. The aim of this study is to characterize VIM-52, a His224Arg variant of VIM-1, identified in a Klebsiella pneumoniae clinical isolate. VIM-52 conferred lower MICs to cefepime and ceftazidime as compared to VIM-1. These results were confirmed by steady state kinetic measurements, where VIM-52 yielded a lower activity towards ceftazidime and cefepime but not against carbapenems. Residue 224 is part of the L10 loop (residues 221-241), which borders the active site. As Arg 224 and Ser 228 are both playing an important and interrelated role in enzymatic activity, stability and substrate specificity for the MBLs, targeted mutagenesis at both positions were performed and further confirmed their crucial role for substrate specificity.

In Gram-negative bacteria, the major mechanism of resistance to β-lactam antibiotics is the production of one or several β-lactamases (BLs), including the highly worrying carbapenemases. Whereas inhibitors of these enzymes were recently marketed, they only target serine-carbapenemases (e.g. KPC-type), and no clinically useful inhibitor is available yet to neutralize the class of metallo-β-lactamases (MBLs). We are developing compounds based on the 1,2,4-triazole-3-thione scaffold, which binds to the di-zinc catalytic site of MBLs in an original fashion, and we previously reported its promising potential to yield broad-spectrum inhibitors. However, up to now only moderate antibiotic potentiation could be observed in microbiological assays and further exploration was needed to improve outer membrane penetration. Here, we synthesized and characterized a series of compounds possessing a diversely functionalized alkyl chain at the 4-position of the heterocycle. We found that the presence of a carboxylic group at the extremity of an alkyl chain yielded potent inhibitors of VIM-type enzymes with Ki values in the μM to sub-μM range, and that this alkyl chain had to be longer or equal to a propyl chain. This result confirmed the importance of a carboxylic function on the 4-substituent of 1,2,4-triazole-3-thione heterocycle. As observed in previous series, active compounds also preferentially contained phenyl, 2-hydroxy-5-methoxyphenyl, naphth-2-yl or m-biphenyl at position 5. However, none efficiently inhibited NDM-1 or IMP-1. Microbiological study on VIM-2-producing E. coli strains and on VIM-1/VIM-4-producing multidrug-resistant K. pneumoniae clinical isolates gave promising results, suggesting that the 1,2,4-triazole-3-thione scaffold worth continuing exploration to further improve penetration. Finally, docking experiments were performed to study the binding mode of alkanoic analogues in the active site of VIM-2.

Background: The aim of this work was to understand the molecular features that trigger the cross-reactivity observed between Der p 5 from Dermatophagoides pteronyssinus, Blo t 5 from Blomia tropicalis, and Der f 5 from D. farinae. Methods: We collected serum from 60 house dust mite (HDM)-allergic patients residing in the Dellys area of Boumerdès province in northern Algeria. The presence of specific IgE to Der p 5, Der f 5, and Blo t 5 was analyzed. We performed in silico analysis of the structure of the different allergens in order to identify epitopes that can elicit the cross-reactivity of the sera. Synthetic peptides corresponding to the linear epitope sequence of Der p 5, Der f 5, and Blo t 5 were used to evaluate its implication in the cross-reactivity between the allergens. We also modified the sequence of the conformational epitope of Der p 5 by site-directed mutagenesis to mimic Blo t 5. Results: Several sera of patients allergic to HDM contained specific IgE antibodies to Der p 5 and Blo t 5. We demonstrated that the linear epitope of Der p 5 and Blo t 5 is not involved in the cross-reactivity of the sera. Furthermore, mutations introduced in the sequence of Der p 5 to mimic Blo t 5 could not modulate the cross-reactivity between them. Conclusions: The major linear IgE epitopes of Der p 5 and Blo t 5 are involved in species-specific recognition. Our results may be useful for the development of a hypoallergenic vaccine against HDM group 5 allergens.

Der p 5 is one of the important house dust mite allergens in Algeria; this allergen is frequently recognized by patients with allergic asthma. However, there is no information on its IgG–binding epitopes. In the present study, rabbits were immunized with recombinant Der p 5 allergen, and serum samples were obtained. Recognition of linear IgG epitopes of Der p 5 was determined using synthesized peptides derived from the allergen sequence. The results showed that serum from immunized rabbits recognized three linear epitopes from Der p 5 (28EDKKHDYQNEFDFLLMERIHEQIK43), (37IHEQIKKGELALFYLQEQ55) and (92LMQRKDLDIFEQYNLEMAKKS112). More interestingly, we observed that the 92L-S112 amino acid sequence is well recognized by both IgE and IgG antibodies. Der p 5 stimulates the synthesis of specific IgG antibodies which recognize common but also novel epitopes compared to IgE antibody binding. Indeed, the potential to induce IgG antibodies can be used to inhibit human IgE binding to allergens which may be part of the mechanism of action of specific immunotherapy. Keywords: Allergen, blocking antibodies, Der p 5, house dust mite, IgG-binding epitope

The chromosome-encoded class C β-lactamase CHE-1 produced by Enterobacter cloacae exhibits a lower sensitivity to avibactam than the P99 enzyme from which it is derived by a 6-residue deletion in the H-10 helix. In the present study, we investigated the sensitivity of CHE-1 to two other β-lactamase inhibitors: LK-157 (or Lek 157), a tricyclic β-lactam, and BAL29880, a bridged monobactam. With both compounds, the second-order rate constants for inactivation were significantly lower for CHE-1, which can thus be considered an inactivator-resistant mutant of P99. However, the second-order rate constant for the inactivation by BAL29880 probably remains adequate for a rather rapid reaction with CHE-1 in the absence of protection by the substrate.

The discovery of new glycoside hydrolases that can be utilized in the chemoenzymatic synthesis of carbohydrates has emerged as a promising approach for various biotechnological processes. In this study, recombinant Ps_Cel5A from Pseudomonas stutzeri A1501, a novel member of the GH5_5 subfamily, was expressed, purified and crystallized. Preliminary experiments confirmed the ability of Ps_Cel5A to catalyze transglycosylation with cellotriose as a substrate. The crystal structure revealed several structural determinants in and around the positive subsites, providing a molecular basis for a better understanding of the mechanisms that promote and favour synthesis rather than hydrolysis. In the positive subsites, two nonconserved positively charged residues (Arg178 and Lys216) were found to interact with cellobiose. This adaptation has also been reported for transglycosylating β-mannanases of the GH5_7 subfamily., SCOPUS: ar.j, info:eu-repo/semantics/published

Functional analysis of the HMA4 protein C-terminal extension reveals a similar and key function of high affinity zinc binding by di-Cys motifs in both Arabidopsis thaliana and A. halleri., The PIB ATPase heavy metal ATPase 4 (HMA4) has a central role in the zinc homeostasis network of Arabidopsis thaliana. This membrane protein loads metal from the pericycle cells into the xylem in roots, thereby allowing root to shoot metal translocation. Moreover, HMA4 is key for zinc hyperaccumulation as well as zinc and cadmium hypertolerance in the pseudometallophyte Arabidopsis halleri. The plant-specific cytosolic C-terminal extension of HMA4 is rich in putative metal-binding residues and has substantially diverged between A. thaliana and A. halleri. To clarify the function of the domain in both species, protein variants with truncated C-terminal extension, as well as with mutated di-Cys motifs and/or a His-stretch, were functionally characterized. We show that di-Cys motifs, but not the His-stretch, contribute to high affinity zinc binding and function in planta. We suggest that the HMA4 C-terminal extension is at least partly responsible for protein targeting to the plasma membrane. Finally, we reveal that the C-terminal extensions of both A. thaliana and A. halleri HMA4 proteins share similar function, despite marginally different zinc-binding capacity.

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used β-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP β-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the β-lactamase activity. This suggests that the nanobody and β-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid β-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has become a major priority of biotech and pharmaceutical industries. Recently, a growing number of modified antibody-based products have emerged including fragments, multi-specific and conjugate antibodies. In this study, using protein engineering, we have functionalized the anti-hen egg-white lysozyme (HEWL) camelid VHH antibody fragment (cAb-Lys3), by insertion into a solvent-exposed loop of the Bacillus licheniformis β-lactamase BlaP. We showed that the generated hybrid protein conserved its enzymatic activity while the displayed nanobody retains its ability to inhibit HEWL with a nanomolar affinity range. Then, we successfully implemented the functionalized cAb-Lys3 in enzyme-linked immunosorbent assay, potentiometric biosensor and drug screening assays. The hybrid protein was also expressed on the surface of phage particles and, in this context, was able to interact specifically with HEWL while the β-lactamase activity was used to monitor phage interactions. Finally, using thrombin-cleavage sites surrounding the permissive insertion site in the β-lactamase, we reported an expression system in which the nanobody can be easily separated from its carrier protein. Altogether, our study shows that insertion into the BlaP β-lactamase constitutes a suitable technology to functionalize nanobodies and allows the creation of versatile tools that can be used in innovative biotechnological assays.

SummaryBackground The allergen Der p 3 is underrepresented in house dust mite (HDM) extracts probably due to autolysis. Recombinant stable molecule of the allergen is thus needed to improve the diagnosis of allergy and the safety and efficacy of immunotherapy. Objective The current study reports the immunological characterization of two recombinant molecules of the HDM allergen Der p 3 as useful tools for diagnosis and immunotherapy. Methods Recombinant mature (rDer p 3) and immature (proDer p 3) Der p 3 and their corresponding S196A mutants were produced in Pichia pastoris and purified. The stability, IgE-binding capacity and allergenicity of the different proteins were analysed and compared with those of the major mite allergen Der p 1 used as a reference. Additionally, the immunogenicity of the different allergens was evaluated in a murine model of Der p 3 sensitization. Results Compared to the IgE reactivity to recombinant and natural Der p 3 (nDer p 3), the mean IgE binding of patient's sera to rDer p 3-S196A (50%) was higher. The poorly binding to nDer p 3 or rDer p 3 was due to autolysis of the allergen. Contrary to Der p 3, proDer p 3 displayed very weak IgE reactivity, as measured by sandwich ELISA and competitive inhibition, rat basophil leukaemia degranulation and human basophil activation assays. Moreover, proDer p 3 induced a TH1-biased immune response that prevented allergic response in mice but retained Der p 3-specific T-cell reactivity. Conclusion rDer p 3-S196A should be used for the diagnosis of HDM allergy elicited by Der p 3, and proDer p 3 may represent a hypoallergen of Der p 3.

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by 31P and 1H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TFHis6) of Escherichia coli. The active fusion enzyme was named TagK-TFHis6. Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TFHis6 enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated.

52nd International Conference on Medicinal Chemistry (RICT) of the French-Medicinal-Chemistry-Society (SCT) - Interfacing Chemical Biology and Drug Discovery -- JUL 06-08, 2016 -- Caen, FRANCE, WOS: 000403905300014, PubMed ID: 28505394, Metallo-beta-lactamases (MBLs) cause resistance of Gram-negative bacteria to beta-lactam antibiotics and are of serious concern, because they can inactivate the last-resort carbapenems and because MBL inhibitors of clinical value are still lacking. We previously identified the original binding mode of 4-amino-2,4-dihydro-5-(2-methylphenyl)-3H-1,2,4-triazole-3-thione (compound IIIA) within the dizinc active site of the L1 MBL. Herein we present the crystallographic structure of a complex of L1 with the corresponding non-amino compound IIIB (1,2-dihydro-5-(2-methylphenyl)-3H-1,2,4-triazole-3-thione). Unexpectedly, the binding mode of IIIB was similar but reverse to that of IIIA. The 3D structures suggested that the triazolethione scaffold was suitable to bind to the catalytic site of dizinc metalloenzymes. On the basis of these results, we synthesized 54 analogues of IIIA or IIIB. Nineteen showed IC50 values in the micromolar range toward at least one of five representative MBLs (i.e., L1, VIM-4, VIM-2, NDM-1, and IMP-1). Five of these exhibited a significant inhibition of at least four enzymes, including NDM-1, VIM2, and IMP-1. Active compounds mainly featured either halogen or bulky bicyclic aryl substituents. Finally, some compounds were also tested on several microbial dinuclear zinc-dependent hydrolases belonging to the MBL-fold superfamily (i.e., endonucleases and glyoxalase II) to explore their activity toward structurally similar but functionally distinct enzymes. Whereas the bacterial tRNases were not inhibited, the best IC50 values toward plasmodial glyoxalase II were in the 10 mm range., French Med Chem Soc, Univ Caen, Agence Nationale de la Recherche ("ANTIMBL") [ANR-14-CE16-0028-01]; Deutsche Forschungsgemeinschaft [BE1540/15-2 within SPP 1710]; Agence Nationale de la Recherche [ANR-06-BLAN-0086], We thank Mr. Pierre Sanchez for mass spectrometry analyses and Wolfram Meyer-Klaucke for advice about tRNase Z. Part of this work was supported by the Agence Nationale de la Recherche ("ANTIMBL", ANR-14-CE16-0028-01, including a fellowship to L.S.), the Deutsche Forschungsgemeinschaft (BE1540/15-2 within SPP 1710 to K.B.), and Agence Nationale de la Recherche ("subtilRNA", ANR-06-BLAN-0086) to C.C.

PER-2 accounts for up to 10% of oxyimino-cephalosporin resistance in Klebsiella pneumoniae and Escherichia coli in Argentina and hydrolyzes both cefotaxime and ceftazidime with high catalytic efficiencies (kcat/Km). Through crystallographic analyses, we recently proposed the existence of a hydrogen bond network connecting Ser70-Gln69-oxyanion water-Thr237-Arg220 that might be important for the activity and inhibition of the enzyme. Mutations at Arg244 in most class A β -lactamases (such as TEM and SHV) reduce susceptibility to mechanism-based inactivators, and Arg220 in PER β -lactamases is equivalent to Arg244. Alterations in the hydrogen bond network of the active site in PER-2, through modifications in key residues such as Arg220 and (to a much lesser extent) Thr237, dramatically impact the overall susceptibility to inactivation, with up to 300-And 500-fold reductions in the rate constant of inactivation (kinact)/Kivalues for clavulanic acid and tazobactam, respectively. Hydrolysis on cephalosporins and aztreonam was also affected, although to different extents compared to with wild-Type PER-2; for cefepime, only an Arg220Gly mutation resulted in a strong reduction in the catalytic efficiency. Mutations at Arg220 entail modifications in the catalytic activity of PER-2 and probably local perturbations in the protein, but not global conformational changes. Therefore, the apparent structural stability of the mutants suggests that these enzymes could be possibly selected in vivo. Fil: Ruggiero, Melina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Curto, Lucrecia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina Fil: Brunetti, Florencia Lourdes. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Sauvage, Eric. Université de Liège; Bélgica Fil: Galleni, Moreno. Université de Liège; Bélgica Fil: Power, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina

Objectives Two natural variants of ADC-type β-lactamases of Acinetobacter spp., ADC-1 and ADC-5, differ by nine mutations in their protein sequence. ADC-5 hydrolyses cefoxitin better than ADC-1 and the opposite is true for ceftazidime. We produced single and combined mutations in ADC-5 and characterized the variants microbiologically and biochemically to determine which amino acid residues are involved in the hydrolysis of β-lactam antibiotics in this family of β-lactamases. Methods Site-directed mutagenesis, with blaADC-5 as a source of DNA, was used to generate nine single mutated and three combined mutated enzymes. The proteins (wild-type and derivatives) were then expressed in isogenic conditions in Escherichia coli. MICs of β-lactams were determined using Etest strips. ADC-1, ADC-5, ADC-5-P167S and ADC-5-P167S/D242G/Q163K/G342R were also purified and the kinetic parameters determined for ceftazidime, cefoxitin, cefalotin and ampicillin. Results Single mutations did not significantly convert the hydrolysis spectrum of the ADC-5 enzyme into that of the ADC-1 enzyme, although among all studied mutants only the quadruple mutant (ADC-5-P167S/D242G/Q163K/G342R) displayed microbiological and biochemical properties consistent with those of ADC-1. Conclusions Although some single mutations are known to affect cefepime hydrolysis in ADC-type β-lactamases, little is known about ceftazidime and cefoxitin hydrolysis in this family of β-lactamases. Hydrolysis of these antibiotics appears to be positively and negatively affected, respectively, by the Q163K, P167S, D242G and G342R amino acid replacements.

We report the first identification of a gene cluster involved in d -tagatose catabolism in Bacillus licheniformis . The pathway is closely related to the d -tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca , in contrast to the d -tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus .

The CphAII protein from the hyperthermophile Aquifex aeolicus shows the five conserved motifs of the metallo-β-lactamase (MBL) superfamily and presents 28% identity with the Aeromonas hydrophila subclass B2 CphA MBL. The gene encoding CphAII was amplified by PCR from the A. aeolicus genomic DNA and overexpressed in Escherichia coli using a pLex-based expression system. The recombinant CphAII protein was purified by a combination of heating (to denature E. coli proteins) and two steps of immobilized metal affinity chromatography. The purified enzyme preparation did not exhibit a β-lactamase activity but showed a metal-dependent phosphodiesterase activity versus bis-p-nitrophenyl phosphate and thymidine 5'-monophosphate p-nitrophenyl ester, with an optimum at 85°C. The circular dichroism spectrum was in agreement with the percentage of secondary structures characteristic of the MBL αββα fold.

Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from diseases (such as Alzheimer's and Parkinson's diseases) to the production (e.g. inclusion bodies), stability, storage and delivery of protein drugs. β-Cyclodextrin (β-CD) is a circular heptasaccharide characterized by a hydrophilic exterior and a hydrophobic interior ring structure. In this research, we studied the effects of a chemically modified β-CD (BCD07056), on the aggregating and refolding properties of BlaPChBD, a hybrid protein obtained by inserting the chitin binding domain of the human macrophage chitotriosidase into the class A β-lactamase BlaP from Bacillus licheniformis 749/I during its thermal denaturation. The results show that BCD07056 strongly increases the refolding yield of BlaPChBD after thermal denaturation and constitutes an excellent additive to stabilize the protein over time at room temperature. Our data suggest that BCD07056 acts early in the denaturation process by preventing the formation of an intermediate which leads to an aggregated state. Finally, the role of β-CD derivatives on the stability of proteins is discussed.

This work analyses the chitin-binding and catalytic domains of the human macrophage chitotriosidase and investigates the physiological role of this glycoside hydrolase in a complex mechanism such as the innate immune system, especially its antifungal activity. Accordingly, we first analyzed the ability of its chitin-binding domain to interact with chitin embedded in fungal cell walls using the β-lactamase activity reporter system described in our previous work. The data showed that the chitin-binding activity was related to the cell wall composition of the fungi strains and that their peptide-N-glycosidase/zymolyase treatments increased binding to fungal by increasing protein permeability. We also investigated the antifungal activity of the enzyme against Candida albicans. The antifungal properties of the complete chitotriosidase were analyzed and compared with those of the isolated chitin-binding and catalytic domains. The isolated catalytic domain but not the chitin-binding domain was sufficient to provide antifungal activity. Furthermore, to explain the lack of obvious pathologic phenotypes in humans homozygous for a widespread mutation that renders chitotriosidase inactive, we postulated that the absence of an active chitotriosidase might be compensated by the expression of another human hydrolytic enzyme such as lysozyme. The comparison of the antifungal properties of chitotriosidase and lysozyme indicated that surprisingly, both enzymes have similar in vitro antifungal properties. Furthermore, despite its more efficient hydrolytic activity on chitin, the observed antifungal activity of chitotriosidase was lower than that of lysozyme. Finally, this antifungal duality between chitotriosidase and lysozyme is discussed in the context of innate immunity.

The metallo-β-lactamase VIM-4, mainly found in Pseudomonas aeruginosa or Acinetobacter baumannii , was produced in Escherichia coli and characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn 2+ at concentrations ranging from 0.4 to 100 μM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 Å. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn 2+ ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure.

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Klebsiella pneumoniae ML0306 was isolated from a patient in the intensive care unit of the Military hospital of Tunis in Tunisia. The strain was resistant to β-lactams, aminoglycosides, quinolones, phenicols and tetracyclines. Crude extract from the isolate showed a broad-substrate profile by hydrolyzing benzylpenicillin, ampicillin, cloxacilline, cephalothin, cefotaxime and ceftriaxone but not ceftazidime, cefoxitin, cefpirome, imipinem and aztreonam. The isolate was identified as a producer of pI 7.6 β-lactamase. Sequencing of the β-lactamase gene showed that it coded for a penicillinase SHV-11. This is the first description of this enzyme in Tunisia. The gene encoding this enzyme was located on the chromosome and a 50 kb conjugative plasmid. The overproduction of SHV-11 was involved in the wading of the hydrolytic spectrum of the strain.

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been described yet, and the zymogen maturation mechanism remains to be elucidated. The Der p 3 zymogen was produced in Pichia pastoris. We demonstrated that the recombinant zymogen is glycosylated at the level of its propeptide. We showed that the activation mechanism of proDer p 3 is intermolecular and is mediated by the house dust mite cysteine protease Der p 1. The primary structure of the proDer p 3 propeptide is associated with a unique zymogen activation mechanism, which is different from those described for the trypsin-like family and relies on the house dust mite papain-like protease Der p 1. This is the first report of a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. Glycosylation of the propeptide was found to decrease the rate of maturation. Finally, we showed that recombinant Der p 3 is inhibited by the free modified prosequence TP1R.

Among the Enterobacteriaceae, Proteus vulgaris is exceptional in the inducible production of a 29-kDa β-lactamase (cefuroximase) with an unusually high activity towards the β-lactamase-stable oximino-cephalosporins (e.g. cefuroxime and cefotaxime). Sequencing of the corresponding gene, cumA, showed that the derived CumA β-lactamase belonged to the molecular class A. The structural gene was under the direct control of gene cumR, which was transcribed backwards and whose initiation codon was 165 bp away from that of the β-lactamase gene. This resembled the arrangement of structural and regulator genes ampC and ampR of the 39-kDa molecular-class-C β-lactamase AmpC present in many enterobacteria. Moreover, cloned genes ampD and ampG for negative modulation and signal transduction of AmpC β-lactamase induction, respectively, were also able to restore constitutively CumA overproducing and non-inducible P. vulgaris mutants to the inducible, wild-type phenotype. The results indicate that controls of the induction phenomena are equivalent for the CumA and AmpC β-lactamase. Very different structural genes can thus be under the control of identical systems.

The major allergen Der p 1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) produced as an inactive precursor and associated with allergic diseases. The propeptide of Der p 1 exhibits a specific fold that makes it unique in the CA1 propeptide family. In this study, we investigated the activation steps involved in the maturation of the recombinant protease Der p 1 expressed in Pichia pastoris and the interaction of the full-length and truncated soluble propeptides with their parent enzyme in terms of activity inhibition and BIAcore interaction analysis. According to our results, the activation of protease Der p 1 is a multistep mechanism that is characterized by at least two intermediates. The propeptide strongly inhibits unglycosylated and glycosylated recombinant Der p 1 ( K D = 7 nM) at neutral pH. This inhibition is pH dependent. It decreases from pH 7 to pH 4 and can be related to conformational changes of the propeptide characterized by an increase of its flexibility and formation of a molten globule state. Our results indicate that activation of the zymogen at pH 4 is a compromise between activity preservation and propeptide unfolding.

Using genetic engineering technologies, the chitin-binding domain (ChBD) of the human macrophage chitotriosidase has been inserted into the host protein BlaP, a class A beta-lactamase produced by Bacillus licheniformis. The product of this construction behaved as a soluble chimeric protein that conserves both the capacity to bind chitin and to hydrolyze beta-lactam moiety. Here we describe the biochemical and biophysical properties of this protein (BlaPChBD). This work contributes to a better understanding of the reciprocal structural and functional effects of the insertion on the host protein scaffold and the heterologous structured protein fragments. The use of BlaP as a protein carrier represents an efficient approach to the functional study of heterologous protein fragments.

Overgrowth of Clostridium perfringens clones with production of one or more of its toxin(s) results in diverse digestive and systemic pathologies in human and animals, such as cattle enterotoxaemia. The so-called beta2 toxin (CPB2) is the most recently described major toxin produced by C. perfringens. In this study, the cpb2 ORF (cpb2FM) from a cattle C. perfringens-associated enterotoxaemia was cloned and sequenced. The cpb2FM and its deduced nucleotide sequence clearly corresponded to the cpb2 allele considered as "consensus" and not to "atypical" allele, despite its "non-porcine" origin. Expression assays of the recombinant toxin CPB2FM were performed in Escherichia coli and Bacillus subtilis with the expression vector pBLTS72, and by genomic integration by double recombination in B. subtilis. Highest level of production was obtained with the expression vector in B. subtilis 168 strain. The recombinant CPB2FM protein was purified and a specific rabbit polyclonal antiserum was produced. Polyclonal antibodies could detect CPB2 production in supernatants of C. perfringens from enterotoxaemic cattle.

This study characterised the genetic environment of the chromosomally encoded bla(KLUA-9) gene from a clinical Kluyvera ascorbata isolate and performed a kinetic characterisation of KLUA-9. Purified KLUA-9 showed the highest catalytic efficacies towards benzylpenicillin, ampicillin, piperacillin, first-generation cephalosporins, cefuroxime and cefoperazone; like other 'cefotaximases', it showed a much higher rate of hydrolysis of cefotaxime than ceftazidime, whilst dicloxacillin, cefoxitin and imipenem behaved as poor substrates. A 9kb insert from K. ascorbata was cloned (Escherichia coli KK68C1) and sequenced. bla(KLUA-9) and its 266bp upstream flanking region (almost identical to the integron-associated bla(CTX-M-2)) are preceded by an aspat variant, a ypdABC-like operon and two open reading frames with unknown functions. Unlike ISCR1-associated bla(CTX-M-2) genes, we failed to detect the putative orf513 recombination sites. Instead, we were able to localise the 5bp target sites for insertion of ISEcp1B, suggesting that this element could be responsible for future (or still undetected) mobilisation of bla(KLUA-9) to more efficiently transferred elements.

Clostridium perfringens has been implicated in a broad array of enteric infections including the fatal haemorrhagic enteritis/enterotoxaemia syndrome in cattle. The beta2 toxin (CPB2), encoded by cpb2 , is suspected to be implicated in this syndrome. However, among C. perfringens isolates from cattle suspected of clostridial disease, an atypical allele was recently found to predominate at the cpb2 locus and atypical corresponding CPB2 proteins were shown to be poorly expressed, thus arguing against a biologically significant role of the beta2 toxin in clostridial diseases in cattle. This study compared genotype and phenotype of the beta2 toxin between C. perfringens isolates from a group of healthy calves ( n = 14, 87 isolates) and from a group of enterotoxaemic calves ( n = 8, 41 isolates). PCR results revealed the exclusive presence of the typical “consensus” cpb2 in the enterotoxaemic group. Western blot analysis demonstrated that the typical variant of CPB2 was often expressed in isolates from enterotoxaemic calves (43.9%) and infrequently in isolates from healthy cattle (6.9%). These data suggest that the typical variant of the CPB2 toxin may play a role in the pathogenesis of cattle enterotoxaemia.

PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases.

Mycobacteria are important causes of infectious diseases. Although Mycobacterium tuberculosis and Mycobacterium leprae, two slow-growing species, are responsible for the most serious diseases, some fast-growing species, such as Mycobacterium avium, Mycobacterium kansasii, Mycobacterium chelonae, and Mycobacterium fortuitum, may cause opportunistic infections among AIDS patients (24). In addition, M. fortuitum has been reported to be responsible for a wide spectrum of clinical diseases, such as skin or soft tissue infections following surgery, pulmonary infections, accidental penetrating trauma, and wounds that come in contact with soil or water contaminated with these mycobacteria, though nosocomial infections are by far the most common (41, 44). The cell wall of mycobacteria contains mycolic acid, arabinogalactan, and peptidoglycan, forming a covalent complex. The influx of small hydrophilic agents through the resulting low-permeability envelope is extremely slow and is thought to be one of the major factors involved in the resistance of mycobacteria to β-lactam antibiotics (21, 41). β-Lactamase production, catalyzing β-lactam antibiotic hydrolysis, appears to be the second mechanism by which mycobacteria express β-lactam resistance (9, 21, 35). Chromosomally encoded β-lactamases have been detected in most, but not all, mycobacterial species, including M. fortuitum and M. tuberculosis (43). The class A β-lactamase of M. fortuitum (MFO) is a chromosomally encoded broad-spectrum β-lactamase hydrolyzing both cephalosporins and penicillins. Its substrate profile includes ureidopenicillins (piperacillin, azlocillin, and mezlocillin), carbenicillin, cephalothin, cefotaxime, and cefuroxime, but not ceftazidime. Cefoxitin, dicloxacillin, imipenem, and aztreonam are poorly recognized by the enzyme, and the protein is inhibited by the penem inhibitor BRL42715 and, to a lesser extent, clavulanic acid. The specificity profile is similar overall to those of CTX-M-type enzymes; some TEM-derived extended-spectrum β-lactamases (ESBLs); the chromosomally encoded ESBLs from Citrobacter sedlakii and Yersinia enterocolitica; and β-lactamases identified in the genera Nocardia, Kluyvera, and Burkholderia (1, 12, 31, 33, 35, 37). At the amino-acid level, the mature protein shares 70% identity with the β-lactamase of Mycobacterium smegmatis and between 40 and 45% with the aforementioned proteins. MFO also shares 41% amino acid identity with the ESBL Toho-1. Many recent studies have been concerned with the structure solution of TEM, SHV, or CTX-M-type ESBLs, but few structural data are available on chromosomally encoded broad-spectrum class A β-lactamases. MFO is the first solved structure of a mycobacterial β-lactamase. Its structure-activity relationship was analyzed on the basis of a comparison between its structure and recently solved ESBL structures (5, 27, 29, 38).

The heterologous production in Escherichia coli , the purification, and the kinetic characterization of four plasmid-encoded class C β-lactamases (ACT-1, MIR-1, CMY-2, and CMY-1) were performed. Except for their instability, these enzymes are very similar to the known chromosomally encoded AmpC β-lactamases. Their kinetic parameters did not show major differences from those obtained for the corresponding chromosomal enzymes. However, the K m values of CMY-2 for cefuroxime, cefotaxime, and oxacillin were significantly decreased compared to those of the chromosomal AmpC enzymes. Finally, the susceptibility patterns of different E. coli hosts producing a plasmid- or a chromosome-encoded class C enzyme toward β-lactam antibiotics are mainly due to the overproduction of the β-lactamase in the periplasmic space of the bacteria rather than to a specific catalytic profile of the plasmid-encoded β-lactamases.

Among class B beta-lactamases, the subclass B2 CphA enzyme is characterized by a unique specificity profile. CphA efficiently hydrolyzes only carbapenems. In this work, we generated site-directed mutants that possess a strongly broadened activity spectrum when compared with the WT CphA. Strikingly, the N116H/N220G double mutant exhibits a substrate profile close to that observed for the broad spectrum subclass B1 enzymes. The double mutant is significantly activated by the binding of a second zinc ion under conditions where the WT enzyme is non-competitively inhibited by the same ion.

A method was developed for the determination of the stoichiometric Zn/protein ratio for the Aeromonas hydrophila metallo beta-lactamase AE036. Capillary electrophoresis (CE) was used to separate free Zn ions from the Zn bound to the metalloprotein, the only Zn-species which is biochemically relevant. An inductively coupled plasma-sector field-mass spectrometer (ICP-SF-MS), operated at a mass resolution of 3000, was used as an element-specific detector. If the primary structure of the protein and thus the number of sulfur-containing amino acids is known, as is the case for Aeromonas hydrophila Zn beta-lactamase, monitoring of both Zn and S with ICP-SF-MS permits the Zn/protein ratio to be determined. The method was optimised in order to obtain short migration times (less than 10 min) for the metallo beta-lactamase and for standards with bare fused silica capillaries. After optimisation, a 75 mmol L−1 bis-Tris background electrolyte buffered at a pH of 6.75 was used for the separation, a voltage of 30 kV was applied over a capillary with a total length of approximately 70 cm and the sample was introduced hydrodynamically at 1000 mbar s. Quantitative results (stoichiometric Zn/protein ratio of 1 and 2, before and after saturation of the enzyme with Zn, respectively) were obtained using external calibration with albumin as a S standard and ZnCl2 as a Zn standard. The method was also demonstrated to be precise with < 5% RSD on the Zn/protein ratio for n = 3. An absolute limit of detection of 8 ng of A. hydrophila Zn beta-lactamase (calculated on the basis of sulfur) was obtained under these conditions.

We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-β-lactamase subclasses, B1 (BcII from Bacillus cereus and BlaB from Chryseobacterium meningosepticum), B2 (CphA from Aeromonas hydrophila), and B3 (L1 from Stenotrophomonas maltophilia). By competition experiments with metal-free apoenzymes and chromophoric zinc chelators or EDTA, we determined the dissociation constants in the absence and presence of substrates. For the formation of the monozinc enzymes we determined constants of 1.8, 5.1, 0.007, and 2.6 nm in the absence and 13.6, 1.8, 1.2, and 5.7 pm in the presence of substrates for BcII, BlaB, CphA, and L1, respectively. A second zinc ion binds in the absence (presence) of substrates with considerably higher dissociation constants, namely 1.8 (0.8), 0.007 (0.025), 50 (1.9), and 0.006 (0.12) μm for BcII, BlaB, CphA, and L1, respectively. We have concluded that the apo form might be the prevailing state of most of the metallo-β-lactamases under physiological conditions in the absence of substrates. Substrate availability induces a spontaneous self-activation due to a drastic decrease of the dissociation constants, resulting in the formation of active mononuclear enzymes already at picomolar free zinc ion concentrations. In the presence of substrates, the binuclear state of the enzymes only exists at unphysiologic high zinc concentrations and might be of no biological relevance. From the competition experiments with EDTA it is further concluded that the reactivation rate does not depend on the pool of free zinc ions but proceeds via the EDTA-Zn(II)-enzyme ternary complexes.

Metallo-β-lactamases are emerging resistance determinants among gram-negative pathogens (15-17, 20). They can contribute to the intrinsic β-lactam resistance of some species that carry resident metallo-β-lactamase genes (e.g., Bacillus cereus, a cluster of Bacteroides fragilis, Stenotrophomonas maltophilia, some Aeromonas spp., Chryseobacterium meningosepticum, Chryseobacterium indologenes, and Legionella gormanii) and to acquired β-lactam resistance in Enterobacteriaceae, pseudomonads, and other nonfastidious gram-negative nonfermenters following acquisition of metallo-β-lactamase determinants carried by mobile genetic elements (3). Metallo-β-lactamases are included in molecular class B and in group 3 of the functional classification (5). Remarkable structural and functional heterogeneities exist among these enzymes. Structural analysis identified three major molecular lineages, subclasses B1, B2, and B3, with a degree of sequence divergence that can be higher than 90% between members of different subclasses (14, 19). Functional analysis revealed variable substrate specificities and heterogeneous kinetic behaviors and identified three different subgroups, determined to be 3a (showing a broad substrate specificity), 3b (acting as specific carbapenemases), and 3c (overall preferring cephalosporin substrates) (22). However, a constant feature of metallo-β-lactamases that contributes to their clinical relevance is the ability to efficiently hydrolyze carbapenem compounds, which are not hydrolyzed by the majority of active-site serine β-lactamases (4). Although some of these enzymes have been subjected to detailed biochemical (2, 11-13, 19) and structural analysis (6, 7, 8, 9), the catalytic mechanism(s), interaction with metal, and structure-function relationships are only partially understood. Additional biochemical and structural analyses are essential to the understanding of these aspects and are also strategic to the development of new metallo-β-lactamase inhibitors. In this work we developed a system for overproduction and purification of the BlaB metallo-β-lactamase of C. meningosepticum, and we carried out a biochemical characterization of the enzyme.

The stability of cefcapene and cefpodoxime, oral antibacterial cephalosporins, toward different classes of beta-lactamases was evaluated. For the class A beta-lactamases, TEM-1, SHV-1, and NMC-A, only the steady-state kinetic parameter ( k(cat)/ Km) values were calculated (3100 - 1.1 x 10(7) M(-1) x s(-1)), because these enzymes have very high Km values for cefpodoxime and cefotaxime. As for class B beta-lactamases L1, IMP-1, and CcrA, in general, similar k(cat)/ Km values were obtained. However, regarding class C beta-lactamases from Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, and Citrobacter freundii, we found major differences in stability between the two compounds. Cefpodoxime acted as a good substrate for the class C beta-lactamases, except for the enzyme from E. cloacae; its k(cat) and Km values were successfully calculated ( k(cat)/ Km, 1.8 x 10(5) - 1.2 x 10(7) M(-1) x s(-1)). On the other hand, cefcapene acted as a poor substrate or an inactivator for class C beta-lactamases; its k(2)/ K value was successfully calculated (8.7 x 10(5) - 7.0 x 10(6) M(-1) x s(-1)). In addition, k(3) values were determined for beta-lactamases from P. aeruginosa (2.3 x 10(-2) x s(-1)) and C. freundii (2.1 x 10(-1) x s(-1)). Even though these values could be calculated, transient inactivation as an enzyme reactivation reaction for all these enzymes was observed. These findings suggest the potential of cephem compounds as inhibitors of class C beta-lactamases.

Klebsiella pneumoniaeML2011, a multiresistant isolate, was isolated from the Military Hospital of Tunis (Tunisia). The determination of the minimal inhibitory concentrations exhibited byK. pneumoniaeML2011 was performed by Etest. The crude extract of the isolates contains four differentβ-lactamases with pI 5.5, 7.3, 7.6, and 8.6. Only theβ-lactamases with pI 7.3 and pI 8.6 were transferred by transformation and conjugation experiment. Molecular characterization of these genes was performed by PCR and sequencing. The chromosomalβ-lactamases are TEM (pI 5.5) and SHV-1 (7.6). CTX-M-28 (pI 8.6) and the novel variant of SHV named SHV-104 (pI 7.3) were encoded byblagene located on a 50 kb highly conjugative plasmid. The SHV-104β-lactamase was produced inE. coliand purified. Its profile of activity was determined. Compared to SHV-1, SHV-104 contains one mutation, R202S. Their kinetic parameters were similar except for cefotaxime. The analysis of the predicted structure of SHV-104 indicated that the R202S mutation suppresses a salt bridge present in SHV-1. Therefore, the overall flexibility of the protein increased and might improve the hydrolysis of cefotaxime. We can conclude that the multiresistant phenotype ofK. pneumoniaeML2011 strain is mainly linked to the production of CTX-M-28 since SHV-104 possesses a narrow spectrum of activity.

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII β-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those β-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 β-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of β-lactamases.

CENTA, a chromogenic cephalosporin, is readily hydrolyzed by β-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of β-lactamase-producing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.

The bla FEZ-1 gene coding for the metallo-β-lactamase of Legionella (Fluoribacter) gormanii ATCC 33297 T was overexpressed via a T7 expression system in Escherichia coli BL21(DE3)(pLysS). The product was purified to homogeneity in two steps with a yield of 53%. The FEZ-1 metallo-β-lactamase exhibited a broad-spectrum activity profile, with a preference for cephalosporins such as cephalothin, cefuroxime, and cefotaxime. Monobactams were not hydrolyzed. The β-lactamase was inhibited by metal chelators. FEZ-1 is a monomeric enzyme with a molecular mass of 29,440 Da which possesses two zinc-binding sites. Its zinc content did not vary in the pH range of 5 to 9, but the presence of zinc ions modified the catalytic efficiency of the enzyme. A model of the FEZ-1 three-dimensional structure was built.

Beta-lactamases are involved in bacterial resistance. Members of the metallo-enzyme class are now found in many pathogenic bacteria and are becoming thus of major clinical importance. Despite the availability of Zn-beta-lactamase X-ray structures their mechanism of action is still unclear. One puzzling observation is the presence of one or two zincs in the active site. To aid in assessing the role of zinc content in beta-lactam hydrolysis, the replacement by Ser of the zinc-liganding residue Cys168 in the Zn-beta-lactamase from Bacillus cereus strain 569/H/9 was carried out: the mutant enzyme (C168S) is inactive in the mono-Zn form, but active in the di-Zn form. The structure of the mono-Zn form of the C168S mutant has been determined at 1.85 A resolution. Ser168 occupies the same position as Cys168 in the wild-type enzyme. The protein residues mostly affected by the mutation are Asp90-Arg91 and His210. A critical factor for the activity of the mono-Zn species is the distance between Asp90 and the Zn ion, which is controlled by Arg91: a slight movement of Asp90 impairs catalysis. The evolution of a large superfamily including Zn-beta-lactamases suggests that they may not all share the same mechanism.

In addition to the BlaB metallo-β-lactamase, Chryseobacterium ( Flavobacterium ) meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine β-lactamase, named CME-1, with an alkaline isoelectric pH. The blaA CME gene that encodes the latter enzyme was isolated from a genomic library constructed in the Escherichia coli plasmid vector pACYC184 by screening for cefuroxime-resistant clones. Sequence analysis revealed that the CME-1 enzyme is a new class A β-lactamase structurally divergent from the other members of this class, being most closely related to the VEB-1 (also named CEF-1) and PER β-lactamases and the Bacteroides chromosomal cephalosporinases. The blaA CME determinant is located on the chromosome and exhibits features typical of those of C. meningosepticum resident genes. The CME-1 protein was purified from an E. coli strain that overexpresses the cloned gene via a T7-based expression system by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. Kinetic parameters for several substrates were determined. CME-1 is a clavulanic acid-susceptible extended-spectrum β-lactamase that hydrolyzes most cephalosporins, penicillins, and monobactams but that does not hydrolyze cephamycins and carbapenems. The enzyme exhibits strikingly different kinetic parameters for different classes of β-lactams, with both K m and k cat values much higher for cephalosporins than for penicillins and monobactams. However, the variability of both kinetic parameters resulted in overall similar acylation rates ( k cat / K m ratios) for all types of β-lactam substrates.

Although β-lactamases have generally been considered as being devoid of peptidase activity, a low but significant hydrolysis of various N-acylated dipeptides was observed with representatives of each class of β-lactamases. The kcat/Km values were below 0.1 M-1˙s-1, but the enzyme rate enhancement factors were in the range 5000-20000 for the best substrates. Not unexpectedly, the best ‘peptidase’ was the class C β-lactamase of Enterobacter cloacae P99, but, more surprisingly, the activity was always higher with the phenylacetyl- and benzoyl-D-Ala-D-Ala dipeptides than with the diacetyl- and α-acetyl-L-Lys-D-Ala-D-Ala tripeptides, which are the preferred substrates of the low-molecular-mass, soluble DD-peptidases. A comparison between the β-lactamases and DD-peptidases showed that it might be as difficult for a DD-peptidase to open the β-lactam ring as it is for the β-lactamases to hydrolyse the peptides, an observation which can be explained by geometric and stereoelectronic considerations.

Background The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation cascade of their corresponding precursor forms and particularly to highlight the interconnection between proteases during this cascade. Methods The cleavage of the four peptides corresponding to the mite zymogen activation sites was studied on the basis of the Forster Resonance Energy Transfer method. The proDer p 6 zymogen was then produced in Pichia pastoris to elucidate its activation mechanism by mite proteases, especially Der p 1. The role of the propeptide in the inhibition of the enzymatic activity of Der p 6 was also examined. Finally, the Der p 1 and Der p 6 proteases were localised via immunolocalisation in D. pteronyssinus. Results All peptides were specifically cleaved by Der p 1, such as proDer p 6. The propeptide of proDer p 6 inhibited the proteolytic activity of Der p 6, but once cleaved, it was degraded by the protease. The Der p 1 and Der p 6 proteases were both localised to the midgut of the mite. Conclusions Der p 1 in either its recombinant form or in the natural context of house dust mite extracts specifically cleaves all zymogens, thus establishing its role as a major activator of both mite cysteine and serine proteases. General significance This finding suggests that Der p 1 may be valuable target against mites. © 2013 Elsevier B.V.

A gene identical to plasmid-borne bla CTX-M-3 is present in the chromosome of one Kluyvera ascorbata strain. It is associated with a structure including an inverted repeat right and an open reading frame 477-like gene probably involved in the mobilization of bla CTX-M-3 . Two other K. ascorbata strains rendered the previously described bla KLUA-9 gene.

Objectives: Our aim was to unravel the inactivation pathway of the class A β-lactamase produced by Bacillus licheniformis BS3 (BS3) by clavulanate. Methods: The interaction between clavulanate and BS3 was studied by X-ray crystallography, pre-steady-state kinetics and mass spectrometry. Results: The analysis of the X-ray structure of the complex yielded by the reaction between clavulanate and BS3 indicates that the transient inactivated form, namely the cis-trans enamine complex, is hydrolysed to an ethane-imine ester covalently linked to the active site serine and a pentan-3-one-5-ol acid. It is the first time that this mechanism has been observed in an inactivated β-lactamase. Furthermore, the ionic interactions made by the carboxylic group of pentan-3-one-5-ol may provide an understanding of the decarboxylation process of the trans-enamine observed in the non-productive complex observed for the interaction between clavulanate and SHV-1 and Mycobacterium tuberculosis β-lactamase (Mtu). Conclusions: This work provides a comprehensive clavulanate hydrolysis pathway accounting for the observed acyl-enzyme structures of class A β-lactamase/clavulanate adducts. Fil: Power, Pablo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Mercuri, Paola. Université de Liège; Bélgica Fil: Herman, Raphaël. Université de Liège; Bélgica Fil: Kerff, Frédéric. Université de Liège; Bélgica Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Dive, Georges. Université de Liège; Bélgica Fil: Galleni, Moreno. Université de Liège; Bélgica Fil: Charlier, Paulette. Université de Liège; Bélgica Fil: Sauvage, Eric. Université de Liège; Bélgica

Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis, mainly in cats, dogs and humans. Proteolytic enzymes have been postulated to be key factors involved in the invasion of the stratum corneum and keratinized epidermal structures. Among these proteases, the secreted subtilisin protease Sub3 was found to be required for adherence of M. canis arthroconidia to feline epidermis. This protease is synthetized as a preproenzyme consisting of a signal peptide followed by the propeptide and the protease domain. In order to assess whether the enzymatic activity of Sub3 could be responsible for the role of the protease in the adherence process, we expressed and characterized the propeptide of Sub3 and demonstrated that this propeptide is a strong inhibitor of its mature enzyme. This propeptide acts as a noncompetitive inhibitor with dissociation constants, Ki and K?i of 170 and 130 nM respectively. When tested for its capacity to inhibit adherence of M. canis to feline epidermis using an ex vivo adherence model made of feline epidermis, the propeptide does not prevent adherence of M. canis arthroconidia because it loses its capacity to inhibit rSub3 following a direct contact with living arthroconidia, presumably through inactivation by fungal membrane-bound proteases. Fil: Baldo, A.. Université de Liège; Bélgica Fil: Chevigné, A.. Université de Liège; Bélgica Fil: Dumez, M. E.. Université de Liège; Bélgica Fil: Mathy, A.. Université de Liège; Bélgica Fil: Power, Pablo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Université de Liège; Bélgica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Tabart, J.. Université de Liège; Bélgica Fil: Cambier, L.. Université de Liège; Bélgica Fil: Galleni, Lorena Alejandra. Université de Liège; Bélgica Fil: Mignon, María Belen. Université de Liège; Bélgica

A beta-lactamase produced by Pseudomonas stutzeri was purified to protein homogeneity, and its physicochemical and catalytic properties were determined. Its profile was unusual since, in addition to penicillins, the enzyme hydrolysed second- and third-generation ‘beta-lactamase-stable’ cephalosporins and monobactams with similar efficiencies. On the basis of the characteristics of the interaction with beta-iodopenicillanic acid, the enzyme could be classified as a class-A beta-lactamase. However, when compared with most class-A beta-lactamases, it exhibited significantly lower kcat./Km values for the compounds usually considered to be the best substrates of these enzymes.

While TEM- and SHV-derived extended-spectrum β-lactamases (ESBLs) arose by point mutations in genes encoding broad-spectrum variants that were extensively disseminated into mobile elements, CTX-M β-lactamases are derived from natural (chromosomal) counterparts that already have oxyimino-cephalosporinase activity (11). To date, the CTX-M/KLU β-lactamases account for nearly 110 representatives clustered in five main groups (http://www.lahey.org/studies/webt.asp): CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25. A hypothetical chromosomal counterpart for the CTX-M-25 subfamily is still missing. Kluyvera georgiana 14751 was isolated from a bloodstream infection in a 62-year-old male patient through the SENTRY Antimicrobial Surveillance Program in Louisville, KY, on 2 December 2002. Biochemical classification, ambiguous between Kluyvera georgiana and Kluyvera ascorbata, was resolved by 16S rRNA gene sequencing (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AM933755","term_id":"193227311","term_text":"AM933755"}}AM933755), displaying 99.7% identity with the sole available sequence from K. georgiana ATCC 51603 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF047186","term_id":"3273703","term_text":"AF047186"}}AF047186). Identity to all deposited K. ascorbata sequences ranged from 97.4 to 98.5% (6). The strain was resistant to ampicillin, gentamicin, nalidixic acid (Table ​(Table1)1) and trimethoprim-sulfamethoxazole (TMP-SMX) (by disc diffusion). Conjugation to Escherichia coli {"type":"entrez-protein","attrs":{"text":"CAG12177","term_id":"47220029","term_text":"CAG12177"}}CAG12177 resulted in E. coli CK10 harboring an IncFII group conjugative plasmid of ca. 30 kb (pTC10; partial sequence deposited in EMBL under accession no. {"type":"entrez-nucleotide","attrs":{"text":"FN568351","term_id":"290964838","term_text":"FN568351"}}FN568351), responsible for TMP-SMX and aminoglycoside resistance (Table ​(Table1)1) by a class 1 integron harboring dfrA17 and aadA5 gene cassettes (previously reported individually in different Kluyvera intermedia isolates; EMBL accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU523051","term_id":"171188351","term_text":"EU523051"}}EU523051 and {"type":"entrez-nucleotide","attrs":{"text":"EU523052","term_id":"171188353","term_text":"EU523052"}}EU523052). Upstream of the integron we found a blaTEM-1b gene followed by a region, including the Tn3-tnpR, the first 91 bp of Tn3-tnpA, and an IS26 which is also found in plasmids carrying some blaCTX-M genes. However, no CTX-M could be recovered from the transferred plasmid. TABLE 1. MICs of antibiotics for Kluyvera georgiana 14751, the recipient strain, and the derived recombinant clone Partially EcoRI-digested chromosomal DNA from K. georgiana 14751 was cloned in a pK19 vector (Kanr) and transformed into E. coli Top10F′ (E. coli TKE14751-1KA2), yielding pTKE-1KA2 with an 8-kb insert (Fig. ​(Fig.1)1) harboring a bla gene (876 bp) which encodes a novel CTX-M-78 (EMBL accession no. {"type":"entrez-nucleotide","attrs":{"text":"AM982522","term_id":"209413817","term_text":"AM982522"}}AM982522) closely related to CTX-M-39 (96.2% amino acid identity) (1) and clustered in the CTX-M-25 subgroup. As seen in Table ​Table1,1, the MIC increases for cefotaxime, while that of ceftazidime is almost unaffected, correlating with the expected (and experimental [data not shown]) kinetics for most CTX-M enzymes. FIG. 1. Schematic representation of the insert of pTKE-1KA2 from Kluyvera georgiana 14751 (top) and comparison with available sequences harboring blaCTX-M-25 (middle) and blaCTX-M-26 (bottom). As in other chromosome-encoded blaCTX-M/KLU, a 1,227-bp att-like gene encoding a putative aspartate aminotransferase is located upstream, with at least 96% identity with other entries from Kluyvera. Downstream, an orf3-like gene similar to a putative ttrR response regulator from Kluyvera georgiana (5) and similar to the orf3 version from the complex class 1 integrons associated with blaCTX-M-2 (found as a fusion gene orf3::qacEΔ1) (7), and an orf4-like gene encoding a putative autotransporter in Kluyvera ascorbata (2), were also found. In contrast to other kluyveras, in which sequences similar to IRr-ISEcp1 were found downstream of blaCTX-M/KLU (8), we did not observe any sequence that could serve as putative right inverted repeats (IRrs) there (Fig. ​(Fig.1).1). A 35-bp sequence seems to represent the boundary between the chromosome-derived information and the plasmid-acquired information. In blaCTX-M-25 and blaCTX-M-26 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF518567","term_id":"37789822","term_text":"AF518567"}}AF518567 and {"type":"entrez-nucleotide","attrs":{"text":"AY455830","term_id":"38373216","term_text":"AY455830"}}AY455830, respectively) (4), the immediate upstream region is occupied by ISEcp1, also associated with other blaCTX-M genes (3). A putative 5-bp target site for ISEcp1B (AATAC) was found immediately upstream of the 35-bp sequence in the chromosomal DNA from K. georgiana 14751. CTX-M-78 possesses high similarity with members of the CTX-M-25 subgroup, making this enzyme the closest representative to be considered one of the probable progenitors of the subfamily.